HK40060453A

HK40060453A - Selection of improved tumor reactive t-cells

Info

Publication number: HK40060453A
Application number: HK62022048292.6A
Authority: HK
Inventors: Simpson-Abelson Michelle; Natarajan Arvind; Chartier-Courtaud Cecile; PAULSON Matt
Original assignee: Iovance Biotherapeutics, Inc.
Priority date: 2018-11-05
Filing date: 2019-11-04
Publication date: 2022-05-20

Description

Improved selection of tumor-reactive T cells

Cross Reference to Related Applications

Priority of the present application is claimed in U.S. provisional patent application No. 62/756,006 filed on 5.11.2018, U.S. provisional patent application No. 62/826,831 filed on 29.3.2019, U.S. provisional patent application No. 62/903,629 filed on 20.9.2019, and U.S. provisional patent application No. 62/924,602 filed on 22.10.2019, which are hereby incorporated by reference in their entireties.

Background

Adoptive transfer of Tumor Infiltrating Lymphocytes (TILs) to treat large refractory cancers represents an effective therapeutic approach to treat patients with poor prognosis. Gattinoni et al, Nature immunology review (Nat. Rev. Immunol.) 2006,6, 383-393. Successful immunotherapy requires large amounts of TIL and commercialization requires robust and reliable processes. This has been a difficult challenge to achieve due to technical, logistical and regulatory issues with cell expansion. Because of its speed and efficiency, IL-2-based TIL amplification followed by a "Rapid amplification Process" (REP) has become the preferred method for TIL amplification. Dudley et al, Science 2002,298,850-54; ducley et al, journal of clinical oncology (j.clin.oncol.) 2005,23, 2346-57; dudley et al, J.Clin.Oncology 2008,26, 5233-39; riddell et al, science 1992,257,238-41; dudley et al, J.Immunotherapy 2003,26, 332-42. REP can produce a 1,000-fold expansion of TIL over a 14 day period, but it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as Monocytes (MNCs)), typically in the form of feeder cells from multiple donors, as well as anti-CD 3 antibody (OKT3) and a high dose of IL-2. Dudley et al, journal of immunotherapeutics 2003,26, 332-42. TILs undergoing the REP program produce successful adoptive cell therapy after host immunosuppression in patients with melanoma. Current infusion acceptance parameters depend on readings of TIL composition (e.g., CD28, CD8, or CD4 positive) and on fold amplification and viability of the REP product.

Current TIL manufacturing processes are limited by length, cost, sterility issues, and other factors described herein, such that the potential for commercializing such processes is severely limited. Although TILs have been characterized, for example, have been shown to express various receptors, including the inhibitory receptor programmed cell death 1 (PD-1; also known as CD279) (see, gross, a. et al, journal of clinical research 124(5): 2246-. There is an urgent need to provide TIL manufacturing processes and therapies based on such processes that are suitable for commercial scale manufacturing and regulatory approval for use in human patients in multiple clinical centers. The present invention fills this need by providing methods for pre-selecting TILs based on PD-1 expression in order to obtain TILs with enhanced tumor-specific killing capabilities (e.g., enhanced cytotoxicity).

Disclosure of Invention

The present invention provides methods for expanding TIL and generating a therapeutic TIL population, the methods comprising a PD-1 status pre-selection step.

In some embodiments, the present invention provides a method for expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(a) Obtaining and/or receiving a first TIL population from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

(b) selecting a PD-1 positive TIL from the first TIL population in (a) to obtain a population of PD-1 enriched TILs;

(c) performing a priming first expansion by culturing the PD-1 enriched TIL population in a cell culture medium comprising IL-2, OKT-3, and Antigen Presenting Cells (APCs) to produce a second TIL population, wherein the priming first expansion is performed in a vessel comprising a first gas permeable surface region, wherein the priming first expansion is performed for a first period of time of about 1 to 7/8 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population;

(d) performing a rapid second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and APCs to produce a third TIL population, wherein the number of APCs added in the rapid second expansion is at least twice the number of APCs added in step (b), wherein the rapid second expansion is performed for a second period of time of about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second expansion is performed in a container comprising a second gas permeable surface region;

(e) Collecting the therapeutic TIL population obtained from step (d); and

(f) transferring the collected TIL population from step (e) to an infusion bag.

a) obtaining and/or receiving a first TIL population from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

b) selecting a PD-1 positive TIL from the first TIL population in (a) to obtain a population of PD-1 enriched TILs;

c) priming a first expansion by culturing the PD-1 enriched TIL population in a cell culture medium comprising IL-2, OKT-3, and optionally Antigen Presenting Cells (APCs) to produce a second TIL population, wherein the priming a first expansion is performed for a first period of time of about 1 to 7/8 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population;

d) performing a rapid second expansion by contacting the second TIL population with a cell culture medium comprising IL-2, OKT-3, and APCs to produce a third TIL population, wherein the rapid second expansion is performed for a second time period of about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and

e) Collecting the therapeutic TIL population obtained from step (d).

In some embodiments, "obtaining" indicates that the TIL employed in the method and/or process may originate directly from a sample (including from a surgical resection, needle biopsy, core biopsy, mini biopsy, or other sample) as part of the method and/or process steps. In some embodiments, "receiving" indicates that the TIL employed in the methods and/or processes may be derived indirectly from the sample (including from a surgical resection, needle biopsy, core biopsy, mini biopsy, or other sample) and then employed in the methods and/or processes, (e.g., at the beginning of step (a), a TIL that has been obtained from the sample by a separation process not included in part (a), such TIL may be referred to as "received").

In some embodiments, in step (b), the cell culture medium further comprises Antigen Presenting Cells (APCs), and wherein the number of APCs in the medium in step (c) is greater than the number of APCs in the medium in step (b).

In some embodiments, in step (b), the cell culture medium further comprises Antigen Presenting Cells (APCs), and wherein the number of APCs in the medium in step (c) is equal to the number of APCs in the medium in step (b).

In some embodiments, the PD-1 positive TIL is a PD-1 high TIL.

(a) performing a priming first expansion by culturing a first TIL population that has been selected to be PD-1 positive in a cell culture medium comprising IL-2, OKT-3, and Antigen Presenting Cells (APCs) to produce a second TIL population, the first TIL population obtainable by tumor digestion processing a tumor sample from a subject and selecting PD-1 positive TILs, wherein the priming first expansion is performed in a container comprising a first gas permeable surface region, wherein the priming first expansion is performed for a first time period of about 1 to 7/8 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population;

(b) performing a rapid second expansion by contacting the second TIL population with cell culture medium of the second TIL population having additional IL-2, OKT-3, and APCs to produce a third TIL population, wherein the number of APCs in the rapid second expansion is at least twice the number of APCs in step (a), wherein the rapid second expansion is performed for a second period of time of about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second expansion is performed in a container comprising a second gas permeable surface region; and

(c) Collecting the therapeutic TIL population obtained from step (b).

(a) performing a primed first expansion of TILs that have been selected to be PD-1 positive by culturing a first TIL population in cell culture medium comprising IL-2, OKT-3, and optionally Antigen Presenting Cells (APCs), to produce a second TIL population, wherein the primed first expansion is performed for a first time period of about 1 to 7/8 days to obtain the second TIL population, wherein the second TIL population is greater in number than the first TIL population;

(b) performing a rapid second expansion by contacting the second TIL population with a cell culture medium comprising IL-2, OKT-3, and APCs to produce a third TIL population, wherein the rapid second expansion is performed for a second time period of about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and

c) collecting the therapeutic TIL population obtained from step (c).

In some embodiments, the PD-1 positive TIL is a PD-1 high TIL.

In some embodiments, the selecting of step (b) comprises the steps of: (i) exposing the first TIL population to an excess of monoclonal anti-PD-1 IgG4 antibody that binds to PD-1 through the N-terminal loop outside the IgV domain of PD-1; (ii) adding an excess of anti-IgG 4 antibody conjugated to a fluorophore; and (iii) performing flow-based cell sorting based on the fluorophore to obtain a population of PD-1 enriched TILs.

In some embodiments, the monoclonal anti-PD-1 IgG4 antibody is nivolumab or a variant, fragment, or conjugate thereof. In some embodiments, the anti-IgG 4 antibody is clone anti-human IgG4, clone HP 6023.

In some embodiments, the ratio of the number of APCs in the rapid second amplification to the number of APCs in the priming first amplification is selected from the range of about 1.5:1 to about 20: 1.

In some embodiments, the ratio is selected from the range of about 1.5:1 to about 10: 1.

In some embodiments, the ratio is selected from the range of about 2:1 to about 5: 1.

In some embodiments, the ratio is selected from the range of about 2:1 to about 3: 1.

In some embodiments, the ratio is about 2: 1.

In some embodiments, the number of APCs in the priming first amplification is selected from about 1 × 10⁸APC to about 3.5X 10⁸A range of APCAnd wherein the number of APCs in said rapid second amplification is selected from about 3.5X 10⁸APC to about 1X 10⁹Range of individual APC.

In some embodiments, the number of APCs in the priming first amplification is selected from about 1.5X 10⁸APC to about 3X 10⁸A range of APCs, and wherein the number of APCs in the rapid second amplification is selected from about 4 x 10⁸APC to about 7.5X 10⁸Range of individual APC.

In some embodiments, the number of APCs in the priming first amplification is selected from about 2 x 10⁸APC to about 2.5X 10⁸A range of APCs, and wherein the number of APCs in the rapid second amplification is selected from about 4.5 x 10⁸APC to about 5.5X 10⁸Range of individual APC.

In some embodiments, about 2.5 x 10 will be used⁸Addition of individual APCs to the priming first amplification, and 5X 10⁸Individual APCs were added to the rapid second amplification.

In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 1.5:1 to about 100: 1.

In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 50: 1.

In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 25: 1.

In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 20: 1.

In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is about 10: 1.

In some embodiments, the second TIL population is at least 50-fold greater in number than the first TIL population.

In some embodiments, the method comprises performing the following additional steps after the step of collecting the therapeutic TIL population: the collected therapeutic TIL populations were transferred to infusion bags.

In some embodiments, the plurality of tumor fragments are distributed into a plurality of separate containers, in each of the separate containers, the second TIL population is obtained from the first TIL population in the step of priming a first expansion and the third TIL population is obtained from the second TIL population in the step of rapid second expansion, and wherein the therapeutic TIL populations obtained from the third TIL population are collected from each of the plurality of containers and combined to produce the collected TIL population.

In some embodiments, the plurality of individual containers comprises at least two individual containers.

In some embodiments, the plurality of individual containers comprises two to twenty individual containers.

In some embodiments, the plurality of individual containers comprises two to ten individual containers.

In some embodiments, the plurality of individual containers comprises two to five individual containers.

In some embodiments, each of the individual containers includes a first gas-permeable surface area.

In some embodiments, the plurality of tumor fragments are dispensed in a single container.

In some embodiments, the single container includes a first gas-permeable surface region.

In some embodiments, in the step of initiating a first expansion, the cell culture medium comprises Antigen Presenting Cells (APCs), and the APCs are layered onto the first gas permeable surface region at an average thickness of about one cell layer to about three cell layers.

In some embodiments, in the step of initiating a first expansion, the APCs are layered onto the first gas permeable surface region at an average thickness of about 1.5 cell layers to about 2.5 cell layers.

In some embodiments, in the step of initiating a first expansion, the APCs are layered onto the first gas permeable surface region at an average thickness of about 2 cell layers.

In some embodiments, in the step of rapid second expansion, the APCs are laminated to the first gas permeable surface region at a thickness of about 3 cell layers to about 5 cell layers.

In some embodiments, in the step of rapid second expansion, the APCs are laminated to the first gas permeable surface region at a thickness of about 3.5 cell layers to about 4.5 cell layers.

In some embodiments, in the step of rapid second expansion, the APCs are laminated to the first gas permeable surface region at a thickness of about 4 cell layers.

In some embodiments, in the step of priming a first amplification, the priming first amplification is performed in a first container comprising a first gas-permeable surface region, and in the step of rapid second amplification, the rapid second amplification is performed in a second container comprising a second gas-permeable surface region.

In some embodiments, the second container is larger than the first container.

In some embodiments, in the step of rapid second expansion, the APCs are laminated to the second gas permeable surface region at an average thickness of about 3 cell layers to about 5 cell layers.

In some embodiments, in the step of rapid second expansion, the APCs are layered onto the second gas permeable surface region at an average thickness of about 3.5 cell layers to about 4.5 cell layers.

In some embodiments, in the step of rapid second expansion, the APCs are layered onto the second gas permeable surface region at an average thickness of about 4 cell layers.

In some embodiments, for each vessel in which said primed first expansion is performed on a first TIL population, said rapid second expansion is performed on said second TIL population produced from such first TIL population in the same vessel.

In some embodiments, each container includes a first gas-permeable surface region.

In some embodiments, in the step of rapid second expansion, the APCs are layered onto the first gas permeable surface region at an average thickness of about 3 cell layers to about 5 cell layers.

In some embodiments, in the step of rapid second expansion, the APCs are layered onto the first gas permeable surface region at an average thickness of about 3.5 cell layers to about 4.5 cell layers.

In some embodiments, in the step of rapid second expansion, the APCs are layered onto the first gas permeable surface region at an average thickness of about 4 cell layers.

In some embodiments, for each container in which the priming first expansion is performed on a first TIL population in the priming first expansion step, the first container comprises a first surface region, the cell culture medium comprises Antigen Presenting Cells (APCs), and the APCs are stacked onto the first gas permeable surface region, and wherein a ratio of an average number of layers of APCs stacked in the priming first expansion step to an average number of layers of APCs stacked in the rapid second expansion step is selected from a range of about 1:1.1 to about 1: 10.

In some embodiments, the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapid second amplification is selected from the range of about 1:1.2 to about 1: 8.

In some embodiments, the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapid second amplification is selected from the range of about 1:1.3 to about 1: 7.

In some embodiments, the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapid second amplification is selected from the range of about 1:1.4 to about 1: 6.

In some embodiments, the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapid second amplification is selected from the range of about 1:1.5 to about 1: 5.

In some embodiments, the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapid second amplification is selected from the range of about 1:1.6 to about 1: 4.

In some embodiments, the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapid second amplification is selected from the range of about 1:1.7 to about 1: 3.5.

In some embodiments, the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapid second amplification is selected from the range of about 1:1.8 to about 1:3.

In some embodiments, the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapid second amplification is selected from the range of about 1:1.9 to about 1: 2.5.

In some embodiments, the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapid second amplification is about 1:2.

In some embodiments, the cell culture medium is supplemented with additional IL-2 after 2 to 3 days in the step of rapid second expansion.

In some embodiments, the method further comprises cryopreserving the collected TIL population using a cryopreservation method in the step of collecting the therapeutic TIL population.

In some embodiments, the method further comprises the step of cryogenically preserving the infusion bag.

In some embodiments, the cryopreservation process is performed using the collected TIL population and cryopreservation media in a ratio of 1: 1.

In some embodiments, the antigen presenting cells are Peripheral Blood Mononuclear Cells (PBMCs).

In some embodiments, the PBMCs are irradiated and allogeneic.

In some embodiments, in the step of priming a first expansion, the cell culture medium comprises Peripheral Blood Mononuclear Cells (PBMCs), and wherein the total number of PBMCs in the cell culture medium in the step of priming a first expansion is 2.5 x 10⁸。

In some embodiments, in the step of rapid second expansion, the Antigen Presenting Cells (APCs) in the cell culture medium are Peripheral Blood Mononuclear Cells (PBMCs), and wherein the total number of PBMCs added to the cell culture medium in the step of rapid second expansion is 5 x 10 ⁸。

In some embodiments, the antigen presenting cell is an artificial antigen presenting cell.

In some embodiments, the collecting in the step of collecting the therapeutic TIL population is performed using a membrane-based cell processing system.

In some embodiments, the collecting in step (d) is performed using a LOVO cell processing system.

In some embodiments, in the step of initiating a first amplification, the plurality of fragments comprises about 60 fragments per container, wherein the volume of each fragment is about 27mm³。

In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, wherein the total volume is about 1300mm³To about 1500mm³。

In some embodiments, the plurality of fragments comprises about 50 fragments, wherein the total volume is about 1350mm³。

In some embodiments, the plurality of chips comprises about 50 chips with a total mass of about 1 gram to about 1.5 grams.

In some embodiments, the cell culture medium is provided in a container selected from the group consisting of a G container and a Xuri cell bag.

In some embodiments, the cell culture medium is supplemented with additional IL-2 after 2 to 3 days in step (d).

In some embodiments, the IL-2 concentration is from about 10,000IU/mL to about 5,000 IU/mL.

In some embodiments, the IL-2 concentration is about 6,000 IU/mL.

In some embodiments, in the step of transferring the collected therapeutic TIL population to an infusion bag, the infusion bag is a hypo thermosol-containing infusion bag.

In some embodiments, the cryopreservation media comprises dimethyl sulfoxide (DMSO).

In some embodiments, the cryopreservation media comprises 7% to 10% DMSO.

In some embodiments, the first time period in the step of priming first amplification and the second time period in the step of rapid second amplification are each performed separately over a time period of 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some embodiments, the first time period in the step of priming first amplification is performed over a period of 5 days, 6 days, or 7 days.

In some embodiments, the second period of time in the step of rapid second amplification is performed over a period of 7 days, 8 days, or 9 days.

In some embodiments, the first time period in the step of priming first amplification and the second time period in the step of rapid second amplification are each performed separately over a 7 day period.

In some embodiments, the step of priming a first expansion is performed over a period of about 14 days to about 16 days by collecting the therapeutic TIL population.

In some embodiments, the step of priming the first expansion is performed over a period of about 15 days to about 16 days by collecting the therapeutic TIL population.

In some embodiments, the step of priming a first expansion is performed over a period of about 14 days by collecting the therapeutic TIL population.

In some embodiments, the step of priming the first expansion is performed over a period of about 15 days by collecting the therapeutic TIL population.

In some embodiments, the step of priming a first expansion is performed over a period of about 16 days by collecting the therapeutic TIL population.

In some embodiments, the method further comprises the step of cryopreserving the collected therapeutic TIL population using a cryopreservation process, wherein the step of initiating the first expansion is performed in 16 days or less by collecting the therapeutic TIL population and cryopreserving.

In some embodiments, the therapeutic TIL population collected in the step of collecting the therapeutic TIL population includes sufficient TIL for a therapeutically effective dose of TIL.

In some embodiments, the amount of TIL sufficient for a therapeutically effective dose is about 2.3 x 10¹⁰To about 13.7X 10¹⁰。

In some embodiments, the third TIL population in the step of rapidly second amplifying provides increased efficacy, increased interferon gamma production, and/or increased polyclonality.

In some embodiments, the third TIL population in the step of rapidly second expanding provides at least one to five or more times interferon gamma production as compared to TILs prepared by a process longer than 16 days.

In some embodiments, the effector T cells and/or central memory T cells obtained from the third TIL population in the step of rapidly expanding second exhibit increased expression of CD8 and CD28 relative to the effector T cells and/or central memory T cells obtained from the second TIL population in the step of priming first expansion.

In some embodiments, the therapeutic TIL population from the step of collecting the therapeutic TIL population is infused into a patient.

In some embodiments, the method further comprises the step of cryopreserving the infusion bag comprising the collected TIL population in step (f) using a cryopreservation process.

In some embodiments, the PBMCs are irradiated and allogeneic.

In some embodiments, the collecting in step (e) is performed using a membrane-based cell processing system.

In some embodiments, the collecting in step (e) is performed using a LOVO cell processing system.

In some embodiments, in step (c), the plurality of fragments comprises about 60 fragments per first gas-permeable surface area, wherein each fragment has a volume of about 27mm³。

In some embodiments, the IL-2 concentration is about 6,000 IU/mL.

In some embodiments, the infusion bag in step (d) is a hypo thermosol-containing infusion bag.

In some embodiments, the cryopreservation media comprises 7% to 10% DMSO.

In some embodiments, the first time period in step (c) and the second time period in step (c) are each performed separately over a period of 5 days, 6 days, or 7 days.

In some embodiments, the first period of time in step (c) is performed over a period of 5 days, 6 days, or 7 days.

In some embodiments, the second period in step (d) is performed over a period of 7 days, 8 days, or 9 days.

In some embodiments, the first time period in step (c) and the second time period in step (c) are each performed separately over a 7 day period.

In some embodiments, steps (a) through (f) are performed over a period of about 14 days to about 16 days.

In some embodiments, steps (a) through (f) are performed over a period of about 15 days to about 16 days.

In some embodiments, steps (a) through (f) are performed over a period of about 14 days.

In some embodiments, steps (a) through (f) are performed over a period of about 15 days.

In some embodiments, steps (a) through (f) are performed over a period of about 16 days.

In some embodiments, steps (a) through (f) and the cryopreservation are performed in 16 days or less.

In some embodiments, the therapeutic TIL population collected in step (f) comprises sufficient TIL for a therapeutically effective dose of TIL.

In some embodiments, the vessel in step (c) is larger than the vessel in step (b).

In some embodiments, the third TIL population in step (d) provides increased efficacy, increased interferon gamma production, and/or increased polyclonality.

In some embodiments, the third TIL population in step (d) provides at least one to five or more times interferon gamma production as compared to TILs prepared by a process longer than 16 days.

In some embodiments, the effector T cells and/or central memory T cells obtained from the third TIL population in step (d) exhibit increased expression of CD8 and CD28 relative to the effector T cells and/or central memory T cells obtained from the second population of cells in step (c).

In some embodiments, the TIL from step (f) is infused into a patient.

In some embodiments, the invention provides a method for treating a subject having cancer, the method comprising administering expanded Tumor Infiltrating Lymphocytes (TILs), the method comprising:

(c) performing a priming first expansion by culturing the PD-1 enriched TIL population in a cell culture medium comprising IL-2, OKT-3, and Antigen Presenting Cells (APCs) to produce a second TIL population, wherein the priming first expansion is performed in a vessel comprising a first gas permeable surface region, wherein the priming first expansion is performed for about 1 to 7 days to obtain the second TIL population, wherein the second TIL population is at least 50-fold more in number than the first TIL population;

(d) performing a rapid second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and APCs to produce a third TIL population, wherein the number of APCs added to the rapid second expansion is at least twice the number of APCs added in step (b), wherein the rapid second expansion is performed for about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the rapid second expansion is performed in a container comprising a second gas permeable surface region;

(e) Collecting the therapeutic TIL population obtained from step (c);

(f) transferring the collected TIL population from step (d) to an infusion bag; and

(g) administering to the subject a therapeutically effective dose of the TIL from step (e).

In some embodiments, the amount of TIL sufficient to administer a therapeutically effective dose in step (g) is about 2.3 x 10¹⁰To about 13.7X 10¹⁰。

In some embodiments, the PD-1 positive TIL is a PD-1 high TIL.

In some embodiments, the monoclonal anti-PD-1 IgG4 antibody is nivolumab or a variant, fragment, or conjugate thereof.

In some embodiments, the anti-IgG 4 antibody is clone anti-human IgG4, clone HP 6023.

In some embodiments, the Antigen Presenting Cells (APCs) are PBMCs.

In some embodiments, the non-myeloablative lymphocyte depletion regimen has been administered to the patient prior to administering the therapeutically effective dose of TIL cells in step (g).

In some embodiments, the non-myeloablative lymphocyte depletion protocol comprises the steps of: at 60 mg/m²Cyclophosphamide was administered for two days at a dose of 25 mg/m²The dose of fludarabine was administered for five days.

In some embodiments, the method further comprises the step of treating the patient with a high-dose IL-2 regimen beginning the day after administering the TIL cells to the patient in step (g).

In some embodiments, the high dose IL-2 regimen comprises administering 600,000 or 720,000IU/kg every eight hours in 15 minute bolus intravenous infusion until tolerated.

In some embodiments, the third TIL population in step (c) provides increased efficacy, increased interferon gamma production, and/or increased polyclonality.

In some embodiments, the cancer is selected from the group consisting of: melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.

In some embodiments, the cancer is selected from the group consisting of: melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In some embodiments, the cancer is melanoma.

In some embodiments, the cancer is HNSCC.

In some embodiments, the cancer is cervical cancer.

In some embodiments, the cancer is NSCLC.

In some embodiments, the cancer is glioblastoma (including GBM).

In some embodiments, the cancer is gastrointestinal cancer.

In some embodiments, the cancer is a highly mutated cancer.

In some embodiments, the cancer is a pediatric hypermutation cancer.

In some embodiments, the container is GREX-10.

In some embodiments, the containment vessel comprises GREX-100.

In some embodiments, the containment vessel comprises GREX-500.

In some embodiments, the subject has been previously treated with an anti-PD-1 antibody.

In some embodiments, the subject has not been previously treated with an anti-PD-1 antibody.

In some embodiments, in step (b), the PD-1 positive TIL is selected from the first TIL population by performing the following steps: performing the step of contacting the first TIL population with an anti-PD-1 antibody to form a first complex of the anti-PD-1 antibody and the TIL cells in the first TIL population; and then performing the step of separating the first complex to obtain the population of PD-1 enriched TILs.

In some embodiments, the anti-PD-1 antibody comprises an Fc region, wherein after the step of forming the first complex and before the step of isolating the first complex, the method further comprises the step of contacting the first complex with an anti-Fc antibody to form a second complex of the anti-Fc antibody and the first complex, the anti-Fc antibody binding to the Fc region of the anti-PD-1 antibody, and wherein the step of isolating the first complex is performed by isolating the second complex.

In some embodiments, the anti-PD-1 antibody used for selection in step (b) is selected from the group consisting of: EH12.2H7, PD1.3.1, M1H4, nivolumab (BMS-936558, Bristol-Myers Squibb);) Pembrolizumab (lambrolizumab), MK03475 or MK-3475 Merck (Merck);) H12.1, PD1.3.1, NAT 105, humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR-042(Tesaro, Inc.), Pidilizumab (Pidilizumab) (anti-PD-1 mAb CT-011, mediwesson medical company (Medivation)), anti-PD-1 monoclonal antibody BGB-a317 (BeiGene)), and/or anti-PD-1 antibody SHR-1210 (ShangHai henderui), human monoclonal antibody REGN2810 (regenerlon)), human monoclonal antibody MDX-1106 (nikamura), humanized anti-PD-1 antibody 4 PDR001 (novanis) and RMP1-14 (catalog number xp) -0146 rat (biox).

In some embodiments, the anti-PD-1 antibody used for selection in step (b) is EH12.2H7.

In some embodiments, the anti-PD-1 antibody selected for use in step (b) binds to an epitope other than nivolumab or pembrolizumab.

In some embodiments, the anti-PD-1 antibody selected for use in step (b) binds the same epitope as EH12.2H7 or nivolumab.

In some embodiments, the anti-PD-1 antibody used for selection in step (b) is nivolumab.

In some embodiments, the subject has been previously treated with a first anti-PD-1 antibody, wherein in step (b), the PD-1 positive TIL is selected by contacting the first TIL population with a second anti-PD-1 antibody, and wherein the second anti-PD-1 antibody does not block binding to the first TIL population by the first anti-PD-1 antibody that is insoluble in the first TIL population.

In some embodiments, the subject has been previously treated with a first anti-PD 1 antibody, wherein in step (b), the PD-1 positive TIL is selected by contacting the first TIL population with a second anti-PD-1 antibody, and wherein the second anti-PD-1 antibody blocks binding to the first TIL population by the first anti-PD-1 antibody that is insoluble in the first TIL population.

In some embodiments, the subject has been previously treated with a first anti-PD 1 antibody, wherein in step (b), the PD-1 positive TIL is selected by performing the following steps: performing the step of contacting the first TIL population with a second anti-PD-1 antibody to form a first complex of the second anti-PD-1 antibody and the first TIL population, wherein the second anti-PD-1 antibody does not block binding to the first TIL population by the first anti-PD-1 antibody that is insoluble in the first TIL population; and then performing the step of separating the first complex to obtain the population of PD-1 enriched TILs.

In some embodiments, the first anti-PD-1 antibody and the second anti-PD-1 antibody comprise Fc regions, wherein after the step of forming the first complex and before the step of isolating the first complex, the method further comprises the step of contacting the first complex with an anti-Fc antibody to form a second complex of the anti-Fc antibody and the first complex, the anti-Fc antibody binding to the Fc regions of the first anti-PD-1 antibody and the second anti-PD-1 antibody, and wherein the step of isolating the first complex is performed by isolating the second complex.

In some embodiments, the second anti-PD-1 antibody comprises an Fc region, and the subject has been previously treated with a first anti-PD 1 antibody, wherein in step (b), the PD-1 positive TIL is selected by performing the steps of: performing the step of contacting the first TIL population with a second anti-PD-1 antibody to form a first complex of the second anti-PD-1 antibody and the first TIL population, wherein the second anti-PD-1 antibody does not block binding to the first TIL population by the first anti-PD-1 antibody that is insoluble in the first TIL population, and wherein after the step of forming the first complex, the method further comprises the steps of: contacting the first complex with an anti-Fc antibody to form a second complex of the anti-Fc antibody and the first complex, the anti-Fc antibody binding to the Fc region of the second anti-PD-1 antibody, and then performing the step of isolating the second complex to obtain a population of TILs enriched in PD-1.

In some embodiments, the subject has been previously treated with a first anti-PD 1 antibody, wherein in step (b), the PD-1 positive TIL is selected by performing the following steps: performing a step of contacting the first TIL population with a second anti-PD-1 antibody to form a first complex of the second anti-PD-1 antibody and the first TIL population, wherein the second anti-PD-1 antibody is blocked from binding to the PD-1 positive TIL by the first anti-PD-1 antibody that is insoluble in the first TIL population, wherein the first anti-PD-1 antibody and the second anti-PD-1 antibody comprise an Fc region, wherein after the step of forming the first complex and before the step of obtaining the PD-1 enriched TIL population, the method further comprises the steps of: contacting the first complex with an anti-Fc antibody to form a second complex of the anti-Fc antibody and the first complex, the anti-Fc antibody binding to the Fc region of the second anti-PD-1 antibody; and contacting the first anti-PD-1 antibody that is insoluble in the first TIL population with the anti-Fc antibody to form a third complex of the anti-Fc antibody and the first anti-PD-1 antibody that is insoluble in the first TIL population, and performing the step of separating the second complex and the third complex to obtain the PD-1 enriched TIL population.

In some embodiments, the PD-1 positive TIL is a PD-1 high TIL.

In some embodiments, the present invention provides a therapeutic Tumor Infiltrating Lymphocyte (TIL) population prepared from PD-1 positive cells selected from a tumor tissue of a patient, wherein the therapeutic TIL population provides increased efficacy and/or increased interferon gamma production.

In some embodiments, the present invention provides a therapeutic Tumor Infiltrating Lymphocyte (TIL) population prepared from PD-1 positive cells selected from a tumor tissue of a patient, wherein the therapeutic TIL population provides increased interferon gamma production.

In some embodiments, the present invention provides a therapeutic Tumor Infiltrating Lymphocyte (TIL) population prepared from PD-1 positive cells selected from a tumor tissue of a patient, wherein the therapeutic TIL population provides increased efficacy.

In some embodiments, the present invention provides a therapeutic Tumor Infiltrating Lymphocyte (TIL) population prepared from PD-1 positive cells selected from a tumor tissue of a patient, wherein the therapeutic TIL population is capable of producing at least one-fold more interferon gamma production as compared to a TIL prepared by a process longer than 16 days.

In some embodiments, the present invention provides a therapeutic Tumor Infiltrating Lymphocyte (TIL) population prepared from PD-1 positive cells selected from a tumor tissue of a patient, wherein the therapeutic TIL population is capable of producing at least one-fold more interferon gamma production as compared to a TIL prepared by a process longer than 16 to 22 days.

In some embodiments, selecting a PD-1 positive TIL from the first TIL population to obtain a population of TILs enriched for PD-1 comprises selecting a population of TILs from the first TIL population that is at least 11.27% to 74.4% of the PD-1 positive TIL.

In some embodiments, the selection of steps comprises the steps of:

(i) exposing the first TIL population and PBMC population to an excess of monoclonal anti-PD-1 IgG4 antibody that binds to PD-1 through the N-terminal loop outside the IgV domain of PD-1;

(ii) adding an excess of anti-IgG 4 antibody conjugated to a fluorophore;

(iii) obtaining the PD-1 enriched TIL population based on the intensity of the fluorophore of the PD-1 positive TIL in the first TIL population compared to the intensity in the PBMC population as performed by Fluorescence Activated Cell Sorting (FACS).

In some embodiments, the intensities of the fluorophores in both the first population and the PBMC population are used to establish FACS gates to establish low, intermediate, and high levels of intensity corresponding to PD-1 negative TIL, PD-1 intermediate TIL, and PD-1 positive TIL, respectively.

In some embodiments, the FACS gates are established after step (a).

In some embodiments, the PD-1 positive TIL is a PD-1 high TIL.

In some embodiments, at least 80% of the population of PD-1-enriched TILs is PD-1 positive TILs.

The present invention also provides a method for expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(b) selecting a PD-1 positive TIL from the first TIL population in (a) to obtain a population of PD-1 enriched TILs, wherein at least in the range of 10% to 80% of the first TIL population are PD-1 positive TILs;

(e) collecting the therapeutic TIL population obtained from step (d); and

(f) transferring the collected TIL population from step (e) to an infusion bag.

In some embodiments, the selecting of step (b) comprises the steps of:

(ii) adding an excess of anti-IgG 4 antibody conjugated to a fluorophore;

In some embodiments, the FACS gates are established after step (a).

In some embodiments, the PD-1 positive TIL is a PD-1 high TIL.

In some embodiments, the third TIL population comprises at least about 1 x 10 in the container⁸And (4) TIL.

In some embodiments, the third TIL population comprises at least about 1 x 10 in the container⁹And (4) TIL.

In some embodiments, the amount of PD-1-enriched TIL in the priming-first amplification is about 1X 10⁴To about 1X 10⁶。

In some embodiments, the amount of PD-1-enriched TIL in the priming-first amplification is about 5X 10⁴To about 1X 10⁶。

In some embodiments, the amount of PD-1-enriched TIL in the priming-first amplification is about 2X 10⁵To about 1X 10⁶。

In some embodiments, the method further comprises the step of performing cryopreservation on the first population of TILs from a tumor resected from the subject prior to performing step (a).

Drawings

FIGS. 1A-1B: A) a comparison between the 2A process for TIL manufacture (about 22 day process) and an example of the Gen 3 process (about 14 to 16 day process) is shown. B) An exemplary process PD-1 Gen 3 map providing an overview of steps a through F (about 14 days to 16 days of process). C) A diagram is provided that summarizes an overview of steps a through F (a process of about 14 days to 16 days) for each of three exemplary Gen 3 processes and three process variations.

FIG. 2: an experimental flow chart is provided for the comparability between GEN 2 (process 2A) and PD-1 GEN 3.

FIGS. 3A-3C: A) l4054-phenotypic characterization of TIL products in the Gen 2 and Gen 3 processes. B) L4055-phenotypic characterization of TIL products in the Gen 2 and Gen 3 processes. C) M1085T-phenotypic characterization of TIL products in Gen 2 and Gen 3 processes.

FIGS. 4A-4C: A) l4054-memory marker analysis for TIL products from Gen 2 and Gen 3 processes. B) L4055-memory marker analysis for TIL products from Gen 2 and Gen 3 processes. C) M1085T-memory marker analysis of TIL products from Gen 2 and Gen 3 processes.

FIG. 5: (A) gating on CD4+, (B) L4054 activation and depletion markers gated on CD8 +.

FIG. 6: (A) (ii) gating on CD4+, (B) L4055 activation and depletion markers gated on CD8 +.

FIG. 7: IFN γ production (pg/mL): (a) L4054, (B) L4055 and (C) M1085T of Gen 2 and Gen 3 processes: each bar graph represented here is the mean + SEM of IFN γ levels for stimulated, unstimulated, and media controls. The optical density was measured at 450 nm.

FIG. 8: ELISA analysis of IL-2 concentration in cell culture supernatants: (A) l4054 and (B) L4055. Each bar graph presented here is the mean value of IL-2 levels on spent medium + SEM. The optical density was measured at 450 nm.

FIG. 9: quantification of glucose and lactate in spent medium (g/L): (A) glucose and (B) lactic acid: in both tumor strains, and in both processes, a glucose reduction was observed throughout the REP amplification. Conversely, as expected, an increase in lactic acid was observed. Glucose reduction and lactic acid increase were comparable between Gen 2 and Gen 3 processes.

FIG. 10: A) quantification of L-Glutamine in spent media of L4054 and L4055. B) Quantification of Glutamax in spent media of L4054 and L4055. C) Quantification of ammonia in the spent media of L4054 and L4055.

FIG. 11: analysis of telomere length: the above RTL values indicate that the mean telomere fluorescence for each chromosome/genome in the Gen 2 and Gen 3 processes is the same as the telomere fluorescence for each chromosome/genome in the control cell line using the DAKO kit (the 1301 leukemia cell line).

FIG. 12: unique CDR3 sequence analysis of TIL end products on L4054 and L4055 under Gen 2 and Gen 3 processes. Bars show 1X 10 collections from Gen 2 (e.g., day 22) and Gen 3 (e.g., days 14-16) process collection days⁶The number of unique TCR B clonotypes identified in individual cells. Based on the number of unique peptide CDRs within the sample, Gen 3 showed higher clonal diversity compared to Gen 2.

FIG. 13: frequency of unique CDR3 sequences on final cell products of L4054 IL collection (Gen 2 process (e.g., day 22) and Gen 3 process (e.g., days 14-16)).

FIG. 14: frequency of unique CDR3 sequences on final cell products (Gen 2 (e.g., day 22) and Gen 3 processes (e.g., days 14-16)) of L4055 TIL collection.

FIG. 15: diversity index of TIL end products on L4054 and L4055 under Gen 2 and Gen 3 processes. The shannon entropy diversity index (shannon entropy diversity index) is a more reliable and commonly used metric for comparison. Gen 3L4054 and L4055 show a slightly higher diversity than Gen 2.

FIG. 16: raw data for cell counts at day 7-Gen 3REP initiation presented in table 22 (see example 5 below).

FIG. 17: raw data for cell counts at day 11-Gen 2REP initiation and Gen 3 upscaling presented in table 22 (see example 5 below).

FIG. 18: raw data for cell counts at day 16-Gen 2 scale-up and Gen 3 collection (e.g., day 16) presented in table 23 (see example 5 below).

FIG. 19: raw data for cell counts at day 22-Gen 2 collection (e.g., day 22) presented in table 23 (see example 5 below). For L4054 Gen 2, the post-LOVO count was extrapolated to 4 flasks because of the total number of studies. 1 flask was contaminated and extrapolated to a total of 6.67E + 10.

FIG. 20: raw data for the flow cytometry results depicted in fig. 3A, 4A and 4B.

FIG. 21: raw data for the flow cytometry results depicted in fig. 3C and 4C.

FIG. 22: raw data for the flow cytometry results depicted in fig. 5 and 6.

FIG. 23: the IFN γ of the L4054 sample depicted in figure 7 produced raw data for the assay results.

FIG. 24: the IFN γ of the L4055 sample depicted in figure 7 produced raw data for the assay results.

FIG. 25: the IFN γ of the M1085T sample depicted in fig. 7 produced raw data for the assay results.

FIG. 26: raw data for the results of the IL-2ELISA assay depicted in figure 8.

FIG. 27 is a schematic view showing: raw data for metabolic substrates and metabolic analysis results presented in fig. 9 and 10.

FIG. 28: raw data of the relative telomere length analysis results presented in fig. 11.

FIG. 29: raw data for the unique CD3 sequence and clone diversity analysis results presented in fig. 12 and 15.

FIG. 30: a comparison between various Gen 2(2A process) and Gen 3.1 process examples is shown.

FIG. 31: charts depicting various features of embodiments of the Gen 2, Gen 2.1, and Gen 3.0 processes.

FIG. 32: summary of the medium conditions of the example of the Gen 3 process (designated Gen 3.1).

FIG. 33: charts depicting various features of embodiments of the Gen 2, Gen 2.1, and Gen 3.0 processes.

FIG. 34: graphs comparing various features of examples of the Gen 2 and Gen 3.0 processes.

FIG. 35: a chart of the media usage in the various embodiments of the amplification process described is provided.

FIG. 36: and (3) phenotype comparison: the Gen 3.0 and Gen 3.1 examples of the process show comparable CD28, CD27, and CD57 expression.

FIG. 37: higher IFN γ production on Gen 3 final product. IFN γ analysis (by ELISA) was evaluated in culture frozen supernatants to compare the two processes. For each tumor, anti-CD 3 coated plates were stimulated overnight with fresh TIL product in each Gen 2 (e.g., day 22) and Gen 3 process (e.g., day 16). Each bar graph represented here is the IFN γ levels for stimulated, unstimulated, and media control.

FIG. 38: the upper diagram: unique CDR3 sequence analysis of TIL end product: the bars show 1X 10 collections from Gen 2 (e.g., day 22) and Gen 3 (e.g., days 14-16) processes⁶The number of unique TCR B clonotypes identified in individual cells. Based on the number of unique peptide CDRs within the sample, Gen 3 showed higher clonal diversity compared to Gen 2. The following figures: diversity index of TIL final product: the shannon entropy diversity index is a more reliable common measure for comparison. Gen 3 shows slightly higher diversity than Gen 2.

FIG. 39: the Gen 3 and Gen 2 end products share 199 sequences between them, corresponding to 97.07% of the first 80% unique CDR3 sequences shared by Gen 2 and Gen 3 end products.

FIG. 40: 1833 sequences were shared between Gen 3 and Gen 2 end products, corresponding to 99.45% of the first 80% unique CDR3 sequences shared by Gen 2 and Gen 3 end products.

FIG. 41: schematic of an exemplary example of the Gen 3 process (16 day process).

FIG. 42: schematic representation of PD-1 selection prior to amplification.

FIG. 43: binding structure of nivolumab (nivolumab) and PD-1. See, FIG. 5 by Tan, S.et al (Tan, S.et al, Nature Communications, 8:14369| DOI:10.1038/ncomms14369 (2017)).

FIG. 44: a binding structure of pembrolizumab to PD-1. See, FIG. 5 by Tan, S.et al (Tan, S.et al, Nature Commission), 8:14369| DOI:10.1038/ncomms14369 (2017)).

FIG. 45: a streamlined protocol was developed to expand PD1+ TIL to clinically relevant levels. Tumors were excised from patients and transported to the research laboratory. Upon arrival, the tumors were digested and single cell suspensions were stained for CD3 and PD 1. PD1+ TIL was sorted by FACS using FX500 instrument (Sony)). PD1+ cell fractions were placed in flasks with anti-human CD3 antibody (OKT 3; 30ng/ml) and allogeneic PBMCs (feeder cells) were irradiated at a ratio of 1:100(TIL: feeder cells) and rapidly expanded for 22 days (REP).

FIG. 46: the frequency of PD1+ TIL varied from tumor sample to tumor sample, but the in vitro amplification process reliably produced more than 10 hundred million TILs. Selected and bulk TILs were amplified from melanoma (n ═ 6), lung (n ═ 7), breast (n ═ 6), and sarcoma (n ═ 3), (a) shows the frequency of PD1+ cells in fresh tumor digests for each individual sample. Horizontal and vertical lines represent mean and standard error, respectively. (B) PD1+ and PD 1-sorted cells and bulk digests were expanded as described in figure 1. Cells were counted at the end of REP and fold expansion for extrapolation of total cell counts (final cell count/seeded cell count) was calculated. For bulk TIL, the percentage of T cells in the tumor digest was used to estimate the seeding cell count. The mean values are plotted as a bar graph and the standard error is shown as a vertical line.

FIG. 47: PD1+ TIL showed a different phenotypic spectrum compared to PD 1-TIL. Prior to sorting, digestive tumors from melanoma (n ═ 2), lung (n ═ 2), and breast (n ═ 2) were phenotypically assessed by flow cytometry. (A) Representative mapping of surface marker expression on TILs from digested melanoma tumors. Samples were first gated on CD3 and a double-axis plot of positive and negative PD1 events was drawn. Both fractions were then subjected to unsupervised viSNE clustering. The top row contains a PD1 positive event and the bottom row contains a PD1 negative event. (B-C) live lymphocytes were gated on CD3+ cells and PD1+ and PD 1-were evaluated. Cell surface expressed (B) activation and (C) depletion markers for the assessed PD1+ and PD 1-populations. The mean values are plotted as a bar graph and the standard error is shown as a vertical line. Statistical significance was assessed by paired student t-test P < 0.0001P < 0.05.

FIG. 48: at PD1⁺PD1 expression decreased when TIL was amplified in vitro. Cell surface expression T cell markers were assessed by flow cytometry for PD1+ pre-sorted TIL and in vitro expanded PD1+ TIL (PD1+ derived TIL) from melanoma (n ═ 1), lung (n ═ 4) and breast (n ═ 2). The bar graph is shown in 2 TI L mean percentage of each subset in the formulation, and the vertical line indicates standard error. Test by paired students t<0.001，**p<Statistical significance was assessed at 0.01.

FIG. 49: the in vitro amplified PD1+ TIL phenotype was similar to that of bulk TIL. Cell surface expression of T cell markers from PD1+ -derived TIL, PD 1-derived TIL and bulk TIL of melanoma (n ═ 5), lung (n ═ 7), breast (n ═ 6) and sarcoma (n ═ 3) was phenotypically assessed by flow cytometry. (A) Four effector/memory subsets were identified based on (CD45RA and CCR7) levels on CD3+ cells. TEM ═ effector memory (CD45RA-, CCR7-), TCM ═ central memory (CD45RA-, CCR7+), TSCM ═ stem cell memory (CD45RA +, CCR7+), TEMRA ═ effector T cells (CD45RA +, CCR 7-). Differentiation markers (B), (C) depletion and (D) activation were also evaluated. The bar graph represents the average percentage of each subset in all 3 TIL formulations, and the vertical line represents the standard error.

FIG. 50: the amplified PD1+ TIL is oligoclonal and comprises a portion of the clone present in the bulk TIL. Selected PD1 and bulk TILs from melanoma (n ═ 2), breast (n ═ 2), and lung (n ═ 2) were analyzed by RNA sequencing. (A) Unique CDR3 (ucrd 3) peptide sequences were numbered and boxed plots were generated using the pandas and mapping database library of Python 3.6.3 from anancoda, Inc. (B) The Shannon Diversity index (Shannon Diversity index) of each sample was calculated by irpertore and boxed plots were generated using the pandas of Python 3.6.3 from arnada ltd and the mapping database library. The bar graph represents the average percentage of each subset and the vertical line represents the standard error. Test by paired student t <0.001，**p<Statistical significance was assessed at 0.01. (C) For each of the samples sequenced, the uCDR3 frequencies were ranked in descending order and reported or summed at the indicated intervals (highest ranking uCDR3, 2-10, 11-20 rankings CDR3, etc.). The frequencies were then averaged by group and plotted using Excel v.1803. (D) In the complementary entities TIL and PD1⁺Shared u CDR3 clones were identified in the source samples. The sum of the frequencies of each shared unique CDR3 clone is reported in the "shared%" column.

FIG. 51: the amplified PD1+ TIL was functional as determined by IFN γ secretion and CD107a mobilization in response to non-specific stimulation. A) PD1+ derived TIL, PD 1-derived TIL and bulk TIL from melanoma (n ═ 5), lung (n ═ 6) and breast (n ═ 6) were stimulated with plate-bound anti-CD 3 for 18 hours. Supernatants were assessed for IFN γ secretion by ELISA. Results for individual samples are plotted. (B) CD107a cell surface expression from PD1+ derived TIL, PD 1-derived TIL and bulk TIL of melanoma (n ═ 5), lung (n ═ 7), breast (n ═ 6) and sarcoma (n ═ 1) was assessed by flow cytometry in response to PMA stimulation on CD4+ and CD8+ cells for 4 hours. Results for individual samples are plotted. The horizontal lines represent the average percentage of each subset and the vertical lines represent the standard error.

FIG. 52: the expanded PD1+ TIL showed increased autologous melanoma cell killing and tumor reactivity relative to PD 1-TIL. Tumor reactivity was assessed on TIL products selected by PD1 from one melanoma sample. (A) Whole tumor digests were cleared using a dead cell removal kit (Miltenyi). 1e5 live cells were plated in each well of a 96-well plate and allowed to adhere in an xCEELLigence instrument (ACEA Biosciences, Inc.) for 18 hours at 37 ℃. 1e5 PD1+ derived and PD 1-derived autologous TIL was added to their respective wells to generate a 1:1(TIL: target) cell ratio and incubated for 48 hours. Killing of autologous target cells was recorded as an increase in impedance caused by cell detachment. Cell killing (% cytolysis) (left-most panel) was calculated using the formula,% cytolysis [1- (NCIst)/(avgcntirt) ] × 100, where NCIst is the normalized cell index of the sample and NCIRt is the average of the normalized cell indices of the matched reference wells (digested alone). The right panel shows the normalized cell index of the sample. (B) 1e5 cells from a whole tumor digest were co-cultured with 1e5 TIL (or digest and TIL alone) for 18 hours. The supernatants were evaluated for IFN γ release by ELISA (R & D systems). The bar graph represents the mean of duplicate wells and the vertical line represents the standard error.

FIG. 53: PD1+ cells were selected from tumor digests using fluorescence activated cell sorting.

FIG. 54: identification of tumor tissue digestion methods.

FIG. 55: reagents available using GMP identify tumor tissue digestion methods.

FIG. 56: reagents available using GMP identify tumor tissue digestion methods.

FIG. 57: reagents available using GMP identify tumor tissue digestion methods.

FIG. 58: sorting yields of fresh tumor digests were higher than frozen tumor digests.

FIG. 59: similar expression of PD1 in fresh and frozen TIL.

FIG. 60: PD1 antibody titration: variable expression PD1 was used from commercially available clones.

FIG. 61: nivolumab inhibited binding of 5 commercially available PD1 staining antibodies.

FIG. 62: pembrolizumab differentially inhibited the binding of 5 commercially available PD1 staining antibodies.

FIG. 63: PD-1MFI decreased significantly when cells were preincubated with pembrolizumab.

FIG. 64: TIL co-incubated with Pembro and Nivo and secondary stained with IgG4 showed similar expression of PD-1 when compared to clone EH12.2H7.

FIG. 65: incubation of TIL with Pembro and Nivo did not alter the ability to detect surface PD1 expression.

FIG. 66: sorting and amplification results of PD1 selection.

FIG. 67: sorting and amplification results of PD1 selection.

FIG. 68: sorting and amplification results of PD1 selection.

FIG. 69: the optimal seeding density for PD1+ derived TIL was greater than 10,000 cells.

FIG. 70: PD1 in contrast to PD1-TIL⁺TILs exhibit different phenotypic spectra.

FIG. 71: PD1 in contrast to PD1-TIL⁺TILs exhibit different phenotypic spectra.

FIG. 72: the frequency of PD1+ TIL varies from tumor sample to tumor sample and requires 2 REP cycles to overcome the low initial proliferation rate.

FIG. 73: the frequency of PD1+ TIL varies from tumor sample to tumor sample and requires 2 REP cycles to overcome the initial proliferative defect.

FIG. 74: the in vitro amplified PD1+ TIL phenotype was similar to that of bulk TIL.

FIG. 75: PD1 expression decreased when PD1+ TIL was amplified in vitro.

FIG. 76: PD1⁺The selected TILs are oligoclonal and impair a portion of the clones present in the bulk TIL.

FIG. 77: PD1⁺The selected TILs are oligoclonal and impair a portion of the clones present in the bulk TIL.

FIG. 78: PD1⁺The selected TILs are oligoclonal and impair a portion of the clones present in the bulk TIL.

FIG. 79: PD1⁺The selected TILs are oligoclonal and impair a portion of the clones present in the bulk TIL.

FIG. 80: PD1 as determined by IFN γ secretion and mobilization of CD107a in response to non-specific stimulation ⁺The native TIL is functional.

FIG. 81: PD1⁺The derived TIL was shown to be similar to PD1 in melanoma^-Enhanced killing of the native TIL compared to the bulk TIL.

FIG. 82: PD1⁺The derived TIL was shown to be associated with PD in melanoma^-Enhanced tumor cell killing compared to autologous TIL.

FIG. 83: illustrative examples of methods for amplifying TIL from hematopoietic malignancies using the Gen 3 amplification platform.

FIG. 84: in several in vitro assays, ex vivo amplified PD1+ TIL showed effector activity. The data indicate that PD1+ selected TILs are antigen specific and have greater effector function.

FIG. 85: schematic of an exemplary embodiment for tumor digestion and PD-1+ selection step (comprising PD-1 high selection).

FIG. 86: PD-1 selected TIL data and information, including uCDR numbers and amplification data.

FIG. 87: TIL sorting strategies and data were selected using PD-1 of EH12.2H7 anti-PD-1 antibody instead of M1H4 anti-PD-1 antibody.

FIG. 88: TIL sorting data for PD-1 selection of populations in a PD-1 high gating strategy using EH12.2H7 anti-PD-1 antibody is shown.

FIG. 89: PD1+ sorting strategy data for the evaluation of anti-PD 1 antibodies for sorting M1H4 anti-PD-1 antibodies and EH12.2H7 anti-PD-1 antibodies are shown.

FIG. 90: PD-1 staining for TIL selection. The data show that EH12.2H7 and M1H4 exhibit different PD1 spectra in PBMC and TIL.

FIG. 91: comparative analysis of M1H 4-derived TIL versus EH12.2H7-derived TIL. The frequency of PD1+ in EH12.2H7 sorted TILs increased.

FIG. 92: fold amplification in PD1+ -derived TIL was reduced during the use of REP1 cloned in M1H 4.

FIG. 93: comparative analysis of TIL from M1H4 and EH12.2H7. Greater oligoclonality (reduced diversity) was observed in M1H 4-sorted TILs. (Shannon entropy is a standard measure reflecting how many different types of species are present.)

FIG. 94: greater oligoclonality (reduced diversity) was observed in PD1+ derived TILs compared to EH12.2H7 clones compared to the bulk TIL with the M1H4 clone. (Shannon entropy is a standard measure reflecting how many different types of species are present.)

FIG. 95: PD1 is shown⁺Selected exemplary data: PD1+ high (PD-1 high) was gated.

FIG. 96: schematic of an exemplary embodiment of a modified Gen 2 process developed for TIL selected via PD 1.

FIG. 97: PD1 is shown⁺Selected exemplary data: for the small (upper panel) and large (lower panel) scales, PD1+ high (PD-1 high) was gated for different tumor samples.

FIG. 98: schematic of an exemplary embodiment of an improved amplification process developed for TIL selected via PD 1.

FIG. 99: early REP collection on day 17 under PD1+ conditions is shown to produce data for 55e9 and 37e9 TIL.

FIG. 100: showing IFN γ secretion from two tumor samples under various amplification process conditions as depicted in figures 96 and 98.

FIG. 101: granzyme B secretion is shown for two tumor samples under various amplification process conditions as depicted in figures 96 and 98.

FIG. 102: the CD3+ CD45+ population of one tumor sample under various amplification process conditions, as depicted in figures 96 and 98, is shown. PD1+ Gen 2 condition > 90% CD3+ CD45 +.

FIG. 103: the CD3+ CD45+ population of one tumor sample under various amplification process conditions, as depicted in figures 96 and 98, is shown. PD1+ Gen 2 condition > 90% CD3+ CD45 +.

FIG. 104: TIL profiles of a tumor sample under various amplification process conditions, as depicted in fig. 96 and 98, are shown. Purity: > 90% TCR a/B + and no detectable NK or monocyte or B cell.

FIG. 105: TIL profiles of a tumor sample under various amplification process conditions, as depicted in fig. 96 and 98, are shown. Purity: > 90% TCR a/B + and no detectable NK or monocyte or B cell.

FIGS. 106A-B: TIL profiles of two tumor samples under various amplification process conditions, as depicted in fig. 96 and 98, are shown. Differentiation: PD1+ Gen 2 differentiation status was comparable.

FIGS. 107A-B: TIL profiles of two tumor samples under various amplification process conditions, as depicted in fig. 96 and 98, are shown. Memory: PD1+ Gen 2 is primarily effector memory TIL.

FIGS. 108A-B: TIL profiles of two tumor samples under various amplification process conditions, as depicted in fig. 96 and 98, are shown. The activation and depletion states on CD4+ are similar.

FIG. 109: TIL profiles of two tumor samples under various amplification process conditions, as depicted in fig. 96 and 98, are shown. The activation and depletion states on CD8+ are similar.

FIG. 110: PD1 is shown⁺Selected exemplary data: different tumor samples were gated for PD1+ elevation (PD-1 elevation) and compared for pre-sorting and post-sorting.

FIG. 111: show thatPD1⁺Selected exemplary data: the L4097 tumor samples were gated for PD1+ elevation (PD-1 elevation).

FIG. 112: PD1 is shown⁺Selected exemplary data: the L4089 tumor sample was gated for PD1+ elevation (PD-1 elevation).

FIG. 113: PD1 is shown⁺Selected exemplary data: PD1+ high (PD-1 high) was gated on H3035 tumor samples.

FIG. 114: PD1 is shown⁺Selected exemplary data: the M1139 tumor samples were gated for PD1+ elevation (PD-1 elevation).

FIG. 115: PD1 is shown⁺Selected exemplary data: the L4100 tumor sample was gated for PD1+ elevation (PD-1 elevation).

FIG. 116: PD1 is shown⁺Selected exemplary data: OV8030 tumor samples were gated for PD1+ elevation (PD-1 elevation).

Fig. 117: PD1 is shown⁺Selected exemplary data: the L4104 tumor samples were gated for PD1+ elevation (PD-1 elevation).

FIG. 118: PD1 is shown⁺Selected exemplary data: the M1132 tumor sample was gated for PD1+ high (PD-1 high).

FIG. 119: PD1 is shown⁺Selected exemplary data: the M1136 tumor samples were gated for PD1+ elevation (PD-1 elevation).

FIG. 120: PD1 is shown⁺Selected exemplary data: PD1+ high (PD-1 high) was gated on H3037 tumor samples.

FIG. 121: PD1 is shown⁺Selected exemplary data: the L4106 tumor samples were gated for PD1+ elevation (PD-1 elevation).

FIG. 122: PD1 is shown⁺Selected exemplary data: the L1141 tumor sample was gated for PD1+ elevation (PD-1 elevation).

FIG. 123: PD1 is shown⁺Selected exemplary data: the L4096 tumor samples were gated for PD1+ elevation (PD-1 elevation).

FIG. 124: PD1 is shown⁺Selected exemplary data: PD1+ high (PD-1 high) was gated on H3038 tumor samples.

FIG. 125: PD1 is shown⁺Selected exemplary data: the L4101 tumor samples were gated for PD1+ elevation (PD-1 elevation). (Note: CD8 in the third panel may have gating problems.)

FIG. 126: PD1 is shown⁺Selected exemplary data: the L4097 tumor samples were gated for PD1+ elevation (PD-1 elevation).

FIG. 127: data showing amplification in various populations of PD-1 selections. The expansion of PD-1 highly expanded cells in REP1 may be reduced.

FIG. 128: summary of sorting and amplification results for PD-1 selection. PD1 Using EH12.2H7 anti-PD-1 antibody^{Height of}The cells were sorted.

FIG. 129: summary of sorting and amplification results for PD-1 selection. PD1 Using EH12.2H7 anti-PD-1 antibody^{Height of}The cells were sorted.

FIG. 130: graphical representation of summarized data for sorting and amplification results of PD-1 selection from figures 128 and 129. PD1 Using EH12.2H7 anti-PD-1 antibody^{Height of}The cells were sorted.

FIG. 131: structures I-A and I-B are provided, and cylinders refer to separate polypeptide binding domains. Structures I-a and I-B include three linearly linked TNFRSF binding domains derived from, for example, 4-1BBL or an antibody that binds to 4-1BB, which fold to form a trivalent protein, which is then linked to a second trivalent protein by IgG1-Fc (comprising CH3 and CH2 domains), which is then used to link the two trivalent proteins together by a disulfide bond (small elongated oval), stabilizing the structure and providing an agonist capable of bringing together the intracellular signaling domains of the six receptors and the signaling protein to form a signaling complex. The TNFRSF binding domain represented in cylindrical form may be an scFv domain, which includes, for example, VH and VL chains linked by linkers that may include hydrophilic residues and flexible Gly and Ser sequences, as well as Glu and Lys with solubility.

FIG. 132: data for a set of 100,000 cells selected for the two drop down menus seen above is shown. Confirming that the cell population was correctly gated. The three populations were differentiated by using PBMC, FMO control and the sample itself, with gates set high, medium and low.

FIG. 133: upper left panel: this is the FMO control. Ensure that int gate and high gate are less than 0.5%. Upper right panel: with a representative drawing in which the separation of the high and middle portions is unclear. The background of the day is high, resulting in a high negative gate. The following figures: a clear representation of the high and middle portions. Data provision may necessitate adjustment of BSC or FSC settings. The voltage of any other channel is not adjusted. The PD1 gate was adjusted as needed.

FIG. 134: unique CDR3v β composition of PD 1-selected and unselected TILs. Libraries of CDR3v β of amplified unselected and PD 1-selected TILs from 2 HNSCCs and 5 NSCLCs were analyzed. The number of unique CDRs 3 β for each individual sample was plotted, labeled ucrd 3 counts (a.) and diversity index expressed in shannon entropy (B.). The paired samples were connected by a colored line. The P values calculated by the paired t test are labeled on their respective graphs.

FIG. 135: a graph showing clonal overlap between PD 1-selected and unselected TILs. Libraries of CDR3v β from amplified TILs from 2 HNSCCs and 5 NSCLCs were analyzed. A. The number of unique CDR3v β shared between the PD 1-selected (blue) and unselected (red) TIL samples is shown at the intersection of the wien plots for each individual tumor sample (b. -D). The percentage and fraction (E.) of unselected and shared TILs among those selected with PD1 were plotted for each individual sample. The paired samples were connected by a colored line. The P values calculated by the paired t test are labeled on their respective graphs.

FIG. 136: frequency of the first 10 TIL clones selected by PD1 in the unselected TIL product. (A. -C.) libraries of CDR3v β of amplified PD 1-selected and unselected TILs from 2 HNSCCs and 5 NSCLCs were analyzed. Unique CDR3v β sequences identified in PD 1-selected TIL products were ranked from most to least frequent. The frequency of each of the first 10 TIL clones selected by PD1 in each paired product was plotted. The mated samples were connected by a straight line. The P values calculated by the paired t test are labeled on their respective graphs.

FIG. 137: description of tumor digests used in these studies.

FIG. 138: detection of PD1 in tumor digests from various histologies⁺A cell. Legend: expression of PD1 in various histologies. For individual samples within each histology, CD3 was plotted⁺PD1 in TIL population⁺Percentage of TIL. The horizontal lines represent the average percentage of each subset and the vertical lines represent the standard error.

FIG. 139: description of PD 1-selected and unselected TILs for this study.

FIG. 140: fold expansion reduction in TIL selected with PD1 during REP1 but not REP 2. Legend: PD 1-sorted and unselected from (a) melanoma, (B) NSCLC and (C) HNSCC were amplified by two 11-day REP. The fold expansion of all tumors determined is shown in (D). Fold expansion in TIL populations was calculated using the total cell counts at completion of REP1 and REP 2. Results for individual samples were plotted, with black dots representing TILs selected by PD1 and grey triangles representing unselected TILs. The horizontal lines represent the average percentage of each subset and the vertical lines represent the standard error. Statistical significance was assessed by paired student t-test; assigned p value < 0.05.

FIG. 141: amplification results from various tumor samples.

FIG. 142: description of PD 1-selected and unselected TILs for this study. Both PD 1-selected and unselected TIL products were obtained from 4 melanomas, 7 NSCLC and 2 HNSCC according to the procedure TMP-18-015. Briefly, whole tumor biopsies were digested with a mixture of dnase, hyaluronidase, and collagenase IV. A portion of the resulting single cell suspension was stained for PD1 and sorted on FX500 instrument (sony, headquarters, new york). PD1 sorted cells and unselected whole tumor digests were subjected to two 11-day Rapid Expansion Phases (REP) to obtain PD1 selected TIL and unselected TIL, respectively.

FIG. 143: both PD 1-selected and unselected TILs produced IFN γ and granzyme B in response to stimulation with activated beads. Legend: secretion of (a) IFN γ and (B) granzyme from PD 1-selected TIL and unselected TIL of 4 melanomas, 7 NSCLC and 2 HNSCC was evaluated. Results for individual samples were plotted, with black dots representing unstimulated conditions and gray triangles representing conditions stimulated with α CD3/α CD28/α 41 BB. The horizontal lines represent the average percentage of each subset and the vertical lines represent the standard error. Statistical significance was assessed by paired student t-test; assigned p value < 0.01.

FIG. 144: both PD 1-selected and unselected TILs mobilize CD107a in response to PMA/ionomycin stimulation. Legend: cell surface expression of PD 1-selected and unselected TILs from 4 melanomas, 5 NSCLCs and 1 HNSCC in response to PMA and ionomycin (BioLegend, ca) stimulated CD107a was assessed by flow cytometry. Results for individual samples are plotted, with black dots representing unstimulated conditions and grey triangles representing conditions stimulated with PMA/ionomycin. The horizontal lines represent the average percentage of each subset and the vertical lines represent the standard error.

FIG. 145: description of PD 1-selected and unselected TILs for this study.

FIG. 146: both PD 1-selected and unselected TILs showed autologous tumor reactivity in vitro. Tumor killing and reactivity were evaluated in PD 1-selected and unselected TILs. The (a) cell index and (B) tumor cell killing (% cytolysis) of melanoma samples are shown. Supernatants from 2 NSCLC and 3 melanoma were assessed for (C) IFN γ release by ELISA. The mean values are plotted as a bar graph and the standard error is shown as a vertical line. Statistical significance was assessed by paired student t-test; assigned p value < 0.01.

FIG. 147: description of PD 1-selected and unselected TILs for example 16. Both PD 1-selected and unselected TIL products were obtained from 4 melanomas, 7 NSCLC and 2 HNSCC according to the procedure TMP-18-015. Briefly, whole tumor biopsies were digested with a mixture of dnase, hyaluronidase, and collagenase IV. A portion of the resulting single cell suspension was stained for PD1 and sorted on FX500 instrument (sony, headquarters, new york). PD 1-selected and unselected TILs were subjected to two 11-day REPs.

Fig. 148: FIG. 1: comparison of CD4 in PD 1-selected and unselected TILs⁺And CD8⁺Levels of T cells. Legend: t cell lineages (CD4 and CD8) of PD 1-selected and unselected TIL from 4 melanomas, 7 NSCLCs and 2 HNSCCs were evaluated using flow cytometry. The results are expressed as CD3⁺Percentage of cells. The mean values are plotted as a bar graph and the standard error is shown as a vertical line.

FIG. 149: will pass through PD1^-The differentiation status of the selected TILs was compared with that of the unselected TILs. Legend: expression of CD27, CD28, CD56, CD57, and KLRG1 of PD 1-selected TIL and unselected TIL from 4 melanomas, 7 NSCLCs, and 2 HNSCCs was evaluated using flow cytometry. The results are expressed as CD3 ⁺Percentage of cells. The mean values are plotted as a bar graph and the standard error is shown as a vertical line. Statistical significance was assessed by paired student t-test; specifying p value<0.05。

FIG. 150: the distribution of memory T cell subsets in PD 1-selected and unselected TILs was compared. Legend: expression of memory markers CD45RA and CCR7 from PD 1-selected TIL and unselected TIL of 4 melanomas, 7 NSCLC and 2 HNSCC was assessed by flow cytometry. As indicated, T cell memory subpopulations were determined and the average percentage of each subpopulation was plotted as black bars for the PD 1-selected TILs and gray bars for the unselected TILs. The standard error is shown as a vertical line.

FIG. 151: activation status of PD 1-selected TILs and unselected TILs were compared. Legend: expression of CD25, CD69, CD134 and CD137 from PD 1-selected TIL and unselected TIL from 4 melanomas, 7 NSCLCs and 2 HNSCCs was evaluated. Will CD3⁺The average percentage of T cells was plotted as black bars for the TILs selected with PD1 and gray bars for the unselected TILs. The standard error is shown as a vertical line. Statistical significance was assessed by paired student t-test; specifying p value <0.05。

FIG. 152: expression of depletion/inhibition markers in PD 1-selected and unselected TILs was compared. Legend: expression of LAG3, PD1, TIM3 and CD101 from PD 1-selected TIL and unselected TIL of 4 melanomas, 7 NSCLCs and 2 HNSCCs was assessed by flow cytometry. The mean values are plotted as a bar graph and the standard error is shown as a vertical line. Statistical significance was assessed by paired student t-test; indicates p value < 0.001.

Fig. 153: expression of resident memory T cell markers in PD 1-selected and unselected TILs was compared. Expression of CD39, CD49a and CD103 from PD 1-selected TIL and unselected TIL of 4 melanomas, 7 NSCLCs and 2 HNSCCs was assessed by flow cytometry. The mean values are plotted as a bar graph and the standard error is shown as a vertical line. Statistical significance was assessed by paired student t-test; indicates p value < 0.01.

FIG. 154: example of a full scale process for PD1 TIL culture.

FIG. 155: summary of small-scale processes: PD1-a is the condition for using the nivolumab staining procedure outlined in this protocol. PD1-B is the condition for using the anti-PD 1-PE (clone No. EH12.2H7) staining method. Bulk conditions were used as controls.

FIG. 156: the purity (PD-1 +%) after sorting all three tumors met the criterion (A. and B.) of 80% or more. A slightly lower purity of melanoma tumors was observed relative to head and neck tumors, most likely due to lower expression of PD-1+ cells upon sorting.

FIG. 157: detection of PD-1 in tumor digests from various histologies⁺A cell. Expression of PD-1 in various histologies. For individual samples within each histology, CD3 was plotted⁺PD-1 in the TIL population⁺Percentage of TIL. The horizontal lines represent the average percentage of each subset and the vertical lines represent the standard error.

FIGS. 158A-158P: FACS data plots.

FIG. 159: flow cytometry was used to assess the T cell lineages of PD-1-selected TILs (CD4 and CD8) sorted using nivolumab or EH12.2H7 to identify PD-1+ TILs from 1 ovary, 1 melanoma, and 1 HNSCC. Results are expressed as percentage of CD3+ cells. The mean values are plotted as a bar graph and the standard error is shown as a vertical line.

FIG. 160: expression of memory markers CD45RA and CCR7 from PD-1 selected TIL from 1 ovarian, 1 melanoma, and 1 HNSCC tumor sample sorted using nivolumab or EH12.2H7 to identify PD-1+ TIL was assessed by flow cytometry. As indicated, T cell memory subpopulations (TN/TSCM) were determined and the average percentage of each subpopulation was plotted as black bars for nivolumab selected TIL-1 and gray bars for EH12.2H7 selected TIL-1. The standard error is shown as a vertical line.

Fig. 161A: evaluation results from sorting using nivolumab or EH12.2H7 to identify PD-1⁺PD-1-sorted TIL expression of 1 ovary of TIL, 1 melanoma, and 1 HNSCC before and after amplification. Use of post-sort purity of PD-1 sorted products to determine pre-amplification PD-1⁺Percentage of (c). The mean values are plotted as a bar graph and the standard error is shown as a vertical line. Statistical significance was assessed by paired student t-test; indicates p value<0.01。

Fig. 161B: secretion of (a) IFN γ and (B) granzyme B from PD-1 selected TIL using nivolumab or EH12.2H7 sorting to identify 1 ovary, 1 melanoma, and 1 HNSCC of PD-1+ TIL was evaluated. Results for individual samples were plotted, with black dots representing unstimulated conditions and gray triangles representing conditions stimulated with α CD3/α CD28/α 41 BB. The horizontal lines represent the average percentage of each subset and the vertical lines represent the standard error.

FIG. 162: PD-1 levels in pre-sorted nivolumab and EH12.2H7-stained TIL. Whole tumor digests were split and stained with nivolumab or EH12.2H7 and evaluated by flow cytometry. PD-1 from 1 ovary, 1 melanoma, and 1 HNSCC identified with each antibody was then sorted using an FX500 cell sorter (Sony, New York) ⁺A cell.

FIG. 163: PD-1 levels in nivolumab and EH12.2H7-stained TIL after sorting.

FIG. 164: the whole tumor digest is divided, andstaining was performed with nivolumab or EH12.2H7 and evaluated by flow cytometry. PD-1 from 1 ovary, 1 melanoma, and 1 HNSCC identified with each antibody was then sorted using an FX500 cell sorter (Sony, New York)⁺A cell.

FIG. 165: detection of PD-1 in tumor digests from various histologies⁺A cell. Expression of PD-1 in various histologies. The percentage of PD-1+ TIL in the CD3+ TIL population was plotted for individual samples within each histology. The horizontal lines represent the average percentage of each subset and the vertical lines represent the standard error.

FIG. 166: fold amplification reduction in PD-1 selected TILs during the activation phase rather than REP. PD-1 sorted TIL and whole tumor digests from 4 melanoma, 7 NSCLC and 2 HNSCC tumor samples were amplified using a two-step procedure consisting of an 11-day activation step followed by 11-day REP. Fold expansion of all tumors assayed is shown. Fold expansion in TIL populations was calculated using the total cell count at the completion of the activation step and REP step. Results for individual samples were plotted, with black dots representing TILs selected by PD-1 and grey triangles representing unselected TILs. The horizontal lines represent the average percentage of each subset and the vertical lines represent the standard error.

FIG. 167: CD4 in PD-1-selected and unselected TILs⁺And CD8⁺Levels of T cells. Flow cytometry was used to assess T cell lineages (CD4 and CD8) of PD-1-selected and unselected TIL from 4 melanoma, 7 NSCLC and 2 HNSCC tumor samples. The results are expressed as CD3⁺Percentage of cells. The mean values are plotted as a bar graph and the standard error is shown as a vertical line.

FIG. 168: the distribution of memory T cell subsets in PD-1-selected TILs and unselected TILs was compared. Expression of memory markers CD45RA and CCR7 of PD-1 selected TIL and unselected TIL from 4 melanoma, 6 NSCLC and 2 HNSCC tumor samples was assessed by flow cytometry. As indicated, T cell memory subpopulations were determined and the average percentage of each subpopulation was plotted as black bars for PD-1-selected TILs and gray bars for unselected TILs. The standard error is shown as a vertical line.

FIG. 169: before and after amplification, in the presence of PD-1⁺PD-1 expression in sorted TILs and unselected TILs. PD-1-sorted TIL and whole tumor digests from 3 melanoma, 7 NSCLC and 2 HNSCC tumor samples were evaluated for PD-1 expression before and after amplification. Using PD1 ⁺Determination of Pre-amplification PD-1 by post-sort purity of sorted products⁺Percentage of TIL. The mean values are plotted as a bar graph and the standard error is shown as a vertical line. Statistical significance was assessed by paired student t-test; indicates p values<0.001 and<0.0001。

FIG. 170: frequency of the first 10 PD-1-selected TCRv β clones in unselected TILs. Legend: libraries of amplified PD-1-selected and unselected TILs CDR3v β from 2 HNSCC and 5 NSCLC tumor samples were analyzed. Unique CDR3v β sequences identified in PD-1 selected TIL products were ranked from most to least frequent. The frequency of "top 10" (i.e., 10 most frequently cloned) TIL clones selected by PD-1 in each matched pair was plotted. The mated samples were connected by a straight line. The P values calculated by the paired t test are labeled on their respective graphs.

FIG. 171: PD-1 selected TILs demonstrated superior autologous tumor reactivity compared to matched unselected TILs. PD-1 selected and matched unselected TILs obtained from 3 melanoma, 2 NSCLC, 1 PC and 1 TNBC samples were tested for IFN γ secretion by ELISA in response to 18-24 hours of incubation with autologous tumor digests. The difference in IFN γ concentration measured for each individual sample with and without HLAI-type blocking antibody is shown. Positive values reflect HLA-specific anti-tumor responses, while negative or negative values reflect non-specific responses.

FIG. 172: PD-1-selected and unselected TILs demonstrated autologous tumor killing. Tumor killing and reactivity were assessed in PD-1-selected and unselected TILs using an xCELLigence real-time cell analysis system. The (a) cell index and (B) tumor cell killing (% cytolysis) of melanoma samples are shown.

FIG. 172: PD-1 levels in nivolumab and EH12.2H7-stained TIL. Whole tumor digests were split and stained with nivolumab or EH12.2H7 and evaluated by flow cytometry. PD-1 from 1 ovary, 1 melanoma, and 1 HNSCC identified with each antibody was then sorted using an FX500 cell sorter (Sony, New York)⁺A cell.

FIG. 173: final product yields of nivolumab and EH12.2H7 stained PD-1 sorted TIL. A PD-1 sorted TIL derived from tins stained with nivolumab and EH12.2H7 from 1 ovary, 1 melanoma, and 1 HNSCC was amplified using a 11 day activation step followed by 11 days REP. Shows inoculated CD3⁺Number of cells, fold expansion and extrapolated/actual cell count. Ovarian and melanoma tumors designated by x are small scale experiments, and HNSCC designated by x was performed at full scale.

FIG. 174: CD4+ and CD8+ TIL were expressed in PD-1 selected TIL using EH12.2H7 and nivolumab. Evaluation of PD-1 Using nivolumab or EH12.2H7 sorting to identify from 1 ovary, 1 melanoma, and 1 HNSCC Using flow cytometry⁺T cell lineages of PD-1 selected TIL for TIL (CD4 and CD 8). The results are expressed as CD3⁺Percentage of cells. The mean values are plotted as a bar graph and the standard error is shown as a vertical line.

FIG. 175: EH12.2H7 and nivolumab sorted PD-1+ TIL. Evaluation of PD-1 from sorting using nivolumab or EH12.2H7 by flow cytometry⁺Expression of memory markers CD45RA and CCR7 for PD-1 selected TIL in 1 ovarian, 1 melanoma, and 1 HNSCC tumor sample of TIL. As indicated, T cell memory subpopulations were determined and the average percentage of each subpopulation was plotted as black bars for nivolumab with PD-1 selected TIL and gray bars for EH12.2H7 with PD-1 selected TIL. The standard error is shown as a vertical line.

FIG. 176: PD-1-expressed TIL expression in PD-1-sorted TIL generated using EH12.2H7 and nivolumab before and after amplification. Evaluation results from sorting using nivolumab or EH12.2H7 to identify PD-1 ⁺PD-1-sorted TIL expression of 1 ovary of TIL, 1 melanoma, and 1 HNSCC before and after amplification. Use of post-sort purity of PD-1 sorted products to determine pre-amplification PD-1⁺Percentage of (c). The mean values are plotted as a bar graph and the standard error is shown as a vertical line. Statistical significance was assessed by paired student t-test; indicates p value<0.01。

FIG. 177: PD-1 sorted using EH12.2H7 and nivolumab⁺TIL-generated PD-1-selected TIL produced IFN γ and granzyme B responded to non-specific stimulation. Evaluation results from sorting using nivolumab or EH12.2H7 to identify PD-1⁺Secretion of (a) IFN γ and (B) granzyme B from PD-1 selected TIL of 1 ovary, 1 melanoma and 1 HNSCC of TIL. Results for individual samples were plotted, with black dots representing unstimulated conditions and gray triangles representing conditions stimulated with α CD3/α CD28/α 41 BB. The horizontal lines represent the average percentage of each subset and the vertical lines represent the standard error.

FIG. 178: summary of examples of PD-1+ high Gen-2 processes.

FIG. 179: FACS plots data.

Brief description of the sequence listing

SEQ ID NO 1 is the amino acid sequence of the heavy chain of Moluomamab (muromonab).

SEQ ID NO 2 is the amino acid sequence of the light chain of Moluomamab.

SEQ ID NO 3 is the amino acid sequence of recombinant human IL-2 protein.

SEQ ID NO 4 is the amino acid sequence of aldesleukin (aldesleukin).

SEQ ID NO 5 is the amino acid sequence of recombinant human IL-4 protein.

SEQ ID NO 6 is the amino acid sequence of recombinant human IL-7 protein.

SEQ ID NO 7 is the amino acid sequence of recombinant human IL-15 protein.

SEQ ID NO 8 is the amino acid sequence of recombinant human IL-21 protein.

SEQ ID NO 9 is the amino acid sequence of human 4-1 BB.

SEQ ID NO 10 is the amino acid sequence of murine 4-1 BB.

SEQ ID NO:11 is the heavy chain of the 4-1BB agonist monoclonal antibody, Utomilumab (PF-05082566).

SEQ ID NO:12 is the light chain of the 4-1BB agonist monoclonal antibody, Utomoluumab (PF-05082566).

SEQ ID NO:13 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody, Utomoluzumab (PF-05082566).

SEQ ID NO:14 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody, Utomoluzumab (PF-05082566).

SEQ ID NO. 15 is the heavy chain CDRL of the 4-1BB agonist monoclonal antibody, urotropinumab (PF-05082566).

SEQ ID NO 16 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody, urotropinumab (PF-05082566).

SEQ ID NO:17 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody, urotropinumab (PF-05082566).

18 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody Utomoluumab (PF-05082566).

SEQ ID NO:19 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody, Utomorrugamab (PF-05082566).

SEQ ID NO:20 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody, Utomorrugamab (PF-05082566).

SEQ ID NO:21 is the heavy chain of the 4-1BB agonist monoclonal antibody Urelumab (BMS-663513).

SEQ ID NO. 22 is the light chain of the 4-1BB agonist monoclonal antibody Uruguzumab (BMS-663513).

SEQ ID NO:23 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody Uruguzumab (BMS-663513).

SEQ ID NO. 24 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody Uruguzumab (BMS-663513).

SEQ ID NO:25 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody Uruguzumab (BMS-663513).

SEQ ID NO:26 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody Uruguzumab (BMS-663513).

SEQ ID NO:27 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody Uruguzumab (BMS-663513).

SEQ ID NO 28 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody Uruguzumab (BMS-663513).

SEQ ID NO:29 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody Uruguzumab (BMS-663513).

SEQ ID NO 30 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody Uruguzumab (BMS-663513).

31 is the Fc domain of a TNFRSF agonist fusion protein.

SEQ ID NO 32 is a linker for TNFRSF agonist fusion proteins.

SEQ ID NO 33 is a linker for TNFRSF agonist fusion proteins.

SEQ ID NO 34 is a linker for TNFRSF agonist fusion proteins.

35 is a linker for TNFRSF agonist fusion proteins.

SEQ ID NO 36 is a linker for TNFRSF agonist fusion proteins.

SEQ ID NO 37 is a linker for TNFRSF agonist fusion proteins.

SEQ ID NO 38 is a linker for TNFRSF agonist fusion proteins.

SEQ ID NO 39 is a linker for TNFRSF agonist fusion proteins.

SEQ ID NO 40 is a linker for TNFRSF agonist fusion proteins.

SEQ ID NO 41 is a linker for TNFRSF agonist fusion proteins.

42 is the Fc domain of TNFRSF agonist fusion protein.

SEQ ID NO 43 is a linker for TNFRSF agonist fusion proteins.

SEQ ID NO 44 is a linker for TNFRSF agonist fusion proteins.

SEQ ID NO 45 is a linker for TNFRSF agonist fusion proteins.

SEQ ID NO 46 is the 4-1BB ligand (4-1BBL) amino acid sequence.

47 is the soluble portion of the 4-1BBL polypeptide.

SEQ ID NO:48 is the heavy chain variable region (VH) of 4-1BB agonist antibody 4B4-1-1 version 1.

SEQ ID NO:49 is the light chain variable region (VL) of 4-1BB agonist antibody 4B4-1-1 version 1.

50 is the heavy chain variable region (VH) of 4-1BB agonist antibody 4B4-1-1 version 2.

SEQ ID NO:51 is the light chain variable region (VL) of 4-1BB agonist antibody 4B4-1-1 version 2.

SEQ ID NO:52 is the heavy chain variable region (VH) of the 4-1BB agonist antibody H39E 3-2.

SEQ ID NO:53 is the light chain variable region (VL) of 4-1BB agonist antibody H39E 3-2.

54 is the amino acid sequence of human OX 40.

SEQ ID NO:55 is the amino acid sequence of murine OX 40.

SEQ ID NO:56 is the heavy chain of the OX40 agonist monoclonal antibody tamsulizumab (Tavolixizumab) (MEDI-0562).

57 is the light chain of the OX40 agonist monoclonal antibody tavalizumab (MEDI-0562).

SEQ ID NO:58 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody tavalizumab (MEDI-0562).

SEQ ID NO:59 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody tavalizumab (MEDI-0562).

SEQ ID NO:60 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tavalizumab (MEDI-0562).

SEQ ID NO 61 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tamsulzumab (MEDI-0562).

SEQ ID NO:62 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tavalizumab (MEDI-0562).

63 is the light chain CDR1 of the OX40 agonist monoclonal antibody tamsulizumab (MEDI-0562).

SEQ ID NO:64 is the light chain CDR2 of the OX40 agonist monoclonal antibody tavalizumab (MEDI-0562).

SEQ ID NO:65 is the light chain CDR3 of the OX40 agonist monoclonal antibody tavalizumab (MEDI-0562).

66 is the heavy chain of OX40 agonist monoclonal antibody 11D 4.

67 is the light chain of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO 68 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO:69 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 11D 4.

70 is the heavy chain CDR1 of OX40 agonist monoclonal antibody 11D 4.

71 is the heavy chain CDR2 of OX40 agonist monoclonal antibody 11D 4.

72 is the heavy chain CDR3 of OX40 agonist monoclonal antibody 11D 4.

73 is the light chain CDR1 of OX40 agonist monoclonal antibody 11D 4.

74 is the light chain CDR2 of OX40 agonist monoclonal antibody 11D 4.

75 is the light chain CDR3 of OX40 agonist monoclonal antibody 11D 4.

76 is the heavy chain of OX40 agonist monoclonal antibody 18D 8.

77 is the light chain of OX40 agonist monoclonal antibody 18D 8.

78 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO:79 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 18D 8.

80 is the heavy chain CDR1 of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO 81 is the heavy chain CDR2 of OX40 agonist monoclonal antibody 18D 8.

82 is the heavy chain CDR3 of OX40 agonist monoclonal antibody 18D 8.

83 is the light chain CDR1 of OX40 agonist monoclonal antibody 18D 8.

84 is the light chain CDR2 of OX40 agonist monoclonal antibody 18D 8.

85 is the light chain CDR3 of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO 86 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody Hu 119-122.

SEQ ID NO:87 is the light chain variable region (VL) of OX40 agonist monoclonal antibody Hu 119-122.

88 is the heavy chain CDR1 of OX40 agonist monoclonal antibody Hu 119-122.

89 is the heavy chain CDR2 of OX40 agonist monoclonal antibody Hu 119-122.

SEQ ID NO 90 is the heavy chain CDR3 of OX40 agonist monoclonal antibody Hu 119-122.

91 is the light chain CDR1 of OX40 agonist monoclonal antibody Hu 119-122.

92 is the light chain CDR2 of OX40 agonist monoclonal antibody Hu 119-122.

93 is the light chain CDR3 of OX40 agonist monoclonal antibody Hu 119-122.

SEQ ID NO 94 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO 95 is the light chain variable region (VL) of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO 96 is the heavy chain CDR1 of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO 97 is the heavy chain CDR2 of OX40 agonist monoclonal antibody Hu 106-222.

98 is the heavy chain CDR3 of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO 99 is the light chain CDR1 of OX40 agonist monoclonal antibody Hu 106-222.

100 is the light chain CDR2 of OX40 agonist monoclonal antibody Hu 106-222.

101 is the light chain CDR3 of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO:102 is the OX40 ligand (OX40L) amino acid sequence.

103 is a soluble portion of an OX40L polypeptide.

104 is an alternative soluble portion of the OX40L polypeptide.

105 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 008.

106 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 008.

107 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 011.

108 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 011.

109 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 021.

SEQ ID NO:110 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 021.

111 is the heavy chain variable region (VH) of OX40 agonist monoclonal antibody 023.

SEQ ID NO:112 is the light chain variable region (VL) of OX40 agonist monoclonal antibody 023.

113 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

SEQ ID NO:114 is the light chain variable region (VL) of the OX40 agonist monoclonal antibody.

SEQ ID NO:115 is the heavy chain variable region (VH) of the OX40 agonist monoclonal antibody.

116 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

SEQ ID NO:117 is the heavy chain variable region (VH) of the humanized OX40 agonist monoclonal antibody.

118 is the heavy chain variable region (VH) of the humanized OX40 agonist monoclonal antibody.

SEQ ID NO:119 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

120 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

121 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.

122 is the heavy chain variable region (VH) of the humanized OX40 agonist monoclonal antibody.

123 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

124 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.

125 is the heavy chain variable region (VH) of an OX40 agonist monoclonal antibody.

126 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.

I. Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned herein are incorporated by reference in their entirety.

The term "in vivo" refers to an event that occurs in a subject.

The term "in vitro" refers to an event that occurs outside of the body of a subject. In vitro assays encompass cell-based assays, wherein viable or dead cells are employed; and also cell-free assays, where intact cells are not employed, can be contemplated.

The term "ex vivo" refers to an event that involves a treatment or performance of a procedure on a cell, tissue, and/or organ that has been removed from a subject. Suitably, the cells, tissues and/or organs may be returned to the subject during surgery or a method of treatment.

The term "rapid expansion" means that the amount of antigen-specific TIL is increased at least about 3 fold (or 4 fold, 5 fold, 6 fold, 7 fold, 8 fold or 9 fold) over a period of one week, more preferably at least about 10 fold (or 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold or 90 fold) over a period of one week or most preferably at least about 100 fold over a period of one week. A number of rapid amplification protocols are outlined below.

By "tumor infiltrating lymphocytes" or "TILs" herein is meant a population of cells, originally obtained as leukocytes, that have left the subject's bloodstream and migrated into the tumor. TIL includes but is not limited to CD8⁺Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4⁺T cells, natural killer cells, dendritic cells and M1 macrophages. The TILs include both primary and secondary TILs. "Primary TIL" is derived fromThose TILs obtained from patient tissue samples as outlined (sometimes referred to as "freshly obtained" or "freshly isolated"), and a "secondary TIL" is any TIL cell population that has been expanded or propagated as discussed herein, including but not limited to bulk TIL and expanded TIL ("REP TIL" or "post-REP TIL"). The TIL cell population may comprise a genetically modified TIL.

By "cell population" (comprising TILs) herein is meant a number of cells that share a common trait. Typically, the number of populations is typically 1 × 10⁶To 1X 10¹⁰Wherein different TIL populations comprise different amounts. For example, initial growth of primary TIL in the presence of IL-2 would result in a tissue having a size of approximately 1X 10⁸A bulk TIL population of individual cells. Amplification of REP is generally accomplished to provide a DNA with a size of 1.5X 10⁹To 1.5X 10¹⁰A population of individual cells for infusion.

By "cryopreserved TIL" herein is meant TIL (primary, bulk or amplified (REP TIL)) processed or stored in the range of about-150 ℃ to-60 ℃. General methods for cryopreservation are also described elsewhere herein (included in the examples). For clarity, "cryopreserved TILs" may be distinguished from frozen tissue samples that may be used as a source of primary TILs.

By "thawed cryopreserved TILs" herein is meant a population of TILs that have been previously cryopreserved and subsequently processed to return to room temperature or higher (including but not limited to cell culture temperature or temperature at which TILs may be administered to a patient).

TILs can be generally defined biochemically using cell surface markers, or functionally by their ability to infiltrate a tumor and effect treatment. TILs can be generally classified by expressing one or more of the following biomarkers: CD4, CD8, TCR α β, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD 25. Additionally and alternatively, TIL may be functionally defined by its ability to infiltrate a solid tumor after reintroduction into the patient.

The term "cryopreserved medium" means a culture medium that can be stored at a low temperatureAny medium used for cryopreservation of cells. Such media may comprise 7% to 10% DMSO media. Exemplary media include CryoStor CS10, hyperthermosol, and combinations thereof. The term "CS 10" refers to cryopreserved media obtained from stem cell Technologies, inc (Stemcell Technologies) or Biolife Solutions, inc (Biolife Solutions). CS10 Medium may be under the trade name "CS10 ". CS10 medium is serum-free animal component-free medium including DMSO.

The term "central memory T cell" refers to a CD45R0+ in humans and constitutively expresses CCR7(CCR 7)^hi) And CD62L (CD 62)^hi) A subpopulation of T cells. The surface phenotype of central memory T cells also includes TCR, CD3, CD127(IL-7R) and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2 and BMI 1. After TCR triggering, central memory T cells secrete mainly IL-2 and CD40L as effector molecules. Central memory T cells are predominantly present in the CD4 compartment in the blood and are proportionally abundant in lymph nodes and tonsils in humans.

The term "effector memory T cells" refers to e.g. central memory T cells that are CD45R0+, but have lost constitutive expression of CCR7(CCR 7) ^lo) And are heterogeneous or low for CD62L expression (CD 62L)^lo) Of human or mammalian T cells. The surface phenotype of central memory T cells also includes TCR, CD3, CD127(IL-7R) and IL-15R. The transcription factor of central memory T cells includes BLIMP 1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines including interferon gamma, IL-4 and IL-5 following antigen stimulation. Effector memory T cells are predominantly present in the CD8 compartment in the blood and are proportionally enriched in the lung, liver and intestinal tract in humans. CD8+ effector memory T cells carry large amounts of perforin.

The term "closed system" refers to a system that is closed to the external environment. The method of the present invention may employ any closed system suitable for cell culture methods. The closed system comprises, for example but not limited to, a closed G-vessel. Once the tumor section is added to the closed system, the system is not open to the external environment until the TIL is ready to be administered to the patient.

The terms "fragmenting", "fragmenting" and "fragmented" as used herein to describe the process of destroying a tumor encompass mechanical fragmentation methods such as crushing, cutting, disrupting and mincing tumor tissue and any other method for destroying the physical structure of tumor tissue.

The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with a circular nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen presenting cells (PBMCs are a type of antigen presenting cells), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

The terms "peripheral blood lymphocytes" and "PBLs" refer to T cells expanded from peripheral blood. In some embodiments, the PBLs are isolated from whole blood or apheresis (apheresis) products from a donor. In some embodiments, PBLs are isolated from whole blood or apheresis products from a donor by positive or negative selection for a T cell phenotype (e.g., a T cell phenotype of CD3+ CD45 +).

The term "anti-CD 3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody, directed against the CD3 receptor among T cell antigen receptors of mature T cells, and includes human, humanized, chimeric, or murine antibodies. The anti-CD 3 antibody comprises OKT-3, also known as molobuzumab. The anti-CD 3 antibody also comprises the UHCT1 clone, also known as T3 and CD3 epsilon. Other anti-CD 3 antibodies include, for example, oxizumab (otelixizumab), telizumab (teplizumab), and vislizumab (visilizumab).

The term "OKT-3" (also referred to herein as "OKT 3") refers to a monoclonal antibody or biosimilar or variant thereof directed against CD3 receptor in the T cell antigen receptor of mature T cells, including human, humanized, chimeric, or murine antibodies, and including commercially available forms such as OKT-3(30ng/mL, pure MACS GMP CD3, Miltenyi Biotech, inc., San Diego, CA, USA) and molo mab or a variant, conservative amino acid substitution, glycoform, or biosimilar thereof. The amino acid sequences of the heavy and light chains of Moluomamab are given in Table 1 (SEQ ID NO:1and SEQ ID NO: 2). Hybridomas capable of producing OKT-3 are deposited by the American Type Culture Collection and assigned ATCC accession number CRL 8001. Hybridomas capable of producing OKT-3 are also deposited by the European Collection of Authenticated Cell Cultures (ECACC) and are assigned catalog number 86022706.

Table 1: amino acid sequence of Moluomamab.

The term "IL-2" (also referred to herein as "IL 2") refers to a T cell growth factor known as interleukin-2, and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biological analogs and variants thereof. IL-2 is described, for example, in Nelson, J.Immunol. (R) 2004,172,3983-88 and Malek, Annu.Rev.Immunol. (R) 2008,26,453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant forms of IL-2, such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers, 2200 thousand IU per single use vial), as well as recombinant IL-2 forms sold by CellGenix corporation of celtomason town, new hampshire (CellGenix, inc., Portsmouth, NH, USA) (cellgirro GMP) or profec-Tany TechnoGene ltd, East bronzee, new jersey (profec-Tany TechnoGene ltd., East unswick, NJ, USA) (catalog number CYT-209-b) and other commercial equivalents from other suppliers. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is given in Table 2 (SEQ ID NO: 4). The term IL-2 also encompasses pegylated forms of IL-2 as described herein, comprising pegylated IL2 prodrug NKTR-214, available from nkta Therapeutics, South San Francisco, CA, USA, in San Francisco, South, njac, california. NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in U.S. patent application publication No. US 2014/0328791A 1and International patent application publication No. WO 2012/065086 Al, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. patent nos. 4,766,106, 5,206,344, 5,089,261 and 4902,502, the disclosures of which are incorporated herein by reference. IL-2 formulations suitable for use in the present invention are described in U.S. Pat. No. 6,706,289, the disclosure of which is incorporated herein by reference.

Table 2: amino acid sequence of interleukin.

The term "IL-4" (also referred to herein as "IL 4") refers to a cytokine called interleukin 4, which is produced by Th 2T cells and by eosinophils, basophils, and mast cells. IL-4 regulates differentiation of naive helper T cells (Th0 cells) into Th 2T cells. Steinke and Borish, "respiratory study (respir. res.), 2001,2, 66-70. Following activation by IL-4, Th 2T cells subsequently produced additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and MHC class II expression, and induces class switching to IgE and IgG of B cells₁And (4) expressing. Recombinant human IL-4 suitable for use in the present invention is commercially available from a variety of suppliers, including ProSpec-Tany TechnoGene, Inc., of east Broensvick, N.J. (Cat. CYT-211) and Semmerfel technologies, Inc., of Waltherm, Mass. (human IL-15 recombinant protein, Cat. Gibco CTP 0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 5).

The term "IL-7" (also referred to herein as "IL 7") refers to a glycosylated tissue-derived cytokine called interleukin 7, which can be obtained from stromal and epithelial cells, as well as dendritic cells. Fry and Mackall, Blood (Blood) 2002,99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor (a heterodimer consisting of the IL-7 receptor alpha and the common gamma chain receptor, among a series of signals critical for T cell development in the thymus and survival in the periphery). Recombinant human IL-7 suitable for use in the present invention is commercially available from a variety of suppliers, including ProSpec-Tany TechnoGene, Inc., of east Bronstick, N.J. (Cat. CYT-254) and Semmerman Feishel science, Inc., of Waltherm, Mass. (human IL-15 recombinant protein, Cat. Gibco PHC 0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 6).

The term "IL-15" (also referred to herein as "IL 15") refers to a T cell growth factor known as interleukin-15, and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biological analogs and variants thereof. IL-15 is described, for example, in Fehniger and Caligiuri, blood 2001,97,14-32, the disclosures of which are incorporated herein by reference. IL-15 shares beta and gamma signaling receptor subunits with IL-2. Recombinant human IL-15 is a 12.8kDa molecular weight non-glycosylated polypeptide chain of 114 amino acids (and an N-terminal methionine). Recombinant human IL-15 is commercially available from a variety of suppliers, including ProSpec-Tany TechnoGene, Inc., of east Bronstick, N.J. (Cat. CYT-230-b), and Seimer Feishel science, Inc., of Waltham, Massachusetts (human IL-15 recombinant protein, Cat. 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 7).

The term "IL-21" (also referred to herein as "IL 21") refers to a pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biological analogs and variants thereof. IL-21 is described, for example, in Spolski and Leonard, Nature review-pharmaceuticals In research and development (nat. rev. drug. disc.), 2014,13,379-95, the disclosures of which are incorporated herein by reference. IL-21 is mainly composed of natural killer T cells and activated human CD4⁺T cell production. Recombinant human IL-21 is a 132 amino acid-containing, non-glycosylated polypeptide chain with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from a variety of suppliers, including ProSpec-Tany TechnoGene, Inc., Voronesvick, N.J. (Cat. CYT-408-b), and Semmerfel scientific, Inc., Volserm, Mass., U.S.A. (human IL-21 recombinant protein, Cat. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 8).

When an "anti-tumor effective amount", "tumor inhibiting effective amount", or "therapeutic amount" is indicated, the precise amount of the composition of the invention to be administered can be determined by a physician considering individual differences in age, weight, tumor size, extent of infection or metastasis, and the condition of the patient (subject). It can be generally stated that a pharmaceutical composition comprising a tumor infiltrating lymphocyte (e.g., a secondary TIL or a genetically modified cytotoxic lymphocyte) as described herein can be present at 10 ⁴To 10¹¹Dosage per kilogram body weight (e.g., 10)⁵To 10⁶、10⁵To 10¹⁰、10⁵To 10¹¹、10⁶To 10¹⁰、10⁶To 10¹¹、10⁷To 10¹¹、10⁷To 10¹⁰、10⁸To 10¹¹、10⁸To 10¹⁰、10⁹To 10¹¹Or 10⁹To 10¹⁰Individual cells per kilogram body weight) (including all integer values within these ranges). Compositions of tumor infiltrating lymphocytes (in some cases comprising genetically modified cytotoxic lymphocytes) can also be administered multiple times at these doses. Tumor-infiltrating lymphocytes, in some cases genetically contained, can be administered by using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J.of Med.) (319: 1676,1988). Optimal dosages and treatment regimens for a particular patient may be determined by the skill in the medical artsThe operator can easily determine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

The terms "hematological malignancy", or related terms refer to mammalian cancers and tumors of hematopoietic and lymphoid tissues, including but not limited to blood, bone marrow, lymph nodes, and tissues of the lymphatic system. Hematological malignancies are also known as "liquid tumors". Hematological malignancies include, but are not limited to, Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Lymphoma (CLL), Small Lymphocytic Lymphoma (SLL), Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B cell hematologic malignancy" refers to a hematologic malignancy that affects B cells.

The term "solid tumor" refers to an abnormal tissue mass that generally does not contain cysts or fluid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic or cancerous solid tumor. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung, breast, prostate, colon, rectal, and bladder cancers. The tissue structure of solid tumors comprises interdependent tissue compartments, including parenchyma tissue (cancer cells) and supporting stromal cells in which cancer cells are dispersed and which can provide a supporting microenvironment.

The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as Marrow Infiltrating Lymphocytes (MILs). TILs obtained from liquid tumors (including liquid tumors circulating in peripheral blood) may also be referred to herein as PBLs. The terms MIL, TIL and PBL are used interchangeably herein and differ only based on the type of tissue from which the cells are derived.

The term "microenvironment" as used herein may refer to a solid or hematologic tumor microenvironment as a whole or to individual cell subsets within the microenvironment. Tumor microenvironment as used herein refers to a complex mixture of cells, soluble factors, signaling molecules, extracellular matrix and mechanical cues that "promote neoplastic transformation, support tumor growth and invasion, protect tumor host immunity, promote treatment resistance and provide for metastasis of dominant cancers to a growing niche" as described in Swartz et al, Cancer research (Cancer Res.) 2012,72, 2473. Although tumors express antigens that should be recognized by T cells, clearance of the tumor by the immune system is rare due to immunosuppressive effects of the microenvironment.

In an embodiment, the invention comprises a method of treating cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to infusion of a TIL according to the invention. In some embodiments, a population of TILs may be provided wherein a patient is pre-treated with non-myeloablative chemotherapy prior to infusion of a TIL according to the invention. In the examples, the non-myeloablative chemotherapy was cyclophosphamide 60mg/kg/d for 2 days (day 27 and 26 before TIL infusion) and fludarabine 25mg/m2/d for 5 days (day 27 to day 23 before TIL infusion). In the examples, following non-myeloablative chemotherapy and infusion of TIL according to the invention (day 0), patients received intravenous infusion of 720,000IU/kg IL-2 every 8 hours until physiological tolerance.

Experimental results indicate that lymphocyte depletion plays a key role in enhancing the efficacy of treatment by eliminating competing elements of the regulatory T cells and the immune system ("cytokine sink") prior to adoptive transfer of tumor-specific T lymphocytes. Thus, some embodiments of the invention employ a lymphocyte depletion step (sometimes also referred to as "immunosuppression modulation") on the patient prior to introducing the rTILs of the invention.

The terms "co-administration", "administration in combination with … …", "administration in combination with … …", "simultaneous" and "simultaneous" as used herein encompass the administration of two or more active pharmaceutical ingredients (in a preferred embodiment of the invention, e.g. at least one potassium channel agonist in combination with a plurality of TILs) to a subject such that both active pharmaceutical ingredients and/or their metabolites are present in the subject at the same time. Co-administration comprises simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. It is preferred that the administration is simultaneous in the form of separate compositions and in the form of a composition in which both agents are present.

The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds sufficient to effect the intended use (including but not limited to treatment of disease) as described herein. The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo) or the subject and disease condition to be treated (e.g., weight, age, and sex of the subject), the severity of the disease condition, or the mode of administration. The term also applies to doses that will induce a particular response (e.g., reduced platelet adhesion and/or cell migration) in the target cells. The specific dosage will vary depending upon the particular compound selected, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which the compound is administered, and the physical delivery system in which the compound is carried.

The terms "treatment (therapy)", "treating (therapy)" and the like refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or a symptom thereof, and/or may be therapeutic in terms of a partial or complete cure for a disease and/or a side effect attributable to a disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing a disease from occurring in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) alleviating the disease, i.e., causing regression of the disease and/or alleviating one or more symptoms of the disease. "treating" is also intended to encompass delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition (e.g., in the case of a vaccine).

The term "heterologous" when used in reference to portions of a nucleic acid or protein indicates that the nucleic acid or protein includes two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically recombinantly produced, with two or more sequences from unrelated genes arranged to produce a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source or a coding region from a different source. Similarly, a heterologous protein indicates that a protein includes two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

The terms "sequence identity", "percent identity", and "percent sequence identity" (or synonyms thereof, such as "99% identical") in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, regardless of any conservative amino acid substitutions as part of sequence identity, when compared and aligned for maximum correspondence (introducing gaps, if necessary). Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, the BLAST program suite available from the National Center for Biotechnology Information BLAST website (u.s.goverment's National Center for Biotechnology BLAST web site) of the united states government. Comparisons between two sequences can be made using the BLASTN or BLASTP algorithms. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, southern san Francisco, Calif.) or MegAlign available from DNASTAR are other publicly available software programs that can be used to ALIGN sequences. One skilled in the art can determine the appropriate parameters for maximum alignment by the particular alignment software. In certain embodiments, default parameters of the alignment software are used.

As used herein, the term "variant" encompasses, but is not limited to, an antibody or fusion protein that includes an amino acid sequence that differs from the amino acid sequence of a reference antibody by means of one or more substitutions, deletions, and/or additions at certain positions within or adjacent to the amino acid sequence of the reference antibody. A variant may comprise one or more conservative substitutions in its amino acid sequence, as compared to the amino acid sequence of a reference antibody. Conservative substitutions may involve, for example, substitution of similarly charged or uncharged amino acids. The variants retain the ability to specifically bind to the antigen of the reference antibody. The term variant also encompasses pegylated antibodies or proteins.

By "tumor infiltrating lymphocytes" or "TILs" herein is meant a population of cells, originally obtained as leukocytes, that have left the subject's bloodstream and migrated into the tumor. TIL includes but is not limited to CD8⁺Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4⁺T cells, natural killer cells, dendritic cells and M1 macrophages. The TILs include both primary and secondary TILs. "primary TILs" are those TILs obtained from patient tissue samples as outlined herein (sometimes referred to as "freshly obtained" or "freshly isolated"), and "secondary TILs" are any TIL cell population that has been expanded or propagated as discussed herein, including but not limited to bulk TILs, expanded TILs ("REP TILs"), and "REP TILs" as discussed herein. The rep TIL may comprise, for example, a second amplified TIL or a second additional amplified TIL (e.g., those described in step D of fig. 27, comprising a TIL referred to as a rep TIL).

TILs can be generally defined biochemically using cell surface markers, or functionally by their ability to infiltrate a tumor and effect treatment. TILs can be generally classified by expressing one or more of the following biomarkers: CD4, CD8, TCR α β, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD 25. Additionally and alternatively, TIL may be functionally defined by its ability to infiltrate a solid tumor after reintroduction into the patient. The TIL may be further characterized by potency-for example, a TIL may be considered effective if, for example, Interferon (IFN) release is greater than about 50pg/mL, greater than about 100pg/mL, greater than about 150pg/mL, or greater than about 200 pg/mL.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to encompass any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is envisaged. Additional active pharmaceutical ingredients (such as other drugs) may also be incorporated into the described compositions and methods.

The terms "about" and "approximately" mean within a statistically significant range of values. Such ranges may be within an order of magnitude, preferably within 50%, more preferably within 20%, more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variations encompassed by the terms "about" or "approximately" depend on the particular system under study and can be readily understood by one of ordinary skill in the art. Further, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, reflecting tolerances, conversion factors, rounding off, measurement error and the like, as desired, and other factors known to those of skill in the art. Typically, the size, dimensions, formulation, parameters, shape, or other quantities or characteristics are "about" or "approximately" whether or not explicitly stated in the formulation. It should be noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.

The transitional terms "comprising", "consisting essentially of … …" and "consisting of … …" when used in the appended claims, both original and modified form, define the scope of the claims with respect to which additional claim elements or steps, if any, that are not listed are excluded from the scope of one or more claims. The term "comprising" is intended to be inclusive or open-ended and does not exclude any additional unrecited elements, methods, steps, or materials. The term "consisting of … …" excludes any element, step, or material other than those specified in the claims, and in the latter case excludes impurities ordinarily associated with the specified material or materials. The term "consisting essentially of … …" limits the scope of the claims to one or more specified elements, steps or materials and to elements, steps or materials that do not materially affect one or more of the basic and novel characteristics of the claimed invention. In alternative embodiments, all compositions, methods, and kits embodying the invention described herein may be more specifically defined by any of the transitional terms "comprising", "consisting essentially of … … (of), and" consisting of … … (of) ".

The term "PD-1 high" or "PD-1^{Height of}"refers to high levels of PD-1 protein expression by cells (such as, but not limited to, tumor infiltrating lymphocytes or T cells) relative to control cells from a healthy subject. In some embodiments, PD-1 expression levels are determined using standard methods known to those skilled in the art for measuring protein levels present on cells, such as flow cytometry, Fluorescence Activated Cell Sorting (FACS), immunocytochemistry, and the like. In some cases, a high TIL of PD-1 expresses higher levels of PD-1 than immune cells from healthy subjects. In some cases, a PD-1 high TIL population expresses higher levels of PD-1 than a population of immune cells (e.g., peripheral blood mononuclear cells) from a healthy subject or a group of healthy subjects. PD-1 high cells may be referred to as PD-1 bright cells.

The term "PD-1 intermediate" or "PD-1 intermediate" refers to an intermediate or moderate level of PD-1 protein expression by cells (such as, but not limited to, tumor infiltrating lymphocytes or T cells) relative to control cells from a healthy subject. For example, PD-1 intermediate T cells express PD-1 protein at a level or range that is similar or substantially equivalent to the highest range of PD-1 protein expressed by control cells (e.g., peripheral blood mononuclear cells) from healthy subjects. In other words, the PD-1 expression level of the PD-1 intermediate TIL is similar or substantially equivalent to the background level of PD-1 expression of control immune cells from healthy subjects. PD-1 intermediate cells may be referred to as PD-1 dark cells. One skilled in the art recognizes that PD-1 positive TIL may be PD-1 high TIL or PD-1 intermediate TIL.

The term "PD-1 negative" or "PD-1^{Negative of}"refers to negative or low level expression of PD-1 protein by cells such as, but not limited to, tumor infiltrating lymphocytes or T cells, relative to control cells from a healthy subject. For example, PD-1 negative T cells do not express PD-1 protein. In some cases, PD-1 negative T cells express PD-1 protein at a level similar to or substantially equivalent to the lowest level of PD-1 protein expressed by control cells (e.g., peripheral blood mononuclear cells) from healthy subjects. PD-1 negative lymphocytes may express PD-1 at the same level or range as most lymphocytes in the control population.

PD-1 high, PD-1 intermediate and PD-1 negative TIL are different and different subsets of TILs amplified ex vivo according to the methods described herein. In some embodiments, the ex vivo expanded TIL population comprises PD-1 high TIL, PD-1 intermediate TIL, and PD-1 negative TIL.

TIL manufacturing Process (an example of the GEN3 Process, optionally comprising defined Medium)

Without being bound by any particular theory, it is believed that priming a first expansion that primes T cell activation followed by promoting a rapid second expansion of T cell activation as described in the methods of the invention allows for the preparation of expanded T cells that retain a "younger" phenotype, and as such the expanded T cells of the invention are expected to exhibit greater cytotoxicity to cancer cells as compared to T cells expanded by other methods. In particular, it is believed that T cell activation promoted by exposure to anti-CD 3 antibodies (e.g., OKT-3), IL-2, and optionally Antigen Presenting Cells (APCs), as taught by the methods of the invention, and then by subsequent exposure to additional anti-CD 3 antibodies (e.g., OKT-3), IL-2, and APCs, limits or avoids T cell maturation in culture, thereby generating a population of T cells with a less mature phenotype that are depleted by expansion in culture and exhibit greater cytotoxicity to cancer cells. In some embodiments, the step of rapid second amplification is divided into multiple steps to achieve a scaled-up culture by: (a) performing a rapid second expansion by culturing T cells in a small-scale culture in a first container (e.g., a G-REX 100MCS container) for a period of about 3 to 4 days, and then (b) effecting transfer of T cells in the small-scale culture to a second container larger than the first container (e.g., a G-REX 500MCS container) and culturing T cells from the small-scale culture in a larger-scale culture in the second container for a period of about 4 to 7 days. In some embodiments, the step of rapid amplification is divided into multiple steps to achieve the outwardly expanding culture by: (a) performing a rapid second expansion by culturing the T cells in a first small-scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 3 to 4 days, and then (b) effecting transfer and distribution of the T cells from the first small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels of equal size to the first vessel, wherein in each second vessel the portion of the T cells transferred from the first small-scale culture to this second vessel is cultured in the second small-scale culture for a period of about 4 to 7 days. In some embodiments, the step of rapid amplification is divided into multiple steps to achieve the culture of scale-out and scale-up by: (a) performing a rapid second expansion by culturing the T cells in a small-scale culture in a first container (e.g., a G-REX 100MCS container) for a time period of about 3 to 4 days, and then (b) effecting transfer and distribution of the T cells from the small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of a larger size than the first container (e.g., a G-REX 500MCS container), wherein in each second container the portion of the T cells transferred from the large-scale culture to this second container is cultured in a larger-scale culture for a time period of about 4 to 7 days. In some embodiments, the step of rapid amplification is divided into multiple steps to achieve the culture of scale-out and scale-up by: (a) performing a rapid second expansion by culturing the T cells in a small-scale culture in a first container (e.g., a G-REX 100MCS container) for a period of about 4 days, and then (b) effecting transfer and distribution of the T cells from the small-scale culture into at least 2, 3, or 4 second containers of a larger size than the first container (e.g., a G-REX 500MCS container), wherein in each second container, the portion of the T cells transferred from the small-scale culture to this second container is cultured in a larger-scale culture for a period of about 5 days.

In some embodiments, the rapid second expansion is performed after the onset of reduction, attenuation, decline, or regression of T cell activation achieved by the priming first expansion.

The second rapid expansion is performed after the activation of T cells by the priming first expansion has been reduced to or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

In some embodiments, the rapid second expansion is performed after the activation of T cells by priming the first expansion has been reduced to or by a percentage in the range of about 1% to 100%.

In some embodiments, the rapid second expansion is performed after the activation of T cells by the priming first expansion has been reduced to or about a percentage in the range of 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

The second rapid expansion is performed after the activation of T cells by the priming first expansion has been reduced by at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.

The second rapid expansion is performed after the activation of T cells by the priming first expansion has been reduced by at most or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

In some embodiments, the reduction in T cell activation achieved by priming the first expansion is determined by a reduction in the amount of interferon gamma released by the T cells in response to stimulation with the antigen.

In some embodiments, priming first expansion of T cells is performed during a period of up to or about 7 days or about 8 days.

In some embodiments, priming first expansion of T cells is performed during a time period of at most or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.

In some embodiments, priming first expansion of T cells is performed during a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.

In some embodiments, the rapid second expansion of T cells is performed during a period of up to or about 11 days.

In some embodiments, the rapid second expansion of T cells is performed during a time period of at most or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some embodiments, the rapid second expansion of T cells is performed during a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some embodiments, the priming first expansion of T cells is performed during a time period of at or about 1 day to at or about 7 days, and the rapid second expansion of T cells is performed during a time period of at or about 1 day to at or about 11 days.

In some embodiments, the priming first expansion of T cells is performed during a time period of at most or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days, and the rapid second expansion of T cells is performed during a time period of at most or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some embodiments, the priming first expansion of T cells is performed during a time period of from at or about 1 day to at or about 8 days, and the rapid second expansion of T cells is performed during a time period of from at or about 1 day to at or about 9 days.

In some embodiments, priming first expansion of T cells is performed during a period of 8 days, and rapid second expansion of T cells is performed during a period of 9 days.

In some embodiments, the priming first expansion of T cells is performed during a time period of at or about 1 day to at or about 7 days, and the rapid second expansion of T cells is performed during a time period of at or about 1 day to at or about 9 days.

In some embodiments, the priming first expansion of T cells is performed during a 7 day period, and the rapid second expansion of T cells is performed during a 9 day period.

In some embodiments, the T cell is a Tumor Infiltrating Lymphocyte (TIL).

In some embodiments, the T cell is a Marrow Infiltrating Lymphocyte (MIL).

In some embodiments, the T cell is a Peripheral Blood Lymphocyte (PBL).

In some embodiments, the T cells are obtained from a donor having cancer.

In some embodiments, the T cell is a TIL obtained from a tumor resected from a patient having cancer.

In some embodiments, the T cells are MILs obtained from bone marrow of a patient with a hematological malignancy.

In some embodiments, the T cells are Peripheral Blood Mononuclear Cells (PBMCs) obtained from a donor. In some embodiments, the donor has cancer. In some embodiments, the donor has hematological malignancy.

In certain aspects of the disclosure, immune effector cells (e.g., T cells) can be obtained from a blood unit collected from a subject using any number of techniques known to those skilled in the art (e.g., FICOLL isolation). In a preferred aspect, the cells from the circulating blood of the individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one aspect, cells collected by apheresis may be washed to remove plasma fractions and, optionally, placed in an appropriate buffer or culture medium for subsequent processing steps. In one example, cells are washed with Phosphate Buffered Saline (PBS). In alternative embodiments, the wash solution lacks calcium, and may lack magnesium, or may lack many, if not all, divalent cations. In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by PERCOLL gradient centrifugation or by countercurrent centrifugation elutriation.

In some embodiments, the T cells are PBLs isolated from whole blood or an apheresis product enriched for lymphocytes from a donor. In some embodiments, the donor has cancer. In some embodiments, the donor has cancer. In some embodiments, the cancer is a cancer selected from the group consisting of: melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of: melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the donor has a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor has hematological malignancy.

In some embodiments, the T cells are PBLs isolated from whole blood or an apheresis product enriched for lymphocytes from a donor. In some embodiments, the donor has cancer. In some embodiments, the cancer is a cancer selected from the group consisting of: melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of: melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma. In some embodiments, the donor has a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor has hematological malignancy. In some embodiments, PBL is isolated from lymphocyte-enriched whole blood or apheresis products by using a positive or negative selection method, i.e., removing PBL with one or more markers for the T cell phenotype (e.g., CD3+ CD45+) or removing non-T cell phenotype cells, leaving PBL behind. In other embodiments, the PBLs are separated by gradient centrifugation. After isolation of PBLs from donor tissue, a suitable number of isolated PBLs (in some embodiments, about 1 x 10) can be seeded in a priming first expansion culture by a priming first expansion step according to any of the methods described herein ⁷PBL) to initiate priming first amplification of the PBLs.

An exemplary TIL process, referred to as process 3 (also referred to herein as GEN3), containing some of these features is depicted in fig. 1 (specifically, e.g., fig. 1B), and some advantages of this embodiment of the present invention over process 2A are described in fig. 1, 2, 30, and 31 (specifically, e.g., fig. 1B). Two embodiments of process 3 are shown in fig. 1 and 30 (specifically, for example, fig. 1B). Process 2A or Gen 2 is also described in U.S. patent publication No. 2018/0280436, which is incorporated herein by reference in its entirety. The Gen3 process is also described in USSN 62/755,954 (116983-.

As discussed and generally outlined herein, TILs are taken from patient samples and manipulated to amplify their quantity prior to transplantation into a patient using the TIL amplification process described herein and referred to as Gen 3. In some embodiments, as discussed below, TIL may optionally be genetically manipulated. In some embodiments, the TIL may be cryopreserved before or after amplification. After thawing, it may also be restimulated to increase its metabolism prior to infusion into the patient.

In some embodiments, the priming first amplification (comprising a process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in fig. 1 (in particular, e.g., fig. 1B and/or fig. 1C)) is shortened to 1 to 8 days, and the rapid second amplification (comprising a process referred to herein as a rapid amplification protocol (REP) and the process shown as step D in fig. 1 (in particular, e.g., fig. 1B and/or fig. 1C)) is shortened to 1 to 9 days, as discussed in detail below and in the examples and figures. In some embodiments, the priming first amplification (comprising a process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in fig. 1 (in particular, e.g., fig. 1B and/or fig. 1C)) is shortened to 1 to 8 days, and the rapid second amplification (comprising a process referred to herein as a rapid amplification protocol (REP) and the process shown as step D in fig. 1 (in particular, e.g., fig. 1B and/or fig. 1C)) is shortened to 1 to 8 days, as discussed in detail below and in the examples and figures. In some embodiments, the priming first amplification (comprising a process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in fig. 1 (in particular, e.g., fig. 1B and/or fig. 1C)) is shortened to 1 to 7 days, and the rapid second amplification (comprising a process referred to herein as a rapid amplification protocol (REP) and the process shown as step D in fig. 1 (in particular, e.g., fig. 1B and/or fig. 1C)) is shortened to 1 to 9 days, as discussed in detail below and in the examples and figures. In some embodiments, the priming first amplification (comprising a process referred to herein as pre-rapid amplification (pre-REP), and the process shown as step B in fig. 1 (in particular, e.g., fig. 1B and/or fig. 1C)) is from 1 to 7 days, and the rapid second amplification (comprising a process referred to herein as rapid amplification protocol (REP) and the process shown as step D in fig. 1 (in particular, e.g., fig. 1B and/or fig. 1C)) is from 1 to 10 days, as discussed in detail below and in the examples and figures. In some embodiments, the priming first amplification (e.g., the amplification described as step B in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is shortened to 8 days and the rapid second amplification (e.g., the amplification described as step D in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 7 to 9 days. In some embodiments, the priming first amplification (e.g., the amplification described as step B in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 8 days, and the rapid second amplification (e.g., the amplification described as step D in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 8 to 9 days. In some embodiments, the priming first amplification (e.g., the amplification described as step B in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is shortened to 7 days, and the rapid second amplification (e.g., the amplification described as step D in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is between 7 and 8 days. In some embodiments, the priming first amplification (e.g., the amplification described as step B in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is shortened to 8 days, and the rapid second amplification (e.g., the amplification described as step D in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 8 days. In some embodiments, the priming first amplification (e.g., the amplification described as step B in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 8 days and the rapid second amplification (e.g., the amplification described as step D in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 9 days. In some embodiments, the priming first amplification (e.g., the amplification described as step B in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 8 days and the rapid second amplification (e.g., the amplification described as step D in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 10 days. In some embodiments, the priming first amplification (e.g., the amplification described as step B in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 7 days, and the rapid second amplification (e.g., the amplification described as step D in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 7 to 10 days. In some embodiments, the priming first amplification (e.g., the amplification described as step B in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 7 days, and the rapid second amplification (e.g., the amplification described as step D in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 8 to 10 days. In some embodiments, the priming first amplification (e.g., the amplification described as step B in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 7 days, and the rapid second amplification (e.g., the amplification described as step D in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 9 to 10 days. In some embodiments, the priming first amplification (e.g., the amplification described as step B in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is shortened to 7 days, and the rapid second amplification (e.g., the amplification described as step D in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 7 to 9 days. In some embodiments, the combination of priming the first amplification and rapid second amplification (e.g., the amplification described as step B and step D in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is 14-16 days, as discussed in detail below and in the examples and figures. In particular, it is believed that certain embodiments of the invention comprise priming a first amplification step in which TIL is activated by exposure to an anti-CD 3 antibody (e.g., OKT-3) in the presence of IL-2 or to an antigen in the presence of at least IL-2 and an anti-CD 3 antibody (e.g., OKT-3). In certain embodiments, the TIL activated in priming the first amplification step as described above is the first TIL population, i.e. it is a primary cell population.

The following "step" designation A, B, C, and the like, refers to the non-limiting example in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C) and to certain non-limiting embodiments described herein. The order of the steps below and in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C) is exemplary, and any combination or order of steps as well as additional steps, repeated steps, and/or omitted steps are contemplated by the present application and the methods disclosed herein.

A. Step A: obtaining a patient tumor sample

Typically, TILs are initially obtained from a patient tumor sample ("primary TIL") or from circulating lymphocytes (such as peripheral blood lymphocytes, including peripheral blood lymphocytes having TIL-like properties), and then expanded into a larger population for further manipulation as described herein, optionally cryopreserved, and optionally evaluated for phenotypic and metabolic parameters as an indication of TIL health.

Patient tumor samples can be obtained using methods known in the art, typically by surgical resection, needle biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. In general, the tumor sample may be from any solid tumor, including a primary tumor, an invasive tumor, or a metastatic tumor. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematological malignancy. The solid tumor can be any cancer type, including but not limited to breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, stomach cancer, and skin cancer (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, e.g., Head and Neck Squamous Cell Carcinoma (HNSCC) Glioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer, and non-small cell lung cancer. In some embodiments, useful TILs are obtained from malignant melanoma tumors, as these malignant melanoma tumors are reported to have particularly high levels of TIL.

Once obtained, the tumor sample is typically fragmented into 1 to about 8mm using a sharp instrument cut³A tablet of (2-3 mm)³Is particularly useful. TILs were cultured from these fragments using enzymatic tumor digestion. Such tumor digests can be produced by incubation in enzymatic media (e.g., the Roxwell park Command (RPMI)1640 buffer, 2mM glutamic acid, 10mcg/mL gentamicin, 30 units/mL DNase, and 1.0mg/mL collagenase), followed by mechanical dissociation (e.g., using a tissue dissociator). Swelling and swelling treating medicineThe tumor digest can be prepared by placing the tumor in an enzymatic medium and mechanically dissociating the tumor for approximately 1 minute, followed by 37 ℃/5% CO₂Incubation for 30 minutes followed by repeated cycles of mechanical dissociation and incubation under the aforementioned conditions until only a small piece of tissue is present. At the end of the process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides can be used to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. patent application publication No. 2012/0244133a1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods may be used in any of the embodiments of the methods for amplifying TIL or treating cancer described herein.

As indicated above, in some embodiments, the TIL is derived from a solid tumor. In some embodiments, the solid tumor is not fragmented. In some embodiments, the solid tumor is not fragmented and enzymatically digested as an intact tumor. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, dnase and hyaluronidase. In some embodiments, the tumor is digested in an enzyme mixture comprising collagenase, dnase and hyaluronidase for 1-2 hours. In some embodiments, the tumor is treated with 5% CO at 37 ℃ in an enzyme mixture comprising collagenase, DNase, and hyaluronidase₂Digesting for 1-2 hours.

In some embodiments, the tumor is treated with 5% CO at 37 ℃ in an enzyme mixture comprising collagenase, DNase, and hyaluronidase₂Digestion is carried out for 1-2 hours by rotary digestion. In some embodiments, the tumor is digested overnight at constant rotation. In some embodiments, the tumor is treated with 5% CO at 37 ℃₂Digestion was performed overnight at constant rotation. In some embodiments, the whole tumor is combined with an enzyme to form a tumor digestion reaction mixture.

In some embodiments, the tumor is reconstituted with lyophilized enzyme in sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme cocktail comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock of collagenase is a 100mg/ml 10 × working stock.

In some embodiments, the enzyme mixture comprises dnase. In some embodiments, the working stock of DNase is 10,000IU/ml 10 Xworking stock.

In some embodiments, the enzyme mixture comprises a hyaluronidase. In some embodiments, the working stock of hyaluronidase is a 10-mg/ml 10 × working stock.

In some embodiments, the enzyme mixture comprises 10mg/ml collagenase, 1000IU/ml DNase, and 1mg/ml hyaluronidase.

In some embodiments, the enzyme mixture comprises 10mg/ml collagenase, 500IU/ml DNase, and 1mg/ml hyaluronidase.

In some embodiments, the enzyme mixture comprises about 10mg/ml collagenase, about 1000IU/ml DNase, and about 1mg/ml hyaluronidase.

In general, a cell suspension obtained from a tumor is referred to as an "initial cell population" or a "freshly obtained" or "freshly isolated" cell population. In certain embodiments, a freshly obtained TIL cell population is exposed to a cell culture medium comprising antigen presenting cells IL-12 and OKT-3.

In some embodiments, the fragmentation comprises physical fragmentation, including, for example, dissection and digestion. In some embodiments, the fragmentation is physical fragmentation. In some embodiments, the fracturing is slicing. In some embodiments, the fragmentation is performed by digestion. In some embodiments, the TIL may be initially cultured from an enzymatic tumor digest and tumor fragments obtained from the patient. In embodiments, TILs may be initially cultured from enzymatic tumor digests and tumor fragments obtained from patients.

In some embodiments, when the tumor is a solid tumor, the tumor undergoes physical fragmentation (as provided in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) after obtaining the tumor sample, e.g., in step a. In some embodiments, fragmentation is performed prior to cryopreservation. In some embodiments, fragmentation is performed after cryopreservation. In some embodiments, the fragmentation is performed after the tumor is obtained and in the absence of any cryopreservation. In some embodiments, the step of fragmenting is an in vitro or ex vivo process. In some embodiments, the tumor is fragmented and 10, 20, 30, 40, or more fragments or pieces are placed in each container used to prime the first amplification. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container used to prime the first amplification. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container used to prime the first amplification. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, wherein each fragment has a volume of about 27mm ³. In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, wherein the total volume is about 1300mm³To about 1500mm³. In some embodiments, the plurality of fragments comprises about 50 fragments, wherein the total volume is about 1350mm³. In some embodiments, the plurality of chips comprises about 50 chips with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

In some embodiments, the TIL is obtained from a tumor fragment. In some embodiments, the tumor fragments are obtained by sharp dissection. In some embodiments, the tumor fragments are between about 1mm³And 10mm³In the meantime. In some embodiments, the tumor fragments are between about 1mm³And 8mm³In the meantime. In some embodiments, the tumor fragments are about 1mm³. In some embodiments, the tumor fragments are about 2mm³. In some embodiments, the tumor fragments are about 3mm³. In some embodiments, the tumor fragments are about 4mm³. In some embodiments, the tumor fragments are about 5mm³. In some embodiments, the tumor fragments are about 6mm³. In some embodiments, the tumor fragments are about 7mm³. In some embodiments, the tumor fragments are about 8mm ³. In some embodiments, the tumor fragments are about 9mm³. In some embodiments, the tumor fragments are about 10mm³. In some embodiments, the tumor fragments are 1-4mm by 1-4 mm. In some embodiments, the tumor fragments are 1mm by 1 mm. In some embodiments, the tumor fragment is 2mm x 2 mm. In some embodiments, the tumor fragment is 3mm x 3 mm. In some embodiments, the tumor fragments are 4mm x 4 mm.

In some embodiments, the tumor is fragmented in order to minimize the amount of bleeding, necrosis, and/or adipose tissue on each fragment. In some embodiments, the tumor is fragmented in order to minimize the amount of hemorrhagic tissue on each fragment. In some embodiments, the tumor is fragmented in order to minimize the amount of necrotic tissue on each fragment. In some embodiments, the tumor is fragmented in order to minimize the amount of adipose tissue on each fragment. In certain embodiments, the step of disrupting the tumor is an in vitro or ex vivo method.

In some embodiments, tumor disruption is performed in order to maintain tumor internal structure. In some embodiments, tumor disruption is performed without a sawing action with a scalpel. In some embodiments, the TIL is obtained from a tumor digest. In some embodiments, tumor digests are produced by incubation in an enzyme medium (such as, but not limited to, RPMI 1640, 2mM GlutaMAX, 10mg/mL gentamicin, 30U/mL DNase, and 1.0mg/mL collagenase), followed by mechanical dissociation (GentleMeACS, GentleMed whirlpool Biotech, Inc., Orben, Calif.). After placing the tumor in an enzymatic medium, the tumor can be mechanically dissociated for about 1 minute. The solution may then be brought to 5% CO at 37 deg.C ₂The incubation was continued for 30 minutes and again subjected to mechanical disruption for about 1 minute. At 37 deg.C/5% CO₂After a further 30 minutes of incubation, the tumor may be subjected to a third mechanical disruption for about 1 minute. In some embodiments, after the third mechanical disruption, if a larger piece of tissue is present, it may or may not be at 37 ℃/5% CO₂Next, in the case of 30 minutes of incubation again, the sample is subjected to 1 or 2 more mechanical dissociation cycles. In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, it can be performed using FicollDensity gradient separation to remove these cells.

In some embodiments, the cell suspension prior to initiating the first expansion step is referred to as an "initial cell population" or a "freshly obtained" or "freshly isolated" cell population.

In some embodiments, the cells can optionally be frozen after sample isolation (e.g., after obtaining a tumor sample and/or after obtaining a cell suspension from a tumor sample) and stored frozen prior to entering the expansion described in step B, which is described in further detail below and illustrated in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C).

1. core/Mini-biopsy derived TIL

In some embodiments, TILs are initially obtained from patient tumor samples obtained by core biopsy or similar procedure ("initial TILs") and then expanded into larger populations for further manipulation, optionally cryopreservation, and optionally assessment of phenotypic and metabolic parameters as described herein.

In some embodiments, a patient tumor sample can be obtained using methods known in the art, typically by a mini-biopsy, core biopsy, needle biopsy, or other means of obtaining a sample containing a mixture of tumor and TIL cells. In general, the tumor sample may be from any solid tumor, including a primary tumor, an invasive tumor, or a metastatic tumor. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematological malignancy. In some embodiments, the sample may be from a plurality of small tumor samples or biopsies. In some embodiments, the sample may comprise multiple tumor samples from a single tumor of the same patient. In some embodiments, the sample may comprise multiple tumor samples from one, two, three, or four tumors of the same patient. In some embodiments, the sample may comprise multiple tumor samples from multiple tumors of the same patient. The solid tumor can be any cancer type, including but not limited to breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, stomach cancer, and skin cancer (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, e.g., Head and Neck Squamous Cell Carcinoma (HNSCC)), Glioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer (NSCLC). In some embodiments, useful TILs are obtained from malignant melanoma tumors, as these malignant melanoma tumors are reported to have particularly high levels of TIL.

In general, a cell suspension obtained from the core or debris of a tumor is referred to as an "initial cell population" or a "freshly obtained" or "freshly isolated" cell population. In certain embodiments, a freshly obtained TIL cell population is exposed to a cell culture medium comprising antigen presenting cells IL-2 and OKT-3.

In some embodiments, if the tumor is metastatic and has been effective in the past to treat/remove primary lesions, it may be desirable to remove one of the metastatic lesions. In some embodiments, the minimally invasive method is to remove the skin lesion, or lymph nodes on the neck or axillary area, at the time of procurement. In some embodiments, the skin lesion is removed or a small biopsy thereof is removed. In some embodiments, a lymph node or a small biopsy thereof is removed. In some embodiments, lung or liver metastasis, or an intra-abdominal or thoracic lymph node or a small biopsy thereof may be employed.

In some embodiments, the tumor is melanoma. In some embodiments, the small biopsy of melanoma comprises a mole or portion thereof.

In some embodiments, the small biopsy is a drill biopsy. In some embodiments, a drill biopsy is obtained with a circular blade pressed into the skin. In some embodiments, a drill biopsy is obtained with a circular blade pressed into the skin. Suspicious moles are around. In some embodiments, a drill biopsy is obtained with a circular blade pressed into the skin, and a circular piece of skin is removed. In some embodiments, the small biopsy is a drill biopsy and the circular portion of the tumor is removed.

In some embodiments, the small biopsy is an excisional biopsy. In some embodiments, the small biopsy is an excisional biopsy, and all moles or growths are removed. In some embodiments, the small biopsy is an excisional biopsy, and all moles or growths and small borders of the skin that normally appear are removed.

In some embodiments, the small biopsy is a resection biopsy. In some embodiments, the small biopsy is a resection biopsy and only the most irregular part of the moles or growth is acquired. In some embodiments, the small biopsy is a resection biopsy, and is used when other techniques cannot be done, for example, where the suspected moles are very large.

In some embodiments, the small biopsy is a lung biopsy. In some embodiments, the small biopsy is obtained by bronchoscopy. Generally, bronchoscopy places the patient under anesthesia and small tools, which are used to remove some tissue, pass through the nose or mouth, down the larynx and into the bronchial passages. In some embodiments, a transthoracic puncture biopsy may be employed in cases where a tumor or growth cannot be reached by bronchoscopy. Generally, for transthoracic puncture biopsy, the patient is still under anesthesia and a needle is inserted through the skin directly into the suspect site to remove a small tissue sample. In some embodiments, a transthoracic puncture biopsy may require interventional radiology (e.g., using x-ray or CT scanning to guide the needle). In some embodiments, the small biopsy is obtained by needle biopsy. In some embodiments, a small biopsy is obtained by endoscopic ultrasound (e.g., an endoscope with light and placed orally into the esophagus). In some embodiments, the small biopsy is obtained surgically.

In some embodiments, the small biopsy is a head and neck biopsy. In some embodiments, the small biopsy is a resection biopsy. In some embodiments, the small biopsy is a resection biopsy, in which a small piece of tissue is cut from the region where the abnormality is observed. In some embodiments, if the abnormal area is easily accessible, the sample may be taken without hospitalization. In some embodiments, if the tumor is deeper inside the mouth or throat, a biopsy with general anesthesia in the operating room may be required. In some embodiments, the small biopsy is an excisional biopsy. In some embodiments, the small biopsy is an excisional biopsy, wherein the entire region is removed. In some embodiments, the small biopsy is Fine Needle Aspiration (FNA). In some embodiments, the small biopsy is Fine Needle Aspiration (FNA), where an extremely thin needle connected to a syringe is used to extract (aspirate) cells from a tumor or tumor. In some embodiments, the small biopsy is a drill biopsy. In some embodiments, the small biopsy is a punch biopsy, in which a piece of suspicious region is removed using a punch forceps.

In some embodiments, the small biopsy is a cervical biopsy. In some embodiments, the small biopsy is obtained by colposcopy. Typically, colposcopy methods employ the use of an illuminated magnifying instrument connected to a magnifying binocular (colposcope) which is then used to biopsy small portions of the cervical surface. In some embodiments, the small biopsy is a conization/cone biopsy. In some embodiments, the small biopsy is a conization/cone biopsy, where an outpatient procedure may be required to remove a large piece of tissue from the cervix. In some embodiments, the cone biopsy is in addition to helping confirm the diagnosis, the cone biopsy may be used as an initial treatment.

The term "solid tumor" refers to an abnormal tissue mass that generally does not contain cysts or fluid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic or cancerous solid tumor. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, triple negative breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. In some embodiments, the cancer is selected from cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer, and non-small cell lung cancer. The tissue structure of solid tumors comprises interdependent tissue compartments, including parenchyma tissue (cancer cells) and supporting stromal cells in which cancer cells are dispersed and which can provide a supporting microenvironment.

In some embodiments, the sample from the tumor is obtained in the form of a Fine Needle Aspiration (FNA), core biopsy, mini biopsy (including, e.g., a punch biopsy). In some embodiments, the sample is first placed into G-Rex 10. In some embodiments, when there are 1 or 2 core biopsy and/or mini-biopsy samples, the samples are first placed into G-Rex 10. In some embodiments, when there are 3, 4, 5, 6, 8, 9, or 10 or more core and/or mini-biopsy samples, the samples are first placed into G-Rex 100. In some embodiments, when there are 3, 4, 5, 6, 8, 9, or 10 or more core and/or mini-biopsy samples, the samples are first placed into the G-Rex 500.

FNA may be obtained from a tumor selected from the group consisting of: lung, melanoma, head and neck, cervix, ovary, pancreas, glioblastoma, colorectal and sarcoma. In some embodiments, FNAs are obtained from lung tumors, such as lung tumors from patients with non-small cell lung cancer (NSCLC). In some cases, patients with NSCLC have previously undergone surgical treatment.

The TILs described herein may be obtained from a FNA sample. In some cases, FNA samples are obtained or isolated from patients using fine gauge needles ranging from 18 gauge needles to 25 gauge needles. The fine gauge needle may be 18 gauge, 19 gauge, 20 gauge, 21 gauge, 22 gauge, 23 gauge, 24 gauge, or 25 gauge. In some embodiments, the FNA sample from the patient can contain at least 400,000 TILs, e.g., 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs, 800,000 TILs, 850,000 TILs, 900,000 TILs, 950,000 TILs, or more.

In some cases, the TILs described herein are obtained from a core biopsy sample. In some cases, core biopsy samples are obtained or isolated from patients using surgical or medical needles ranging from 11 gauge needles to 16 gauge needles. The needle may be 11 gauge, 12 gauge, 13 gauge, 14 gauge, 15 gauge or 16 gauge. In some embodiments, a core biopsy sample from a patient may contain at least 400,000 TILs, e.g., 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 700,000 TILs, 750,000 TILs, 800,000 TILs, 850,000 TILs, 900,000 TILs, 950,000 TILs, or more.

Typically, the collected cell suspension is referred to as the "initial cell population" or the "freshly collected" cell population.

In some embodiments, the TIL is not obtained from tumor digest. In some embodiments, the solid tumor core is not fragmented.

In some embodiments, the TIL is obtained from a tumor digest. In some embodiments, the tumor digest is generated by incubation in an enzyme medium (such as, but not limited to, RPMI 1640, 2mM GlutaMAX, 10mg/mL gentamicin, 30U/mL DNase, and 1.0mg/mL collagenase), followed by mechanical dissociation (GentleMACCS, American day and whirlpool biotechnology, Orben, Calif.). After placing the tumor in an enzymatic medium, the tumor can be mechanically dissociated for about 1 minute. The solution may then be brought to 5% CO at 37 deg.C₂The incubation was continued for 30 minutes and again subjected to mechanical disruption for about 1 minute. At 37 deg.C/5% CO₂After a further 30 minutes of incubation, the tumor may be subjected to a third mechanical disruption for about 1 minute. In some embodiments, after the third mechanical disruption, if a larger piece of tissue is present, it may or may not be at 37 ℃/5% CO₂Next, in the case of 30 minutes of incubation again, the sample is subjected to 1 or 2 more mechanical dissociation cycles. In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using Ficoll can be performed to remove these cells.

2.Method for expanding Peripheral Blood Lymphocytes (PBLs) from peripheral blood

PBL method 1: in embodiments of the invention, PBLs are amplified using the methods described herein. In an embodiment of the invention, the method comprises obtaining a PBMC sample from whole blood. In embodiments, the method comprises enriching T cells by isolating pure T cells from PBMCs by negative selection using a non-CD 19+ fraction. In embodiments, the method comprises enriching T cells by isolating pure T cells from PBMCs by magnetic bead-based negative selection using a non-CD 19+ fraction.

In an example of the invention, PBL method 1 is performed as follows: on day 0, cryopreserved PBMC samples were thawed and PBMCs were counted. T cells were isolated using a human Pan T cell isolation kit and LS column (american whirlpool biotechnology).

PBL method 2:in an embodiment of the invention, PBL is amplified using PBL method 2, which comprises obtaining a PBMC sample from whole blood. T cells from PBMCs were enriched by incubating PBMCs at 37 ℃ for at least three hours and then isolating non-adherent cells.

In an example of the invention, PBL method 2 was performed as follows: on day 0, cryopreserved PMBC samples were thawed and PBMC cells were seeded at 600 ten thousand cells per well in CM-2 medium in 6-well plates and incubated at 37 degrees celsius for 3 hours. After 3 hours, non-adherent cells as PBLs were removed and counted.

PBL method 3: in an embodiment of the invention, PBL is amplified using PBL method 3, which comprises obtaining a PBMC sample from peripheral blood. B cells were isolated using CD19+ selection and T cells were selected using negative selection of a non-CD 19+ fraction of PBMC samples.

In an embodiment of the invention, PBL method 3 is performed as follows: on day 0, cryopreserved PBMC samples derived from peripheral blood were thawed and counted. CD19+ B cells were sorted using the human CD19 Multisort kit (american whirlpool biotechnology). In the non-CD 19+ cell fraction, T cells were purified using a human Pan T cell isolation kit and LS column (american whirlpool biotechnology).

In an embodiment, PBMCs are isolated from a whole blood sample. In the examples, PBMC samples were used as starting material for PBL amplification. In embodiments, the sample is cryogenically preserved prior to the amplification process. In another embodiment, fresh samples are used as starting material for the amplification of PBLs. In embodiments of the invention, T cells are isolated from PBMCs using methods known in the art. In the examples, T cells were isolated using a human Pan T cell isolation kit and LS column. In embodiments of the invention, T cells are isolated from PBMCs using antibody selection methods known in the art, such as CD19 negative selection.

In embodiments of the invention, the PBMC sample is incubated for a period of time at a desired temperature effective to identify non-adherent cells. In an embodiment of the invention, the incubation time is about 3 hours. In an embodiment of the invention, the temperature is about 37 ℃. Non-adherent cells were then expanded using the process described above.

In some embodiments, the PBMC sample is from a subject or patient that has optionally been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the tumor sample is from a subject or patient that has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient that has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor, that has undergone treatment for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or 1 year or more. In another embodiment, the PBMCs are derived from patients currently undergoing an ITK inhibitor regimen, such as ibrutinib (ibrutinib).

In some embodiments, the PBMC sample is from a subject or patient that has been pre-treated with a regimen comprising a kinase inhibitor or ITK inhibitor and is refractory to treatment with a kinase inhibitor or ITK inhibitor, e.g., ibrutinib.

In some embodiments, the PBMC sample is from a subject or patient that has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor but is no longer undergoing treatment with the kinase inhibitor or ITK inhibitor. In some embodiments, the PBMC sample is from a subject or patient that has been pre-treated with a regimen comprising a kinase inhibitor or an ITK inhibitor but no longer undergoing treatment with the kinase inhibitor or the ITK inhibitor and has not undergone treatment for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year or more. In another embodiment, the PBMCs are derived from a patient that has been previously exposed to an ITK inhibitor, but has not been treated for at least 3 months, at least 6 months, at least 9 months, or at least 1 year.

In an embodiment of the invention, on day 0, cells were selected for CD19+ and sorted accordingly. In embodiments of the invention, antibody-bound beads are used for selection. In an embodiment of the invention, pure T cells are isolated from PBMCs on day 0.

In the inventionIn the examples, for patients not pre-treated with ibrutinib or other ITK inhibitors, 10-15ml of Buffy Coat will yield about 5X 10 ⁹PBMC which in turn will yield about 5.5X 10⁷And PBL.

In embodiments of the invention, the expansion process will yield about 20 x 10 for patients pre-treated with ibrutinib or other ITK inhibitors⁹And PBL. In the examples of the present invention, 40.3X 10⁶One PBMC will yield about 4.7X 10⁵And PBL.

In any of the preceding embodiments, the PBMCs may be derived from a whole blood sample, by apheresis, buffy coat, or any other method known in the art for obtaining PBMCs.

3.Method for expanding Marrow Infiltrating Lymphocytes (MILs) from bone marrow-derived PBMCs

MIL method 3: in an embodiment of the invention, the method comprises obtaining PBMCs from bone marrow. On day 0 PBMCs were selected for CD3+/CD33+/CD20+/CD14+ and sorted, and the non-CD 3+/CD33+/CD20+/CD14+ cell fraction was sonicated and a portion of the sonicated cell fraction was added back to the selected cell fraction.

In an example of the invention, MIL method 3 was performed as follows: on day 0, cryopreserved PBMC samples were thawed and PBMCs were counted. Cells were stained with CD3, CD33, CD20, and CD14 antibodies and sorted using sorted S3e cells (burle (Bio-Rad)). Cells were sorted into two fractions: immune cell fraction (or MIL fraction) (CD3+ CD33+ CD20+ CD14+) and AML blast fraction (non-CD 3+ CD33+ CD20+ CD14 +).

In embodiments of the invention, the PBMCs are obtained from bone marrow. In embodiments, PBMCs are obtained from bone marrow by apheresis, aspiration, needle biopsy, or other similar means known in the art. In embodiments, the PBMCs are fresh. In another embodiment, the PBMCs are cryopreserved.

In the examples of the present invention, MILs were amplified from 10-50ml of bone marrow aspirate. In an embodiment of the invention, 10ml of bone marrow aspirate is obtained from a patient. In another embodiment, 20ml of bone marrow aspirate is obtained from the patient. In another embodiment, 30ml of bone marrow aspirate is obtained from the patient. In another embodiment, 40ml of bone marrow aspirate is obtained from the patient. In another embodiment, 50ml of bone marrow aspirate is obtained from the patient.

In an embodiment of the invention, the number of PBMCs produced from about 10-50ml of bone marrow aspirate is about 5X 10⁷To about 10X 10⁷And (5) PBMCs. In another embodiment, the number of PMBCs generated is about 7 × 10⁷And (5) PBMCs.

In an embodiment of the invention, about 5X 10⁷To about 10X 10⁷Approximately 0.5X 10 PBMC was produced⁶To about 1.5X 10⁶And (3) MIL. In an embodiment of the invention, about 1 × 10 is produced⁶And (3) MIL.

In an embodiment of the invention, 12X 10 derived bone marrow aspirate ⁶Approximately 1.4X 10 PBMC were produced⁵And (3) MIL.

In any of the preceding embodiments, the PBMCs may be derived from a whole blood sample, from bone marrow, by apheresis, from buffy coat, or by any other method known in the art for obtaining PBMCs.

Pre-selection of PD-1 (as exemplified in step A2 of FIG. 1)

According to the method of the invention, TIL was pre-selected to be PD-1 positive (PD-1+) before priming the first amplification.

In some embodiments, a minimum of 3,000 TILs are required to be seeded into the first amplification. In some embodiments, the pre-selection step produces a minimum of 3,000 TILs. In some embodiments, a minimum of 4,000 TILs are required to be seeded into the first amplification. In some embodiments, the pre-selection step produces a minimum of 4,000 TILs. In some embodiments, a minimum of 5,000 TILs are required to be seeded into the first amplification. In some embodiments, the pre-selection step produces a minimum of 5,000 TILs. In some embodiments, a minimum of 6,000 TILs are required to be seeded into the first amplification. In some embodiments, the pre-selection step produces a minimum of 6,000 TILs. In some embodiments, a minimum of 7,000 TILs are required to be seeded into the first amplification. In some embodiments, the pre-selection step produces a minimum of 7,000 TILs. In some embodiments, a minimum of 8,000 TILs are required to be seeded into the first amplification. In some embodiments, the pre-selection step produces a minimum of 8,000 TILs. In some embodiments, a minimum of 9,000 TILs are required to be seeded into the first amplification. In some embodiments, the pre-selection step produces a minimum of 9,000 TILs. In some embodiments, a minimum of 10,000 TILs are required to be seeded into the first amplification. In some embodiments, the pre-selection step produces a minimum of 10,000 TILs. In some embodiments, the cells are grown or expanded to a density of 200,000. In some embodiments, the cells are grown or expanded to a density of 200,000 to provide about 2e8 TILs for initiating a rapid second expansion. In some embodiments, the cells are grown or expanded to a density of 150,000. In some embodiments, the cells are grown or expanded to a density of 150,000 to provide about 2e8 TILs for initiating a rapid second expansion. In some embodiments, the cells are grown or expanded to a density of 250,000. In some embodiments, the cells are grown or expanded to a density of 250,000 to provide about 2e8 TILs for initiating a rapid second expansion. In some embodiments, the minimum cell density is 10,000 cells to provide 10e6 for initiating a rapid second expansion. In some embodiments, a seeding density of 10e6 to initiate rapid second amplification may yield greater than 1e9 TILs.

In some embodiments, the TIL used to prime the first amplification is PD-1 positive (PD-1+) (e.g., after pre-selection and before priming the first amplification). In some embodiments, the TIL used to prime the first amplification is at least 75% PD-1 positive, at least 80% PD-1 positive, at least 85% PD-1 positive, at least 90% PD-1 positive, at least 95% PD-1 positive, at least 98% PD-1 positive, or at least 99% PD-1 positive (e.g., after pre-selection and before priming the first amplification). In some embodiments, the PD-1 population is PD-1 high. In some embodiments, the TIL used to elicit the first amplification is at least 25% PD-1 high, at least 30% PD-1 high, at least 35% PD-1 high, at least 40% PD-1 high, at least 45% PD-1 high, at least 50% PD-1 high, at least 55% PD-1 high, at least 60% PD-1 high, at least 65% PD-1 high, at least 70% PD-1 high, at least 75% PD-1 high, at least 80% PD-1 high, at least 85% PD-1 high, at least 90% PD-1 high, at least 95% PD-1 high, at least 98% PD-1 high, or at least 99% PD-1 high (e.g., after pre-selection and before eliciting the first amplification).

In some embodiments, the pre-selection for PD-1 positive TIL is performed by staining the initial cell population, whole tumor digest, and/or whole tumor cell suspension TIL with an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is a polyclonal antibody, such as a mouse anti-human PD-1 polyclonal antibody, a goat anti-human PD-1 polyclonal antibody, or the like. In some embodiments, the anti-PD-1 antibody is a monoclonal antibody. In some embodiments, anti-PD-1 antibodies include, for example, but are not limited to, EH12.2H7, PD1.3.1, M1H4, nivolumab (BMS-936558, bosch & robert company; ) Pembrolizumab (lambertilizumab, MK03475 or MK-3475, merck corporation;) H12.1, PD1.3.1, NAT 105, humanized anti-PD-1 antibody JS001 (Shanghai Junshi Co.), monoclonal anti-PD-1 antibody TSR-042(Tesaro Co.), pidilizumab (anti-PD-1 mAb CT-011, Madisison healthcare Co.), anti-PD-1 monoclonal antibody BGB-A317 (Baiji Shenzhou Co.) and/or anti-PD-1 antibody SHR-1210 (Shanghai Henry Co.), human monoclonal antibody REGN2810 (Resheng Yuan Co.), human monoclonal antibody MDX-1106 (Becky Messajous Co.) and/or humanized anti-PD-1 IgG4 antibody PDR001 (Noohua Co.). In some embodiments, the PD-1 antibody is from clone: RMP1-14 (rat IgG) -BioXcell catalog number BP 0146. Other suitable antibodies (as exemplified by steps a through F, as described herein) for pre-selecting PD-1 positive TIL for use in amplifying TIL according to the methods of the invention are anti-PD-1 antibodies disclosed in U.S. patent No. 8,008,449, which is incorporated herein by reference. In some embodiments, the anti-PD-1 antibody used for the pre-selection is different from nivolumab (BMS-936558, behcet stan;) Binds to the epitope of (a). In some embodiments, the anti-PD-1 antibody used for pre-selection is conjugated to a second antibody other than pembrolizumab (lambertilizumab, MK03475 or MK-3475, merck corporation; ) Binds to the epitope of (a). In some embodiments, the anti-PD-1 antibody used for the pre-selection binds to an epitope different from the humanized anti-PD-1 antibody JS001 (shanghai junshi). In some embodiments, the anti-PD-1 antibody used for the pre-selection binds to an epitope different from the monoclonal anti-PD-1 antibody TSR-042(Tesaro corporation). In some embodiments, the anti-PD-1 antibody used for pre-selection binds to an epitope different from pidilizumab (anti-PD-1 mAb CT-011, madivison healthcare). In some embodiments, the anti-PD-1 antibody used for pre-selection binds to an epitope different from the anti-PD-1 monoclonal antibody BGB-a317 (baiji state corporation). In some embodiments, the anti-PD-1 antibody used for the pre-selection binds to an epitope different from anti-PD-1 antibody SHR-1210 (shanghai galvano). In some embodiments, the anti-PD-1 antibody used for the pre-selection binds to an epitope different from the human monoclonal antibody REGN2810 (regenerators). In some embodiments, the anti-PD-1 antibody used for the pre-selection binds to an epitope different from that of human monoclonal antibody MDX-1106 (bosch & robusta, behcet). In some embodiments, the anti-PD-1 antibody used for pre-selection binds to an epitope other than the humanized anti-PD-1 IgG4 antibody PDR001 (novartis). In some embodiments, the anti-PD-1 antibody used for pre-selection binds to an epitope different from RMP1-14 (rat IgG) -BioXcell catalog No. BP 0146. The structures of binding of nivolumab and pembrolizumab to PD-1 are known and have been described, for example, in Tan, S. et al (Tan, S. et al, Nature letters), 8:14369| DOI:10.1038/ncomms14369 (2017); which is incorporated herein by reference in its entirety for all purposes). In some embodiments, the anti-PD-1 antibody is EH12.2H7. In some embodiments, the anti-PD-1 antibody is PD1.3.1. In some embodiments, the anti-PD-1 antibody is not PD1.3.1. In some embodiments, the anti-PD-1 antibody is M1H 4. In some embodiments, the anti-PD-1 antibody is not M1H 4.

In some embodiments, the anti-PD-1 antibody used for pre-selection binds to at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of PD-1 expressing cells.

In some embodiments, the patient has been treated with an anti-PD-1 antibody. In some embodiments, the subject has not received anti-PD-1 antibody therapy. In some embodiments, the subject has not been treated with an anti-PD-1 antibody. In some embodiments, the subject has been previously treated with a chemical agent. In some embodiments, the subject has been previously treated with the chemical agent but is no longer treated with the chemical agent. In some embodiments, the subject is post-chemotherapy treatment or post-anti-PD-1 antibody treatment. In some embodiments, the subject is post-chemotherapy treatment and post-anti-PD-1 antibody treatment. In some embodiments, the patient has not received anti-PD-1 antibody therapy. In some embodiments, the subject has an untreated cancer or has received post-chemotherapy treatment but has not received anti-PD-1 antibody treatment. In some embodiments, the subject has not received treatment and has received treatment after chemotherapy but has not received treatment with an anti-PD-1 antibody.

In some embodiments, wherein the patient has been previously treated with a first anti-PD-1 antibody, the pre-selection is performed by staining the initial cell population, whole tumor digest, and/or whole tumor cell suspension TIL with a second anti-PD-1 antibody that does not block binding to PD-1 on the surface of the initial cell population TIL by the first anti-PD-1 antibody.

In some embodiments, wherein the patient has been previously treated with an anti-PD-1 antibody, the pre-selection is performed by staining the initial cell population TIL with an antibody ("anti-Fc antibody") that binds to an Fc region of the anti-PD-1 antibody that is insoluble on the surface of the initial cell population TIL. In some embodiments, the anti-Fc antibody is a polyclonal antibody, such as a mouse anti-human Fc polyclonal antibody, a goat anti-human Fc polyclonal antibody, or the like. In some embodiments, the anti-Fc antibody is a monoclonal antibody. In some embodiments, wherein the patient has been previously treated with an anti-PD-1 human or humanized IgG antibody and the initial cell population TIL is stained with an anti-human IgG antibody. In some embodiments, wherein the patient has been previously treated with an anti-PD-1 human or humanized IgG1 antibody, the initial cell population TIL is stained with an anti-human IgG1 antibody. In some embodiments, wherein the patient has been previously treated with an anti-PD-1 human or humanized IgG2 antibody, the initial cell population TIL is stained with an anti-human IgG2 antibody. In some embodiments, wherein the patient has been previously treated with an anti-PD-1 human or humanized IgG3 antibody, the initial cell population TIL is stained with an anti-human IgG3 antibody. In some embodiments, wherein the patient has been previously treated with an anti-PD-1 human or humanized IgG4 antibody, the initial cell population TIL is stained with an anti-human IgG4 antibody.

In some embodiments, wherein the patient has been previously treated with an anti-PD-1 antibody, the pre-selection is performed by contacting the initial cell population TIL with the same anti-PD-1 antibody and then staining the initial cell population TIL with an anti-Fc antibody that binds to an Fc region of the anti-PD-1 antibody that is insoluble on the surface of the initial cell population TIL.

In some embodiments, the cell sorting method is used for pre-selection. In some embodiments, the cell sorting method is a flow cytometry method, such as Flow Activated Cell Sorting (FACS). In some embodiments, the intensities of the fluorophores in both the first population and the PBMC population are used to establish FACS gates to establish low, intermediate, and high levels of intensity corresponding to PD-1 negative TIL, PD-1 intermediate TIL, and PD-1 positive TIL, respectively. In some embodiments, the cell sorting method is performed such that gates are set high, medium (also referred to as intermediates) and low (also referred to as negative) using PBMCs, FMO controls, and the sample itself to distinguish between these three populations. In some embodiments, PBMCs are used as gating controls. In some embodiments, a PD-1 high population is defined as a population of cells observed in PBMCs that are positive for PD-1 above. In some embodiments, the intermediate PD-1+ population in the TIL encompasses PD-1+ cells in PBMCs. In some embodiments, negatives are gated based on FMO. In some embodiments, the FACS gates are established after the step of obtaining and/or receiving a first TIL population from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments. In some embodiments, each build is performed for a door. In some embodiments, gating is established for each sample of PBMCs. In some embodiments, gating is established for each sample of PBMCs. In some embodiments, the gating template is established from PBMCs every 10, 20, 30, 40, 50, or 60 days. In some embodiments, the gating template is established every 60 days from PBMCs. In some embodiments, the gating template is established for each sample of PBMCs every 10, 20, 30, 40, 50 or 60 days. In some embodiments, a gating template is established every 60 days for each sample of PBMCs.

In some embodiments, the pre-selecting involves selecting a PD-1 positive TIL from the first TIL population to obtain a population of TILs enriched in PD-1 comprises selecting a TIL population from the first TIL population that is at least 11.27% to 74.4% PD-1 positive TIL. In some embodiments, the first population of TILs is at least 20% to 80% PD-1 positive TILs, at least 30% to 80% PD-1 positive TILs, at least 40% to 80% PD-1 positive TILs, at least 50% to 80% PD-1 positive TILs, at least 10% to 70% PD-1 positive TILs, at least 20% to 70% PD-1 positive TILs, at least 30% to 70% PD-1 positive TILs, or at least 40% to 70% PD-1 positive TILs.

In some embodiments, the selecting step (e.g., pre-selecting and/or selecting PD-1 positive cells) comprises the steps of:

(ii) adding an excess of anti-IgG 4 antibody conjugated to a fluorophore;

In some embodiments, a PD-1 positive TIL is a PD-1 high TIL.

In some embodiments, at least 70% of the population of PD-1-enriched TILs is PD-1 positive TILs. In some embodiments, at least 80% of the population of PD-1-enriched TILs is PD-1 positive TILs. In some embodiments, at least 90% of the population of PD-1-enriched TILs is PD-1 positive TILs. In some embodiments, at least 95% of the population of PD-1-enriched TILs is PD-1 positive TILs. In some embodiments, at least 99% of the population of PD-1-enriched TILs is PD-1 positive TILs. In some embodiments, 100% of the population of PD-1-enriched TILs is PD-1 positive TILs.

Different anti-PD-1 antibodies exhibit different binding properties to different epitopes within PD-1. In some embodiments, the anti-PD-1 antibody binds to an epitope other than pembrolizumab. In some embodiments, the anti-PD 1 antibody binds to an epitope in an N-terminal loop outside the IgV domain of PD-1. In some embodiments, the anti-PD 1 antibody binds through the N-terminal loop outside the IgV domain of PD-1. In some embodiments, the anti-PD-1 antibody is an anti-PD-1 antibody that binds to PD-1 through the N-terminal ring outside the IgV domain of PD-1. In some embodiments, the anti-PD-1 antibody is a monoclonal anti-PD-1 antibody that binds to PD-1 through the N-terminal loop outside the IgV domain of PD-1. In some embodiments, the monoclonal anti-PD-1 antibody is an anti-PD-1 IgG4 antibody that binds to PD-1 through the N-terminal loop outside the IgV domain of PD-1. See, e.g., Tan, s., (naturel) volume 8, sections 14369:1-10 (2017).

In some embodiments, the selecting step as illustrated by step a2 of fig. 1 comprises the steps of: (i) exposing the first TIL population to an excess of monoclonal anti-PD-1 IgG4 antibody that binds to PD-1 through the N-terminal loop outside the IgV domain of PD-1; (ii) adding an excess of anti-IgG 4 antibody conjugated to a fluorophore; and (iii) performing flow-based cell sorting based on the fluorophore to obtain a population of PD-1 enriched TILs. In some embodiments, the monoclonal anti-PD-1 IgG4 antibody is nivolumab or a variant, fragment, or conjugate thereof. In some embodiments, the anti-IgG 4 antibody is clone anti-human IgG4, clone HP 6023. In some embodiments, the anti-PD-1 antibody selected for use in step (b) binds the same epitope as EH12.2H7 or nivolumab.

In some embodiments, the PD-1 gating method of WO2019156568 is employed. To determine whether TIL derived from a tumor sample is PD-1 high, one skilled in the art can utilize a reference value corresponding to the expression level of PD-1 in peripheral T cells obtained from a blood sample from one or more healthy human subjects. PD-1 positive cells in a reference sample can be defined using fluorescence minus one control and matching isotype controls. In some embodiments, the expression level of PD-1 is measured in CD3+/PD-1+ peripheral T cells (e.g., reference cells) from healthy subjects for establishing a threshold or cut-off value for the intensity of immunostaining for PD-1 in TILs obtained from tumors. The threshold may be defined as the minimum intensity of PD-1 immunostaining of PD-1 high T cells. Thus, a TIL with PD-1 expression equal to or above the threshold value may be considered a PD-1 high cell. In some cases, PD-1 high TIL indicates those with the highest intensity PD-1 immunostaining corresponding to a maximum of 1% or less of total CD3+ cells. In other cases, PD-1 high TILs indicate those with the highest intensity PD-1 immunostaining corresponding to a maximum of 0.75% or less of total CD3+ cells. In some cases, PD-1 high TIL indicates those with the highest intensity PD-1 immunostaining corresponding to a maximum of 0.50% or less of total CD3+ cells. In one instance, PD-1 high TILs indicate those with the highest intensity PD-1 immunostaining corresponding to a maximum of 0.25% or less of total CD3+ cells.

a. Fluorophores

In some embodiments, the initial population of cells TIL is stained with a cocktail (cocktail) comprising an anti-PD-1 antibody linked to a fluorophore and an anti-CD 3 antibody linked to a fluorophore. In some embodiments, the initial cell population TIL is stained with a mixture comprising an anti-PD-1 antibody linked to a fluorophore (e.g., PE, live/dead purple) and anti-CD 3-FITC. In some embodiments, the initial cell population TIL is stained with a mixture comprising anti-PD-1-PE, anti-CD 3-FITC, and a live/dead blue stain (seemer feishel (ThermoFisher), catalogue No. L23105, massachusetts). In some embodiments, PD-1 positive cells are selected for expansion after incubation with the anti-PD 1 antibody according to priming the first expansion described herein (e.g., in step B).

In some embodiments, fluorophores include, but are not limited to, PE (phycoerythrin), APC (allophycocyanin), PerCP (polyazomorphin chlorophyll protein), DyLight 405, Alexa Fluor 405, Pacific Blue (Pacific Blue), Alexa Fluor 488, FITC (fluorescein isothiocyanate), DyLight 550, Alexa Fluor 647, DyLight 650, and Alexa Fluor 700. In some embodiments, fluorophores include, but are not limited to, PE-Alexa 647、PE-Cy5、PerCP-Cy5.5、PE-Cy5.5、PE-Alexa750. PE-Cy7 and APC-Cy 7. In some embodiments, the fluorophore includes, but is not limited to, a fluorescein dye. Examples of fluorescein dyes include, but are not limited to, 5-carboxyfluorescein, 5-fluorescein isothiocyanate and 6-carboxyfluorescein, 5, 6-dicarboxyifluorescein, 5- (and 6) -sulfofluorescein, sulfone-based fluorescein, succinylfluorescein, 5- (and 6) -carboxySNARF-1, carboxyfluorescein sulfonate, carboxyfluorescein zwitterion, carboxyfluorescein quaternary ammonium salt, carboxyfluorescein phosphonate, carboxyfluorescein GABA, 5'(6') -carboxyfluorescein, carboxyfluorescein-cysteine-Cy 5, and fluorescein glutathione. In some embodiments, the fluorescent moiety is a rhodamine dye. Examples of rhodamine dyes include, but are not limited to, tetramethylrhodamine-6-isothiocyanate, 5-carboxytetramethylrhodamine, 5-carboxyrhodol derivatives, carboxyrhodamine 110, tetramethyl and tetraethylrhodamine, diphenyldimethyl and diphenyldiethylrhodamine, dinaphthylrhodamine, rhodamine 101 sulfonyl chloride (as TEXAS)Trade name of (c). In some embodiments, the fluorescent moiety is a cyanine dye. Examples of cyanine dyes include, but are not limited to, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy 7.

B. And B: priming the first amplification

In some embodiments, the present methods provide a younger TIL, which may provide additional therapeutic benefits relative to an older TIL (i.e., a TIL that has undergone further more rounds of replication prior to administration to a subject/patient). The characteristics of young TILs have been described in the following documents: for example, Donia et al, Scandinavian Journal of Immunology, 75: 157-167 (2012); dudley et al, clinical Cancer research (Clin Cancer Res), 16: 6122-; huang et al, J Immunotherapy, 28(3) 258 (267) (2005); besser et al, clinical cancer research, 19(17) OF1-OF9 (2013); besser et al, journal of immunotherapeutics 32: 415-423 (2009); robbins et al, journal of immunology (J Immunol) 2004; 173: 7125-7130; shen et al, J.Immunotherapy, 30: 123-129 (2007); zhou et al, J.Immunotherapy, 28: 53-62 (2005); and Tran et al, J.Immunotherapy 31: 742-751 (2008), all of which are incorporated herein by reference in their entirety.

After dissecting or digesting (e.g., to obtain a whole tumor digest and/or a whole tumor cell suspension) tumor debris and/or tumor debris, e.g., as described in step a of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), the resulting cells are cultured in serum containing IL-2, OKT-3, and feeder cells (e.g., antigen presenting feeder cells or allogeneic irradiated PBMCs) under conditions that favor the growth of TIL over tumor and other cells. In some embodiments, IL-2, OKT-3, and feeder cells are added with tumor digests and/or tumor debris at the start of culture (e.g., on day 0). In some embodiments, the tumor digest and/or tumor fragments are incubated in containers, wherein each container has up to 60 fragments and has 6000IU/mL of IL-2. In some embodiments, such an initial cell population is cultured for a period of several days (typically 1 to 8 days) to produce a bulk TIL population, typically about 1 × 10 ⁸Individual TIL cells in bulk. In some embodiments, such an initial cell population is cultured for a period of several days (typically 1 to 7 days) to produce a bulk TIL population, typically about 1 × 10⁸Individual TIL cells in bulk.In some embodiments, priming the first amplification is performed for a period of 1 to 8 days, resulting in an bulk TIL population, typically about 1 × 10⁸Individual TIL cells in bulk. In some embodiments, priming the first amplification is performed for a period of 1 to 7 days, resulting in an bulk TIL population, typically about 1 × 10⁸Individual TIL cells in bulk. In some embodiments, this priming first amplification is performed for a period of 5 to 8 days, resulting in a bulk TIL population, typically about 1 × 10⁸Individual TIL cells in bulk. In some embodiments, this priming first amplification is performed for a period of 5 to 7 days, resulting in a bulk TIL population, typically about 1 × 10⁸Individual TIL cells in bulk. In some embodiments, this priming first amplification is performed for a period of about 6 to 8 days, resulting in a bulk TIL population, typically about 1 x 10⁸Individual TIL cells in bulk. In some embodiments, this priming first amplification is performed for a period of about 6 to 7 days, resulting in a bulk TIL population, typically about 1 x 10⁸Individual TIL cells in bulk. In some embodiments, this priming first amplification is performed for a period of about 7 to 8 days, resulting in a bulk TIL population, typically about 1 x 10 ⁸Individual TIL cells in bulk. In some embodiments, this priming of the first amplification is performed for a period of about 7 days, resulting in a bulk TIL population, typically about 1 × 10⁸Individual TIL cells in bulk. In some embodiments, this priming first amplification is performed for a period of about 8 days, resulting in a bulk TIL population, typically about 1 × 10⁸Individual TIL cells in bulk.

In some embodiments of the present invention, the,

any suitable dose of TIL may be administered. In some embodiments, about 2.3 x 10 is administered¹⁰To about 13.7X 10¹⁰TILs of which average about 7.8X 10¹⁰TIL, especially where the cancer is melanoma. In some embodiments, about 1.2 x 10 is administered¹⁰To about 4.3X 10¹⁰And (4) TIL. In some embodiments, about 3 x 10 is administered¹⁰To about 12X 10¹⁰And (4) TIL. In some embodiments, about 4 x 10 is administered¹⁰To about 10X 10¹⁰And (4) TIL. In some embodiments, about 5 x 10 is administered¹⁰To about 8X 10¹⁰And (4) TIL. In some embodiments, about 6 x 10 is administered¹⁰To about 8X 10¹⁰And (4) TIL. In some embodiments, about 7 x 10 is administered¹⁰To about 8X 10¹⁰And (4) TIL. In some embodiments, the therapeutically effective dose is about 2.3 x 10¹⁰To about 13.7X 10¹⁰. In some embodiments, the therapeutically effective dose is about 7.8 x 10¹⁰TIL, especially where the cancer is melanoma. In some embodiments, the therapeutically effective dose is about 1.2 x 10 ¹⁰To about 4.3X 10¹⁰And (4) TIL. In some embodiments, the therapeutically effective dose is about 3 x 10¹⁰To about 12X 10¹⁰And (4) TIL. In some embodiments, the therapeutically effective dose is about 4 x 10¹⁰To about 10X 10¹⁰And (4) TIL. In some embodiments, the therapeutically effective dose is about 5 x 10¹⁰To about 8X 10¹⁰And (4) TIL. In some embodiments, the therapeutically effective dose is about 6 x 10¹⁰To about 8X 10¹⁰And (4) TIL. In some embodiments, the therapeutically effective dose is about 7 x 10¹⁰To about 8X 10¹⁰And (4) TIL.

In some embodiments, the TIL is provided in the pharmaceutical compositions of the invention in an amount of about 1 × 10⁶、2×10⁶、3×10⁶、4×10⁶、5×10⁶、6×10⁶、7×10⁶、8×10⁶、9×10⁶、1×10⁷、2×10⁷、3×10⁷、4×10⁷、5×10⁷、6×10⁷、7×10⁷、8×10⁷、9×10⁷、1×10⁸、2×10⁸、3×10⁸、4×10⁸、5×10⁸、6×10⁸、7×10⁸、8×10⁸、9×10⁸、1×10⁹、2×10⁹、3×10⁹、4×10⁹、5×10⁹、6×10⁹、7×10⁹、8×10⁹、9×10⁹、1×10¹⁰、2×10¹⁰、3×10¹⁰、4×10¹⁰、5×10¹⁰、6×10¹⁰、7×10¹⁰、8×10¹⁰、9×10¹⁰、1×10¹¹、2×10¹¹、3×10¹¹、4×10¹¹、5×10¹¹、6×10¹¹、7×10¹¹、8×10¹¹、9×10¹¹、1×10¹²、2×10¹²、3×10¹²、4×10¹²、5×10¹²、6×10¹²、7×10¹²、8×10¹²、9×10¹²、1×10¹³、2×10¹³、3×10¹³、4×10¹³、5×10¹³、6×10¹³、7×10¹³、8×10¹³And 9X 10¹³And (4) respectively. In the examples, the amount of TIL provided in the pharmaceutical composition of the present invention ranges from 1 × 10⁶To 5X 10⁶、5×10⁶To 1X 10⁷、1×10⁷To 5X 10⁷、5×10⁷To 1X 10⁸、1×10⁸To 5X 10⁸、5×10⁸To 1X 10⁹、1×10⁹To 5X 10⁹、5×10⁹To 1X 10¹⁰、1×10¹⁰To 5X 10¹⁰、5×10¹⁰To 1X 10¹¹、5×10¹¹To 1X 10¹²、1×10¹²To 5X 10¹²And 5X 10¹²To 1X 10¹³。

In preferred embodiments, expansion of TIL may be performed using those described in step B of priming a first amplification step as described below and herein (e.g., fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may comprise a process referred to as pre-REP or priming REP and which contains feeder cells from day 0 and/or culture initiation), followed by rapid second amplification as described below in step D and herein (step D, comprising a process referred to as a rapid amplification protocol (REP) step), followed by optional cryopreservation and followed by a second step D as described below and herein (comprising a process referred to as a restimulation REP step). The TILs obtained from the process may optionally be characterized by phenotypic properties and metabolic parameters as described herein. In some embodiments, the tumor fragments are between about 1mm ³And 10mm³In the meantime.

In some embodiments, the first amplification medium is referred to as "CM," an abbreviation for medium. In some embodiments, the CM of step B consists of RPMI 1640 and GlutaMAX supplemented with 10% human AB serum, 25mM Hepes and 10mg/mL gentamicin.

In some embodiments, there are less than or equal to 240 tumor fragments. In some embodiments, there are less than or equal to 240 tumor fragments placed in less than or equal to 4 receptacles. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, less than or equal to 60 tumor fragments are placed in 1 container. In some embodiments, each vessel comprises less than or equal to 500mL of media per vessel. In some embodiments, the culture medium comprises IL-2. In some embodiments, the medium includes 6000IU/mL IL-2. In some embodiments, the culture medium comprises antigen presenting feeder cells (also referred to herein as "antigen presenting cells"). In some embodiments, the medium comprises 2.5 × 10 per vessel⁸Antigen presenting feeder cells. In some embodiments, the medium comprises OKT-3. In some embodiments, the medium comprises 30ng/mL OKT-3 per vessel. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium includes 6000IU/mL IL-2, 30ng OKT-3, and 2.5X 10 ⁸Antigen presenting feeder cells. In some embodiments, the medium includes 6000IU/mL IL-2, 30ng/mL OKT-3, and 2.5X 10 per vessel⁸Antigen presenting feeder cells.

After preparing tumor debris, whole tumor digests, and/or whole tumor cell suspensions, the resulting cells (i.e., the debris and/or digests of the initial cell population) are cultured in medium containing IL-2, antigen presenting feeder cells, and OKT-3 under conditions that favor the growth of TIL over tumor and other cells and allow TIL priming and accelerated growth from day 0 culture. In some embodiments, the tumor digest and/or tumor fragments are incubated with 6000IU/mL of IL-2, as well as antigen presenting feeder cells and OKT-3. This initial cell population is cultured for a period of several days (typically 1 to 8 days) to produce a bulk TIL population, typically about 1 × 10⁸Individual TIL cells in bulk. In some embodiments, the growth medium that initiates the first expansion period comprises IL-2 or a variant thereof, as well as antigen-presenting feeder cells and OKT-3. In some embodiments, this is doneThe seed initial cell population is cultured for a period of several days (typically 1 to 7 days) to produce a bulk TIL population, typically about 1 × 10 ⁸Individual TIL cells in bulk. In some embodiments, the growth medium that initiates the first expansion period comprises IL-2 or a variant thereof, as well as antigen-presenting feeder cells and OKT-3. In some embodiments, IL-2 is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution has a specific activity of 20-30X 10 for 1mg vials⁶IU/mg. In some embodiments, the IL-2 stock solution has a specific activity of 20X 10 for a 1mg vial⁶IU/mg. In some embodiments, the IL-2 stock solution has a specific activity of 25X 10 for a 1mg vial⁶IU/mg. In some embodiments, the IL-2 stock solution has a specific activity of 30X 10 for a 1mg vial⁶IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4-8X 10⁶IU/mg of IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7X 10⁶IU/mg of IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6X 10⁶IU/mg of IL-2. In some embodiments, IL-2 stock solutions are prepared as described in example C. In some embodiments, the priming first amplification medium comprises about 10,000IU/mL IL-2, about 9,000IU/mL IL-2, about 8,000IU/mL IL-2, about 7,000IU/mL IL-2, about 6000IU/mL IL-2, or about 5,000IU/mL IL-2. In some embodiments, the priming first amplification medium comprises about 9,000IU/mL IL-2 to about 5,000IU/mL IL-2. In some embodiments, the priming first amplification medium comprises about 8,000IU/mL IL-2 to about 6,000IU/mL IL-2. In some embodiments, the priming first amplification medium comprises about 7,000IU/mL IL-2 to about 6,000IU/mL IL-2. In some embodiments, the priming first amplification medium comprises about 6,000IU/mL of IL-2. In embodiments, the cell culture medium further comprises IL-2. In some embodiments, the priming first expanded cell culture medium comprises about 3000IU/mL of IL-2. In embodiments, the priming first expanded cell culture medium further comprises IL-2. In a preferred embodiment, the priming first expanded cell culture medium comprises about 3000IU/mL IL-2. In an embodiment, priming the first expanded cell culture medium comprises about 1000IU/mL, about 1500IU/mL, about 2000IU ™ based on blood glucose mL, about 2500IU/mL, about 3000IU/mL, about 3500IU/mL, about 4000IU/mL, about 4500IU/mL, about 5000IU/mL, about 5500IU/mL, about 6000IU/mL, about 6500IU/mL, about 7000IU/mL, about 7500IU/mL, or about 8000IU/mL of IL-2. In embodiments, the priming first expansion cell culture medium comprises IL-2 of between 1000 and 2000IU/mL, between 2000 and 3000IU/mL, between 3000 and 4000IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000IU/mL, between 6000 and 7000IU/mL, between 7000 and 8000IU/mL, or about 8000 IU/mL.

In some embodiments, the priming first amplification medium comprises about 500IU/mL IL-15, about 400IU/mL IL-15, about 300IU/mL IL-15, about 200IU/mL IL-15, about 180IU/mL IL-15, about 160IU/mL IL-15, about 140IU/mL IL-15, about 120IU/mL IL-15, or about 100IU/mL IL-15. In some embodiments, the priming first amplification medium comprises about 500IU/mL of IL-15 to about 100IU/mL of IL-15. In some embodiments, the priming first amplification medium comprises about 400IU/mL of IL-15 to about 100IU/mL of IL-15. In some embodiments, the priming first amplification medium comprises about 300IU/mL of IL-15 to about 100IU/mL of IL-15. In some embodiments, the priming first amplification medium comprises about 200IU/mL of IL-15. In some embodiments, the priming first expanded cell culture medium comprises about 180IU/mL of IL-15. In one embodiment, the priming first expanded cell culture medium further comprises IL-15. In a preferred embodiment, the priming first expanded cell culture medium comprises about 180IU/mL of IL-15.

In some embodiments, the priming first amplification medium comprises about 20IU/mL IL-21, about 15IU/mL IL-21, about 12IU/mL IL-21, about 10IU/mL IL-21, about 5IU/mL IL-21, about 4IU/mL IL-21, about 3IU/mL IL-21, about 2IU/mL IL-21, about 1IU/mL IL-21, or about 0.5IU/mL IL-21. In some embodiments, the priming first amplification medium comprises about 20IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the priming first amplification medium comprises about 15IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the priming first amplification medium comprises about 12IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the priming first amplification medium comprises about 10IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the priming first amplification medium comprises about 5IU/mL IL-21 to about 1IU/mL IL-21. In some embodiments, the priming first amplification medium comprises about 2IU/mL IL-21. In some embodiments, the priming first expanded cell culture medium comprises about 1IU/mL of IL-21. In some embodiments, the priming first expanded cell culture medium comprises about 0.5IU/mL of IL-21. In embodiments, the cell culture medium further comprises IL-21. In a preferred embodiment, the priming first expanded cell culture medium comprises about 1IU/mL of IL-21.

In embodiments, the priming first expansion cell culture medium comprises an OKT-3 antibody. In some embodiments, the priming first expanded cell culture medium comprises about 30ng/mL of OKT-3 antibody. In an embodiment, the priming first expansion cell culture medium comprises OKT-3 antibody at about 0.1ng/mL, about 0.5ng/mL, about 1ng/mL, about 2.5ng/mL, about 5ng/mL, about 7.5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, about 100ng/mL, about 200ng/mL, about 500ng/mL, and about 1 μ g/mL. In embodiments, the cell culture medium comprises between 0.1ng/mL and 1ng/mL, between 1ng/mL and 5ng/mL, between 5ng/mL and 10ng/mL, between 10ng/mL and 20ng/mL, between 20ng/mL and 30ng/mL, between 30ng/mL and 40ng/mL, between 40ng/mL and 50ng/mL, and between 50ng/mL and 100ng/mL of OKT-3 antibody. In an embodiment, the cell culture medium comprises between 15ng/mL and 30ng/mL of the OKT-3 antibody. In an embodiment, the cell culture medium comprises 30ng/mL of the OKT-3 antibody. In some embodiments, the OKT-3 antibody is molobumab.

Table 3: amino acid sequence of Moluomamab (exemplary OKT-3 antibody)

In some embodiments, priming the first expanded cell culture medium comprises cell culture medium comprising one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of: uluzumab, Utomilluzumab, EU-101, a fusion protein, and fragments, derivatives, variants, biological analogs and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium between 0.1 μ g/mL and 100 μ g/mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 20 μ g/mL and 40 μ g/mL.

In some embodiments, the priming first expanded cell culture medium further comprises IL-2 at an initial concentration of about 3000IU/mL and OKT-3 antibody at an initial concentration of about 30ng/mL in addition to the one or more TNFRSF agonists, and wherein the one or more TNFRSF agonists comprises a 4-1BB agonist. In some embodiments, the priming first expanded cell culture medium further comprises IL-2 at an initial concentration of about 6000IU/mL and OKT-3 antibody at an initial concentration of about 30ng/mL in addition to the one or more TNFRSF agonists, and wherein the one or more TNFRSF agonists comprises a 4-1BB agonist.

In some embodiments, the priming first amplification medium is referred to as "CM", an abbreviation for medium. In some embodiments, it is referred to as CM1 (medium 1). In some embodiments, CM consists of RPMI 1640 and GlutaMAX supplemented with 10% human AB serum, 25mM Hepes, and 10mg/mL gentamicin. In some embodiments, the CM is CM1 described in the examples, see example a. In some embodiments, priming the first expansion is performed in the initial cell culture medium or the first cell culture medium. In some embodiments, the priming first expansion medium or initial cell culture medium or first cell culture medium comprises IL-2, OKT-3, and antigen presenting feeder cells (also referred to herein as feeder cells).

In some embodiments, the medium used in the amplification processes disclosed herein is a serum-free medium or a defined medium. In some embodiments, the serum-free or defined medium comprises a basal cell medium and a serum supplement and/or serum replacement. In some embodiments, serum-free or defined media are used to prevent and/or reduce experimental variation due in part to batch-to-batch variation of serum-containing media.

In some embodiments, the serum-free or defined medium comprises a basal cell medium and a serum supplement and/or serum replacement. In some embodiments, the basal cell culture medium includes, but is not limited to, CTS ^TM OpTmizer^TMT cell expansion basal Medium, CTS^TM OpTmizer^TMT cell expansion SFM, CTS^TMAIM-V Medium, CTS^TM AIM-V SFM、LymphoONE^TMT cell expansion xeno-free Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, Minimal Essential Medium (α MEM), Glasgow's Minimal Essential Medium (G-MEM), RPMI growth Medium, and Issufu Modified Dulbecco's Medium.

In some embodiments, the serum supplement or serum replacement includes, but is not limited to, one or more of the following: CTS^TMOpTsizer T cell expansion serum supplement, CTS^TMImmune cell serum replacement, one or more albumins or albumin replacements, one or more amino acids, one or more vitamins, one or more transferrins or transferrin replacements, one or more antioxidants, one or more insulins or insulin replacements, one or more collagen precursors, one or more antibiotics, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and Containing trace element part Ag⁺、Al³⁺、Ba²⁺、Cd²⁺、Co²⁺、Cr³⁺、Ge⁴⁺、Se⁴⁺、Br、T、Mn²⁺、P、Si⁴⁺、V⁵⁺、Mo⁶⁺、Ni²⁺、Rb⁺、Sn²⁺And Zr⁴⁺The compound of (1). In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some embodiments, CTS^TMOpTmizer^TMT cell immune cell serum replacement for use in conventional growth media, including but not limited to CTS^TM OpTmizer^TMT cell expansion basal Medium, CTS^TM OpTmizer^TMT cell expansion SFM, CTS^TMAIM-V Medium, CST^TM AIM-V SFM、LymphoONE^TMT cell expansion xeno-free medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimal essential medium (α MEM), Glasgow's minimal essential medium (G-MEM), RPMI growth medium, and Iskifun's modified Dulbecco's medium.

In some embodiments, the total serum replacement concentration (% by volume) in the serum-free or defined medium is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% by volume of the total serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 3% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 5% of the total volume of the serum-free or defined medium. In some embodiments, the total serum replacement concentration is about 10% of the total volume of the serum-free or defined medium.

In some embodiments, the serum-free or defined medium is CTS^TM OpTmizer^TMT cell expansion SFM (seimer feishell science). CTS^TM OpTmizer^TMAny of the formulations of (a) can be used in the present invention. CTS^TM OpTmizer^TMT cell expansion SFM is 1L CTS mixed together prior to use^TM OpTmizer^TMT cell expansion basal Medium and 26mL CTS^TM OpTmizer^TMA combination of T cell expansion supplements. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Seimer Feishell science) supplements CTS^TM OpTmizer^TMT cells expand SFM. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Seimer Feishell science) and CTS supplemented with 55mM 2-mercaptoethanol^TM OpTmizer^TMT cells expand SFM. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Seimer Feishell science) supplements CTS^TM OpTmizer^TMT cells expanded SFM and the final concentration of 2-mercaptoethanol in the medium was 55. mu.M.

In some embodiments, the defined medium is CTS^TM OpTmizer^TMT cell expansion SFM (seimer feishell science). CTS^TM OpTmizer^TMAny of the formulations of (a) can be used in the present invention. CTS^TM OpTmizer^TMT cell expansion SFM is 1L CTS mixed together prior to use^TM OpTmizer^TMT cell expansion basal Medium and 26mL CTS^TMOpTmizer^TMA combination of T cell expansion supplements. In some embodiments, about 3% CTS is used ^TMImmune cell Serum Replacement (SR) (Seimer Feishell science) and CTS supplemented with 55mM 2-mercaptoethanol^TM OpTmizer^TMT cells expand SFM. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Saimer Feishell science), 55mM 2-mercaptoethanol and 2mM L-glutamine supplemented with CTS^TMOpTmizer^TMT cells expand SFM. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Saimer Feishell science), 55mM 2-thiolEthanol base and 2mM L-Glutamine supplementing CTS^TMOpTmizer^TMThe T cells expand SFM and further comprise about 1000IU/mL to about 8000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Saimer Feishell science), 55mM 2-mercaptoethanol and 2mM L-glutamine supplemented with CTS^TMOpTmizer^TMThe T cells expand SFM and further include about 3000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Saimer Feishell science), 55mM 2-mercaptoethanol and 2mM L-glutamine supplemented with CTS^TMOpTmizer^TMThe T cells expand SFM and further include about 6000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Seimer Feishell science) and CTS supplemented with 55mM 2-mercaptoethanol ^TMOpTmizer^TMThe T cells expand SFM and further comprise about 1000IU/mL to about 8000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Seimer Feishell science) and CTS supplemented with 55mM 2-mercaptoethanol^TMOpTmizer^TMThe T cells expand SFM and further include about 3000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Seimer Feishell science) and CTS supplemented with 55mM 2-mercaptoethanol^TMOpTmizer^TMThe T cells expand the SFM and further comprise about 1000IU/mL to about 6000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Saimer Feishell science) and approximately 2mM glutamine supplemented CTS^TMOpTmizer^TMThe T cells expand SFM and further comprise about 1000IU/mL to about 8000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Saimer Feishell science) and approximately 2mM glutamine supplemented CTS^TMOpTmizer^TMThe T cells expand SFM and further include about 3000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Saimer Feishale)Science & technology company) and about 2mM glutamine supplemented CTS^TMOpTmizer^TMThe T cells expand SFM and further include about 6000IU/mL of IL-2. In some embodiments, about 3% CTS is used ^TMImmune cell Serum Replacement (SR) (Seimer Feishell science) supplements CTS^TM OpTmizer^TMT cells expanded SFM and the final concentration of 2-mercaptoethanol in the medium was 55. mu.M.

In some embodiments, glutamine is used at a concentration of about 0.1mM to about 10mM, 0.5mM to about 9mM, 1mM to about 8mM, 2mM to about 7mM, 3mM to about 6mM, or 4mM to about 5mM (i.e.,) Serum-free medium or defined medium. In some embodiments, glutamine is used at a concentration of about 2mM (i.e.,) Serum-free medium or defined medium.

In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 5mM to about 150mM, 10mM to about 140mM, 15mM to about 130mM, 20mM to about 120mM, 25mM to about 110mM, 30mM to about 100mM, 35mM to about 95mM, 40mM to about 90mM, 45mM to about 85mM, 50mM to about 80mM, 55mM to about 75mM, 60mM to about 70mM, or about 65 mM. In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM. In some embodiments, the final concentration of 2-mercaptoethanol in the culture medium is 55 μ M.

In some embodiments, defined media described in International PCT publication No. WO/1998/030679, which is incorporated herein by reference, may be used in the present invention. In said publication, serum-free eukaryotic cell culture media are described. Serum-free eukaryotic cell culture media comprises basal cell culture media supplemented with serum-free supplements capable of supporting cell growth in serum-free culture. Serum-free eukaryotic cell culture media supplements comprising or obtained by combining one or more components selected from the group consisting of Obtaining: one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or β -mercaptoethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of: one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and a trace element containing fraction Ag ⁺、Al³⁺、Ba²⁺、Cd²⁺、Co²⁺、Cr³⁺、Ge⁴⁺、Se⁴⁺、Br、T、Mn²⁺、P、Si⁴⁺、V⁵⁺、Mo⁶⁺、Ni²⁺、Rb⁺、Sn²⁺And Zr⁴⁺The compound of (1). In some embodiments, the basal cell culture medium is selected from the group consisting of: dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimal essential medium (α MEM), Glasgow's minimal essential medium (G-MEM), RPMI growth medium, and Iskifuku's modified Dulbecco's medium.

In some embodiments, the concentration of glycine in the defined medium is between about 5-200mg/LIn the range of about 5-250 mg/L-histidine, about 5-300 mg/L-isoleucine, about 5-200 mg/L-methionine, about 5-400 mg/L-phenylalanine, about 1-1000 mg/L-proline, about 1-45 mg/L-hydroxyproline, about 1-250 mg/L-serine, about 10-500 mg/L-threonine, about 2-110 mg/L-tryptophan, about 3-175 mg/L-tyrosine, about 5-500 mg/L-valine, thiamine in a concentration of about 1-20mg/L, reduced glutathione in a concentration of about 1-20mg/L, L-ascorbic acid-2-phosphate in a concentration of about 1-200mg/L, iron-saturated transferrin in a concentration of about 1-50mg/L, insulin in a concentration of about 1-100mg/L, sodium selenite in a concentration of about 0.000001-0.0001mg/L, and albumin (e.g., I) Is about 5000-50,000 mg/L.

In some embodiments, the non-trace element fraction components in the defined medium are present in concentration ranges listed in the column under the heading "concentration range in 1X medium" in table a below. In other examples, the non-trace element fraction components of the defined media are present at the final concentrations listed in the following column under the heading "preferred examples of 1X media" in table a below. In other embodiments, the defined medium is a basal cell medium comprising a serum-free supplement. In some of these embodiments, the serum-free supplement includes non-trace element fraction components of the following types and at concentrations listed in the column under the heading "preferred embodiments of the supplement" in table a below.

Table a: concentration of non-trace element fraction component

In some embodiments, the osmolarity of the defined media is between about 260 and 350 mOsmol. In some embodiments, the osmolarity is between about 280 and 310 mOsmol. In some embodiments, defined media is supplemented with up to about 3.7g/L or about 2.2g/L sodium bicarbonate. The defined medium may be further supplemented with L-glutamine (final concentration of about 2mM), one or more antibiotics, non-essential amino acids (NEAA; final concentration of about 100. mu.M), 2-mercaptoethanol (final concentration of about 100. mu.M).

In some embodiments, defined media described in Smith et al, "Ex vivo expansion of human T cells for adoptive immunotherapy using novel Xeno-free CTS Immune Cell Serum Replacement" using novel Xeno-free CTS Immune Cell Serum Replacement, "clinical transformation Immunology (Clin Transl immunization), 4(1)2015(doi:10.1038/cti.2014.31) may be used in the present invention. Briefly, RPMI or CTS^TM OpTmizer^TMUsed as basal cell culture medium and with 0, 2%, 5% or 10% CTS^TMImmune cell serum replacement supplementation.

In embodiments, the cell culture medium in the first and/or second gas permeable container is not filtered. The use of unfiltered cell culture medium can simplify the procedures necessary to expand the cell number. In an embodiment, the cell culture medium in the first and/or second gas permeable container is devoid of beta-mercaptoethanol (BME or beta ME; also known as 2-mercaptoethanol, CAS 60-24-2).

In some embodiments, the processes that prime the first amplification (including processes such as those described in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those sometimes referred to as pre-REP or priming REP) are from 1 to 8 days, as discussed in the examples and figures. In some embodiments, the processes that prime the first amplification (including, for example, those processes described in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those processes sometimes referred to as pre-REP or priming REP) are from 2 to 8 days. In some embodiments, the processes that prime the first amplification (including, for example, those processes described in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those processes sometimes referred to as pre-REP or priming REP) are from 3 to 8 days. In some embodiments, the process that elicits the first amplification (including processes such as those described in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those sometimes referred to as pre-REP or eliciting REP) is from 4 to 8 days, as discussed in the examples and figures. In some embodiments, the process that elicits the first amplification (including processes such as those described in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those sometimes referred to as pre-REP or eliciting REP) is from 1 to 7 days, as discussed in the examples and figures. In some embodiments, the processes that prime the first amplification (including, for example, those processes described in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those processes sometimes referred to as pre-REP or priming REP) are from 2 to 8 days. In some embodiments, the processes that prime the first amplification (including processes such as those described in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those sometimes referred to as pre-REP or priming REP) are from 2 to 7 days. In some embodiments, the process that primes the first amplification (including processes such as those described in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those processes sometimes referred to as pre-REP or priming REP) is from 3 to 8 days. In some embodiments, the process that primes the first amplification (including processes such as those described in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those processes sometimes referred to as pre-REP or priming REP) is from 3 to 7 days. In some embodiments, the processes that prime the first amplification (including processes such as those described in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those sometimes referred to as pre-REP or priming REP) are from 4 to 8 days. In some embodiments, the processes that prime the first amplification (including processes such as those described in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those sometimes referred to as pre-REP or priming REP) are from 4 to 7 days. In some embodiments, the processes that prime the first amplification (including processes such as those described in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those sometimes referred to as pre-REP or priming REP) are from 5 to 8 days. In some embodiments, the processes that prime the first amplification (including processes such as those described in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those sometimes referred to as pre-REP or priming REP) are from 5 to 7 days. In some embodiments, the processes that prime the first amplification (including processes such as those described in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those sometimes referred to as pre-REP or priming REP) are from 6 to 8 days. In some embodiments, the processes that prime the first amplification (including processes such as those described in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those sometimes referred to as pre-REP or priming REP) are from 6 to 7 days. In some embodiments, the processes that prime the first amplification (including, for example, those processes provided in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those processes sometimes referred to as pre-REP or priming REP) are from 7 to 8 days. In some embodiments, the process that elicits the first amplification (including processes such as those provided in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those processes sometimes referred to as pre-REP or eliciting REP) is 8 days. In some embodiments, the process that elicits the first amplification (including processes such as those provided in step B of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), which may include those processes sometimes referred to as pre-REP or eliciting REP) is 7 days.

In some embodiments, priming the first TIL amplification may be performed for 1 day to 8 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, priming the first TIL amplification may be performed for 1 day to 7 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, priming the first TIL amplification may be performed for 2 days to 8 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, priming the first TIL amplification may be performed for 2 days to 7 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, priming the first TIL amplification may be performed for 3 days to 8 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, priming the first TIL amplification may be performed for 3 days to 7 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, priming the first TIL amplification may be performed for 4 to 8 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, priming the first TIL amplification may be performed for 4 to 7 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, priming the first TIL amplification may be performed for 5 to 8 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, priming the first TIL amplification may be performed for 5 to 7 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, priming the first TIL amplification may be performed for 6 to 8 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, priming the first TIL amplification may be performed for 6 to 7 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, priming the first TIL amplification may be performed for 7 to 8 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, priming the first TIL amplification may be performed for 8 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, priming the first TIL amplification may be performed for 7 days from when fragmentation occurs and/or the first priming amplification step is initiated.

In some embodiments, priming first amplification of TILs may be performed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days. In some embodiments, the first TIL amplification may be performed for 1 day to 8 days. In some embodiments, the first TIL amplification may be performed for 1 day to 7 days. In some embodiments, the first TIL amplification may be performed for 2 days to 7 days. In some embodiments, the first TIL amplification may be performed for 3 days to 7 days. In some embodiments, the first TIL amplification may be performed for 4 days to 7 days. In some embodiments, the first TIL amplification may be performed for 5 days to 7 days. In some embodiments, the first TIL amplification may be performed for 6 days to 7 days. In some embodiments, the first TIL amplification may be performed for 2 days to 8 days. In some embodiments, the first TIL amplification may be performed for 3 days to 8 days. In some embodiments, the first TIL amplification may be performed for 4 days to 8 days. In some embodiments, the first TIL amplification may be performed for 5 days to 8 days. In some embodiments, the first TIL amplification may be performed for 6 days to 8 days. In some embodiments, the first TIL amplification may be performed for 2 days to 9 days. In some embodiments, the first TIL amplification may be performed for 3 days to 9 days. In some embodiments, the first TIL amplification may be performed for 4 days to 9 days. In some embodiments, the first TIL amplification may be performed for 5 days to 9 days. In some embodiments, the first TIL amplification may be performed for 6 days to 9 days. In some embodiments, the first TIL amplification may be performed for 2 days to 10 days. In some embodiments, the first TIL amplification may be performed for 3 days to 10 days. In some embodiments, the first TIL amplification may be performed for 4 days to 10 days. In some embodiments, the first TIL amplification may be performed for 5 days to 10 days.

In some embodiments, the first TIL amplification may be performed for 6 days to 10 days. In some embodiments, the first TIL amplification may be performed for 2 days to 11 days. In some embodiments, the first TIL amplification may be performed for 3 days to 11 days. In some embodiments, the first TIL amplification may be performed for 4 days to 11 days. In some embodiments, the first TIL amplification may be performed for 5 days to 11 days. In some embodiments, the first TIL amplification may be performed for 6 days to 11 days. In some embodiments, the first TIL amplification may be performed for 7 days. In some embodiments, the first TIL amplification may be performed for 8 days. In some embodiments, the first TIL amplification may be performed for 9 days. In some embodiments, the first TIL amplification may be performed for 10 days. In some embodiments, the first TIL amplification may be performed for 11 days.

In some embodiments, combinations of IL-2, IL-7, IL-15, and/or IL-21 are employed in combination during priming of the first amplification. In some embodiments, IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof, may be included during priming of the first amplification, including, for example, during the step B process according to FIG. 1 (specifically, e.g., FIG. 1B), and during the periods described herein. In some embodiments, during priming the first amplification, a combination of IL-2, IL-15, and IL-21 is employed in combination. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the step B process according to FIG. 1 (specifically, e.g., FIG. 1B and/or FIG. 1C), and as described herein.

In some embodiments, priming the first amplification is performed in a closed system bioreactor, such as step B according to fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C). In some embodiments, a closed system is used for TIL amplification, as described herein. In some embodiments, a bioreactor is employed. In some embodiments, the bioreactor serves as a container. In some embodiments, the bioreactor employed is, for example, G-REX-10 or G-REX-100. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-10.

1. Feeder cells and antigen presenting cells

In embodiments, priming a first expansion procedure described herein (e.g., expansions comprising those expansions as described in step B from fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), and those expansions referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the initiation of TIL expansion, but rather are added during priming of the first expansion (priming of REP). In embodiments, priming the first expansion procedure described herein (e.g., expansions comprising those expansions as described in step B from fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), and those expansions referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the onset of TIL expansion, but rather are added at any time during days 4-8 during priming of the first expansion. In embodiments, priming the first expansion procedure described herein (e.g., expansions comprising those expansions as described in step B from fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), and those expansions referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the onset of TIL expansion, but rather are added at any time during days 4-7 during priming of the first expansion. In embodiments, priming the first expansion procedure described herein (e.g., expansions comprising those expansions as described in step B from fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), and those expansions referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the onset of TIL expansion, but rather are added at any time during days 5-8 during priming the first expansion. In embodiments, priming the first expansion procedure described herein (e.g., expansions comprising those expansions as described in step B from fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), and those expansions referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the onset of TIL expansion, but rather are added at any time during days 5-7 during priming of the first expansion. In embodiments, priming the first expansion procedure described herein (e.g., expansions comprising those expansions as described in step B from fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), and those expansions referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the onset of TIL expansion, but rather are added at any time during days 6-8 during priming the first expansion. In embodiments, priming the first expansion procedure described herein (e.g., expansions comprising those expansions as described in step B from fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), and those expansions referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the onset of TIL expansion, but rather are added at any time during days 6-7 during priming of the first expansion. In embodiments, priming the first expansion procedure described herein (e.g., expansions comprising those expansions as described in step B from fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), and those expansions referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the onset of TIL expansion, but rather are added at any time during day 7 or day 8 during priming of the first expansion. In embodiments, priming the first expansion procedure described herein (e.g., expansions comprising those expansions as described in step B from fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), and those expansions referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the onset of TIL expansion, but rather are added at any time during day 7 during priming of the first expansion. In embodiments, priming the first expansion procedure described herein (e.g., expansions comprising those expansions as described in step B from fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), and those expansions referred to as pre-REP or priming REP) does not require feeder cells (also referred to herein as "antigen presenting cells") at the onset of TIL expansion, but rather are added at any time during day 8 during priming of the first expansion.

In embodiments, priming a first expansion procedure described herein (e.g., expansions comprising those expansions as described in step B from fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), and those expansions referred to as pre-REP or priming REP) requires feeder cells (also referred to herein as "antigen presenting cells") at the initiation of TIL expansion and during priming the first expansion. In many embodiments, the feeder cells are Peripheral Blood Mononuclear Cells (PBMCs) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In some embodiments, 2.5 × 10 is used during priming of the first amplification⁸And (4) feeding cells. In some embodiments, 2.5 × 10 per vessel is used during priming of the first amplification⁸And (4) feeding cells. In some embodiments, 2.5X 10 is used per GREX-10 during priming of the first amplification⁸And (4) feeding cells. In some embodiments, 2.5X 10 is used per GREX-100 during priming of the first amplification⁸And (4) feeding cells.

Typically, allogeneic PBMCs are inactivated by irradiation or heat treatment and used in the REP procedure, which provides an exemplary protocol for assessing replication incompetence of irradiated allogeneic PBMCs, as described in the examples.

In some embodiments, a PBMC is considered replication-incompetent and approved for use in the TIL expansion procedure described herein if the total number of viable cells at day 14 is less than the initial number of viable cells placed in culture at day 0 of priming the first expansion.

In some embodiments, PBMCs are considered replication-incompetent and approved for use in the TIL expansion procedure described herein if the total number of viable cells on day 7 in culture in the presence of OKT3 and IL-2 is not increased compared to the initial number of viable cells placed in culture on day 0 of priming the first expansion. In some embodiments, PBMCs are cultured in the presence of 30ng/mL OKT3 antibody and 3000IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 30ng/ml OKT3 antibody and 6000IU/ml IL-2.

In some embodiments, PBMCs are considered replication-incompetent and approved for use in the TIL expansion procedure described herein if the total number of viable cells on day 7 in culture in the presence of OKT3 and IL-2 is not increased compared to the initial number of viable cells placed in culture on day 0 of priming the first expansion. In some embodiments, PBMCs are cultured in the presence of 5-60ng/mL OKT3 antibody and 1000-6000IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50ng/mL OKT3 antibody and 2000-5000IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40ng/mL OKT3 antibody and 2000-4000IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 25-35ng/mL OKT3 antibody and 2500-. In some embodiments, PBMCs are cultured in the presence of 30ng/mL OKT3 antibody and 6000IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 15ng/mL OKT3 antibody and 3000IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 15ng/mL OKT3 antibody and 6000IU/mL IL-2.

In some embodiments, the antigen presenting feeder cells are PBMCs. In some embodiments, the antigen presenting feeder cells are artificial antigen presenting feeder cells. In embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:50 and 1: 300. In embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:100 and 1: 200.

In embodiments, the priming of the first amplification procedure described herein requires about 2.5 × 10⁸Each feeder cell is about 100X 10⁶The ratio of TILs. In another embodiment, the priming of the first amplification procedure described herein requires about 2.5X 10⁸Each feeder cell is about 50X 10⁶The ratio of TILs. In yet another embodiment, priming a first amplification described herein requires about 2.5X 10⁸One feeder cell and about 25X 10⁶And (4) TIL. In yet another embodiment, priming a first amplification described herein requires about 2.5X 10 ⁸And (4) feeding cells. In yet another embodiment, one quarter, one third, five twelfth or one half of the number of feeder cells needed for rapid second expansion is primed for first expansion.

In some embodiments, the medium in which the first amplification is initiated comprises IL-2. In some embodiments, the medium in priming the first amplification comprises 6000IU/mL IL-2. In some embodiments, the medium in which the first expansion is initiated comprises antigen presenting feeder cells. In some embodiments, the medium in initiating the first amplification comprises 2.5 × 10 per vessel⁸Antigen presenting feeder cells. In some embodiments, the medium in which the first amplification is initiated comprises OKT-3. In some embodiments, the medium includes 30ng of OKT-3 per vessel. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium includes 6000IU/mL IL-2, 30ng/mL OKT-3, and 2.5X 10⁸Antigen presenting feeder cells. In some embodiments, the medium includes 6000IU/mL IL-2, 30ng/mL OKT-3, and 2.5X 10 per vessel⁸Antigen presenting feeder cells. In some embodiments, the medium comprises 500mL of medium per vessel and 15 μ g of OKT-3 per 2.5 × 10 ⁸Antigen presenting feeder cells. In some embodiments, the medium comprises 500mL of medium and 15 μ g of OKT-3 per vessel. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium comprises 500mL of medium and 6000IU/mLIL-2, 30ng/mL OKT-3 and 2.5X 10⁸Antigen presenting feeder cells. In some embodiments, the medium comprises 500mL of medium and 6000IU/mL of IL-2, 15 μ g of OKT-3, and 2.5X 10 per vessel⁸Antigen presenting feeder cells. In some embodiments, the medium comprises 500mL of medium per vessel and 15 μ g of OKT-3 per 2.5 × 10⁸Antigen presenting feeder cells.

In one embodiment, priming the first expansion procedure described herein requires an excess of feeder cells relative to TIL during the second expansion. In many embodiments, the feeder cells are Peripheral Blood Mononuclear Cells (PBMCs) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In the examples, artificial antigen presenting (aAPC) cells were used instead of PBMCs.

Typically, allogeneic PBMCs are inactivated by irradiation or heat treatment and used in the TIL amplification procedures described herein, including the exemplary procedures described in the figures and examples.

In embodiments, artificial antigen presenting cells are used in place of PBMCs or in combination with them in priming the first expansion.

2. Cytokine

The expansion methods described herein generally use media with high doses of cytokines (specifically, IL-2), as is known in the art.

Alternatively, it is additionally possible to use a combination of cytokines for priming the first expansion of TIL, wherein the combination of two or more of IL-2, IL-15 and IL-21 is as generally outlined in International publication Nos. WO 2015/189356 and WO 2015/189357, which are hereby expressly incorporated by reference in their entirety. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15 and IL-21, with the latter being particularly suitable in many embodiments. The use of a combination of cytokines is particularly advantageous for the production of lymphocytes, and in particular T cells as described herein.

Table 4: amino acid sequence of interleukin.

C. And C: priming the transition from a first amplification to a Rapid second amplification

In some cases, an bulk TIL population obtained from priming a first amplification (which may comprise what is sometimes referred to as a pre-REP amplification), comprising a TIL population obtained, for example, from step B as indicated in, for example, fig. 1 (specifically, for example, fig. 1B and/or fig. 1C), may be subjected to a rapid second amplification (which may comprise what is sometimes referred to as a rapid amplification protocol (REP)) and then subjected to cryopreservation as discussed below. Similarly, where the genetically modified TIL is to be used in therapy, the amplified TIL population from the priming first amplification or the amplified TIL population from the rapid second amplification may be genetically modified for appropriate treatment prior to the amplification step or after the priming first amplification and prior to the rapid second amplification.

In some embodiments, the TILs obtained from priming the first amplification (e.g., from step B as indicated in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) are stored until phenotyping is performed for selection. In some embodiments, the TIL obtained from priming the first amplification (e.g., from step B as indicated in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) is not stored and the rapid second amplification is performed directly. In some embodiments, the TIL obtained from priming the first amplification is not cryopreserved after priming the first amplification and before the rapid second amplification. In some embodiments, the transition from priming the first amplification to the second amplification is performed about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days from when tumor disruption occurred and/or the first priming amplification step was initiated. In some embodiments, the transition from priming the first amplification to rapid second amplification occurs between about 3 days and 7 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, the transition from priming the first amplification to rapid second amplification occurs between about 3 days and 8 days from when fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, the transition from priming the first amplification to the second amplification occurs between about 4 days and 7 days from when the fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, the transition from priming the first amplification to the second amplification occurs from about 4 days to 8 days from when the fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, the transition from priming the first amplification to the second amplification occurs between about 5 days and 7 days from when the fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, the transition from priming the first amplification to the second amplification occurs from about 5 days to 8 days from when the fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, the transition from priming the first amplification to the second amplification occurs between about 6 days and 7 days from when the fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, the transition from priming the first amplification to the second amplification occurs between about 6 days and 8 days from when the fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, the transition from priming the first amplification to the second amplification occurs from about 7 days to 8 days from when the fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, the transition from priming the first amplification to the second amplification occurs about 7 days from when the fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, the transition from priming the first amplification to the second amplification occurs about 8 days from when the fragmentation occurs and/or the first priming amplification step is initiated.

In some embodiments, the transition from priming the first amplification to rapid second amplification occurs 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days from when the fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, the transition from priming the first amplification to rapid second amplification occurs between 1 day and 7 days of the occurrence of fragmentation and/or initiation of the first priming amplification step. In some embodiments, the transition from priming the first amplification to rapid second amplification occurs between 1 day and 8 days of the occurrence of fragmentation and/or initiation of the first priming amplification step. In some embodiments, the transition from priming the first amplification to the second amplification occurs between 2 days and 7 days of the occurrence of fragmentation and/or initiation of the first priming amplification step. In some embodiments, the transition from priming the first amplification to the second amplification occurs between 2 days and 8 days after fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, the transition from priming the first amplification to the second amplification occurs between 3 days and 7 days of the occurrence of fragmentation and/or initiation of the first priming amplification step. In some embodiments, the transition from priming the first amplification to the second amplification occurs between 3 days and 8 days of the occurrence of fragmentation and/or initiation of the first priming amplification step. In some embodiments, the transition from priming the first amplification to rapid second amplification occurs between 4 days and 7 days of fragmentation and/or initiation of the first priming amplification step. In some embodiments, the transition from priming the first amplification to rapid second amplification occurs between 4 days and 8 days of fragmentation and/or initiation of the first priming amplification step. In some embodiments, the transition from priming the first amplification to rapid second amplification occurs between 5 days and 7 days of the occurrence of fragmentation and/or initiation of the first priming amplification step. In some embodiments, the transition from priming the first amplification to rapid second amplification occurs between 5 days and 8 days of the occurrence of fragmentation and/or initiation of the first priming amplification step. In some embodiments, the transition from priming the first amplification to rapid second amplification occurs between 6 days and 7 days of the occurrence of fragmentation and/or initiation of the first priming amplification step. In some embodiments, the transition from priming the first amplification to rapid second amplification occurs between 6 days and 8 days from the occurrence of fragmentation and/or initiation of the first priming amplification step. In some embodiments, the transition from priming the first amplification to rapid second amplification occurs between 7 days and 8 days after fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, the transition from priming the first amplification to rapid second amplification occurs 7 days after fragmentation occurs and/or the first priming amplification step is initiated. In some embodiments, the transition from priming the first amplification to rapid second amplification occurs 8 days after fragmentation occurs and/or the first priming amplification step is initiated.

In some embodiments, the TIL is not stored after the initial first amplification and before the rapid second amplification, and the TIL is directly subjected to the rapid second amplification (e.g., in some embodiments, there is no storage during the transition from step B to step D as shown in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)). In some embodiments, the transformation is performed in a closed system, as described herein. In some embodiments, the TILs from the priming first amplification (second TIL population) are directly subjected to a rapid second amplification without a transition phase.

In some embodiments, the transition to initiate the first amplification to the rapid second amplification is performed in a closed system bioreactor, such as step C according to fig. 1 (specifically, e.g., fig. 1B). In some embodiments, a closed system is used for TIL amplification, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, a single bioreactor is used such as GREX-10 or GREX-100. In some embodiments, the closed system bioreactor is a single bioreactor. In some embodiments, initiating the transition from the first amplification to the rapid second amplification involves an expansion of the container size. In some embodiments, priming the first amplification is performed in a smaller vessel than the rapid second amplification. In some embodiments, a priming first amplification is performed in GREX-100 and a rapid second amplification is performed in GREX-500.

In some embodiments, up to 1 × 10 is obtained at the end of priming the first amplification⁶Individual cells TIL. 0.1X 10 is obtained at the end of the priming first amplification⁶0.2 x 10⁶0.3 x 10⁶0.4 x 10⁶0.5 x 10⁶0.6 x 10⁶0.7 x 10⁶0.8 x 10⁶0.9 x 10⁶1.0 × 10⁶1.1 × 10⁶1.2 x 10⁶1.3 x 10⁶1.4 x 10⁶Or 0.5X 10⁶And (4) TIL. In some embodiments, the TIL at the end of priming the first amplification is about 9% to about 40% PD-1 +. In some embodiments, the TIL at the end of priming the first amplification is about 10% to about 40% PD-1 +. In some embodiments, the TIL at the end of priming the first amplification is about 15% to about 30% PD-1 +. In some embodiments, the TIL at the end of priming the first amplification is about 20% to about 40% PD-1 +. In some embodiments, the TIL at the end of priming the first amplification is about 20% to about 30% PD-1 +. In some embodiments, the TIL at the end of priming the first amplification is about 10% to about 20% PD-1 +. In some embodiments, the TIL at the end of priming the first amplification is about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% PD-1 +. In some embodiments, the TIL at the end of priming the first amplification is about 9% to about 40% PD-1 high. In some embodiments, the TIL at the end of priming the first amplification is about 15% to about 30% PD-1 high. In some embodiments, the TIL at the end of priming the first amplification is about 20% to about 40% PD-1 high. In some embodiments, the TIL at the end of priming the first amplification is about 20% to about 30% PD-1 high. In some embodiments, the TIL at the end of priming the first amplification is about 10% to about 20% PD-1 high. In some embodiments, the TIL at the end of priming the first amplification is about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or about 40% PD-1 high.

D. Step D: rapid second amplification

In some embodiments, after collection and priming of the first expansion, after step a and step B, and a shift referred to as step C, the number of TIL cell populations is further expanded, as indicated in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C). The further amplification, referred to herein as rapid second amplification, may comprise an amplification process (rapid amplification protocol or REP; and a process as indicated in step D of FIG. 1 (specifically, e.g., FIG. 1B and/or FIG. 1C)) commonly referred to in the art as a rapid amplification process. Rapid second expansion is typically accomplished using a medium comprising a plurality of components including feeder cells, a source of cytokines, and an anti-CD 3 antibody in a gas permeable container. In some embodiments, the TIL is transferred to a larger volume vessel 1 day, 2 days, 3 days, or 4 days after initiation of the rapid second amplification (i.e., at day 8, 9, 10, or 11 days of the overall Gen 3 process).

In some embodiments, up to 1 × 10 is added at the beginning of the rapid second amplification⁶Individual cells TIL. Addition of 0.1X 10 at the beginning of the Rapid second amplification⁶0.2 x 10⁶0.3 x 10⁶0.4 x 10 ⁶0.5 x 10⁶0.6 x 10⁶0.7 x 10⁶0.8 x 10⁶0.9 x 10⁶1.0 × 10⁶1.1 × 10⁶1.2 x 10⁶1.3 x 10⁶1.4 x 10⁶Or 0.5X 10⁶And (4) TIL. In some embodiments, the maximum cell density from priming the first expansion is 1e6 cells to provide 1e9 for initiating a rapid second expansion.

In some embodiments, the rapid second amplification of TIL (which may comprise amplification sometimes referred to as REP; and the process as indicated in step D of FIG. 1 (specifically, e.g., FIG. 1B and/or FIG. 1C)) may be performed using any TIL flask or vessel known to those of skill in the art. In some embodiments, the second TIL amplification may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 1 day to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 1 day to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 2 days to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 2 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 3 days to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 3 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 4 days to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 4 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 5 days to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 5 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 6 days to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 6 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 7 days to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 7 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 8 days to about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 8 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 9 days to about 10 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed about 1 day after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 2 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 3 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 4 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 5 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 6 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 7 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 8 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 9 days after the rapid second amplification is initiated. In some embodiments, the second TIL amplification may be performed for about 10 days after the rapid second amplification is initiated.

In embodiments, the methods of the present disclosure may be used to perform a rapid second amplification (including, for example, an amplification referred to as REP; and a process as indicated in step D of FIG. 1 (specifically, e.g., FIG. 1B and/or FIG. 1C)) in a gas-permeable container. In some embodiments, TIL is expanded in the presence of IL-2, OKT-3, and feeder cells (also referred to herein as "antigen presenting cells") in a rapid second expansion. In some embodiments, the TIL is expanded in the presence of IL-2, OKT-3, and feeder cells in a rapid second expansion, wherein the feeder cells are added to a final concentration that is two-fold, 2.4-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold the concentration of feeder cells present in the priming first expansion. For example, TIL can be rapidly amplified using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulation can include, for example, anti-CD 3 antibodies, such as about 30ng/ml OKT3, mouse monoclonal anti-CD 3 antibody (commercially available from Ormez pharmaceuticals (Ortho-McNeil, Raritan, NJ) of Laritan, N.J.) or American Sun Biotech company (Miltenyi Biotech, Auburn, CA) of Orben, Calif.) or UHCT-1 (commercially available from BioLegend, san Diego, Calif., U.S.A.). The TIL may be amplified to induce further stimulation of TIL in vitro by including one or more antigens of the cancer, including antigenic portions thereof, such as one or more epitopes, during the second expansion, optionally expressed by a carrier, such as a human leukocyte antigen a2(HLA-a2) binding peptide, e.g. 0.3 μ Μ MART-1:26-35(27L) or gpl00: 209-. Other suitable antigens may include, for example, NY-ESO-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2 or antigenic portions thereof. TIL can also be rapidly expanded by re-stimulation with one or more of the same antigens of cancer pulsed on HLA-A2 expressing antigen presenting cells. Alternatively, TIL may be further restimulated with, for example, irradiated autologous lymphocytes or with irradiated HLA-A2+ allogenic lymphocytes and IL-2. In some embodiments, the restimulation is performed as part of a second amplification. In some embodiments, the second expansion is performed in the presence of irradiated autologous lymphocytes or in the presence of irradiated HLA-a2+ allogeneic lymphocytes and IL-2.

In embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000IU/mL of IL-2. In embodiments, the cell culture medium comprises about 1000IU/mL, about 1500IU/mL, about 2000IU/mL, about 2500IU/mL, about 3000IU/mL, about 3500IU/mL, about 4000IU/mL, about 4500IU/mL, about 5000IU/mL, about 5500IU/mL, about 6000IU/mL, about 6500IU/mL, about 7000IU/mL, about 7500IU/mL, or about 8000IU/mL of IL-2. In embodiments, the cell culture medium comprises IL-2 between 1000 and 2000IU/mL, between 2000 and 3000IU/mL, between 3000 and 4000IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000IU/mL, between 6000 and 7000IU/mL, between 7000 and 8000IU/mL, or between 8000 IU/mL.

In embodiments, the cell culture medium comprises an OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30ng/mL of the OKT-3 antibody. In an embodiment, the cell culture medium comprises about 0.1ng/mL, about 0.5ng/mL, about 1ng/mL, about 2.5ng/mL, about 5ng/mL, about 7.5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, about 100ng/mL, about 200ng/mL, about 500ng/mL, and about 1 μ g/mL of OKT-3 antibody. In embodiments, the cell culture medium comprises between 0.1ng/mL and 1ng/mL, between 1ng/mL and 5ng/mL, between 5ng/mL and 10ng/mL, between 10ng/mL and 20ng/mL, between 20ng/mL and 30ng/mL, between 30ng/mL and 40ng/mL, between 40ng/mL and 50ng/mL, and between 50ng/mL and 100ng/mL of OKT-3 antibody. In an embodiment, the cell culture medium comprises between 30ng/mL and 60ng/mL of the OKT-3 antibody. In an embodiment, the cell culture medium comprises about 60ng/mL OKT-3. In some embodiments, the OKT-3 antibody is molobumab.

In some embodiments, the medium in the rapid second expansion comprises IL-2. In some embodiments, the medium includes 6000IU/mL IL-2. In some embodiments, the medium in the rapid second expansion comprises antigen presenting feeder cells. In some embodiments, the medium in the rapid second amplification comprises 7.5X 10 per vessel⁸Antigen presenting feeder cells. In some embodiments, the medium in the rapid second expansion comprises OKT-3. In some embodiments, the medium in the rapid second amplification comprises 500mL of medium and 30 μ g of OKT-3 per vessel. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium in the rapid second expansion comprises 6000IU/mL IL-2, 60ng/mL OKT-3, and 7.5X 10⁸Antigen presenting feeder cells. In some embodiments, the medium comprises 500mL of medium and 6000IU/mL of IL-2, 30 μ g of OKT-3, and 7.5X 10 per vessel⁸Antigen presenting feeder cells.

In some embodiments, the medium in the rapid second expansion comprises IL-2. In some embodiments, the medium includes 6000IU/mL IL-2. In some embodiments, the medium in the rapid second expansion comprises antigen presenting feeder cells. In some embodiments, the medium comprises at 5X 10 per vessel ⁸And 7.5X 10⁸Between antigen presenting feeder cells. In some embodiments, the medium in the rapid second expansion comprises OKT-3. In some embodiments, the medium in the rapid second amplification comprises 500mL of medium and 30 μ g of OKT-3 per vessel. In some embodiments, the container is a GREX100 MCS flask. In some embodiments, the medium in the rapid second expansion comprises 6000IU/mL IL-2, 60ng/mL OKT-3, and at 5X 10⁸And 7.5X 10⁸Between antigen presenting feeder cells. In some embodiments, the medium in the rapid second expansion comprises 500mL of medium and 6000IU/mL of IL-2 per vessel, 30 μ g of OKT-3, and at 5X 10⁸And 7.5X 10⁸Between antigen presenting feeder cells.

In some embodiments, the cell culture medium comprises a cell culture medium comprising one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of: uluzumab, Utomilluzumab, EU-101, a fusion protein, and fragments, derivatives, variants, biological analogs and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium between 0.1 μ g/mL and 100 μ g/mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 20 μ g/mL and 40 μ g/mL.

In some embodiments, the cell culture medium further comprises IL-2 at an initial concentration of about 3000IU/mL and OKT-3 antibody at an initial concentration of about 30ng/mL in addition to the one or more TNFRSF agonists, and wherein the one or more TNFRSF agonists comprises a 4-1BB agonist.

In some embodiments, combinations of IL-2, IL-7, IL-15, and/or IL-21 are employed in combination during the second amplification. In some embodiments, IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof, may be included during the second amplification, including, for example, during the step D process according to FIG. 1 (specifically, e.g., FIG. 1B and/or FIG. 1C), as well as during the periods described herein. In some embodiments, during the second amplification, a combination of IL-2, IL-15, and IL-21 is employed in combination. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during the step D process according to FIG. 1 (specifically, e.g., FIG. 1B and/or FIG. 1C), and as described herein.

In some embodiments, the second expansion can be performed in a supplemented cell culture medium comprising IL-2, OKT-3, antigen presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion is performed in a supplemented cell culture medium. In some embodiments, the supplemented cell culture medium comprises IL-2, OKT-3, and antigen presenting feeder cells. In some embodiments, the second cell culture medium comprises IL-2, OKT-3, and antigen presenting cells (APCs; also referred to as antigen presenting feeder cells). In some embodiments, the second expansion is performed in cell culture medium comprising IL-2, OKT-3, and antigen presenting feeder cells (i.e., antigen presenting cells).

In some embodiments, the second amplification medium comprises about 500IU/mL IL-15, about 400IU/mL IL-15, about 300IU/mL IL-15, about 200IU/mL IL-15, about 180IU/mL IL-15, about 160IU/mL IL-15, about 140IU/mL IL-15, about 120IU/mL IL-15, or about 100IU/mL IL-15. In some embodiments, the second amplification medium comprises about 500IU/mL of IL-15 to about 100IU/mL of IL-15. In some embodiments, the second amplification medium comprises about 400IU/mL of IL-15 to about 100IU/mL of IL-15. In some embodiments, the second amplification medium comprises about 300IU/mL of IL-15 to about 100IU/mL of IL-15. In some embodiments, the second amplification medium comprises about 200IU/mL IL-15. In some embodiments, the cell culture medium comprises about 180IU/mL of IL-15. In embodiments, the cell culture medium further comprises IL-15. In a preferred embodiment, the cell culture medium comprises about 180IU/mL IL-15.

In some embodiments, the second amplification medium comprises about 20IU/mL IL-21, about 15IU/mL IL-21, about 12IU/mL IL-21, about 10IU/mL IL-21, about 5IU/mL IL-21, about 4IU/mL IL-21, about 3IU/mL IL-21, about 2IU/mL IL-21, about 1IU/mL IL-21, or about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises about 20IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the second amplification medium comprises about 15IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the second amplification medium comprises about 12IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises about 10IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the second amplification medium comprises about 5IU/mL IL-21 to about 1IU/mL IL-21. In some embodiments, the second amplification medium comprises about 2IU/mL IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5IU/mL IL-21. In embodiments, the cell culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1IU/mL IL-21.

In some embodiments, the antigen presenting feeder cells (APCs) are PBMCs. In embodiments, the ratio of TILs to PBMCs and/or antigen presenting cells in the rapid expansion and/or the second expansion is about 1 to 10, about 1 to 15, about 1 to 20, about 1 to 25, about 1 to 30, about 1 to 35, about 1 to 40, about 1 to 45, about 1 to 50, about 1 to 75, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In embodiments, the ratio of TIL to PBMC in the rapid amplification and/or the second amplification is between 1:50 and 1: 300. In embodiments, the ratio of TIL to PBMC in the rapid amplification and/or the second amplification is between 1:100 and 1: 200.

In an example, REP and/or rapid second expansion is performed in flasks, wherein bulk TIL is mixed in 150mL of medium with 100-fold or 200-fold excess inactivated feeder cells, 30ng/mL OKT3 anti-CD 3 antibody, and 6000IU/mL IL-2, wherein the feeder cell concentration is at least 1.1-fold (1.1X), 1.2X, 1.3X, 1.4X, 1.5X, 1.6X, 1.7X, 1.8X, 2X, 2.1X, 2.2X, 2.3X, 2.4X, 2.5X, 2.6X, 2.7X, 2.8X, 2.9X, 3.0X, 3.1X, 3.2X, 3.3X, 3.4X, 3.5X, 3.6X, 3.7X, 3.8X, 3.9X, 3.0X, 3.1X, 3.2X, 3.3.3X, 3.4X, 3.5X, 3.6X, 3.7X, 3.8X, 3.9X, or 0X. Media replacement (2/3 media replacement is typically done by aspirating 2/3 spent media and replacing with an equal volume of fresh media) is performed until the cells are transferred to an alternative growth chamber. An alternative growth chamber comprises a G-REX flask and a gas permeable container, as discussed more fully below.

In some embodiments, the rapid second amplification (which may comprise a process referred to as the REP process) is from 7 to 9 days, as discussed in the examples and figures. In some embodiments, the second amplification is 7 days. In some embodiments, the second amplification is 8 days. In some embodiments, the second amplification is 9 days.

In an embodiment, a second amplification (which may comprise an amplification referred to as REP, and those referred to in step D of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) may be performed in a gas permeable flask (G-Rex100, commercially available from Wilson walf Corporation, New bright, MN, USA) having a 500mL capacity with a 100cm gas permeable silica gel bottom (which may be obtained from Wilson Wolf Manufacturing Corporation, New breton, MN, USA), which may comprise an amplification referred to as REP, and which may be 5 x10⁶Or 10X 10⁶TILs were cultured with PBMC in 400mL 50/50 medium supplemented with 5% human AB serum, 3000IU per mL IL-2 and 60ng per mL anti-CD 3(OKT 3). Can be at 37 ℃/5% CO₂Next, G-Rex100 flasks were incubated. On day 5, 250mL of the supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500rpm (491 Xg) for 10 minutes. The TIL pellet can be resuspended in 150mL fresh medium with 5% human AB serum, 6000IU per mL IL-2 and added back to the original GREX-100 flask. When TIL was continuously amplified in GREX-100 flasks, the TIL could be moved to a larger flask, such as GREX-500, on day 10 or day 11. Cells may be harvested on day 14 of culture. Cells may be harvested on day 15 of culture. Can be collected on day 16 of culture And (4) collecting cells. In some embodiments, media replacement is performed until the cells are transferred to an alternative growth chamber. In some embodiments, 2/3 of media is replaced by aspirating 2/3 used media and replacing with an equal volume of fresh media. In some embodiments, the alternative growth chamber comprises a GREX flask and a gas permeable container, as discussed more fully below.

In some embodiments, the serum-free or defined medium comprises a basal cell medium and a serum supplement and/or serum replacement. In some embodiments, the basal cell culture medium includes, but is not limited to, CTS^TM OpTmizer^TMT cell expansion basal Medium, CTS^TM OpTmizer^TMT cell expansion SFM, CTS^TMAIM-V Medium, CTS^TM AIM-V SFM、LymphoONE^TMT cell expansion xeno-free medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimal essential medium (α MEM), Glasgow's minimal essential medium (G-MEM), RPMI growth medium, and Iskifun's modified Dulbecco's medium.

In some embodiments, the serum supplement or serum replacement includes, but is not limited to, one or more of the following: CTS^TMOpTsizer T cell expansion serum supplement, CTS^TMImmune cell serum replacement, one or more albumins or albumin substitutes, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more anti-inflammatory agents, one or a combination thereof, and/or a combination thereof,One or more antibiotics and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and a trace element containing fraction Ag⁺、Al³⁺、Ba²⁺、Cd²⁺、Co²⁺、Cr³⁺、Ge⁴⁺、Se⁴⁺、Br、T、Mn²⁺、P、Si⁴⁺、V⁵⁺、Mo⁶⁺、Ni²⁺、Rb⁺、Sn²⁺And Zr⁴⁺The compound of (1). In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or 2-mercaptoethanol.

In some embodiments, the serum-free or defined medium is CTS^TM OpTmizer^TMT cell expansion SFM (seimer feishell science). CTS^TM OpTmizer^TMAny of the formulations of (a) can be used in the present invention. CTS^TM OpTmizer^TMT cell expansion SFM is 1L CTS mixed together prior to use^TM OpTmizer^TMT cell expansion basal Medium and 26mL CTS^TM OpTmizer^TMA combination of T cell expansion supplements. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Seimer Feishell science) and CTS supplemented with 55mM 2-mercaptoethanol^TM OpTmizer^TMT cells expand SFM.

In some embodiments, the defined medium is CTS^TM OpTmizer^TMT cell expansion SFM (seimer feishell science). CTS^TM OpTmizer^TMAny of the formulations of (a) can be used in the present invention. CTS^TM OpTmizer^TMT cell expansion SFM is 1L CTS mixed together prior to use^TM OpTmizer^TMT cell expansion basal Medium and 26mL CTS^TMOpTmizer^TMA combination of T cell expansion supplements. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Seimer Feishell science) and CTS supplemented with 55mM 2-mercaptoethanol^TM OpTmizer^TMT cells expand SFM. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Saimer Feishell science), 55mM 2-mercaptoethanol and 2mM L-glutamine supplemented with CTS ^TMOpTmizer^TMT cells expand SFM. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Sai)Mufeishiel scientific Co., Ltd.), 55mM of 2-mercaptoethanol and 2mM of L-glutamine supplemented with CTS^TMOpTmizer^TMThe T cells expand SFM and further comprise about 1000IU/mL to about 8000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Saimer Feishell science), 55mM 2-mercaptoethanol and 2mM L-glutamine supplemented with CTS^TMOpTmizer^TMThe T cells expand SFM and further include about 3000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Saimer Feishell science), 55mM 2-mercaptoethanol and 2mM L-glutamine supplemented with CTS^TMOpTmizer^TMThe T cells expand SFM and further include about 6000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Seimer Feishell science) and CTS supplemented with 55mM 2-mercaptoethanol^TMOpTmizer^TMThe T cells expand SFM and further comprise about 1000IU/mL to about 8000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Seimer Feishell science) and CTS supplemented with 55mM 2-mercaptoethanol ^TMOpTmizer^TMThe T cells expand SFM and further include about 3000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Seimer Feishell science) and CTS supplemented with 55mM 2-mercaptoethanol^TMOpTmizer^TMThe T cells expand the SFM and further comprise about 1000IU/mL to about 6000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Saimer Feishell science) and approximately 2mM glutamine supplemented CTS^TMOpTmizer^TMThe T cells expand SFM and further comprise about 1000IU/mL to about 8000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Saimer Feishell science) and approximately 2mM glutamine supplemented CTS^TMOpTmizer^TMThe T cells expand SFM and further include about 3000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmunity fineSerum Replacement (SR) (Saimer Feishell science) and glutamine supplement CTS at about 2mM^TMOpTmizer^TMThe T cells expand SFM and further include about 6000IU/mL of IL-2. In some embodiments, about 3% CTS is used^TMImmune cell Serum Replacement (SR) (Seimer Feishell science) supplements CTS^TM OpTmizer^TMT cells expanded SFM and the final concentration of 2-mercaptoethanol in the medium was 55. mu.M. In some embodiments, glutamine is used at a concentration of about 0.1mM to about 10mM, 0.5mM to about 9mM, 1mM to about 8mM, 2mM to about 7mM, 3mM to about 6mM, or 4mM to about 5mM (i.e., ) Serum-free medium or defined medium. In some embodiments, glutamine is used at a concentration of about 2mM (i.e.,) Serum-free medium or defined medium.

In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 5mM to about 150mM, 10mM to about 140mM, 15mM to about 130mM, 20mM to about 120mM, 25mM to about 110mM, 30mM to about 100mM, 35mM to about 95mM, 40mM to about 90mM, 45mM to about 85mM, 50mM to about 80mM, 55mM to about 75mM, 60mM to about 70mM, or about 65 mM. In some embodiments, the serum-free medium or defined medium is supplemented with 2-mercaptoethanol at a concentration of about 55 mM.

In some embodiments, defined media described in International PCT publication No. WO/1998/030679, which is incorporated herein by reference, may be used in the present invention. In said publication, serum-free eukaryotic cell culture media are described. Serum-free eukaryotic cell culture media comprises basal cell culture media supplemented with serum-free supplements capable of supporting cell growth in serum-free culture. The serum-free eukaryotic cell culture medium supplement comprises or is obtained by combining one or more components selected from the group consisting of: one or more albumins or albumin substitutes, one or more amino groups Acid, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, one or more trace elements, and one or more antibiotics. In some embodiments, the defined medium further comprises L-glutamine, sodium bicarbonate, and/or β -mercaptoethanol. In some embodiments, the defined medium comprises albumin or an albumin substitute and one or more components selected from the group consisting of: one or more amino acids, one or more vitamins, one or more transferrin or transferrin substitutes, one or more antioxidants, one or more insulin or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the defined medium comprises albumin and one or more components selected from the group consisting of: glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin and a trace element containing fraction Ag ⁺、Al³⁺、Ba²⁺、Cd²⁺、Co²⁺、Cr³⁺、Ge⁴⁺、Se⁴⁺、Br、T、Mn²⁺、P、Si⁴⁺、V⁵⁺、Mo⁶⁺、Ni²⁺、Rb⁺、Sn²⁺And Zr⁴⁺The compound of (1). In some embodiments, the basal cell culture medium is selected from the group consisting of: dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), eagle's Basal Medium (BME), RPMI 1640, F-10, F-12, minimal essential medium (α MEM), Glasgow's minimal essential medium (G-MEM), RPMI growth medium, and Iskifuku's modified Dulbecco's medium.

In some embodiments, the concentration of glycine in the defined medium is in the range of about 5-200mg/L, the concentration of L-histidine is about 5-250mg/L, and the concentration of L-isoleucine isAbout 5-300mg/L, L-methionine concentration of about 5-200mg/L, L-phenylalanine concentration of about 5-400mg/L, L-proline concentration of about 1-1000mg/L, L-hydroxyproline concentration of about 1-45mg/L, L-serine concentration of about 1-250mg/L, L-threonine concentration of about 10-500mg/L, L-tryptophan concentration of about 2-110mg/L, L-tyrosine concentration of about 3-175mg/L, L-valine concentration of about 5-500mg/L, thiamine concentration of about 1-20mg/L, reduced glutathione concentration of about 1-20mg/L, the concentration of L-ascorbic acid-2-phosphate is about 1-200mg/L, the concentration of iron-saturated transferrin is about 1-50mg/L, the concentration of insulin is about 1-100mg/L, the concentration of sodium selenite is about 0.000001-0.0001mg/L, and albumin (e.g., I) Is about 5000-50,000 mg/L.

Table a: concentration of non-trace element fraction component

In some embodiments, defined media described in Smith et al, "ex vivo expansion of human T cells for adoptive immunotherapy using a novel xeno-free CTS immune cell serum replacement," clinical transformation immunology, 4(1)2015(doi: 10.1038/ct.2014.31) may be used in the present invention. Briefly, RPMI or CTS^TM OpTmizer^TMUsed as basal cell culture medium and with 0, 2%, 5% or 10% CTS^TMImmune cell serum replacement supplementation.

In the examples, a rapid second amplification (comprising an amplification referred to as REP) is performed and further comprises a step in which TIL is selected to obtain superior tumor reactivity. Any selection method known in the art may be used. For example, the methods described in U.S. patent application publication No. 2016/0010058a1 (the disclosure of which is incorporated herein by reference) can be used to select TILs with excellent tumor reactivity.

Optionally, cell viability assays can be performed after the rapid second amplification (including amplification referred to as REP amplification) using standard assays known in the art. For example, a trypan blue exclusion assay (trypan blue exclusion assay) can be performed on samples with bulk TIL that selectively labels dead cells and allows viability assessment. In some embodiments, TIL samples can be counted and viability determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA, Lawrence). In some embodiments, viability is determined according to the standard Cellometer K2 image cytometer automated cell counter protocol.

The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited but large number of gene segments. These gene segments: v (variable), D (diverse), J (linked) and C (constant) determine the binding specificity and downstream applications of immunoglobulins to T Cell Receptors (TCRs). The present invention provides a method for producing TILs that exhibit and increase T cell bank diversity. In some embodiments, the TILs obtained by the methods of the invention exhibit an increase in diversity of the T cell pool. In some embodiments, the TIL obtained in the second expansion exhibits an increase in T cell bank diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity of immunoglobulins is in immunoglobulin heavy chains. In some embodiments, the immunoglobulin diversity is in an immunoglobulin light chain. In some embodiments, the diversity is in a T cell receptor. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of: alpha receptors, beta receptors, gamma receptors, and delta receptors. In some embodiments, expression of T Cell Receptors (TCR) α and/or β is increased. In some embodiments, expression of T Cell Receptor (TCR) α is increased. In some embodiments, expression of T Cell Receptor (TCR) β is increased. In some embodiments, expression of TCRab (i.e., TCR α/β) is increased.

In some embodiments, the rapid second expansion medium (e.g., sometimes referred to as CM2 or second cell culture medium) includes IL-2, OKT-3, and antigen presenting feeder cells (APCs), as discussed in more detail below. In some embodiments, the rapid second expansion medium (e.g., sometimes referred to as CM2 or second cell culture medium) includes 6000IU/mL IL-2, 30 micrograms/flask OKT-3, and 7.5X 10⁸Individual antigen presenting feeder cells (APCs), as discussed in more detail below. In some embodiments, the rapid second expansion medium (e.g., sometimes referred to as CM2 or second cell culture medium) comprises IL-2, OKT-3, and antigen presenting feederCells (APCs), as discussed in more detail below. In some embodiments, the rapid second expansion medium (e.g., sometimes referred to as CM2 or second cell culture medium) includes 6000IU/mL IL-2, 30 micrograms/flask OKT-3, and 5X 10⁸Individual antigen presenting feeder cells (APCs), as discussed in more detail below.

In some embodiments, the rapid second amplification is performed in a closed system bioreactor, such as according to step D of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C). In some embodiments, a closed system is used for TIL amplification, as described herein. In some embodiments, a bioreactor is employed. In some embodiments, the bioreactor serves as a container. In some embodiments, the bioreactor employed is, for example, G-REX-100 or G-REX-500. In some embodiments, the bioreactor used is G-REX-100. In some embodiments, the bioreactor used is G-REX-500.

1. Feeder cells and antigen presenting cells

In embodiments, the rapid second expansion procedures described herein (e.g., amplifications comprising those amplifications as described in step D from fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), as well as those amplifications referred to as REP) require an excess of feeder cells during the REP TIL expansion and/or during the rapid second expansion. In many embodiments, the feeder cells are Peripheral Blood Mononuclear Cells (PBMCs) obtained from standard whole blood units of healthy blood donors. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation.

In some embodiments, a PBMC is considered replication-incompetent and approved for use in the TIL expansion procedure described herein if the total number of viable cells at day 7 or day 14 is less than the initial number of viable cells placed in culture at day 0 for REP and/or day 0 for the second expansion (i.e., the day on which the second expansion begins).

In some embodiments, PBMCs are considered replication incompetent and approved for use in the TIL expansion procedure described herein if, in culture in the presence of OKT3 and IL-2, the total number of viable cells on days 7 and 14 is not increased compared to the initial number of viable cells placed in culture on day 0 of REP and/or on day 0 of the second expansion (i.e., on the day of the second expansion start). In some embodiments, PBMCs are cultured in the presence of 30ng/ml OKT3 antibody and 3000IU/ml IL-2. In some embodiments, PBMCs are cultured in the presence of 60ng/ml OKT3 antibody and 6000IU/ml IL-2. In some embodiments, PBMCs are cultured in the presence of 60ng/ml OKT3 antibody and 3000IU/ml IL-2. In some embodiments, PBMCs are cultured in the presence of 30ng/ml OKT3 antibody and 6000IU/ml IL-2.

In some embodiments, PBMCs are considered replication incompetent and approved for use in the TIL expansion procedure described herein if, in culture in the presence of OKT3 and IL-2, the total number of viable cells on days 7 and 14 is not increased compared to the initial number of viable cells placed in culture on day 0 of REP and/or on day 0 of the second expansion (i.e., on the day of the second expansion start). In some embodiments, PBMCs are cultured in the presence of 30-60ng/ml OKT3 antibody and 1000-6000IU/ml IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60ng/ml OKT3 antibody and 2000-5000IU/ml IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60ng/ml OKT3 antibody and 2000-4000IU/ml IL-2. In some embodiments, PBMCs are cultured in the presence of 30-60ng/ml OKT3 antibody and 2500-. In some embodiments, PBMCs are cultured in the presence of 30-60ng/ml OKT3 antibody and 6000IU/ml IL-2.

In some embodiments, the antigen presenting feeder cells are PBMCs. In some embodiments, the antigen presenting feeder cells are artificial antigen presenting feeder cells. In embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is about 1 to 10, about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:50 and 1: 300. In embodiments, the ratio of TIL to antigen presenting feeder cells in the second expansion is between 1:100 and 1: 200.

In an embodiment, the second amplification procedure described herein requires about 5 × 10⁸Each feeder cell is about 100X 10⁶The ratio of TILs. In an embodiment, the second amplification procedure described herein requires about 7.5 × 10⁸Each feeder cell is about 100X 10⁶The ratio of TILs. In another embodiment, the second amplification procedure described herein requires about 5X 10⁸Each feeder cell is about 50X 10⁶The ratio of TILs. In another embodiment, the second amplification procedure described herein requires about 7.5X 10⁸Each feeder cell is about 50X 10⁶The ratio of TILs. In yet another embodiment, the second amplification procedure described herein requires about 5 × 10⁸One feeder cell and about 25X 10⁶And (4) TIL. In yet another embodiment, the second amplification procedure described herein requires about 7.5X 10⁸One feeder cell and about 25X 10⁶And (4) TIL. In yet another embodiment, the number of feeder cells required for rapid secondary expansion is twice that of rapid secondary expansion. In yet another embodiment, about 2.5X 10 is required when priming the first amplification described herein⁸About 5X 10 is required for rapid second expansion of individual feeder cells⁸And (4) feeding cells. In yet another embodiment, about 2.5X 10 is required when priming the first amplification described herein ⁸For one feeder cell, about 7.5X 10 is required for rapid second expansion⁸And (4) feeding cells. In yet another embodiment, the number of feeder cells required for rapid second expansion is twice (2.0X), 2.5X, 3.0X, 3.5X, or 4.0X as the number that would initiate the first expansion.

In some embodiments, the second amplification procedure described herein requires about 5x 10⁸Each feeder cell is about 100X 10⁶The ratio of TILs. In an embodiment, the second amplification procedure described herein requires about 7.5 × 10⁸Each feeder cell is about 100X 10⁶The ratio of TILs. In another embodiment, the second amplification procedure described herein requires about 5X 10⁸Each feeder cell is about 50X 10⁶The ratio of TILs. In another embodiment, the second amplification procedure described herein requires about 7.5X 10⁸Each feeder cell is about 50X 10⁶The ratio of TILs. In yet another embodiment, the second amplification procedure described herein requires about 5 × 10⁸One feeder cell and about 25X 10⁶And (4) TIL. In yet another embodiment, the second amplification procedure described herein requires about 7.5X 10⁸One feeder cell and about 25X 10⁶And (4) TIL. In yet another embodiment, the rapid second expansion requires the same number of feeder cells as the rapid second expansion. In yet another embodiment, about 2.5X 10 is required when priming the first amplification described herein ⁸For a single feeder cell, a rapid second expansion of about 2.5X 10 is required⁸And (4) feeding cells. In yet another embodiment, about 5X 10 is required when priming the first amplification described herein⁸About 5X 10 is required for rapid second expansion of individual feeder cells⁸And (4) feeding cells. In yet another embodiment, about 7.5X 10 is required when priming the first amplification described herein⁸For one feeder cell, about 7.5X 10 is required for rapid second expansion⁸And (4) feeding cells. In yet another embodiment, the number of feeder cells required for rapid second expansion is twice (2.0X), 2.5X, 3.0X, 3.5X, or 4.0X as the number that would initiate the first expansion.

In some embodiments, the second amplification procedure described herein requires about 5x 10⁸Each feeder cell is about 100X 10⁶The ratio of TILs. In an embodiment, the second amplification procedure described herein requires about 7.5 × 10⁸Each feeder cell is about 100X 10⁶The ratio of TILs. In another embodiment, the second amplification procedure described herein requires about 5X 10⁸Each feeder cell is about 50X 10⁶The ratio of TILs. In another embodiment, the second amplification procedure described herein requires about 7.5X 10⁸Each feeder cell is about 50X 10⁶The ratio of TILs. In yet another embodiment, the second amplification procedure described herein requires about 5 × 10 ⁸One feeder cell and about 25X 10⁶And (4) TIL. In yet another embodiment, the second amplification procedure described herein requires about 7.5X 10⁸One feeder cell and about 25X 10⁶And (4) TIL. In yet another embodiment, the rapid second expansion requires the same number of feeder cells as the rapid second expansion. In yet another embodiment, about 2.5X 10 is required when priming the first amplification described herein⁸For a single feeder cell, a rapid second expansion of about 2.5X 10 is required⁸And (4) feeding cells. In yet another embodiment, about 5X 10 is required when priming the first amplification described herein⁸About 5X 10 is required for rapid second expansion of individual feeder cells⁸And (4) feeding cells. In yet another embodiment, about 7.5X 10 is required when priming the first amplification described herein⁸For one feeder cell, about 7.5X 10 is required for rapid second expansion⁸And (4) feeding cells.

In embodiments, the rapid second expansion procedure described herein requires an excess of feeder cells during the rapid second expansion. In many embodiments, the feeder cells are Peripheral Blood Mononuclear Cells (PBMCs) obtained from standard whole blood units of allogeneic healthy blood donors. PBMCs are obtained using standard methods, such as Ficoll-Paque gradient separation. In the examples, artificial antigen presenting (aAPC) cells were used instead of PBMCs. In some embodiments, PBMCs are added to the rapid second amplification at twice the concentration of PBMCs added to prime the first amplification.

In embodiments, artificial antigen presenting cells are used in place of or in combination with PBMCs in the rapid second expansion.

2. Cytokine

The rapid second amplification methods described herein generally use media with high doses of cytokines (specifically, IL-2), as is known in the art.

Alternatively, it is additionally possible to use a combination of cytokines for rapid second expansion of TIL, wherein the combination of two or more of IL-2, IL-15 and IL-21 is as generally outlined in International publication Nos. WO 2015/189356 and WO 2015/189357, which are hereby expressly incorporated by reference in their entirety. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15 and IL-21, with the latter being particularly suitable in many embodiments. The use of a combination of cytokines is particularly advantageous for the production of lymphocytes, and in particular T cells as described herein.

E. Step E: collecting TIL

After the rapid second amplification step, cells may be harvested. In some embodiments, the TIL is collected after one, two, three, four, or more amplification steps, e.g., as provided in fig. 1 (in particular, e.g., fig. 1B and/or fig. 1C). In some embodiments, the TIL is collected after two amplification steps, e.g., as provided in fig. 1 (in particular, e.g., fig. 1B and/or fig. 1C). In some embodiments, the TIL is collected after two amplification steps (one priming first amplification and one rapid second amplification) as provided, for example, in fig. 1 (in particular, e.g., fig. 1B).

TIL may be collected in any suitable and sterile manner, including, for example, by centrifugation. Methods for TIL collection are well known in the art and any such known method may be used in the methods of the invention. In some embodiments, the TIL is collected using an automated system.

Cell harvesters and/or cell processing systems are commercially available from a number of sources, including, for example, fisher ews cabi (Fresenius Kabi), Tomtec Life sciences (Tomtec Life sciences), Perkin Elmer, and einotak Biosystems International limited (Inotech Biosystems International, Inc). Any cell-based collector can be used in the methods of the invention. In some embodiments, the cell harvester and/or the cell processing system is a membrane-based cell harvester. In some embodiments, cell collection is performed by a cell processing system, such as the LOVO system (manufactured by the company ferwenskarb). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump a solution comprising cells to a membrane or filter (e.g., a rotating membrane or rotating filter) in a sterile and/or closed system environment, allowing for continuous flow and cell processing to remove supernatant or cell culture media without sedimentation. In some embodiments, the cell harvester and/or cell processing system can perform cell separation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed, sterile system.

In some embodiments, step E according to fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C) is performed according to a process described herein. In some embodiments, the closed system is accessed by a syringe under sterile conditions to maintain the sterility and tightness of the system. In some embodiments, a closed system as described herein is employed.

In some embodiments, the TIL is collected according to the methods described herein. In some embodiments, TILs between day 14 and day 16 are collected using methods as described herein. In some embodiments, TIL is collected using a method as described herein at 14 days. In some embodiments, TIL is collected at 15 days using the methods as described herein. In some embodiments, TIL is collected using the methods as described herein at 16 days.

F. Step F: final formulation/transfer to infusion bag

After completing steps a through E as provided in an exemplary sequence in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C) and as outlined above and in detail herein, the cells are transferred into a container for administration to a patient. In some embodiments, once a therapeutically sufficient amount of TIL is obtained using the amplification methods described above, it is transferred to a container for administration to a patient.

In embodiments, the TIL amplified using the methods of the present disclosure is administered to a patient in the form of a pharmaceutical composition. In the examples, the pharmaceutical composition is a suspension of TIL in sterile buffer. TILs amplified as disclosed herein may be administered by any suitable route as known in the art. In some embodiments, the TIL is administered as a single intra-arterial or intravenous infusion, preferably for about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic.

PBMC feeder cell ratio

In some embodiments, the medium used in the amplification methods described herein (see, e.g., fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) comprises an anti-CD 3 antibody, e.g., OKT-3. Binding of anti-CD 3 antibody to IL-2 induces T cell activation and cell division in the TIL population. This effect can be seen with full length antibodies and Fab and F (ab')2 fragments, the former being generally preferred; see, e.g., Tsoukas et al, journal of immunology 1985,135,1719, which is hereby incorporated by reference in its entirety.

In the examples, the number of PBMC feeder layers was calculated as follows:

A.T volume of cells (10 μm diameter): v ═ r (4/3) pi³＝523.6μm³

B. Volume of G-Rex 100(M) with a height of 40 μ M (4 cells): v ═ r (4/3) pi³＝4×10¹²μm³

C. Number of cells required for packing column B: 4X 10¹²μm³/523.6μm³＝7.6×10⁸μm³*0.64＝4.86×10⁸

D. Number of cells that can be optimally activated in 4D space: 4.86X 10⁸/24＝20.25×10⁶E. Number of feeder cells and TILs extrapolated to G-Rex 500: TIL: 100 x 10⁶And feeder cells: 2.5X 10⁹

In the calculation, pairs with 100cm are used²Activation of TILs in the cylinders of the base provides an approximation of the number of monocytes required for the icosahedral geometry. The experimental results calculated to give a threshold activation of T cells closely reflecting the NCI experimental data were about 5X 10⁸。⁽¹⁾(C) Multiplier (0.64) is any packing density of the equivalent sphere as calculated by Jaeger and Nagel in 1992⁽²⁾. (D) The divisor 24 is "Newton number" that can touch the 4-dimensional space "Number of equivalent spheres of similar objects in (1)⁽³⁾。

⁽¹⁾Jin, Jianjian et al, a Simplified Method of growing Human Tumor Infiltrating Lymphocytes (TIL) in breathable Flasks to the number required for Patient Treatment (Simplified Method of the Growth of Human Tumor Infiltrating Lymphocytes (TIL) in Gas-Permeable flames to Numbers fed for Patient Treatment), journal of immunotherapy 2012, month 4; 35(3):283-292.

⁽²⁾Jaeger HM, Nagel SR., "Physics of the granular State" (science), 1992, 20/3; 255(5051):1523-31.

⁽³⁾"problems of The twenty-five spheres (The proplem of The western-five spheres"), Russian mathematics research (Russ. Math. Surv.) 58(4), 794-.

In embodiments, the number of exogenously supplied antigen-presenting feeder cells during the priming first expansion is about one-half the number of exogenously supplied antigen-presenting feeder cells during the rapid second expansion. In certain embodiments, the method comprises priming the first expansion in a cell culture medium comprising about 50% fewer antigen presenting cells than the rapidly second expanded cell culture medium.

In another embodiment, the number of exogenously supplied antigen presenting feeder cells (APCs) during the rapid second expansion is greater than the number of exogenously supplied APCs during the priming first expansion.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 20: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 10: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 9: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 8: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 7: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 6: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 5: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 4: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 3: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.9: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.8: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.7: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.6: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.5: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.4: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.3: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.2: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.1: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 2:1 to at or about 10: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 2:1 to at or about 5: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 2:1 to at or about 4: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 2:1 to at or about 3: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 2:1 to at or about 2.9: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 2:1 to at or about 2.8: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 2:1 to at or about 2.7: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 2:1 to at or about 2.6: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 2:1 to at or about 2.5: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 2:1 to at or about 2.4: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 2:1 to at or about 2.3: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 2:1 to at or about 2.2: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is selected from the range of at or about 2:1 to at or about 2.1: 1.

In another embodiment, the ratio of the number of exogenously supplied APCs during the rapid second amplification to the number of exogenously supplied APCs during priming the first amplification is at or about 2: 1.

In another embodiment, the ratio of the number of APCs exogenously supplied during the rapid second amplification to the number of APCs exogenously supplied during the priming first amplification is at or about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.1:1, 4.1, 4:1, 4.5:1, 4.6:1, 4:1, 4.7:1, 4.8:1, 4:1, 4.9:1, 4:1, 4.1, 4:1, 4.6:1, 4:1, 4.8:1, or 4: 1.

In another embodiment, the number of APCs exogenously supplied during priming the first amplification is at or about 1X 10⁸、1.1×10⁸、1.2×10⁸、1.3×10⁸、1.4×10⁸、1.5×10⁸、1.6×10⁸、1.7×10⁸、1.8×10⁸、1.9×10⁸、2×10⁸、2.1×10⁸、2.2×10⁸、2.3×10⁸、2.4×10⁸、2.5×10⁸、2.6×10⁸、2.7×10⁸、2.8×10⁸、2.9×10⁸、3×10⁸、3.1×10⁸、3.2×10⁸、3.3×10⁸、3.4×10⁸Or 3.5X 10⁸(ii) APCs, and the number of APCs exogenously supplied during the rapid second amplification is at or about 3.5X 10⁸、3.6×10⁸、3.7×10⁸、3.8×10⁸、3.9×10⁸、4×10⁸、4.1×10⁸、4.2×10⁸、4.3×10⁸、4.4×10⁸、4.5×10⁸、4.6×10⁸、4.7×10⁸、4.8×10⁸、4.9×10⁸、5×10⁸、5.1×10⁸、5.2×10⁸、5.3×10⁸、5.4×10⁸、5.5×10⁸、5.6×10⁸、5.7×10⁸、5.8×10⁸、5.9×10⁸、6×10⁸、6.1×10⁸、6.2×10⁸、6.3×10⁸、6.4×10⁸、6.5×10⁸、6.6×10⁸、6.7×10⁸、6.8×10⁸、6.9×10⁸、7×10⁸、7.1×10⁸、7.2×10⁸、7.3×10⁸、7.4×10⁸、7.5×10⁸、7.6×10⁸、7.7×10⁸、7.8×10⁸、7.9×10⁸、8×10⁸、8.1×10⁸、8.2×10⁸、8.3×10⁸、8.4×10⁸、8.5×10⁸、8.6×10⁸、8.7×10⁸、8.8×10⁸、8.9×10⁸、9×10⁸、9.1×10⁸、9.2×10⁸、9.3×10⁸、9.4×10⁸、9.5×10⁸、9.6×10⁸、9.7×10⁸、9.8×10⁸、9.9×10⁸Or 1X 10⁹And (4) APC.

In another embodiment, the number of APCs exogenously supplied during priming of the first amplification is selected from at or about 1.5X 10⁸APC to at or about 3X 10⁸(ii) a range of APCs, and the number of APCs exogenously supplied during the rapid second amplification is selected from at or about 4X 10 ⁸APC to at or about 7.5X 10⁸Range of individual APC.

In another embodiment, outside the period of priming the first amplificationThe number of APCs supplied by the source is selected from at or about 2X 10⁸APC to at or about 2.5X 10⁸(ii) a range of APCs, and the number of APCs exogenously supplied during the rapid second amplification is selected from at or about 4.5X 10⁸APC to at or about 5.5X 10⁸Range of individual APC.

In another embodiment, the number of APCs exogenously supplied during priming the first amplification is at or about 2.5X 10⁸(ii) APCs, and the number of APCs exogenously supplied during the rapid second amplification is at or about 5X 10⁸And (4) APC.

In embodiments, the number of APCs (comprising, e.g., PBMCs) added on day 0 of priming the first amplification is about one-half of the number of PBMCs added on day 7 of priming the first amplification (e.g., day 7 of the method). In certain embodiments, the method comprises adding antigen presenting cells to the first TIL population on day 0 of priming the first expansion and adding antigen presenting cells to the second TIL population on day 7, wherein the number of antigen presenting cells added on day 0 is about 50% of the number of antigen presenting cells added on day 7 of priming the first expansion (e.g., day 7 of the method).

In another embodiment, the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification is greater than the number of PBMCs exogenously supplied on day 0 of priming the first amplification.

In another embodiment, the APC to be exogenously supplied in priming the first amplification is selected to be at or about 1.0X 10⁶APC/cm²To or about 4.5 x 10⁶APC/cm²In a culture flask.

In another embodiment, the APC to be exogenously supplied in priming the first amplification is selected from at or about 1.5X 10⁶APC/cm²To or about 3.5 x 10⁶APC/cm²In a culture flask.

In another embodiment, the APC to be exogenously supplied in priming the first amplification is selected to be at or about 2X 10⁶APC/cm²To or about 3 x 10⁶APC/cm²In a culture flask.

In another embodiment, the APC to be exogenously supplied in priming the first amplification is at or about 2X 10⁶APC/cm²Is inoculated in a culture flask.

In another embodiment, the APC to be exogenously supplied in priming the first amplification is at or about 1.0X 10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶Or 4.5X 10⁶APC/cm²Is inoculated in a culture flask.

In another embodiment, the APC to be supplied exogenously in the rapid second amplification is selected to be at or about 2.5X 10⁶APC/cm²To or about 7.5 x 10⁶APC/cm²In a culture flask.

In another embodiment, the APC supplied exogenously in the rapid second amplification is selected from at or about 3.5X 10⁶APC/cm²To about 6.0X 10⁶APC/cm²In a culture flask.

In another embodiment, the APC supplied exogenously in the rapid second amplification is selected from at or about 4.0X 10⁶APC/cm²To about 5.5X 10⁶APC/cm²In a culture flask.

In another embodimentIn the example, the APC supplied exogenously in the rapid second amplification is selected from at or about 4.0X 10⁶APC/cm²In a culture flask.

In another embodiment, the APC to be supplied exogenously in the rapid second amplification is at or about 2.5X 10⁶APC/cm²、2.6×10⁶APC/cm²、2.7×10⁶APC/cm²、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.1×10⁶、6.2×10⁶、6.3×10⁶、6.4×10⁶、6.5×10⁶、6.6×10⁶、6.7×10⁶、6.8×10⁶、6.9×10⁶、7×10⁶、7.1×10⁶、7.2×10⁶、7.3×10⁶、7.4×10⁶Or 7.5X 10⁶APC/cm²Is inoculated in a culture flask.

In another embodiment, the APC to be exogenously supplied in priming the first amplification is at or about 1.0X 10⁶、1.1×10⁶、1.2×10⁶、1.3×10⁶、1.4×10⁶、1.5×10⁶、1.6×10⁶、1.7×10⁶、1.8×10⁶、1.9×10⁶、2×10⁶、2.1×10⁶、2.2×10⁶、2.3×10⁶、2.4×10⁶、2.5×10⁶、2.6×10⁶、2.7×10⁶、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶Or 4.5X 10⁶APC/cm²Is inoculated in a culture flask, and the APC supplied exogenously in the rapid second amplification is at or about 2.5X 10 ⁶APC/cm²、2.6×10⁶APC/cm²、2.7×10⁶APC/cm²、2.8×10⁶、2.9×10⁶、3×10⁶、3.1×10⁶、3.2×10⁶、3.3×10⁶、3.4×10⁶、3.5×10⁶、3.6×10⁶、3.7×10⁶、3.8×10⁶、3.9×10⁶、4×10⁶、4.1×10⁶、4.2×10⁶、4.3×10⁶、4.4×10⁶、4.5×10⁶、4.6×10⁶、4.7×10⁶、4.8×10⁶、4.9×10⁶、5×10⁶、5.1×10⁶、5.2×10⁶、5.3×10⁶、5.4×10⁶、5.5×10⁶、5.6×10⁶、5.7×10⁶、5.8×10⁶、5.9×10⁶、6×10⁶、6.1×10⁶、6.2×10⁶、6.3×10⁶、6.4×10⁶、6.5×10⁶、6.6×10⁶、6.7×10⁶、6.8×10⁶、6.9×10⁶、7×10⁶、7.1×10⁶、7.2×10⁶、7.3×10⁶、7.4×10⁶Or 7.5X 10⁶APC/cm²Is inoculated in a culture flask.

In another embodiment, the APC exogenously supplied in priming the first amplification is selected from at or about 1.0X 10⁶APC/cm²To or about 4.5 x 10⁶APC/cm²Is inoculated in a culture flask, and APCs supplied exogenously in the rapid second amplification are selected fromIs at or about 2.5X 10⁶APC/cm²To or about 7.5 x 10⁶APC/cm²In a culture flask.

In another embodiment, the APC exogenously supplied in priming the first amplification is selected from at or about 1.5X 10⁶APC/cm²To or about 3.5 x 10⁶APC/cm²Is inoculated in a culture flask, and the APC supplied exogenously in the rapid second amplification is selected to be at or about 3.5X 10⁶APC/cm²To or about 6 x 10⁶APC/cm²In a culture flask.

In another embodiment, the APC exogenously supplied in priming the first amplification is selected from at or about 2X 10⁶APC/cm²To or about 3 x 10⁶APC/cm²Is inoculated in a culture flask, and the APC supplied exogenously in the rapid second amplification is selected to be at or about 4X 10 ⁶APC/cm²To or about 5.5 x 10⁶APC/cm²In a culture flask.

In another embodiment, the APC to be exogenously supplied in priming the first amplification is at or about 2X 10⁶APC/cm²Is inoculated in a culture flask, and the APC supplied exogenously in the rapid second amplification is at or about 4X 10⁶APC/cm²Is inoculated in a culture flask.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of PBMCs exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 20: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of PBMCs exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 10: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of PBMCs exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 9: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 8: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 7: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 6: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 5: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 4: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 3: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.9: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.8: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.7: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.6: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.5: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.4: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.3: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.2: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2.1: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 1.1:1 to at or about 2: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 2:1 to at or about 10: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 2:1 to at or about 5: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 2:1 to at or about 4: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 2:1 to at or about 3: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 2:1 to at or about 2.9: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 2:1 to at or about 2.8: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 2:1 to at or about 2.7: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 2:1 to at or about 2.6: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 2:1 to at or about 2.5: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 2:1 to at or about 2.4: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 2:1 to at or about 2.3: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 2:1 to at or about 2.2: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is selected from the range of at or about 2:1 to at or about 2.1: 1.

In another embodiment, the ratio of the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of priming the first amplification is at or about 2: 1.

In another embodiment, the ratio of the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification to the number of APCs (including, e.g., PBMCs) exogenously supplied on day 0 of initiating the first amplification is or is about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.1, 1, 4:1, 4.6:1, 3.7:1, 4:1, 4.4:1, 4:1, 4.6:1, 4:1, or 4: 1.

In another embodiment, the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 that initiates the first amplification is at or about 1X 10⁸、1.1×10⁸、1.2×10⁸、1.3×10⁸、1.4×10⁸、1.5×10⁸、1.6×10⁸、1.7×10⁸、1.8×10⁸、1.9×10⁸、2×10⁸、2.1×10⁸、2.2×10⁸、2.3×10⁸、2.4×10⁸、2.5×10⁸、2.6×10⁸、2.7×10⁸、2.8×10⁸、2.9×10⁸、3×10⁸、3.1×10⁸、3.2×10⁸、3.3×10⁸、3.4×10⁸Or 3.5X 10⁸(ii) an APC, and the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification is at or about 3.5X 10⁸、3.6×10⁸、3.7×10⁸、3.8×10⁸、3.9×10⁸、4×10⁸、4.1×10⁸、4.2×10⁸、4.3×10⁸、4.4×10⁸、4.5×10⁸、4.6×10⁸、4.7×10⁸、4.8×10⁸、4.9×10⁸、5×10⁸、5.1×10⁸、5.2×10⁸、5.3×10⁸、5.4×10⁸、5.5×10⁸、5.6×10⁸、5.7×10⁸、5.8×10⁸、5.9×10⁸、6×10⁸、6.1×10⁸、6.2×10⁸、6.3×10⁸、6.4×10⁸、6.5×10⁸、6.6×10⁸、6.7×10⁸、6.8×10⁸、6.9×10⁸、7×10⁸、7.1×10⁸、7.2×10⁸、7.3×10⁸、7.4×10⁸、7.5×10⁸、7.6×10⁸、7.7×10⁸、7.8×10⁸、7.9×10⁸、8×10⁸、8.1×10⁸、8.2×10⁸、8.3×10⁸、8.4×10⁸、8.5×10⁸、8.6×10⁸、8.7×10⁸、8.8×10⁸、8.9×10⁸、9×10⁸、9.1×10⁸、9.2×10⁸、9.3×10⁸、9.4×10⁸、9.5×10⁸、9.6×10⁸、9.7×10⁸、9.8×10⁸、9.9×10⁸Or

In another embodiment, the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 that initiates the first amplification is selected from at or about 1X 10⁸An APC (comprising, e.g., PBMC) to or about 3.5X 10⁸(ii) a range of APCs (including, e.g., PBMCs), and the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification is selected from at or about 3.5 x 10 ⁸An APC (comprising, e.g., PBMC) to or about 1X 10⁹Range of individual APCs (including, e.g., PBMCs).

In another embodiment, the APCs (including, e.g., APCs supplied exogenously on day 0 that initiated the first amplificationPBMC) is selected from the group consisting of at or about 1.5X 10⁸APC to at or about 3X 10⁸(ii) a range of APCs (including, e.g., PBMCs), and the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification is selected from at or about 4X 10⁸An APC (comprising, e.g., PBMC) to or about 7.5X 10⁸Range of individual APCs (including, e.g., PBMCs).

In another embodiment, the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 that initiates the first amplification is selected from at or about 1X 10⁸An APC (comprising, e.g., PBMC) to or about 3.5X 10⁸(ii) a range of APCs (including, e.g., PBMCs), and the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification is selected from at or about 3.5 x 10⁸An APC (comprising, e.g., PBMC) to or about 1X 10⁹Range of individual APCs (including, e.g., PBMCs).

In another embodiment, the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 that initiates the first amplification is selected from at or about 1.5X 10 ⁸APC to at or about 3X 10⁸(ii) a range of APCs (including, e.g., PBMCs), and the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification is selected from at or about 4X 10⁸An APC (comprising, e.g., PBMC) to or about 7.5X 10⁸Range of individual APCs (including, e.g., PBMCs).

In another embodiment, the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 that initiates the first amplification is selected from at or about 2X 10⁸An APC (comprising, e.g., PBMC) to or about 2.5X 10⁸(ii) a range of APCs (including, e.g., PBMCs), and the number of APCs (including, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification is selected from at or about 4.5 x 10⁸An APC (comprising, e.g., PBMC) to or about 5.5X 10⁸Range of individual APCs (including, e.g., PBMCs).

In another embodiment, the number of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 that initiates the first amplification is at or about 2.5X 10⁸Individual APCs (comprising, e.g., PBMCs), and in a rapid second expansionThe number of APCs (including, e.g., PBMCs) exogenously supplied on increased day 7 is at or about 5X 10⁸APC (including, e.g., PBMC).

In one embodiment, the number of APC (including, e.g., PBMC) layers added on day 0 that initiates the first amplification is about one-half the number of APC (including, e.g., PBMC) layers added on day 7 of the rapid second amplification. In certain embodiments, the method comprises adding a layer of antigen presenting cells to the first TIL population on day 0 of priming the first expansion and adding a layer of antigen presenting cells to the second TIL population on day 7, wherein the amount of antigen presenting cell layer added on day 0 is about 50% of the amount of antigen presenting cell layer added on day 7.

In another embodiment, the number of layers of APCs (comprising, e.g., PBMCs) exogenously supplied on day 7 of the rapid second amplification is greater than the number of layers of APCs (comprising, e.g., PBMCs) exogenously supplied on day 0 of the priming first amplification.

In another embodiment, day 0 of priming the first expansion occurs in the presence of stacked APCs (comprising, e.g., PBMCs) with an average thickness of at or about 2 cell layers, and day 7 of the rapid second expansion occurs in the presence of stacked APCs (comprising, e.g., PBMCs) with an average thickness of at or about 4 cell layers.

In another embodiment, day 0 of priming the first expansion occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having an average thickness of at or about one cell layer, and day 7 of the rapid second expansion occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having an average thickness of at or about 3 cell layers.

In another embodiment, day 0 of priming the first expansion occurs in the presence of stacked APCs (comprising, e.g., PBMCs) with an average thickness of at or about 1.5 cell layers to at or about 2.5 cell layers, and day 7 of the rapid second expansion occurs in the presence of stacked APCs (comprising, e.g., PBMCs) with an average thickness of at or about 3 cell layers.

In another embodiment, day 0 of priming the first expansion occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having an average thickness of at or about one cell layer, and day 7 of the rapid second expansion occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having an average thickness of at or about 2 cell layers.

In another embodiment, the 0 th day of priming the first expansion occurs in the presence of a stacked APC (including, e.g., PBMC) having an average thickness of or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 cell layers, and the 7 th day of rapid second expansion occurs in the presence of a stacked APC (including, e.g., PBMC) having an average thickness of or about 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5, 5.2, 4.3, 4.4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.6, 6, 7.6, 6, 7, 6, 6.6, 7, 6, 7.6, 6, 5, 6, 7, 6, 5, 6, 7.6, 6, or 4.6.6.6, 6, 7, 4.6, 6, 4.6, 4.7, or 4.6, 6, 4.7, 6, 4.7, 6, or 4.7, 6, 4.7, 4.6, 4.7, 6, 4, or 4.7, 6, 4.7, 4.6, 6, 2, 4, or 4, 6, 4.7, e.7, 6, e.7, 6, 2, or 4, 6, or 4, e.6, 6, 4, 2, e.7, 2, 6, or 4, 3, 2, or 4, 6, 2, or 4, 2, e.7, 3, 4, 3.7, 2, 4, or 4, 6, 4, 6, or 3, or 4, 2, or 4, 6, 4, 2, 3, 2, 4, or 4, or 3, or 4, 2, 6, 2, 4, 6, 4, or 4, 6, 4, or 4, 2, 3, 4, 6, or 4, 6, respectively, 6, or 4, 2, or 4, e.7, 4, e cells.

In another embodiment, day 0 of initiating the first expansion occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having an average thickness of from or about 1 cell layer to or about 2 cell layers, and day 7 of the rapid second expansion occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having an average thickness of from or about 3 cell layers to or about 10 cell layers.

In another embodiment, day 0 of priming the first expansion occurs in the presence of stacked APCs (comprising, e.g., PBMCs) with an average thickness of from or about 2 cell layers to or about 3 cell layers, and day 7 of the rapid second expansion occurs in the presence of stacked APCs (comprising, e.g., PBMCs) with an average thickness of from or about 4 cell layers to or about 8 cell layers.

In another embodiment, day 0 of priming the first expansion occurs in the presence of stacked APCs (comprising, e.g., PBMCs) with an average thickness of at or about 2 cell layers, and day 7 of the rapid second expansion occurs in the presence of stacked APCs (comprising, e.g., PBMCs) with an average thickness of at or about 4 cell layers to at or about 8 cell layers.

In another embodiment, day 0 of priming the first expansion occurs in the presence of stacked APCs (including, e.g., PBMCs) having an average thickness of at or about 1, 2, or 3 cell layers, and day 7 of the rapid second expansion occurs in the presence of stacked APCs (including, e.g., PBMCs) having an average thickness of at or about 3, 4, 5, 6, 7, 8, 9, or 10 cell layers.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.1 to at or about 1: 10.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.1 to at or about 1: 8.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.1 to at or about 1: 7.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.1 to at or about 1: 6.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.1 to at or about 1: 5.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.1 to at or about 1: 4.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.1 to at or about 1: 3.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.1 to at or about 1: 2.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.2 to at or about 1: 8.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.3 to at or about 1: 7.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.4 to at or about 1: 6.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.5 to at or about 1: 5.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.6 to at or about 1: 4.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.7 to at or about 1: 3.5.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.8 to at or about 1:3.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the range of at or about 1:1.9 to at or about 1: 2.5.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is at or about 1:2.

In another embodiment, day 0 of priming the first amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a first average thickness equal to a first number of layers of APCs (comprising, e.g., PBMCs), and day 7 of the rapid second amplification occurs in the presence of stacked APCs (comprising, e.g., PBMCs) having a second average thickness equal to a second number of layers of APCs (comprising, e.g., PBMCs), wherein the ratio of the first number of layers of APCs (comprising, e.g., PBMCs) to the second number of layers of APCs (comprising, e.g., PBMCs) is selected from the group consisting of or about 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:1.7, 1:1.8, 1:1.9, 1:2.2, 1:2.1, 1:2.2, 1: 2.2.3, 1:3, 1:2.3, 1:2.4, 1:2.5, 1:3, 1.6, 1:3, 1.3, 3, 1:3, 3: 2.3, 1:3, 1.4, 3, 1:3, 1.4, 1:1.5, 1.4, 1.5, 1.3, 1.4, 1.3, 1:1.3, 1.4, 1:1.3, 1.4, 1.3, 1:1.3, 1.4, 1.3, 1.4, 1.3, 1.4, 1.3, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7, 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, or 1: 10.

In some embodiments, the number of APCs in priming the first amplification is selected from about 1.0X 10⁶APC/cm²To about 4.5X 10⁶APC/cm²And the number of APCs in the rapid second amplification is selected from about 2.5X 10⁶APC/cm²To about 7.5X 10⁶APC/cm²The range of (1).

In some embodiments, the number of APCs in priming the first amplification is selected from about 1.5X 10⁶APC/cm²To about 3.5X 10⁶APC/cm²And the number of APCs in the rapid second amplification is selected from about 3.5X 10⁶APC/cm²To about 6.0X 10⁶APC/cm²The range of (1).

In some embodiments, the number of APCs in priming the first amplification is selected from about 2.0X 10⁶APC/cm²To about 3.0X 10⁶APC/cm²And the number of APCs in the rapid second amplification is selected from about 4.0X 10⁶APC/cm²To about 5.5X 10⁶APC/cm²The range of (1).

H. Optional cell culture Medium Components

1. anti-CD 3 antibodies

In some embodiments, the medium used in the amplification methods described herein (see, e.g., fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) comprises an anti-CD 3 antibody. Binding of anti-CD 3 antibody to IL-2 induces T cell activation and cell division in the TIL population. This effect can be seen with full length antibodies and Fab and F (ab')2 fragments, the former being generally preferred; see, e.g., Tsoukas et al, journal of immunology 1985,135,1719, which is hereby incorporated by reference in its entirety.

As will be appreciated by those skilled in the art, there are a variety of suitable anti-human CD3 antibodies for use in the present invention, including anti-human CD3 polyclonal and monoclonal antibodies from a variety of mammals, including but not limited to murine, human, primate, rat, and canine antibodies. In particular embodiments, an OKT3 anti-CD 3 antibody (commercially available from almi pharmaceutical, laritan, new jersey or amantan, whirlwind biotechnology, inc, of oriben, california) is used.

Table 5: amino acid sequence of Moluomamab (exemplary OKT-3 antibody)

2.4-1 BB (CD137) agonists

In embodiments, the cell culture medium that elicits the first expansion and/or the rapid second expansion comprises a TNFRSF agonist. In embodiments, the TNFRSF agonist is a 4-1BB (CD137) agonist. The 4-1BB agonist may be any 4-1BB binding molecule known in the art. The 4-1BB binding molecule may be a monoclonal antibody or a fusion protein capable of binding to human or mammalian 4-1 BB. The 4-1BB agonist or 4-1BB binding molecule may comprise any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin heavy chain. The 4-1BB agonist or 4-1BB binding molecule may have both a heavy chain and a light chain. As used herein, the term binding molecule also includes antibodies (including full length antibodies), monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human, humanized or chimeric antibodies and antibody fragments, e.g., Fab fragments, F (ab') fragments, fragments produced by Fab expression libraries, epitope-binding fragments of any of the above, and engineered forms of antibodies, e.g., scFv molecules, that bind to 4-1 BB. In embodiments, the 4-1BB agonist is an antigen binding protein that is a fully human antibody. In embodiments, the 4-1BB agonist is an antigen binding protein that is a humanized antibody. In some embodiments, the 4-1BB agonists used in the methods and compositions of the present disclosure comprise anti-4-1 BB antibodies, human anti-4-1 BB antibodies, mouse anti-4-1 BB antibodies, mammalian anti-4-1 BB antibodies, monoclonal anti-4-1 BB antibodies, polyclonal anti-4-1 BB antibodies, chimeric anti-4-1 BB antibodies, anti-4-1 BB fibronectin, anti-4-1 BB domain antibodies, single chain anti-4-1 BB fragments, heavy chain anti-4-1 BB fragments, light chain anti-4-1 BB fragments, anti-4-1 BB fusion proteins, and fragments, derivatives, conjugates, variants, or biological analogs thereof. Agonistic anti-4-1 BB antibodies are known to induce a stronger immune response. Lee et al, "public science library Integrated (PLOS One) 2013,8, e 69677. In a preferred embodiment, the 4-1BB agonist is an agonistic anti-4-1 BB humanized or fully human monoclonal antibody (i.e., a monoclonal antibody derived from a single cell line). In embodiments, the 4-1BB agonist is EU-101(Eutilex, ltd), urotropinumab or ulirubizumab, or a fragment, derivative, conjugate, variant, or biological analog thereof. In a preferred embodiment, the 4-1BB agonist is urotremirumab or Uriruzumab, or a fragment, derivative, conjugate, variant or biological analog thereof.

In a preferred embodiment, the 4-1BB agonist or 4-1BB binding molecule may also be a fusion protein. In preferred embodiments, multimeric 4-1BB agonists, such as trimeric or hexamer 4-1BB agonists (having three or six ligand binding domains) can induce superior receptor (4-1BBL) clustering and internal cell signaling complex formation compared to agonistic monoclonal antibodies, which typically have two ligand binding domains. Fusion proteins comprising three TNFRSF binding domains and IgG1-Fc and optionally further linking two or more of these fusion proteins, which are trimeric (trivalent) or hexameric (or hexavalent) or larger, are described, for example, in giefers et al, molecular cancer Therapeutics 2013,12, 2735-47.

Agonistic 4-1BB antibodies and fusion proteins are known to induce a strong immune response. In preferred embodiments, the 4-1BB agonist is a monoclonal antibody or fusion protein that specifically binds to 4-1BB antigen in a manner sufficient to reduce toxicity. In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abrogates antibody-dependent cellular cytotoxicity (ADCC), such as NK cell cytotoxicity. In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abrogates antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the 4-1BB agonist is an agonist 4-1BB monoclonal antibody or fusion protein that abrogates Complement Dependent Cytotoxicity (CDC). In some embodiments, the 4-1BB agonist is an agonist 4-1BB monoclonal antibody or fusion protein that abrogates the functionality of the Fc region.

In some embodiments, the 4-1BB agonist is characterized by binding to human 4-1BB (SEQ ID NO:9) with high affinity and agonistic activity. In embodiments, the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO: 9). In the examples, the 4-1BB agonist is a binding molecule that binds murine 4-1BB (SEQ ID NO: 10). The amino acid sequences of the 4-1BB antigens bound by the 4-1BB agonists or binding molecules are summarized in Table 6.

Table 6: 4-1BB antigen.

In some embodiments, the described compositions, processes, and methods comprise a K of about 100pM or less_DK at about 90pM or less in combination with human or murine 4-1BB_DK at about 80pM or less in combination with human or murine 4-1BB_DK at about 70pM or less in combination with human or murine 4-1BB_DK at about 60pM or less in combination with human or murine 4-1BB_DK at about 50pM or less in combination with human or murine 4-1BB_DK at about 40pM or less in combination with human or murine 4-1BB_DK binding to human or murine 4-1BB or at about 30pM or less_D4-1BB agonists that bind to human or murine 4-1 BB.

In some embodiments, the described compositions, processes, and methods comprise a treatment at about 7.5 x 10⁵1/M.s or faster k _assocBinding to human or murine 4-1BB at a rate of about 7.5X 10⁵1/M.s or faster k_assocBinding to human or murine 4-1BB at about 8X 10⁵1/M.s or faster k_assocBinding to human or murine 4-1BB at about 8.5X 10⁵1/M.s or faster k_assocBinding to human or murine 4-1BB at about 9X 10⁵1/M.s or faster k_assocBinding to human or murine 4-1BB at a rate of about 9.5X 10⁵1/M.s or faster k_assocBinding to human or murine 4-1BB or at about 1X 10⁶1/M.s or faster k_assoc4-1BB agonists that bind to human or murine 4-1 BB.

In some embodiments, the described compositions, processes, and methods comprise a treatment at about 2 x 10^-5K of 1/s or slower_dissocBinding to human or murine 4-1BB at about 2.1X 10^-5K of 1/s or slower_dissocBinding to human or murine 4-1BB at a rate of about 2.2X 10^-5K of 1/s or less_dissocBinding to human or murine 4-1BB at a rate of about 2.3X 10^-5K of 1/s or less_dissocBinding to human or murine 4-1BB at about 2.4X 10^-5K of 1/s or less_dissocBinding to human or murine 4-1BB at a rate of about 2.5X 10^-5K of 1/s or less_dissocBinding to human or murine 4-1BB at a rate of about 2.6X 10^-5K of 1/s or slower_dissocBinding to human or murine 4-1BB or at about 2.7X 10^-5K of 1/s or slower_dissocBinding to human or murine 4-1BB at a rate of about 2.8X 10^-5K of 1/s or less_dissocBinding to human or murine 4-1BB at a rate of about 2.9X 10 ^-5K of 1/s or less_dissocBinding to human or murine 4-1BB or at about 3X 10^-5K of 1/s or less_dissoc4-1BB agonists that bind to human or murine 4-1 BB.

In some embodiments, the described compositions, processes, and methods comprise an IC of about 10nM or less₅₀IC at about 9nM or less for binding to human or murine 4-1BB₅₀IC at about 8nM or less for binding to human or murine 4-1BB₅₀IC at about 7nM or less for binding to human or murine 4-1BB₅₀IC at about 6nM or less for binding to human or murine 4-1BB₅₀With humans or miceClass 4-1BB binding, IC at about 5nM or lower₅₀IC at about 4nM or less for binding to human or murine 4-1BB₅₀IC at about 3nM or less for binding to human or murine 4-1BB₅₀IC at about 2nM or less for binding to human or murine 4-1BB₅₀IC binding to human or murine 4-1BB or at about 1nM or less₅₀4-1BB agonists that bind to human or murine 4-1 BB.

In a preferred embodiment, the 4-1BB agonist is Utomimilumab (also known as PF-05082566 or MOR-7480) or a fragment, derivative, conjugate, variant or biological analog thereof. Urotropin is available from Pfizer, Inc. Utomoluumab is immunoglobulin G2-lambda, anti [ homo sapiens TNFRSF9 (tumor necrosis factor receptor (TNFR) superfamily member 9, 4-1BB, T cell antigen ILA, CD137) ]Homo sapiens (fully human) monoclonal antibody. The amino acid sequence of urotropinumab is set forth in table 7. Urotropinumab comprises glycosylation sites at Asn59 and Asn 292; in positions 22-96 (V)_H-V_L)、143-199(C_H1-C_L)、256-316(C_H2) And 362-420 (C)_H3) An intrachain disulfide bridge of (a); in positions 22'-87' (V)_H-V_L) And 136'-195' (C)_H1-C_L) A disulfide bridge in the light chain; interchain heavy chain-heavy chain disulfide bridges at the IgG2A isomer positions 218, 219-219, 222-222 and 225, at the IgG2A/B isomer positions 218-, 219, 222-222 and 225 and at the IgG2B isomer positions 219-130(2), 222-222 and 225-225; and interchain heavy-light disulfide bridges at the IgG2A isomer positions 130-213'(2), the IgG2A/B isomer positions 218-213' and 130-213', and at the IgG2B isomer position 218-213' (2). The preparation and properties of urotropinumab and variants and fragments thereof are described in U.S. patent No. 8,821,867; 8,337,850 No; and 9,468,678, and international patent application publication No. WO 2012/032433 a1, the disclosure of each of which is incorporated herein by reference. Preclinical properties of urotropinumab are described in Fisher et al, "Cancer immunology and immunotherapy (Cancer Immunolog). &Motherwort 2012,61, 1721-33. Utomorrukin for treating various blood and solid tumorsCurrent clinical trials in response to the disease include national Institutes of Health (u.s.national Institutes of Health) clinical trial database identifiers NCT02444793, NCT01307267, NCT02315066, and NCT 02554812.

In an embodiment, the 4-1BB agonist comprises a heavy chain given by SEQ ID NO. 11 and a light chain given by SEQ ID NO. 12. In embodiments, the 4-1BB agonist comprises heavy and light chains having the sequences set forth in SEQ ID NO 11 and SEQ ID NO 12, respectively, or antigen-binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates thereof. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 99% identical to the sequences set forth in SEQ ID NO 11 and SEQ ID NO 12, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 98% identical to the sequences set forth in SEQ ID NO 11 and SEQ ID NO 12, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 97% identical to the sequences set forth in SEQ ID NO 11 and SEQ ID NO 12, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 96% identical to the sequences set forth in SEQ ID NO 11 and SEQ ID NO 12, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 95% identical to the sequences set forth in SEQ ID NO 11 and SEQ ID NO 12, respectively.

In embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of urotropinumab. In the examples, the 4-1BB agonist heavy chain variable region (V)_H) Comprising the sequence shown in SEQ ID NO:13 and a 4-1BB agonist light chain variable region (V)_L) Including the sequence shown in SEQ ID NO. 14 and conservative amino acid substitutions thereof. In embodiments, the 4-1BB agonist comprises V that is at least 99% identical to the sequences set forth in SEQ ID NO 13 and SEQ ID NO 14, respectively_HAnd V_LAnd (4) a zone. In embodiments, the 4-1BB agonist comprises V that is at least 98% identical to the sequences set forth in SEQ ID NO 13 and SEQ ID NO 14, respectively_HAnd V_LAnd (4) a zone. In embodiments, the 4-1BB agonist comprises V that is at least 97% identical to the sequences set forth in SEQ ID NO 13 and SEQ ID NO 14, respectively_HAnd V_LAnd (4) a zone. In the examples, 4-1The BB agonist comprises a V that is at least 96% identical to the sequences set forth in SEQ ID NO 13 and SEQ ID NO 14, respectively_HAnd V_LAnd (4) a zone. In embodiments, the 4-1BB agonist comprises V that is at least 95% identical to the sequences set forth in SEQ ID NO:13 and SEQ ID NO:14, respectively_HAnd V_LAnd (4) a zone. In embodiments, the 4-1BB agonist comprises an scFv antibody comprising V that is at least 99% identical to each of the sequences set forth in SEQ ID NO:13 and SEQ ID NO:14 _HAnd V_LAnd (4) a zone.

In embodiments, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains and conservative amino acid substitutions thereof having the sequences shown in SEQ ID No. 15, SEQ ID No. 16, and SEQ ID No. 17, respectively, and light chain CDR1, CDR2, and CDR3 domains and conservative amino acid substitutions thereof having the sequences shown in SEQ ID No. 18, SEQ ID No. 19, and SEQ ID No. 20, respectively.

In embodiments, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by the drug regulatory authority (drug regulatory authorities) with reference to utomitumumab. In embodiments, the biosimilar monoclonal antibody comprises a 4-1BB antibody comprising an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) to an amino acid sequence of a reference drug or reference biological product and comprising one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is utomisumab. In some embodiments, the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation and truncation. In some embodiments, the biological analog is a 4-1BB agonist antibody that is authorized or submitted for authorization, wherein the 4-1BB agonist antibody is provided in a formulation that is different from a formulation of a reference drug or reference biological product, wherein the reference drug or reference biological product is utomitrumab. The 4-1BB agonist antibody may be authorized by a drug regulatory agency (e.g., the U.S. FDA and/or European Union EMA). In some embodiments, the biosimilar is provided in a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in a reference drug or a reference biological product, wherein the reference drug or reference biological product is utomisumab. In some embodiments, the biosimilar is provided in a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in a reference drug or a reference biological product, wherein the reference drug or reference biological product is utomisumab.

Table 7: amino acid sequence of a 4-1BB agonist antibody related to urotropinumab.

In a preferred embodiment, the 4-1BB agonist is the monoclonal antibody Uluzumab (also known as BMS-663513 and 20H4.9.h4a) or a fragment, derivative, variant, or biological analog thereof. Urru mab is available from Beckmann Shi Guibao and Creative biological laboratories, Inc. Urru monoclonal antibody is immunoglobulin G4-kappa, anti [ homo sapiens TNFRSF9 (tumor necrosis factor receptor superfamily member 9, 4-1BB, T cell antigen ILA, CD137)]Homo sapiens (fully human) monoclonal antibody. The amino acid sequence of the udeluumab is set forth in table EE. Urru mab includes an N-glycosylation site at position 298 (and 298'); in positions 22-95 (V)_H-V_L)、148-204(C_H1-C_L)、262-322(C_H2) And 368-_H3) Heavy chain intrachain disulfide bridges at (and at positions 22"-95", 148"-204", 262"-322" and 368 "-426"); in positions 23'-88' (V)_H-V_L) And 136'-196' (C)_H1-C_L) Light chain intrachain disulfide bridges at (and at positions 23"'-88"' and 136 "'-196"'); interchain heavy chain-heavy chain disulfide bridges at positions 227-; and interchain heavy-light disulfide bridges at 135-216 'and 135 "-216'". The preparation and properties of Uruguzumab, and variants and fragments thereof, are described in U.S. Pat. Nos. 7,288,638 and 8,962,804, the disclosures of which are incorporated herein by reference. Clinical application of Urru mab Pre-and clinical characteristics are described in Segal et al, clinical cancer research 2016, available from the following websites: http:/dx.doi.org/10.1158/1078-0432. CCR-16-1272. Current clinical trials of udersumab in a variety of hematologic and solid tumor indications include the american national institutes of health clinical trial database identifiers NCT01775631, NCT02110082, NCT02253992, and NCT 01471210.

In an embodiment, the 4-1BB agonist comprises a heavy chain given by SEQ ID NO. 21 and a light chain given by SEQ ID NO. 22. In embodiments, the 4-1BB agonist comprises heavy and light chains having the sequences shown in SEQ ID NO:21 and SEQ ID NO:22, respectively, or antigen binding fragments, Fab fragments, single chain variable fragments (scFv), variants, or conjugates thereof. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain each at least 99% identical to the sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 98% identical to the sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 97% identical to the sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 96% identical to the sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively. In embodiments, the 4-1BB agonist comprises a heavy chain and a light chain that are each at least 95% identical to the sequences set forth in SEQ ID NO:21 and SEQ ID NO:22, respectively.

In embodiments, the 4-1BB agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of udeuzumab. In the examples, the 4-1BB agonist heavy chain variable region (V)_H) Comprising the sequence shown in SEQ ID NO:23, and a 4-1BB agonist light chain variable region (V)_L) Including the sequence shown in SEQ ID NO. 24 and conservative amino acid substitutions thereof. In embodiments, the 4-1BB agonist comprises V that is at least 99% identical to the sequences set forth in SEQ ID NO:23 and SEQ ID NO:24, respectively_HAnd V_LAnd (4) a zone. In embodiments, the 4-1BB agonist comprises V that is at least 98% identical to the sequence set forth in SEQ ID NO:23 and SEQ ID NO:24, respectively_HAnd V_LAnd (4) a zone. In the examples, 4-1BBThe agonist comprises a V that is at least 97% identical to the sequences set forth in SEQ ID NO 23 and SEQ ID NO 24, respectively_HAnd V_LAnd (4) a zone. In embodiments, the 4-1BB agonist comprises V that is at least 96% identical to the sequences set forth in SEQ ID NO:23 and SEQ ID NO:24, respectively_HAnd V_LAnd (4) a zone. In embodiments, the 4-1BB agonist comprises V that is at least 95% identical to the sequences set forth in SEQ ID NO:23 and SEQ ID NO:24, respectively_HAnd V_LAnd (4) a zone. In embodiments, the 4-1BB agonist comprises an scFv antibody comprising V that is at least 99% identical to each of the sequences set forth in SEQ ID NO:23 and SEQ ID NO:24 _HAnd V_LAnd (4) a zone.

In embodiments, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains and conservative amino acid substitutions thereof having the sequences shown in SEQ ID No. 25, SEQ ID No. 26, and SEQ ID No. 27, respectively, and light chain CDR1, CDR2, and CDR3 domains and conservative amino acid substitutions thereof having the sequences shown in SEQ ID No. 28, SEQ ID No. 29, and SEQ ID No. 30, respectively.

In an embodiment, the 4-1BB agonist is a 4-1BB agonist bio-analog monoclonal antibody approved by the drug regulatory agency with reference to wureluzumab. In embodiments, the bioanalog monoclonal antibody comprises a 4-1BB antibody comprising an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) to an amino acid sequence of a reference drug or reference biological product and comprising one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is ulirubizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation and truncation. In some embodiments, the biological analog is a 4-1BB agonist antibody that is authorized or submitted for authorization, wherein the 4-1BB agonist antibody is provided in a formulation that is different from a formulation of a reference drug or reference biological product, wherein the reference drug or reference biological product is Uluzumab. The 4-1BB agonist antibody may be authorized by a drug regulatory agency (e.g., the U.S. FDA and/or European Union EMA). In some embodiments, the biological analog is provided in a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in a reference drug or a reference biological product, wherein the reference drug or the reference biological product is Uluruzumab. In some embodiments, the biological analog is provided in a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in a reference drug or a reference biological product, wherein the reference drug or the reference biological product is Uluruzumab.

Table 8: amino acid sequence of 4-1BB agonist antibody related to Urru mab.

In embodiments, the 4-1BB agonist is selected from the group consisting of: 1D8, 3Elor, 4B4(BioLegend 309809), H4-1BB-M127(BD Pharmingen 552532), BBK2 (Sermer Feishal (Thermo Fisher) MS621PABX), 145501(Leinco Technologies B591), antibodies produced by the cell line deposited as ATCC number HB-11248 and disclosed in U.S. Pat. No. 6,974,863, 5F4(BioLegend 311503), C65-485 (Pharmingen 559446), antibodies disclosed in U.S. patent application publication No. US 2005/0095244, antibodies disclosed in U.S. patent No. 7,288,638 (e.g., 20H4.9-IgG1 (BioLegend 4628)), antibodies disclosed in U.S. patent No. 9 (e.g., 4E9 or BMS-554271), antibodies disclosed in U.S. patent No. 7,214,493, antibodies disclosed in U.S. patent No. 6,303,121, antibodies disclosed in U.S. patent publication No. 6,569,997, antibodies disclosed in U.S. Pat. No. 367, BMS. 36874-554271, or BMS. 9 (BMS. 9-9), Antibodies disclosed in U.S. Pat. No. 6,362,325 (e.g., 1D8 or BMS-469492; 3H3 or BMS-469497; or 3El), antibodies disclosed in U.S. Pat. No. 6,974,863 (e.g., 53A 2); antibodies disclosed in U.S. patent No. 6,210,669 (e.g., 1D8, 3B8, or 3El), antibodies described in U.S. patent No. 5,928,893, antibodies disclosed in U.S. patent No. 6,303,121, antibodies disclosed in U.S. patent No. 6,569,997, antibodies disclosed in international patent application publication nos. WO 2012/177788, WO 2015/119923, and WO 2010/042433, and fragments, derivatives, conjugates, variants, or biological analogs thereof, wherein the disclosure of each of the foregoing patents or patent application publications is incorporated herein by reference.

In the examples, the 4-1BB agonist is a 4-1BB agonist fusion protein described in: international patent application publication nos. WO 2008/025516 a1, WO 2009/007120 a1, WO 2010/003766 a1, WO 2010/010051 a1 and WO 2010/078966 a 1; U.S. patent application publication nos. US 2011/0027218 a1, US 2015/0126709 a1, US 2011/0111494 a1, US 2015/0110734 a1, and US 2015/0126710 a 1; and U.S. patent nos. 9,359,420, 9,340,599, 8,921,519, and 8,450460, the disclosures of which are incorporated herein by reference.

In embodiments, as provided in figure 131, the 4-1BB agonist is a 4-1BB agonist fusion protein or fragment, derivative, conjugate, variant, or biological analog thereof as depicted in structure I-a (C-terminal Fc antibody fragment fusion protein) or structure I-B (N-terminal Fc antibody fragment fusion protein).

In structures I-A and I-B, cylinders refer to the individual polypeptide binding domains. Structures I-A and I-B include three linearly linked TNFRSF binding domains derived from, for example, 4-1BBL (4-1BB ligand, CD137 ligand (CD137L), or tumor necrosis factor superfamily member 9(TNFSF9), or an antibody that binds to 4-1BB, which fold to form a trivalent protein, followed by IgG1-Fc (comprising C) _H3 and C_H2 domain) to a second trivalent protein, which in turn serves to link the two trivalent proteins together by disulfide bonds (small elongated ovoids), stabilizing the structure and providing an agonist capable of bringing together the intracellular signaling domains of the six receptors and the signaling protein to form a signaling complex. The TNFRSF binding domain represented in cylindrical form may be an scFv domain, including, for example, a scFv domain composed of a sequence of Gly and Ser, which may include hydrophilic residues and have flexibility, and G having solubilitylinker-linked V of lu and Lys_HAnd V_LAnd (3) a chain. Any scFv domain design may be used, such as de Marco, Microbial Cell Factories (Microbial cells Factories) 2011,10, 44; ahmad et al, clinical and developmental immunology (Clin.&Dev.)) 2012,980250; monnier et al, Antibodies (Antibodies), 2013,2, 193-208; or incorporated by reference elsewhere herein. Fusion protein structures of this form are described in U.S. patent nos. 9,359,420, 9,340,599, 8,921,519 and 8,450,460, the disclosures of which are incorporated herein by reference.

The amino acid sequences of the other polypeptide domains of structure I-A are given in Table 9. The Fc domain preferably comprises the entire constant domain (amino acids 17-230 of SEQ ID NO: 31), the entire hinge domain (amino acids 1-16 of SEQ ID NO: 31), or a portion of the hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 31). Preferred linkers for linking the C-terminal Fc antibody may be selected from the examples given in SEQ ID NO:32 to SEQ ID NO:41, including linkers suitable for fusing further polypeptides.

Table 9: amino acid sequence of TNFRSF agonist fusion protein (comprising 4-1BB agonist fusion protein) with C-terminal Fc antibody fragment fusion protein design (Structure I-A).

The amino acid sequences of the other polypeptide domains of structure I-B are given in Table 10. If the Fc antibody fragment is fused to the N-terminus of the TNRFSF agonist fusion protein as in structure I-B, the sequence of the Fc module is preferably that shown in SEQ ID NO:42 and the linker sequence is preferably selected from those examples shown in SED ID NO:43 through SEQ ID NO: 45.

Table 10: amino acid sequence of TNFRSF agonist fusion protein (comprising 4-1BB agonist fusion protein) with N-terminal Fc antibody fragment fusion protein design (Structure I-B).

In embodiments, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains selected from the group consisting of: the variable heavy chain and the variable light chain of urothelimab, the variable heavy chain and the variable light chain selected from the variable heavy chain and the variable light chain described in table 10, any combination of the foregoing variable heavy chain and variable light chain, and fragments, derivatives, conjugates, variants, and biological analogs thereof.

In embodiments, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains comprising a 4-1BBL sequence. In an embodiment, a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains comprising a sequence according to SEQ ID NO. 46. In embodiments, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains comprising a soluble 4-1BBL sequence. In an embodiment, a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains comprising a sequence according to SEQ ID NO: 47.

In embodiments, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains that are V-binding domains comprising at least 95% identity to the sequences set forth in SEQ ID NO:13 and SEQ ID NO:14, respectively_HAnd V_LA scFv domain of region wherein V_HAnd V_LThe domains are connected by a linker. In embodiments, a 4-1BB agonist fusion protein according to structures I-A or I-B includes one or more 4-1BB binding domains that are V-binding domains comprising at least 95% identity to the sequences set forth in SEQ ID NO:23 and SEQ ID NO:24, respectively _HAnd V_LA scFv domain of region wherein V_HAnd V_LThe domains are connected by a linker. In embodiments, a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains, which are encompassed by each of the groups in Table 11Given V_HAnd V_LV with at least 95% sequence identity_HAnd V_LA scFv domain of region wherein V_HAnd V_LThe domains are connected by a linker.

Table 11: can be used as a 4-1BB binding domain in a fusion protein or as an additional polypeptide domain of an scFv 4-1BB agonist antibody.

In embodiments, the 4-1BB agonist is a 4-1BB agonistic single chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, further comprising an additional domain at the N-terminus and/or C-terminus, and wherein the additional domain is a Fab or Fc fragment domain. In embodiments, the 4-1BB agonist is a 4-1BB agonistic single chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, further comprising an additional domain at the N-terminus and/or C-terminus, wherein the additional domain is a Fab or Fc fragment domain, wherein each of the soluble 4-1BB domains lacks a stem region (that facilitates trimerization and provides a distance to the cell membrane, but is not part of the 4-1BB binding domain) and the first and second peptide linkers independently have a length of 3-8 amino acids.

In an embodiment, the 4-1BB agonist is a 4-1BB agonistic single chain fusion polypeptide comprising (i) a first soluble Tumor Necrosis Factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain, (iv) a second peptide linker, and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stem region and the first and second peptide linkers independently have a length of 3-8 amino acids, and wherein each TNF superfamily cytokine domain is a 4-1BB binding domain.

In embodiments, the 4-1BB agonist is a 4-1BB agonistic scFv antibody comprising any of the foregoing V_HDomains and any of the foregoing V_LThe domains are linked.

In the examples, the 4-1BB agonist is BPS Bioscience 4-1BB agonist antibody catalog No. 79097-2, commercially available from BPS Bioscience (BPS Bioscience, San Diego, Calif., USA) of San Diego, Calif., USA. In one embodiment, the 4-1BB agonist is the creative biological laboratory company catalog number MOM-18179 for the 4-1BB agonist antibody, commercially available from creative biological laboratory company, Hiland, N.Y..

OX40(CD134) agonists

In embodiments, the TNFRSF agonist is an OX40(CD134) agonist. The OX40 agonist can be any OX40 binding molecule known in the art. The OX40 binding molecule can be a monoclonal antibody or fusion protein capable of binding to human or mammalian OX 40. The OX40 agonist or OX40 binding molecule can include immunoglobulin heavy chains of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or sub-class of immunoglobulin molecules. An OX40 agonist or OX40 binding molecule may have both heavy and light chains. As used herein, the term binding molecule also includes antibodies (including full length antibodies), monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human, humanized or chimeric antibodies and antibody fragments, e.g., Fab fragments, F (ab') fragments, fragments produced by Fab expression libraries, epitope-binding fragments of any of the above, and engineered forms of antibodies, e.g., scFv molecules, that bind to OX 40. In embodiments, the OX40 agonist is an antigen binding protein that is a fully human antibody. In embodiments, the OX40 agonist is an antigen binding protein that is a humanized antibody. In some embodiments, the OX40 agonists for use in the methods and compositions of the present disclosure comprise anti-OX 40 antibodies, human anti-OX 40 antibodies, mouse anti-OX 40 antibodies, mammalian anti-OX 40 antibodies, monoclonal anti-OX 40 antibodies, polyclonal anti-OX 40 antibodies, chimeric anti-OX 40 antibodies, anti-OX 40 fibronectin, anti-OX 40 domain antibodies, single chain anti-OX 40 fragments, heavy chain anti-OX 40 fragments, light chain anti-OX 40 fragments, anti-OX 40 fusion proteins, and fragments, derivatives, conjugates, variants, or biological analogs thereof. In preferred embodiments, the OX40 agonist is an agonist anti-OX 40 humanized or fully human monoclonal antibody (i.e., a monoclonal antibody derived from a single cell line).

In preferred embodiments, the OX40 agonist or OX40 binding molecule may also be a fusion protein. OX40 fusion proteins comprising an Fc domain fused to OX40L are described, for example, in Sadun et al, J.Immunotherapy, 2009,182,1481-89. In preferred embodiments, multimeric OX40 agonists, such as trimeric or hexameric OX40 agonists (having three or six ligand binding domains) can induce superior receptor (OX40L) clustering and internal cell signaling complex formation compared to agonist monoclonal antibodies that typically have two ligand binding domains. Fusion proteins comprising three TNFRSF binding domains and IgG1-Fc and optionally further linking two or more of these fusion proteins, which are trimeric (trivalent) or hexameric (or hexavalent) or larger, are described, for example, in giefers et al, molecular cancer Therapeutics 2013,12, 2735-47.

Agonist OX40 antibodies and fusion proteins are known to induce a stronger immune response. Curti et al, cancer research 2013,73, 7189-98. In preferred embodiments, the OX40 agonist is a monoclonal antibody or fusion protein that specifically binds to OX40 antigen in a manner sufficient to reduce toxicity. In some embodiments, the OX40 agonist is an agonist OX40 monoclonal antibody or fusion protein that abrogates Antibody Dependent Cellular Cytotoxicity (ADCC), e.g., NK cell cytotoxicity. In some embodiments, the OX40 agonist is an agonist OX40 monoclonal antibody or fusion protein that abrogates antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the OX40 agonist is an agonist OX40 monoclonal antibody or fusion protein that abrogates Complement Dependent Cytotoxicity (CDC). In some embodiments, the OX40 agonist is an agonist OX40 monoclonal antibody or fusion protein that abrogates Fc region functionality.

In some embodiments, the OX40 agonist is characterized by binding to human OX40(SEQ ID NO:54) with high affinity and agonist activity. In embodiments, the OX40 agonist is a binding molecule that binds to human OX40(SEQ ID NO: 54). In embodiments, the OX40 agonist is a binding molecule that binds to murine OX40(SEQ ID NO: 55). The amino acid sequences of the OX40 antigen to which the OX40 agonist or binding molecule binds are summarized in Table 12.

Table 12: amino acid sequence of OX40 antigen.

In some embodiments, the described compositions, processes, and methods comprise a K of about 100pM or less_DK binding to human or murine OX40 at about 90pM or less_DBinds to human or murine OX40 at a K of about 80pM or less_DK binding to human or murine OX40 at about 70pM or less_DBinds to human or murine OX40 at a K of about 60pM or less_DBinds to human or murine OX40 at a K of about 50pM or less_DBinds human or murine OX40 at a K of about 40pM or less_DBinds to human or murine OX40 or at a K of about 30pM or less_DAn OX40 agonist that binds to human or murine OX 40.

In some embodiments, the described compositions, processes, and methods comprise a treatment at about 7.5 x 10⁵1/M.s or faster k_assocBinds to human or murine OX40 at about 7.5X 10 ⁵1/M.s or faster k_assocBinds to human or murine OX40 at about 8X 10⁵1/M.s or faster k_assocBinds to human or murine OX40 at about 8.5X 10⁵1/M.s or faster k_assocBinds to human or murine OX40 at about 9X 10⁵1/M.s or faster k_assocBinds to human or murine OX40 at about 9.5X 10⁵1/M.s or faster k_assocBinds to human or murine OX40 or at about 1X 10⁶1/M.s or faster k_assocAn OX40 agonist that binds to human or murine OX 40.

In some embodiments, the described compositions, processes, and methods comprise a treatment at about 2 x 10^-5K of 1/s or slower_dissocBinds to human or murine OX40 at about 2.1X 10^-5K of 1/s or slower_dissocBinds to human or murine OX40 at about 2.2X 10^-5K of 1/s or less_dissocBinds to human or murine OX40 at about 2.3X 10^-5K of 1/s or less_dissocBinds to human or murine OX40 at about 2.4X 10^-5K of 1/s or less_dissocBinds to human or murine OX40 at about 2.5X 10^-5K of 1/s or less_dissocBinds to human or murine OX40 at about 2.6X 10^-5K of 1/s or slower_dissocBinds to human or murine OX40 or at about 2.7X 10^-5K of 1/s or slower_dissocBinds to human or murine OX40 at about 2.8X 10^-5K of 1/s or less_dissocBinds to human or murine OX40 at about 2.9X 10^-5K of 1/s or less_dissocBinds to human or murine OX40 or at about 3X 10 ^-5K of 1/s or less_dissocAn OX40 agonist that binds to human or murine OX 40.

In some embodiments, the described compositions, processes, and methods comprise an IC of about 10nM or less₅₀IC at about 9nM or less for binding to human or murine OX40₅₀IC at about 8nM or less for binding to human or murine OX40₅₀IC at about 7nM or less for binding to human or murine OX40₅₀IC at about 6nM or less for binding to human or murine OX40₅₀IC at about 5nM or less for binding to human or murine OX40₅₀IC at about 4nM or less for binding to human or murine OX40₅₀IC at about 3nM or less for binding to human or murine OX40₅₀IC at about 2nM or less for binding to human or murine OX40₅₀Binds to human or murine OX40 or with an IC of about 1nM or less₅₀An OX40 agonist that binds to human or murine OX 40.

In some embodiments, the OX40 agonist is tavollizumab, also known as MEDI0562 or MEDI-0562. Tavollizumab is available from medical immunology Inc. (MedI) of Aslicon Incmmune sublidiary of AstraZeneca, Inc.). Tavalizumab is immunoglobulin G1-kappa, anti [ homo sapiens TNFRSF4 (tumor necrosis factor receptor (TNFR) superfamily member 4, OX40, CD134)]Humanized and chimeric monoclonal antibodies. The amino acid sequence of tavollizumab is set forth in table 13. Tavollizumab includes N-glycosylation sites at positions 301 and 301", with fucosylated complex bi-antennary CHO-type glycans; in positions 22-95 (V) _H-V_L)、148-204(C_H1-C_L)、265-325(C_H2) And 371-_H3) Heavy chain intrachain disulfide bridges at (and at positions 22"-95", 148"-204", 265"-325" and 371 "-429"); in positions 23'-88' (V)_H-V_L) And 134'-194' (C)_H1-C_L) Light chain intrachain disulfide bridges at (and at positions 23"'-88"' and 134 "'-194"'); interchain heavy-heavy disulfide bridges at positions 230-230 "and 233-233"; and interchain heavy-light disulfide bridges at 224-. Current clinical trials of tavollizumab in a variety of solid tumor indications include the american national institutes of health clinical trial database identifiers NCT02318394 and NCT 02705482.

In embodiments, the OX40 agonist comprises a heavy chain as given by SEQ ID NO. 56 and a light chain as given by SEQ ID NO. 57. In embodiments, the OX40 agonist includes a heavy chain and a light chain having sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 99% identical to the sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 98% identical to the sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 97% identical to the sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 96% identical to the sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 95% identical to the sequences set forth in SEQ ID NO:56 and SEQ ID NO:57, respectively.

In embodiments, the OX40 agonist includes the heavy and light chain CDRs or Variable Regions (VRs) of tavollizumab. In embodiments, the OX40 agonist heavy chain variable region (V)_H) Includes the sequence shown in SEQ ID NO:58, and OX40 agonist light chain variable region (V)_L) Including the sequence shown in SEQ ID NO 59 and conservative amino acid substitutions thereof. In embodiments, the OX40 agonist comprises a V that is at least 99% identical to the sequences set forth in SEQ ID NO:58 and SEQ ID NO:59, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 98% identical to the sequences set forth in SEQ ID NO:58 and SEQ ID NO:59, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 97% identical to the sequences set forth in SEQ ID NO:58 and SEQ ID NO:59, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 96% identical to the sequences set forth in SEQ ID NO:58 and SEQ ID NO:59, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 95% identical to the sequences set forth in SEQ ID NO:58 and SEQ ID NO:59, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises an scFv antibody comprising V that is at least 99% identical to the sequences set forth in SEQ ID NO:58 and SEQ ID NO:59, respectively _HAnd V_LAnd (4) a zone.

In embodiments, the OX40 agonist includes heavy chain CDR1, CDR2, and CDR3 domains and conservative amino acid substitutions thereof having the sequences shown in SEQ ID No. 60, SEQ ID No. 61, and SEQ ID No. 62, respectively, and light chain CDR1, CDR2, and CDR3 domains and conservative amino acid substitutions thereof having the sequences shown in SEQ ID No. 63, SEQ ID No. 64, and SEQ ID No. 65, respectively.

In embodiments, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by the drug regulatory agency with reference to tavollizumab. In embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) to an amino acid sequence of a reference drug or reference biological product and comprising one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is tavollizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation and truncation. In some embodiments, the biological analog is an authorized or submitted authorized OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation that is different from a formulation of a reference drug or reference biological product, wherein the reference drug or reference biological product is tavollizumab. OX40 agonist antibodies may be authorized by drug regulatory agencies (e.g., the U.S. FDA and/or eu EMA). In some embodiments, the biological analog is provided in the form of a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in a reference drug or a reference biological product, wherein the reference drug or the reference biological product is tavollizumab. In some embodiments, the biological analog is provided in the form of a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in a reference drug or a reference biological product, wherein the reference drug or the reference biological product is tavollizumab.

Table 13: amino acid sequence of an OX40 agonist antibody related to tavollizumab.

In some embodiments, the OX40 agonist is 11D4, which is a fully human antibody obtained from pfeiy corporation. The preparation and properties of 11D4 are described in U.S. patent No. 7,960,515; 8,236,930 No; and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 11D4 is set forth in table 14.

In embodiments, the OX40 agonist comprises a heavy chain as given by SEQ ID NO. 66 and a light chain as given by SEQ ID NO. 67. In embodiments, the OX40 agonist includes a heavy chain and a light chain having sequences set forth in SEQ ID NO:66 and SEQ ID NO:67, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 99% identical to the sequences set forth in SEQ ID NO:66 and SEQ ID NO:67, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 98% identical to the sequences set forth in SEQ ID NO:66 and SEQ ID NO:67, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 97% identical to the sequences set forth in SEQ ID NO:66 and SEQ ID NO:67, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 96% identical to the sequences set forth in SEQ ID NO:66 and SEQ ID NO:67, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 95% identical to the sequences set forth in SEQ ID NO:66 and SEQ ID NO:67, respectively.

In embodiments, the OX40 agonist includes the heavy and light chain CDRs or Variable Regions (VRs) of 11D 4. In embodiments, the OX40 agonist heavy chain variable region (V)_H) Including the sequence shown in SEQ ID NO:68, and OX40 agonist light chain variable region (V)_L) Including the sequence shown in SEQ ID NO:69 and conservative amino acid substitutions thereof. In embodiments, the OX40 agonist comprises a V that is at least 99% identical to the sequences set forth in SEQ ID NO:68 and SEQ ID NO:69, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises V that is at least 98% identical to the sequences set forth in SEQ ID NO:68 and SEQ ID NO:69, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 97% identical to the sequences set forth in SEQ ID NO:68 and SEQ ID NO:69, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 96% identical to the sequences set forth in SEQ ID NO:68 and SEQ ID NO:69, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 95% identical to the sequences set forth in SEQ ID NO:68 and SEQ ID NO:69, respectively_HAnd V_LAnd (4) a zone.

In embodiments, the OX40 agonist includes heavy chain CDR1, CDR2, and CDR3 domains and conservative amino acid substitutions thereof having the sequences shown in SEQ ID NO 70, SEQ ID NO 71, and SEQ ID NO 72, respectively, and light chain CDR1, CDR2, and CDR3 domains and conservative amino acid substitutions thereof having the sequences shown in SEQ ID NO 73, SEQ ID NO 74, and SEQ ID NO 75, respectively.

In embodiments, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by the drug regulatory agency reference 11D 4. In embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) to an amino acid sequence of a reference drug or reference biological product and comprising one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is 11D 4. In some embodiments, the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation and truncation. In some embodiments, the biological analog is an authorized or submitted authorized OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation that is different from a formulation of a reference drug or reference biological product, wherein the reference drug or reference biological product is 11D 4. OX40 agonist antibodies may be authorized by drug regulatory agencies (e.g., the U.S. FDA and/or eu EMA). In some embodiments, the biosimilar is provided in a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in a reference drug or a reference biological product, wherein the reference drug or reference biological product is 11D 4. In some embodiments, the biosimilar is provided in a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients included in a reference drug or a reference biological product, wherein the reference drug or reference biological product is 11D 4.

Table 14: amino acid sequence of an OX40 agonist antibody related to 11D 4.

In some embodiments, the OX40 agonist is 18D8, which is a fully human antibody obtained from pfeiy corporation. The preparation and properties of 18D8 are described in U.S. patent No. 7,960,515; 8,236,930 No; and 9,028,824, the disclosures of which are incorporated herein by reference. The amino acid sequence of 18D8 is set forth in table 15.

In embodiments, the OX40 agonist comprises a heavy chain as given by SEQ ID NO. 76 and a light chain as given by SEQ ID NO. 77. In embodiments, the OX40 agonist includes a heavy chain and a light chain having sequences set forth in SEQ ID NO:76 and SEQ ID NO:77, respectively, or an antigen-binding fragment, Fab fragment, single-chain variable fragment (scFv), variant, or conjugate thereof. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 99% identical to the sequences set forth in SEQ ID NO:76 and SEQ ID NO:77, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 98% identical to the sequences set forth in SEQ ID NO:76 and SEQ ID NO:77, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 97% identical to the sequences set forth in SEQ ID NO:76 and SEQ ID NO:77, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 96% identical to the sequences set forth in SEQ ID NO:76 and SEQ ID NO:77, respectively. In embodiments, the OX40 agonist comprises a heavy chain and a light chain that are each at least 95% identical to the sequences set forth in SEQ ID NO:76 and SEQ ID NO:77, respectively.

In embodiments, the OX40 agonist includes heavy and light chain CDRs or Variable Regions (VRs) of 18D 8. In embodiments, the OX40 agonist heavy chain variable region (V)_H) Including the sequence shown in SEQ ID NO:78, and OX40 agonist light chain variable region (V)_L) Including the sequence shown in SEQ ID NO. 79 and conservative amino acid substitutions thereof. In embodiments, the OX40 agonist comprises a V that is at least 99% identical to the sequences set forth in SEQ ID NO:78 and SEQ ID NO:79, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 98% identical to the sequences set forth in SEQ ID NO:78 and SEQ ID NO:79, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 97% identical to the sequences set forth in SEQ ID NO:78 and SEQ ID NO:79, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 96% identical to the sequences set forth in SEQ ID NO:78 and SEQ ID NO:79, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 95% identical to the sequences set forth in SEQ ID NO:78 and SEQ ID NO:79, respectively_HAnd V_LAnd (4) a zone.

In embodiments, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO:82, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in SEQ ID NO:83, SEQ ID NO:84, and SEQ ID NO:85, respectively, and conservative amino acid substitutions thereof.

In embodiments, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by the drug regulatory agency reference 18D 8. In embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) to an amino acid sequence of a reference drug or reference biological product and comprising one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is 18D 8. In some embodiments, the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation and truncation. In some embodiments, the biological analog is an authorized or submitted authorized OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation that is different from a formulation of a reference drug or reference biological product, wherein the reference drug or reference biological product is 18D 8. OX40 agonist antibodies may be authorized by drug regulatory agencies (e.g., the U.S. FDA and/or eu EMA). In some embodiments, the biosimilar is provided in a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in a reference drug or a reference biological product, wherein the reference drug or reference biological product is 18D 8. In some embodiments, the biosimilar is provided in a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients included in a reference drug or a reference biological product, wherein the reference drug or reference biological product is 18D 8.

Table 15: amino acid sequence of an OX40 agonist antibody related to 18D 8.

In some embodiments, the OX40 agonist is Hu119-122, which is a humanized antibody obtained from GlaxoSmithKline plc. The preparation and properties of Hu119-122 are described in U.S. patent nos. 9,006,399 and 9,163,085, and international patent publication No. WO 2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu119-122 is set forth in Table 16.

In embodiments, an OX40 agonist includes the heavy and light chain CDRs or Variable Regions (VRs) of Hu 119-122. In embodiments, the OX40 agonist heavy chain variable region (V)_H) Including the sequence shown in SEQ ID NO:86, and OX40 agonist light chain variable region (V)_L) Including the sequence shown in SEQ ID NO:87 and conservative amino acid substitutions thereof. In embodiments, the OX40 agonist comprises a V that is at least 99% identical to the sequences set forth in SEQ ID NO:86 and SEQ ID NO:87, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 98% identical to the sequences set forth in SEQ ID NO:86 and SEQ ID NO:87, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 97% identical to the sequences set forth in SEQ ID NO:86 and SEQ ID NO:87, respectively _HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 96% identical to the sequences set forth in SEQ ID NO:86 and SEQ ID NO:87, respectively_HAnd V_LAnd (4) a zone. In embodiments, OX40 agonists includeFrom a V at least 95% identical to the sequences shown in SEQ ID NO 86 and SEQ ID NO 87, respectively_HAnd V_LAnd (4) a zone.

In embodiments, the OX40 agonist includes heavy chain CDR1, CDR2, and CDR3 domains and conservative amino acid substitutions thereof having the sequences shown in SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90, respectively, and light chain CDR1, CDR2, and CDR3 domains and conservative amino acid substitutions thereof having the sequences shown in SEQ ID NO:91, SEQ ID NO:92, and SEQ ID NO:93, respectively.

In embodiments, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by the drug regulatory agency with reference to Hu 119-122. In embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) to an amino acid sequence of a reference drug or reference biological product and comprising one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu 119-122. In some embodiments, the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation and truncation. In some embodiments, the biological analog is an authorized or submitted authorized OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation that is different from a formulation of a reference drug or reference biological product, wherein the reference drug or reference biological product is Hu 119-122. OX40 agonist antibodies may be authorized by drug regulatory agencies (e.g., the U.S. FDA and/or eu EMA). In some embodiments, the biosimilar is provided in a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is Hu 119-122. In some embodiments, the biosimilar is provided in a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is Hu 119-122.

Table 16: amino acid sequence of an OX40 agonist antibody related to Hu 119-122.

In some embodiments, the OX40 agonist is Hu106-222, which is a humanized antibody obtained from glatiramer. The preparation and properties of Hu106-222 are described in U.S. patent nos. 9,006,399 and 9,163,085, and international patent publication No. WO 2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu106-222 is set forth in Table 17.

In embodiments, an OX40 agonist includes the heavy and light chain CDRs or Variable Regions (VRs) of Hu 106-222. In embodiments, the OX40 agonist heavy chain variable region (V)_H) Including the sequence shown in SEQ ID NO:94, and OX40 agonist light chain variable region (V)_L) Including the sequence shown in SEQ ID NO. 95 and conservative amino acid substitutions thereof. In embodiments, the OX40 agonist comprises a V that is at least 99% identical to the sequences set forth in SEQ ID NO:94 and SEQ ID NO:95, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 98% identical to the sequences set forth in SEQ ID NO:94 and SEQ ID NO:95, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 97% identical to the sequences set forth in SEQ ID NO:94 and SEQ ID NO:95, respectively _HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 96% identical to the sequences set forth in SEQ ID NO:94 and SEQ ID NO:95, respectively_HAnd V_LAnd (4) a zone. In embodiments, the OX40 agonist comprises a V that is at least 95% identical to the sequences set forth in SEQ ID NO:94 and SEQ ID NO:95, respectively_HAnd V_LAnd (4) a zone.

In embodiments, the OX40 agonist includes heavy chain CDR1, CDR2, and CDR3 domains and conservative amino acid substitutions thereof having the sequences shown in SEQ ID NO 96, SEQ ID NO 97, and SEQ ID NO 98, respectively, and light chain CDR1, CDR2, and CDR3 domains and conservative amino acid substitutions thereof having the sequences shown in SEQ ID NO 99, SEQ ID NO 100, and SEQ ID NO 101, respectively.

In embodiments, the OX40 agonist is an OX40 agonist bio-analog monoclonal antibody approved by the drug regulatory agency with reference to Hu 106-222. In embodiments, the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) to an amino acid sequence of a reference drug or reference biological product and comprising one or more post-translational modifications as compared to the reference drug or reference biological product, wherein the reference drug or reference biological product is Hu 106-222. In some embodiments, the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation and truncation. In some embodiments, the biological analog is an authorized or submitted authorized OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation that is different from a formulation of a reference drug or reference biological product, wherein the reference drug or reference biological product is Hu 106-222. OX40 agonist antibodies may be authorized by drug regulatory agencies (e.g., the U.S. FDA and/or eu EMA). In some embodiments, the biosimilar is provided in a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is Hu 106-222. In some embodiments, the biosimilar is provided in a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients contained in a reference drug or reference biological product, wherein the reference drug or reference biological product is Hu 106-222.

Table 17: amino acid sequence of an OX40 agonist antibody related to Hu 106-222.

In some embodiments, the OX40 agonist antibody is MEDI6469 (also referred to as 9B 12). MEDI6469 is a murine monoclonal antibody. Weinberg et al, journal of immunotherapy 2006,29,575 and 585. In some embodiments, the OX40 agonist is an antibody produced by the 9B12 hybridoma deposited by Biovest, malvern, massachusetts, usa, as described in Weinberg et al, journal of immunotherapeutics 2006,29,575-585, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the antibody comprises CDR sequences of MEDI 6469. In some embodiments, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of MEDI 6469.

In embodiments, the OX40 agonist is L106 BD (Pharmingen product No. 340420). In some embodiments, the OX40 agonist comprises the CDRs of antibody L106(BD Pharmingen product No. 340420). In some embodiments, the OX40 agonist comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody L106(BD Pharmingen product No. 340420). In an embodiment, the OX40 agonist is ACT35 (Santa Cruz Biotechnology, catalog No. 20073). In some embodiments, the OX40 agonist comprises the CDRs of antibody ACT35 (santa cruz biotechnology company, catalog number 20073). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody ACT35 (santa cruz biotechnology company, catalog number 20073). In the examples, the OX40 agonist was murine monoclonal antibody anti-mCD 134/mOX40 (clone OX86) commercially available from BioXcell corporation of West libamon, new hampshire, InVivoMAb (InVivoMAb, BioXcell Inc, West Lebanon, NH).

In embodiments, the OX40 agonist is selected from the group consisting of OX40 agonists described in: international patent application publication nos. WO 95/12673, WO 95/21925, WO 2006/121810, WO 2012/027328, WO 2013/028231, WO 2013/038191 and WO 2014/148895; european patent application EP 0672141; U.S. patent application publication nos. US 2010/136030, US 2014/377284, US 2015/190506 and US 2015/132288 (including clones 20E5 and 12H 3); and U.S. patent nos. 7,504,101, 7,550,140, 7,622,444, 7,696,175, 7,960,515, 7,961,515, 8,133,983, 9,006,399, and 9,163,085, the disclosures of which are incorporated herein by reference in their entirety.

In embodiments, the OX40 agonist is an OX40 agonist fusion protein as depicted in structure I-a (C-terminal Fc antibody fragment fusion protein) or structure I-B (N-terminal Fc antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or biological analog thereof. The nature of structures I-A and I-B is described above and in U.S. Pat. Nos. 9,359,420, 9,340,599, 8,921,519 and 8,450,460, the disclosures of which are incorporated herein by reference. The amino acid sequence of the polypeptide domain of structure I-A is given in Table 9. The Fc domain preferably comprises the entire constant domain (amino acids 17-230 of SEQ ID NO: 31), the entire hinge domain (amino acids 1-16 of SEQ ID NO: 31), or a portion of the hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 31). Preferred linkers for linking the C-terminal Fc antibody may be selected from the examples given in SEQ ID NO:32 to SEQ ID NO:41, including linkers suitable for fusing further polypeptides. Similarly, the amino acid sequence of the polypeptide domain of structure I-B is given in Table 10. If the Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein as in structure I-B, the sequence of the Fc module is preferably the sequence shown in SEQ ID NO:42 and the linker sequence is preferably selected from those examples shown in SED ID NO:43 through SEQ ID NO: 45.

In embodiments, an OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40 binding domains selected from the group consisting of: the variable heavy and variable light chain of tavalizumab, the variable heavy and variable light chain of 11D4, the variable heavy and variable light chain of 18D8, the variable heavy and variable light chain of Hu119-122, the variable heavy and variable light chain of Hu106-222, the variable heavy and variable light chain selected from the variable heavy and variable light chains described in table 17, any combination of the foregoing variable heavy and variable light chains, and fragments, derivatives, conjugates, variants, and biosimilars thereof.

In embodiments, an OX40 agonist fusion protein according to structure I-A or I-B includes one or more OX40 binding domains comprising an OX40L sequence. In embodiments, an OX40 agonist fusion protein according to structure I-A or I-B includes one or more OX40 binding domains comprising a sequence according to SEQ ID NO: 102. In embodiments, an OX40 agonist fusion protein according to structure I-A or I-B includes one or more OX40 binding domains comprising a soluble OX40L sequence. In embodiments, an OX40 agonist fusion protein according to structure I-A or I-B includes one or more OX40 binding domains comprising a sequence according to SEQ ID NO: 103. In embodiments, an OX40 agonist fusion protein according to structure I-A or I-B includes one or more OX40 binding domains comprising a sequence according to SEQ ID NO: 104.

In embodiments, OX40 agonist fusion proteins according to structure I-A or I-B include one or more OX40 binding domains that are V-comprising sequences at least 95% identical to the sequences set forth in SEQ ID NO:58 and SEQ ID NO:59, respectively_HAnd V_LA scFv domain of region wherein V_HAnd V_LThe domains are connected by a linker. In embodiments, OX40 agonist fusion proteins according to structure I-A or I-B include one or more OX40 binding domains that are V-comprising sequences at least 95% identical to the sequences set forth in SEQ ID NO:68 and SEQ ID NO:69, respectively_HAnd V_LA scFv domain of region wherein V_HAnd V_LThe domains are connected by a linker. In embodiments, OX40 agonist fusion proteins according to structure I-A or I-B include one or more OX40 binding domains that are V-comprising sequences at least 95% identical to the sequences set forth in SEQ ID NO:78 and SEQ ID NO:79, respectively_HAnd V_LA scFv domain of region wherein V_HAnd V_LThe domains are connected by a linker. In embodiments, OX40 agonist fusion proteins according to structure I-A or I-B include one or more OX40 binding domains that are V-comprising sequences at least 95% identical to the sequences set forth in SEQ ID NO:86 and SEQ ID NO:87, respectively _HAnd V_LA scFv domain of region wherein V_HAnd V_LThe domains are connected by a linker. In embodiments, an OX40 agonist fusion protein according to structure I-A or I-B includes aOne or more OX40 binding domains that are V comprising at least 95% identity to the sequences set forth in SEQ ID NO:94 and SEQ ID NO:95, respectively_HAnd V_LA scFv domain of region wherein V_HAnd V_LThe domains are connected by a linker. In embodiments, OX40 agonist fusion proteins according to structure I-A or I-B include one or more OX40 binding domains that are fusion proteins comprising V each as set forth in Table 14_HAnd V_LV with at least 95% sequence identity_HAnd V_LA scFv domain of region wherein V_HAnd V_LThe domains are connected by a linker.

Table 18: can be used as additional polypeptide domains of OX40 binding domains (e.g., structures I-A and I-B) or scFv OX40 agonist antibodies in fusion proteins.

In embodiments, the OX40 agonist is an OX40 agonist single chain fusion polypeptide comprising (i) a first soluble OX40 binding domain, (ii) a first peptide linker, (iii) a second soluble OX40 binding domain, (iv) a second peptide linker, and (v) a third soluble OX40 binding domain, further comprising an additional domain at the N-terminus and/or C-terminus, and wherein the additional domain is a Fab or Fc fragment domain. In embodiments, the OX40 agonist is an OX40 agonist single chain fusion polypeptide comprising (i) a first soluble OX40 binding domain, (ii) a first peptide linker, (iii) a second soluble OX40 binding domain, (iv) a second peptide linker, and (v) a third soluble OX40 binding domain, further comprising additional domains at the N-terminus and/or C-terminus, wherein the additional domains are Fab or Fc fragment domains, wherein each of the soluble OX40 domains lacks a stem region (which facilitates trimerization and provides a distance to the cell membrane, but is not part of the OX40 binding domain) and the first and second peptide linkers independently have a length of 3-8 amino acids.

In embodiments, the OX40 agonist is an OX40 agonist single chain fusion polypeptide comprising (i) a first soluble Tumor Necrosis Factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain, (iv) a second peptide linker, and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stem region and the first and second peptide linkers independently have a length of 3-8 amino acids, and wherein the TNF superfamily cytokine domain is an OX40 binding domain.

In some embodiments, the OX40 agonist is MEDI 6383. MEDI6383 is an OX40 agonist fusion protein and can be prepared as described in U.S. patent No. 6,312,700, the disclosure of which is incorporated herein by reference.

In embodiments, the OX40 agonist is an OX40 agonist scFv antibody, comprising any of the foregoing V_HDomains and any of the foregoing V_LThe domains are linked.

In an example, the OX40 agonist is the inventive biological laboratory Co. OX40 agonist monoclonal antibody MOM-18455, commercially available from inventive biological laboratory Co. Hill, N.Y.USA.

In one embodiment, the OX40 agonist is the OX40 agonist antibody clone Ber-ACT35, commercially available from BioLegend, Inc., san Diego, Calif.

I. Optional cell viability assay

Optionally, cell viability assays may be performed after priming the first amplification (sometimes referred to as initial bulk amplification) using standard assays known in the art.For example, it is possible to use a TIL with an ontologyThe samples were subjected to a trypan blue exclusion assay, which selectively labeled dead cells and allowed viability assessment. Other assays for testing viability may include, but are not limited to, Alamar blue assay (Alamar blue assay); and MTT assay.

1. Cell counting, viability, flow cytometry

In some embodiments, cell count and/or viability is measured. Expression of markers such as, but not limited to, CD3, CD4, CD8, and CD56, as well as any other marker disclosed or described herein, can be achieved by use of facscan to with antibodies such as, but not limited to, those commercially available from BD Biosciences (BD Biosciences, San Jose, CA) of San Jose, california^TMFlow cytometry (BD biosciences) performed flow cytometry for measurement. Cells may be counted manually using a disposable c-chip hemocytometer (VWR, bardavia, illinois) and viability may be assessed using any method known in the art, including but not limited to trypan blue staining. Cell viability may also be determined based on USSN 15/863,634, which is incorporated herein by reference in its entirety. Cell viability may also be determined based on U.S. patent publication No. 2018/0280436 or international patent publication No. WO/2018/081473, both of which are incorporated herein in their entirety for all purposes.

In some cases, the bulk TIL population may be cryopreserved immediately using the protocol discussed below. Alternatively, the bulk TIL population may be subjected to REP and then cryopreserved as discussed below. Similarly, where a genetically modified TIL is to be used in therapy, the bulk or REP TIL population may be genetically modified for appropriate treatment.

2. Cell culture

In embodiments, methods for expanding TIL (including those discussed above and illustrated in fig. 1, specifically, e.g., fig. 1B and/or fig. 1C) may comprise using about 5,000mL to about 25,000mL of cell culture medium, about 5,000mL to about 10,000mL of cell culture medium, or about 5,800mL to about 8,700mL of cell culture medium. In some embodiments, the medium is a serum-free mediumAnd (5) nutrient base. In some embodiments, the medium in priming the first amplification is serum-free. In some embodiments, the medium in the second amplification is serum-free. In some embodiments, the culturing in the first amplification and the second amplification (also referred to as rapid second amplification) is initiatedIs serum-free. In embodiments, no more than one type of cell culture medium is used to expand the amount of TIL. Any suitable cell culture medium may be used, for example AIM-V cell culture medium (L-glutamine, 50 μ M streptomycin sulfate and 10 μ M gentamicin sulfate) cell culture medium (Invitrogen, carlsbad, ca). In this regard, the methods of the invention advantageously reduce the amount of medium and the amount of type of medium required to expand the amount of TIL. In an embodiment, expanding the amount of TIL may comprise feeding the cells at a frequency of no more than every three days or every four days. Expanding the number of cells in the gas permeable container simplifies the procedure necessary to expand the number of cells by reducing the feeding frequency necessary to expand the cells.

In embodiments, the cell culture medium in the first and/or second gas permeable container is not filtered. The use of unfiltered cell culture medium can simplify the procedures necessary to expand the cell number. In embodiments, the cell culture medium in the first and/or second gas permeable container is devoid of beta-mercaptoethanol (BME).

In an embodiment, the duration of the method comprises obtaining a tumor tissue sample from the mammal; culturing the tumor tissue sample in a first gas-permeable container containing cell culture medium comprising IL-2, 1X antigen-presenting feeder cells, and OKT-3 for a duration of about 1 to 8 days, e.g., about 8 days, as a priming first expansion; transferring the TIL to a second gas permeable container, and expanding the amount of TIL in the second gas permeable container containing cell culture medium comprising IL-2, 2X antigen presenting feeder cells, and OKT-3 for a duration of about 7 to 9 days, e.g., about 7 days, about 8 days, or about 9 days.

In an embodiment, the duration of the method comprises obtaining a tumor tissue sample from the mammal; culturing the tumor tissue sample in a first gas-permeable container containing cell culture medium comprising IL-2, 1X antigen-presenting feeder cells, and OKT-3 for a duration of about 1 to 7 days, e.g., about 7 days, as a priming first expansion; transferring the TIL to a second gas permeable container, and expanding the amount of TIL in the second gas permeable container containing cell culture medium comprising IL-2, 2X antigen presenting feeder cells, and OKT-3 for a duration of about 7 to 9 days, e.g., about 7 days, about 8 days, or about 9 days.

In an embodiment, the duration of the method comprises obtaining a tumor tissue sample from the mammal; culturing the tumor tissue sample in a first gas-permeable container containing cell culture medium comprising IL-2, 1X antigen-presenting feeder cells, and OKT-3 for a duration of about 1 to 7 days, e.g., about 7 days, as a priming first expansion; transferring the TIL to a second gas permeable container, and expanding the amount of TIL in the second gas permeable container containing cell culture medium comprising IL-2, 2X antigen presenting feeder cells, and OKT-3 for a duration of about 7 to 10 days, e.g., about 7 days, about 8 days, about 9 days, or about 10 days.

In an embodiment, the TIL is amplified in a gas permeable container. Gas permeable containers have been used to amplify TIL using methods, compositions, and devices known in the art using PBMCs, including those described in U.S. patent application publication No. 2005/0106717A1, the disclosure of which is incorporated herein by reference. In an embodiment, the TIL is amplified in a gas permeable bag. In an example, TIL is amplified using a cell expansion system that expands TIL in a gas permeable bag, such as Xuri cell expansion system W25 (GE Healthcare). In an embodiment, TIL is amplified using a cell expansion system that expands TIL in a gas permeable bag, such as the WAVE bioreactor system, also known as Xuri cell expansion system W5 (general electric medical group). In an embodiment, the cell expansion system comprises a gas permeable cell bag having a volume selected from the group consisting of: about 100mL, about 200mL, about 300mL, about 400mL, about 500mL, about 600mL, about 700mL, about 800mL, about 900mL, about 1L, about 2L, about 3L, about 4L, about 5L, about 6L, about 7L, about 8L, about 9L, and about 10L.

In the examples, TIL may be amplified in G-Rex flasks (commercially available from Wilson Walf, Inc.). Such embodiments allow for a cell population from about 5 × 10⁵Individual cell/cm²Amplification to 10X 10⁶And 30X 10⁶Individual cell/cm²In the meantime. In an embodiment, this does not require feeding. In the examples, this does not require feeding, as long as the medium resides at a height of about 10cm in the G-Rex flask. In embodiments, this does not require feeding but requires the addition of one or more cytokines. In embodiments, the cytokine may be added in a bolus without mixing the cytokine with the culture medium. Such containers, devices, and methods are known in the art and have been used to amplify TILs, and include the containers, devices, and methods described in: U.S. patent application publication No. US 2014/0377739A1, International publication No. WO 2014/210036A 1, U.S. patent application publication No. US 2013/0115617A 1, International publication No. WO 2013/188427A 1, U.S. patent application publication No. US 2011/0136228A 1, U.S. patent No. US 8,809,050B 2, International publication No. WO 2011/072088A 2, U.S. patent application publication No. US 2016/0208216 a1, U.S. patent application publication No. US 2012/0244133 a1, international publication No. WO 2012/129201 a1, U.S. patent application publication No. US 2013/0102075 a1, U.S. patent No. US 8,956,860B 2, international publication No. WO 2013/173835 a1, U.S. patent application publication No. US 2015/0175966 a1, the disclosures of which are incorporated herein by reference. Such processes are also described in Jin et al, journal of immunotherapy 2012,35: 283-.

Optional genetic engineering of TIL

In some embodiments, the amplification of TILs of the invention is further manipulated, before, during, or after the amplification step, during a closed, sterile manufacturing process, each as provided herein, in order to alter protein expression in a transient manner. In some embodiments, the transiently altered protein expression is due to transient gene editing. In some embodiments, the amplified TILs of the invention are treated with Transcription Factors (TFs) and/or other molecules that are capable of transiently altering the expression of proteins in the TILs. In some embodiments, TF and/or other molecules capable of transiently altering protein expression effect altered tumor antigen expression and/or alteration of the number of tumor antigen-specific T cells in a TIL population.

In certain embodiments, the method comprises gene editing a TIL population. In certain embodiments, the method comprises gene editing the first TIL population, the second TIL population, and/or the third TIL population.

In some embodiments, the invention comprises gene editing by nucleotide insertion, such as by ribonucleic acid (RNA) insertion, comprising inserting messenger RNA (mrna) or small (or short) interfering RNA (sirna) into a TIL population to promote expression of or inhibit expression of one or more proteins, as well as promoting simultaneous combination of both one set of proteins with inhibiting another set of proteins.

In some embodiments, the amplified TILs of the invention undergo transient changes in protein expression. In some embodiments, the transient change in protein expression occurs in the bulk TIL population prior to the first amplification, including, for example, in the TIL population obtained from step a, e.g., as shown in fig. 1 (specifically, fig. 1B and 1C). In some embodiments, the transient change in protein expression occurs during a first amplification, including for example in the TIL population amplified in step B as shown, for example, in fig. 1 (e.g., fig. 1B). In some embodiments, the transient change in protein expression occurs after the first amplification, in a TIL population (e.g., a second TIL population as described herein) that is comprised, for example, in a transition between the first amplification and the second amplification, a TIL population obtained from, for example, step B and comprised in step C as shown in fig. 1. In some embodiments, the transient change in protein expression occurs in the bulk TIL population prior to the second amplification, including, for example, in the TIL population obtained from, for example, step C and prior to its amplification in step D as shown in fig. 1. In some embodiments, the transient change in protein expression occurs during a second amplification, comprised in a TIL population (e.g., a third TIL population) amplified, for example, in step D shown in fig. 1. In some embodiments, the transient change in protein expression occurs after the second amplification, e.g., as in a TIL population obtained from amplification in step D, e.g., as shown in fig. 1.

In embodiments, the method of transiently altering protein expression in a TIL population comprises the step of electroporation. Electroporation methods are known in the art and are described, for example, in Tsong, journal of biophysics (biophysis. j.) 1991,60,297-306, and U.S. patent application publication No. 2014/0227237 a1, the disclosures of each of which are incorporated herein by reference. In an embodiment, the method of transiently altering protein expression in a TIL population comprises the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating and endocytosis) are known in the art and are described in Graham and van der Eb, Virology (Virology) 1973,52, 456-467; wigler et al, Proc. Natl. Acad. Sci., 1979,76, 1373-1376; and Chen and Okayarea, molecular cell biology (mol. cell. biol.) 1987,7, 2745-2752; and U.S. patent No. 5,593,875, the disclosures of each of which are incorporated herein by reference. In an embodiment, the method of transiently altering protein expression in a TIL population comprises the step of lipofection. Lipofectin, such as 1:1(w/w) liposome formulations of the cationic lipid N- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA) with Dioleoylphosphatidylethanolamine (DOPE) in filtered water, are known in the art and described in Rose et al, Biotech (Biotechniques) 1991,10, 520-7417 and Felgner et al, Proc. Natl. Acad. Sci. USA, 1987,84,7413-7417 and U.S. Pat. No. 5,279,833; 5,908,635 No; 6,056,938 No; 6,110,490 No; 6,534,484 No; and 7,687,070, the disclosures of each of which are incorporated herein by reference. In embodiments, methods of transiently altering protein expression in a TIL population comprise the use of U.S. patent No. 5,766,902; 6,025,337 No; 6,410,517 No; 6,475,994 No; and No. 7,189,705; the methods described in (a), the disclosure of each of which is incorporated herein by reference.

In some embodiments, the transient alteration in protein expression results in an increase in stem memory T cells (TSCMs). TSCM is an early progenitor of central memory T cells that undergo antigen. TSCMs typically exhibit the ability to define long-term survival, self-renewal, and pluripotency of stem cells, and are generally desirable for producing effective TIL products. TSCM has shown enhanced anti-tumor activity compared to other T cell subsets in a mouse model of adoptive cell metastasis (Gattinoni et al, Nature medicine (Nat Med) 2009,2011; Gattinoni, Nature Rev. cancer reviews, 2012; Cieri et al, Blood (Blood) 2013). In some embodiments, transient changes in protein expression result in TIL populations with compositions that include a high proportion of TSCMs. In some embodiments, the transient alteration in protein expression results in an increase in the percentage of TSCM of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, the transient alteration in protein expression results in an increase in TSCM in the TIL population of at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold. In some embodiments, the transient alteration in protein expression produces a TIL population having at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCM. In some embodiments, the transient alteration in protein expression produces a therapeutic TIL population having at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% TSCM.

In some embodiments, the transient alteration in protein expression results in T cell regeneration (rejuvenation) that undergoes antigen. In some embodiments, regeneration comprises, for example, increased proliferation, increased T cell activation, and/or increased antigen recognition.

In some embodiments, the transient change in protein expression alters expression of a majority of T cells in order to preserve the tumor-derived TCR repertoire. In some embodiments, the transient alteration in protein expression does not alter the tumor-derived TCR repertoire. In some embodiments, the transient change in protein expression maintains a tumor-derived TCR repertoire.

In some embodiments, the transient alteration of the protein results in altered expression of a particular gene. In some embodiments, transiently altered targeted genes of protein expression include, but are not limited to, PD-1 (also referred to as PDCD1 or CC279), TGFBR2, CCR4/5, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2ICD, TIM3, LAG3, TIGIT, TGF β, CCR2, CCR4, CCR5, CXCR1, 2, CSCR3, CCL2(MCP-1), CCL3(MIP-1 α), CCL4(MIP1- β), CCL5(RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, and/or cAMP protein kinase a (pka). In some embodiments, the transient alteration in protein expression targets a gene selected from the group consisting of: PD-1, TGFBR2, CCR4/5, CBLB (CBL-B), CISH, CCR (chimeric costimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2ICD, TIM3, LAG3, TIGIT, TGF β, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2(MCP-1), CCL3(MIP-1 α), CCL4(MIP1- β), CCL5(RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1 and/or cAMP Protein Kinase A (PKA). In some embodiments, the transient alteration in protein expression targets PD-1. In some embodiments, the transient change in protein expression targets TGFBR 2. In some embodiments, the transient alteration of protein expression targets CCR 4/5. In some embodiments, the transient change in protein expression targets CBLB. In some embodiments, the transient alteration in protein expression targets CISH. In some embodiments, the transient alteration of protein expression targets CCR (chimeric co-stimulatory receptor). In some embodiments, the transient alteration in protein expression targets IL-2. In some embodiments, the protein expression transient change targeting IL-12. In some embodiments, the transient alteration in protein expression targets IL-15. In some embodiments, the transient alteration in protein expression targets IL-21. In some embodiments, the transient change in protein expression targets NOTCH 1/2 ICD. In some embodiments, transient changes in protein expression target TIM 3. In some embodiments, the transient alteration in protein expression targets LAG 3. In some embodiments, the transient alteration in protein expression targets TIGIT. In some embodiments, the transient alteration in protein expression targets TGF β. In some embodiments, the transient alteration of protein expression targets CCR 1. In some embodiments, the transient alteration of protein expression targets CCR 2. In some embodiments, the transient alteration of protein expression targets CCR 4. In some embodiments, the transient alteration of protein expression targets CCR 5. In some embodiments, the transient alteration in protein expression targets CXCR 1. In some embodiments, the transient alteration in protein expression targets CXCR 2. In some embodiments, the transient alteration in protein expression targets CSCR 3. In some embodiments, the transient alteration in protein expression targets CCL2 (MCP-1). In some embodiments, the transient change in protein expression targets CCL3(MIP-1 α). In some embodiments, the transient change in protein expression targets CCL4(MIP1- β). In some embodiments, the transient alteration in protein expression targets CCL5 (RANTES). In some embodiments, the transient change in protein expression targets CXCL 1. In some embodiments, the transient change in protein expression targets CXCL 8. In some embodiments, the transient alteration in protein expression targets CCL 22. In some embodiments, the transient alteration in protein expression targets CCL 17. In some embodiments, the transient alteration of protein expression targets VHL. In some embodiments, the transient alteration in protein expression targets CD 44. In some embodiments, the transient alteration in protein expression targets PIK3 CD. In some embodiments, the transient alteration in protein expression targets SOCS 1. In some embodiments, the transient change in protein expression targets cAMP protein kinase a (pka).

In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of a chemokine receptor. In some embodiments, chemokine receptors that are overexpressed by transient protein expression comprise receptors with ligands including, but not limited to, CCL2(MCP-1), CCL3(MIP-1 α), CCL4(MIP1- β), CCL5(RANTES), CXCL1, CXCL8, CCL22, and/or CCL 17.

In some embodiments, the transient alteration in protein expression results in a decrease in PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGF β R2, and/or TGF β and/or a decrease in expression (including, for example, causing blockade of the TGF β pathway). In some embodiments, the transient alteration in protein expression results in a reduction in CBLB (CBL-B) and/or a reduction in expression. In some embodiments, the transient alteration in protein expression results in a decrease in CISH and/or decreased expression.

In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of chemokine receptors, for example, to improve TIL trafficking or movement to a tumor site. In some embodiments, transient changes in protein expression result in increased and/or overexpression of CCR (chimeric co-stimulatory receptor). In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of a chemokine receptor selected from the group consisting of: CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, and/or CSCR 3.

In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of an interleukin. In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of an interleukin selected from the group consisting of: IL-2, IL-12, IL-15 and/or IL-21.

In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of NOTCH 1/2 ICD. In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of VHL. In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of CD 44. In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of PIK3 CD. In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of SOCS 1.

In some embodiments, the transient change in protein expression results in a decrease in cAMP protein kinase a (pka) and/or decreased expression.

In some embodiments, the transient alteration in protein expression results in a decrease in expression and/or a decrease in expression of a molecule selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease in expression and/or a decrease in expression of two molecules selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease in PD-1 and a molecule selected from the group consisting of: LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the transient alteration in protein expression results in a reduction in PD-1, LAG-3, CISH, CBLB, TIM3, and combinations thereof and/or a decrease in expression. In some embodiments, the transient alteration in protein expression results in a reduction in one of PD-1 and LAG3, CISH, CBLB, TIM3, and combinations thereof and/or decreased expression. In some embodiments, the transient alteration in protein expression results in a reduction in PD-1 and LAG3 and/or a decrease in expression. In some embodiments, the transient alteration in protein expression results in a reduction in PD-1 and CISH and/or a decrease in expression. In some embodiments, the transient alteration in protein expression results in a reduction in PD-1 and CBLB and/or a decrease in expression. In some embodiments, the transient alteration in protein expression results in a reduction in LAG3 and CISH and/or decreased expression. In some embodiments, the transient alteration in protein expression results in a reduction in LAG3 and CBLB and/or a decrease in expression. In some embodiments, the transient alteration in protein expression results in a reduction in CISH and CBLB and/or a decrease in expression. In some embodiments, the transient alteration in protein expression results in a reduction in TIM3 and PD-1 and/or decreased expression. In some embodiments, the transient alteration in protein expression results in a reduction in TIM3 and LAG3 and/or decreased expression. In some embodiments, the transient alteration in protein expression results in a reduction in TIM3 and CISH and/or a decrease in expression. In some embodiments, the transient change in protein expression results in a reduction in TIM3 and CBLB and/or a decrease in expression.

In some embodiments, an adhesion molecule selected from the group consisting of seq id no, or any combination thereof is inserted into the first TIL population, the second TIL population, or the harvested TIL population by a gammaretrovirus or lentivirus method (e.g., increasing expression of the adhesion molecule): CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

In some embodiments, the transient alteration in protein expression results in a decrease in expression and/or a decrease in expression of a molecule selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3) and combinations thereof, and the increase and/or expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1 and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease in expression and/or a decrease in expression of a molecule selected from the group consisting of: PD-1, LAG3, TIM3, CISH, CBLB and combinations thereof, and the increase and/or expression enhancement of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1 and combinations thereof.

In some embodiments, the reduction in expression is about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the reduction in expression is at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the reduction in expression is at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the reduction in expression is at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, the reduction in expression is at least about 85%, about 90%, or about 95%. In some embodiments, the reduction in expression is at least about 80%. In some embodiments, the reduction in expression is at least about 85%. In some embodiments, the reduction in expression is at least about 90%. In some embodiments, the reduction in expression is at least about 95%. In some embodiments, the reduction in expression is at least about 99%.

In some embodiments, the increase in expression is about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the increase in expression is at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the increase in expression is at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the increase in expression is at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, the increase in expression is at least about 85%, about 90%, or about 95%. In some embodiments, the increase in expression is at least about 80%. In some embodiments, the increase in expression is at least about 85%. In some embodiments, the increase in expression is at least about 90%. In some embodiments, the increase in expression is at least about 95%. In some embodiments, the increase in expression is at least about 99%.

In some embodiments, the transient change in protein expression is induced by treating the TIL with a Transcription Factor (TF) and/or other molecule capable of transiently changing protein expression in the TIL. In some embodiments, the SQZ vector-free microfluidic platform is used for intracellular delivery of Transcription Factors (TFs) and/or other molecules capable of transiently altering protein expression. Such methods have been described that demonstrate the ability to deliver proteins (including transcription factors) to a variety of naive human cells (including T cells) (Sharei et al, journal of the american national academy of sciences 2013, and Sharei et al, public science library synthesis 2015 and Greisbeck et al, journal of immunology 195, 2015); see, for example, international patent publications WO 2013/059343a1, WO 2017/008063a1, and WO 2017/123663a1, all of which are incorporated herein by reference in their entirety. Such methods as described in international patent publications WO 2013/059343a1, WO 2017/008063a1 and WO 2017/123663a1 may be used in the present invention to expose a TIL population to Transcription Factors (TFs) and/or other molecules capable of inducing transient protein expression, wherein the TFs and/or other molecules capable of inducing transient protein expression achieve increased tumor antigen expression and/or an increase in the number of tumor antigen-specific T cells in the TIL population, thereby causing reprogramming of the TIL population and an increase in the efficacy of reprogramming the TIL population as compared to a non-reprogrammed TIL population. In some embodiments, reprogramming results in an increase in a subpopulation of effector T cells and/or central memory T cells relative to an initial or previous population of TILs (i.e., prior to reprogramming), as described herein.

In some embodiments, Transcription Factors (TF) include, but are not limited to, TCF-1, NOTCH 1/2 ICD, and/or MYB. In some embodiments, the Transcription Factor (TF) is TCF-1. In some embodiments, the Transcription Factor (TF) is NOTCH 1/2 ICD. In some embodiments, the Transcription Factor (TF) is MYB. In some embodiments, the Transcription Factor (TF) is administered with an induced pluripotent stem cell culture (iPSC), such as a commercially available knockout serum replacement (Gibco/Saimer Feishale), in some embodiments, Transcription Factors (TF) are administered with the iPSC mixture to induce additional TIL reprogramming, in some embodiments, the Transcription Factor (TF) is administered without the iPSC mixture in some embodiments, in some embodiments, reprogramming results in an increase in the percentage of TSCM of about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% TSCM.

In some embodiments, as described above, the method of transiently altering protein expression may be combined with a method of genetically modifying a TIL population comprising the step of stably incorporating a gene for producing one or more proteins. In certain embodiments, the method comprises the step of genetically modifying the TIL population. In certain embodiments, the method comprises genetically modifying the first TIL population, the second TIL population, and/or the third TIL population. In one embodiment, the method of genetically modifying a TIL population comprises the step of retroviral transduction. In one embodiment, the method of genetically modifying a TIL population comprises the step of lentiviral transduction. Levine transduction systems are known in the art and are described, for example, in Levine et al, Proc. Natl. Acad. Sci. USA 2006,103,17372-77; zufferey et al, Nature Biotechnology (nat. Biotechnol.) 1997,15, 871-75; dull et al, journal of virology (j.virology) 1998,72,8463-71, and U.S. patent No. 6,627,442, the disclosures of each of which are incorporated herein by reference. In an embodiment, the method of genetically modifying a TIL population comprises the step of gamma retroviral transduction. Gamma retrovirus transduction systems are known in the art and are described, for example, in Cepko and Pear, Current protocols for molecular biology (Cur. prot. mol. biol.) 1996,9.9.1-9.9.16, the disclosures of which are incorporated herein by reference. In embodiments, the method of genetically modifying a TIL population comprises the step of transposon-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art and include the following: wherein the transposase is provided in the form of a DNA expression vector or in the form of expressible RNA or protein such that long term expression of the transposase does not occur in the transgenic cell, e.g., a transposase provided in the form of mRNA (e.g., mRNA including cap and poly-a tail). Suitable transposon-mediated gene transfer systems comprising salmonidae fish type Tel-like transposases (SB/Sleeping Beauty transposases), such as SB10, SB11 and SB100x, and engineered enzymes with increased enzymatic activity are described, for example, in hacett et al, molecular therapy (mol. therapy) 2010,18,674-83 and U.S. patent No. 6,489,458, the disclosures of each of which are incorporated herein by reference.

In some embodiments, the transient alteration in protein expression is a decrease in expression induced by self-delivered RNA interference (sdRNA) with a high percentage of 2' -OH substitutions (typically fluoro or-OCH)₃) The chemically synthesized asymmetric siRNA double helix of (1), comprising a 20 nucleotide antisense (guide) strand and a 13 to 15 base sense (passenger) strand conjugated to cholesterol at its 3' end using a tetraethylene glycol (TEG) linker. In some embodiments, the methods comprise transiently altering protein expression in a TIL population, including the use of self-delivering RNA interference (sdRNA) with a high percentage of 2-OH substitution (usually fluorine or-OCH)₃) The chemically synthesized asymmetric siRNA duplex of (1), comprising a 20 nucleotide antisense (guide) strand and a 13 to 15 base sense (passenger) strand conjugated to cholesterol at its 3' end using a tetraethylene glycol (TEG) linker. Methods of using sdRNA have been described in Khvorova and Watts, Nature Biotechnology 2017,35, 238-248; byrne et al, J.Ocular. Pharmacol. The.) -2013, 29, 855-864; and Ligtenberg et al, molecular therapy 2018 (in print), the disclosures of which are incorporated herein by reference. In embodiments, delivery of sdRNA to a TIL population is accomplished without the use of electroporation, SQZ, or other methods, instead using a1 to 3 day time period, wherein the TIL population is exposed to sdRNA in culture medium at a concentration of 1 μ Μ/10,000 TILs. In certain embodiments, the method comprises delivering sdRNA to a TIL population comprising exposing the TIL population to sdRNA in culture medium at a concentration of 1 μ Μ per 10,000 TILs for a period of between 1 and 3 days. In embodiments, delivery of sdRNA to a TIL population is accomplished using a period of 1 to 3 days, wherein the TIL population is exposed to sdRNA in culture medium at a concentration of 10 μ Μ per 10,000 TILs. In embodiments, delivery of sdRNA to a TIL population is accomplished using a period of 1 to 3 days, wherein the TIL population is exposed to sdRNA in culture medium at a concentration of 50 μ Μ per 10,000 TILs. In embodiments, delivery of sdRNA to the TIL population is accomplished using a period of 1 to 3 days, wherein the TIL population is exposed to sdRNA in the culture medium at a concentration of between 0.1 μ Μ/10,000 TILs and 50 μ Μ/10,000 TILs. In embodiments, the delivery of sdRNA to the TIL population is accomplished using a period of 1 to 3 days, wherein the TIL population is exposed to sdRNA in a culture medium at a concentration of between 0.1 μ Μ/10,000 TILs and 50 μ Μ/10,000 TILs, wherein the exposure to sdRNA is performed two, three, four or five times by adding fresh sdRNA to the culture medium. Other suitable processes are described, for example, in U.S. patent application publication nos. US 2011/0039914 a1, US 2013/0131141 a1, and US 2013/0131142 a1, and US 9,080,171, the disclosures of which are incorporated herein by reference.

In some embodiments, the sdRNA is inserted into the TIL population during manufacture. In some embodiments, the sdRNA encodes RNA that interferes with NOTCH 1/2ICD, PD-1, CTLA-4 TIM-3, LAG-3, TIGIT, TGF β, TGFBR2, cAMP Protein Kinase A (PKA), BAFF BR3, CISH, and/or CBLB. In some embodiments, the reduction in expression is determined based on the percentage of gene silencing, e.g., as assessed by flow cytometry and/or qPCR. In some embodiments, the reduction in expression is about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the reduction in expression is at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the reduction in expression is at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the reduction in expression is at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, the reduction in expression is at least about 85%, about 90%, or about 95%. In some embodiments, the reduction in expression is at least about 80%. In some embodiments, the reduction in expression is at least about 85%. In some embodiments, the reduction in expression is at least about 90%. In some embodiments, the reduction in expression is at least about 95%. In some embodiments, the reduction in expression is at least about 99%.

Self-deliverable RNAi technology based on chemically modified siRNA can be used in the methods of the invention to successfully deliver sdRNA to TILs as described herein. The combination of backbone modifications with asymmetric siRNA structures and hydrophobic ligands (see, e.g., Ligtenberg et al, molecular therapy, 2018 and US20160304873) allows sdRNA to permeate cultured mammalian cells with its nuclease stability by simple addition to the culture medium without additional formulations and methods. This stability allows to support a constant level of RNAi-mediated reduction of target gene activity by merely maintaining the active concentration of sdRNA in the culture medium. While not being bound by theory, the backbone of sdRNA stabilizes to achieve extended reduction in gene expression effects, which can last for months in non-dividing cells.

In some embodiments, greater than 95% TIL transfection efficiency and reduced expression of the target by each particular sdRNA occurs. In some embodiments, sdrnas containing several unmodified ribose residues are replaced with fully modified sequences to increase the efficacy and/or longevity of the RNAi effect. In some embodiments, the reduction in expression effect is maintained for 12 hours, 24 hours, 36 hours, 48 hours, 5 days, 6 days, 7 days, or 8 days or more. In some embodiments, the reduction in expression effect is reduced 10 days or more after sdRNA treatment of TIL. In some embodiments, the reduction in expression maintaining target expression is greater than 70%. In some embodiments, the reduction in expression of target expression by TIL is maintained by more than 70%. In some embodiments, the reduced expression in the PD-1/PD-L1 pathway allows the TIL to exhibit more potent in vivo effects, in some embodiments, due to the avoidance of inhibitory effects of the PD-1/PD-L1 pathway. In some embodiments, decreased expression of PD-1 by sdRNA results in increased proliferation of TIL.

Small interfering RNA (siRNA) (sometimes referred to as short interfering RNA or silencing RNA) are double-stranded RNA molecules, typically 19-25 base pairs in length. siRNA is used in RNA interference (RNAi), where it interferes with the expression of a specific gene having a complementary nucleotide sequence.

Double-stranded dna (dsrna) may generally be used to define any molecule comprising a pair of complementary RNA strands, typically the sense (passenger) and antisense (guide) strands, and may comprise a single-stranded overhang region. In contrast to siRNA, the term dsRNA generally refers to a precursor molecule comprising the sequence of an siRNA molecule that is released from a larger dsRNA molecule by the action of a lytic enzyme system comprising Dicer.

sdRNA (self-deliverable RNA) is a new class of covalently modified RNAi compounds that do not require delivery of a vehicle into the cell and have improved pharmacology compared to traditional siRNA. "self-deliverable RNA" or "sdRNA" is a hydrophobically modified RNA interference-antisense hybrid that has proven to be highly effective both in initial cells in vitro and following topical administration in vivo. Robust absorption and/or silencing has been demonstrated without toxicity. sdRNA is typically an asymmetric chemically modified nucleic acid molecule with a minimal double-stranded region. sdRNA molecules typically contain single-stranded and double-stranded regions, and can contain various chemical modifications within both the single-stranded and double-stranded regions of the molecule. In addition, the sdRNA molecule can be linked to a hydrophobic conjugate, e.g., conventional and higher sterol-type molecules, as described herein. sdRNA and related methods for making such sdRNA have also been described extensively, e.g., in US20160304873, WO 2010033246, WO 2017070151, WO 2009102427, WO 2011119887, WO 2010033247a2, WO 2009045457, WO 2011119852, all of which are incorporated herein by reference in their entirety for all purposes. To optimize sdRNA structure, chemical reactions, targeting location, sequence preferences, etc., proprietary algorithms were developed and used for sdRNA potency prediction (see, e.g., US 20160304873). Based on these analyses, functional sdRNA sequences are generally defined as expression decreases by more than 70% at a concentration of 1. mu.M, with a probability of more than 40%.

In some embodiments, the sdRNA sequences used in the invention exhibit a 70% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit a 75% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit an 80% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit 85% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit a 90% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit a 95% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit 99% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 0.25 μ Μ to about 4 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 0.25 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 0.5 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 0.75 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 1.0 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 1.25 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 1.5 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 1.75 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 2.0 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 2.25 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 2.5 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 2.75 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 3.0 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 3.25 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 3.5 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 3.75 μ Μ. In some embodiments, the sdRNA sequences used in the invention exhibit reduced expression of the target gene when delivered at a concentration of about 4.0 μ Μ.

In some embodiments, the oligonucleotide agent includes one or more modifications to increase the stability and/or effectiveness of the therapeutic agent and to achieve effective delivery of the oligonucleotide to the cell or tissue to be treated. Such modifications may comprise 2' -O-methyl modifications, 2' -O-fluoro modifications, phosphorodithioate modifications, 2' F modified nucleotides, 2' -O-methyl modified nucleotides and/or 2' deoxynucleotides. In some embodiments, the oligonucleotide is modified to include one or more hydrophobic modifications, including, for exampleSterols, cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan and/or phenyl. In further particular embodiments, the chemically modified nucleotide is a combination of phosphorothioate, 2 '-O-methyl, 2' deoxy, hydrophobic modification, and phosphorothioate. In some embodiments, the saccharide may be modified and the modified saccharide may include, but is not limited to, D-ribose, 2' -O-alkyl (including 2' -O-methyl and 2' -O-ethyl) (i.e., 2' -alkoxy), 2' -amino, 2' -S-alkyl, 2' -halo (including 2' -fluoro), T-methoxyethoxy, 2' -allyloxy (-OCH), and the like₂CH＝CH₂) 2 '-propargyl, 2' -propyl, ethynyl, ethenyl, propenyl, cyano and the like. In one embodiment, the sugar moiety may be a hexose and incorporated into an oligonucleotide as described (Augustyns, K. et al, nucleic acids research (Nucl. acids. Res.) 18:4711 (1992)).

In some embodiments, the double-stranded oligonucleotides of the invention are double-stranded over their entire length, i.e., do not have a protruding single-stranded sequence at either end of the molecule, i.e., are blunt-ended. In some embodiments, individual nucleic acid molecules may have different lengths. In other words, the double-stranded oligonucleotide of the present invention is not double-stranded over its entire length. For example, when two separate nucleic acid molecules are used, one of the molecules (e.g., the first molecule comprising the antisense sequence) may be longer than the second molecule to which it hybridizes (leaving a portion of the single-stranded molecule). In some embodiments, when a single nucleic acid molecule is used, a portion of the molecule at either end may remain single stranded.

In some embodiments, a double-stranded oligonucleotide of the invention contains mismatches and/or loops or bulges, but is double-stranded over at least about 70% of the length of the oligonucleotide. In some embodiments, a double-stranded oligonucleotide of the invention is double-stranded over at least about 80% of the length of the oligonucleotide. In another embodiment, a double-stranded oligonucleotide of the invention is double-stranded over at least about 90% -95% of the length of the oligonucleotide. In some embodiments, a double-stranded oligonucleotide of the invention is double-stranded over at least about 96-98% of the length of the oligonucleotide. In some embodiments, a double-stranded oligonucleotide of the invention contains at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.

In some embodiments, oligonucleotides can be substantially protected from nucleases, e.g., by modifying the 3 'or 5' linkage (e.g., U.S. Pat. No. 5,849,902 and WO 98/13526). For example, oligonucleotides can be made resistant by incorporating "blocking groups". The term "end capping group" as used herein refers to a protecting or coupling group that may serve as a protecting group for synthesis (e.g., FITC, propyl (CH)₂-CH₂-CH₃) Ethylene glycol (-O-CH)₂-CH₂-O-) Phosphate (PO)₃ ^2") Hydrogen phosphonates or amino phosphites) to the oligonucleotide or to the substituents of the core monomer (e.g., in addition to the OH group). "end capping groups" may also comprise "terminal end capping groups" or "exonuclease end capping groups" which protect the 5 'and 3' ends of oligonucleotides, comprising modified nucleotide and non-nucleotide exonuclease resistant structures.

In some embodiments, at least a portion of consecutive polynucleotides within the sdRNA are connected by substitution linkages, such as phosphorothioate linkages.

In some embodiments, the chemical modification can result in an increase in cellular uptake of at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500. In some embodiments, at least one of the C or U residues comprises a hydrophobic modification. In some embodiments, a plurality of C and U contain hydrophobic modifications. In some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95% of C and U may contain hydrophobic modifications. In some embodiments, all C and U contain hydrophobic modifications.

In some embodiments, the sdRNA or sd-rxRNA exhibits enhanced endosomal release of the sd-rxRNA molecule through incorporation of a protonatable amine. In some embodiments, the protonatable amine is incorporated in the sense strand (in a portion of the molecule that is discarded after RISC loading). In some embodiments, the sdRNA compounds of the invention include asymmetric compounds comprising a duplex region (required for efficient RISC entry with 10-15 base lengths) and a single-stranded region 4-12 nucleotides long; there are 13 nucleotide duplexes. In some embodiments, a 6 nucleotide single stranded region is employed. In some embodiments, the single-stranded region of the sdRNA includes 2-12 phosphorothioate internucleotide linkages (referred to as phosphorothioate modifications). In some embodiments, 6-8 phosphorothioate internucleotide linkages are employed. In some embodiments, the sdRNA compounds of the invention also comprise unique chemical modification patterns that provide stability and are compatible with RISC entry.

For example, the guide strand may also be modified by any chemical modification that confirms stability without interfering with RISC entry. In some embodiments, the chemical modification pattern in the guide strand comprises a majority of C and U nucleotides modified by 2'F and phosphorylated at the 5' end.

In some embodiments, at least 30% of the nucleotides in the sdRNA or sd-rxRNA are modified. In some embodiments, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotides in the sdRNA or sd-rxRNA are modified. In some embodiments, 100% of the nucleotides in the sdRNA or sd-rxRNA are modified.

In some embodiments, the sdRNA molecule has a minimal double-stranded region. In some embodiments, the region of the double-stranded molecule is in the range of 8-15 nucleotides long. In some embodiments, the region of the double-stranded molecule is 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the double-stranded region is 13 nucleotides long. There may be 100% complementarity between the guide strand and the passenger strand, or one or more mismatches between the guide strand and the passenger strand. In some embodiments, at one end of a double-stranded molecule, the molecule is blunt-ended or has a nucleotide overhang. In some embodiments, the single-stranded region of the molecule is between 4-12 nucleotides in length. In some embodiments, the single-stranded region can be 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length. In some embodiments, the single-stranded region can also be less than 4 or greater than 12 nucleotides in length. In certain embodiments, the single-stranded region is 6 or 7 nucleotides long.

In some embodiments, the sdRNA molecule has increased stability. In some cases, the half-life of the chemically modified sdRNA or sd-rxRNA molecule in the culture medium is longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more than 24 hours (including any intermediate values). In some embodiments, the half-life of the sd-rxRNA in the culture medium is longer than 12 hours.

In some embodiments, the sdRNA is optimized for increased potency and/or reduced toxicity. In some embodiments, the nucleotide length of the guide strand and/or the passenger strand and/or the number of phosphorothioate modifications in the guide strand and/or the passenger strand may affect the efficacy of the RNA molecule in some aspects, while replacing the 2 '-fluoro (2' F) modification with a 2 '-O-methyl (2' OMe) modification may affect the toxicity of the molecule in some aspects. In some embodiments, the 2' F content of the molecule is predicted to be reduced to reduce the toxicity of the molecule. In some embodiments, the number of phosphorothioate modifications in the RNA molecule may affect the uptake of the molecule into the cell, e.g., the efficiency of passive uptake of the molecule into the cell. In some embodiments, the sdRNA does not have a 2' F modification, but is characterized by the same efficacy in terms of cellular uptake and tissue penetration.

In some embodiments, the guide strand is about 18-19 nucleotides in length and has about 2-14 phosphate modifications. For example, the guide strand may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 phosphate modified nucleotides. The guide strand may contain one or more modifications that confer increased stability without interfering with RISC entry. The phosphate modified nucleotides (e.g., phosphorothioate modified nucleotides) can be located at the 3 'end, the 5' end, or dispersed throughout the guide strand. In some embodiments, the 10 nucleotides at the 3' terminus of the guide strand contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphorothioate modified nucleotides. The guide strand may also contain 2'F and/or 2' OMe modifications, which may be located throughout the molecule. In some embodiments, the nucleotide at one position in the guide strand (the nucleotide at the closest 5 'position of the guide strand) is modified and/or phosphorylated by a 2' OMe. The C and U nucleotides in the guide strand may be modified by 2' F. For example, the C and U nucleotides at positions 2-10 of a 19nt guide strand (or corresponding positions in guide strands of different lengths) may be modified with 2' F. The C and U nucleotides within the guide strand may also be modified by 2' OMe. For example, the C and U nucleotides at positions 11-18 of a 19nt guide strand (or corresponding positions in guide strands of different lengths) may be modified by a 2' OMe. In some embodiments, the nucleotide closest to the 3' end of the guide strand is unmodified. In certain embodiments, most of the C and U within the guide strand are modified with 2'F, and the 5' end of the guide strand is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 are modified with a 2'OMe, and the 5' end of the guide strand is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 is modified with a 2' OMe, the 5' end of the guide strand is phosphorylated, and the C or U at positions 2-10 is modified with a 2' F.

Self-deliverable RNAi technology provides a method for transfecting cells directly with RNAi agents without the need for additional formulations or techniques. The ability to transfect difficult-to-transfect cell lines, high in vivo activity and ease of use are features of compositions and methods that present significant functional advantages over traditional siRNA-based techniques, and as such the sdRNA method is employed in several embodiments in connection with the methods of reduced expression of target genes in TILs of the invention. The sdRNAi approach allows the direct delivery of chemically synthesized compounds to a wide range of initial cells and tissues (both ex vivo and in vivo). The sdRNAs described herein in some embodiments of the invention are commercially available from Advirna Inc. (Advirna LLC, Worcester, MA, USA), of Worcester, Mass.

sdRNA is formed as a hydrophobically modified siRNA-antisense oligonucleotide hybrid structure and is disclosed, for example, in Byrne et al, 12.2013, J. Ocular Pharmacology and therapeutics, 29(10): 855-.

In some embodiments, sdRNA oligonucleotides can be delivered to TILs described herein using sterile electroporation. In certain embodiments, the method comprises sterile electroporation of the TIL population to deliver the sdRNA oligonucleotides.

In some embodiments, the oligonucleotide may be delivered to the cell in combination with a transmembrane delivery system. In some embodiments, such transmembrane delivery systems include lipids, viral vectors, and the like. In some embodiments, the oligonucleotide agent is a self-delivering RNAi agent that does not require any delivery agent. In certain embodiments, the method comprises delivering the sdRNA oligonucleotides to the TIL population using a transmembrane delivery system.

Oligonucleotides and oligonucleotide compositions are contacted with (e.g., contacted with, also referred to herein as administered or delivered to) and absorbed by TILs as described herein, including passive absorption by TILs. sdRNA can be added to a TIL as described herein during a first amplification (e.g., step B), after the first amplification (e.g., during step C), before or during a second amplification (e.g., before or during step D), after step D and before collection in step E, during or after collection in step F, before or during final formulation and/or transfer to an infusion bag in step F, and before any optional cryopreservation step in step F. Furthermore, sdRNA can be added after thawing from any cryopreservation step in step F. In one embodiment, one or more sdrnas targeting genes as described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium comprising TIL and other agents at a concentration selected from the group consisting of: 100nM to 20mM, 200nM to 10mM, 500nM to 1mM, 1. mu.M to 100. mu.M, and 1. mu.M to 100. mu.M. In embodiments, sdrnas targeting one or more genes (including PD-1, LAG-3, TIM-3, CISH, and CBLB) as described herein may be added to a cell culture medium comprising TIL and other agents in an amount selected from the group consisting of: mu.M sdRNA/10,000 TILs/100. mu.L medium, 0.5. mu.M sdRNA/10,000 TILs/100. mu.L medium, 0.75. mu.M sdRNA/10,000 TILs/100. mu.L medium, 1. mu.M sdRNA/10,000 TILs/100. mu.L medium, 1.25. mu.M sdRNA/10,000 TILs/100. mu.L medium, 1.5. mu.M sdRNA/10,000 TILs/100. mu.L medium, 2. mu.M sdRNA/10,000 TILs/100. mu.L medium, 5. mu.M sdRNA/10,000 TILs/100. mu.L medium or 10. mu.M sdRNA/10,000 TILs/100. mu.L medium. In embodiments, sdRNA targeting one or more genes (comprising PD-1, LAG-3, TIM-3, CISH, and CBLB) as described herein may be added to the TIL culture twice a day, once a day, every two days, every three days, every four days, every five days, every six days, or every seven days during the REP period.

The oligonucleotide compositions of the invention (comprising sdRNA) can be contacted with a TIL as described herein during an amplification process, for example, by dissolving a high concentration of sdRNA in a cell culture medium and allowing sufficient time for passive uptake. In certain embodiments, the methods of the invention comprise contacting a TIL population with an oligonucleotide composition as described herein. In certain embodiments, the method comprises dissolving an oligonucleotide (e.g., sdRNA) in a cell culture medium and contacting the cell culture medium with a TIL population. The TIL may be a first population, a second population, and/or a third population as described herein.

In some embodiments, delivery of the oligonucleotide into the cell can be enhanced by suitable techniques, including calcium phosphate, DMSO, glycerol, or dextran, electroporation, or by transfection using methods known in the art (e.g., using cationic, anionic, or neutral lipid compositions or liposomes) (see, e.g., WO 90/14074; WO 91/16024; WO 91/17424; U.S. Pat. No. 4,897,355; Bergan et al 1993. nucleic acid research 21: 3567).

In some embodiments, more than one sdRNA is used to reduce expression of a target gene. In some embodiments, sdRNAs targeting PD-1, TIM-3, CBLB, LAG3 and/or CISH are used together. In some embodiments, PD-1sdRNA is used with one or more of TIM-3, CBLB, LAG3, and/or CISH to reduce expression of more than one gene target. In some embodiments, LAG3 sdRNA is used in combination with CISH-targeting sdRNA to reduce gene expression of both targets. In some embodiments, sdRNAs herein that target one or more of PD-1, TIM-3, CBLB, LAG3 and/or CISH are commercially available from Advirna, Worcester, Mass.

In some embodiments, the sdRNA targets a gene selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the sdRNA targets a gene selected from the group consisting of: PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, one sdRNA targets PD-1 and the other sdRNA targets a gene selected from the group consisting of: LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the sdRNA targets a gene selected from the group consisting of: PD-1, LAG-3, CISH, CBLB, TIM3 and combinations thereof. In some embodiments, the sdRNA targets a gene selected from PD-1 and one of LAG3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, one sdRNA targets PD-1 and one sdRNA targets LAG 3. In some embodiments, one sdRNA targets PD-1 and one sdRNA targets CISH. In some embodiments, one sdRNA targets PD-1 and one sdRNA targets CBLB. In some embodiments, one sdRNA targets LAG3 and one sdRNA targets CISH. In some embodiments, one sdRNA targets LAG3 and one sdRNA targets CBLB. In some embodiments, one sdRNA targets CISH and one sdRNA targets CBLB. In some embodiments, one sdRNA targets TIM3 and one sdRNA targets PD-1. In some embodiments, one sdRNA targets TIM3 and one sdRNA targets LAG 3. In some embodiments, one sdRNA targets TIM3 and one sdRNA targets CISH. In some embodiments, one sdRNA targets TIM3 and one sdRNA targets CBLB.

As discussed above, embodiments of the present invention provide Tumor Infiltrating Lymphocytes (TILs) that have been genetically modified by gene editing to enhance their therapeutic effect. Embodiments of the invention include gene editing by nucleotide insertion (RNA or DNA) into a TIL population to promote expression of one or more proteins and inhibit expression of one or more proteins, and combinations thereof. Embodiments of the invention also provide methods for expanding TILs into therapeutic populations, wherein the methods comprise genetically editing TILs. There are several gene editing techniques that can be used to genetically modify the TIL population, which are suitable for use in accordance with the present invention.

In some embodiments, the methods include a method of genetically modifying a TIL population comprising the step of stably incorporating a gene for producing one or more proteins. In one embodiment, the method of genetically modifying a TIL population comprises the step of retroviral transduction. In one embodiment, the method of genetically modifying a TIL population comprises the step of lentiviral transduction. Levine transduction systems are known in the art and are described, for example, in Levine et al, Proc. Natl. Acad. Sci. USA 2006,103,17372-77; zufferey et al, Nature Biotechnology 1997,15, 871-75; dull et al, journal of virology 1998,72,8463-71, and U.S. Pat. No. 6,627,442, the disclosures of each of which are incorporated herein by reference. In an embodiment, the method of genetically modifying a TIL population comprises the step of gamma retroviral transduction. Gamma retroviral transduction systems are known in the art and are described, for example, in Cepko and Pear, modern molecular biology protocols 1996,9.9.1-9.9.16, the disclosures of which are incorporated herein by reference. In embodiments, the method of genetically modifying a TIL population comprises the step of transposon-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art and include the following: wherein the transposase is provided in the form of a DNA expression vector or in the form of expressible RNA or protein such that long term expression of the transposase does not occur in the transgenic cell, e.g., a transposase provided in the form of mRNA (e.g., mRNA including cap and poly-a tail). Suitable transposon-mediated gene transfer systems comprising salmonidae fish-type Tel-like transposases (sleeping beauty transposases), such as SB10, SB11, and SB100x, and engineered enzymes with increased enzymatic activity are described, for example, in hacett et al, molecular therapy 2010,18,674-83, and U.S. patent No. 6,489,458, the disclosures of each of which are incorporated herein by reference.

In embodiments, the methods comprise methods of genetically modifying a TIL population (e.g., a first population, a second population, and/or a third population as described herein). In some embodiments, a method of genetically modifying a TIL population comprises the step of stably incorporating a gene for producing or inhibiting (e.g., silencing) one or more proteins. In an embodiment, the method of genetically modifying a TIL population comprises the step of electroporation. Electroporation methods are known in the art and are described, for example, in Tsong, journal of biophysics 1991,60,297 & 306, and U.S. patent application publication No. 2014/0227237a1, the disclosures of each of which are incorporated herein by reference. Other electroporation methods known in the art may be used, such as those described in U.S. Pat. nos. 5,019,034; 5,128,257 No; 5,137,817 No; 5,173,158 No; 5,232,856 No; U.S. Pat. No. 5,273,525; 5,304,120 No; U.S. Pat. No. 5,318,514; 6,010,613 and 6,078,490, the disclosures of which are incorporated herein by reference. In an embodiment, the electroporation method is a sterile electroporation method. In an embodiment, the electroporation method is a pulsed electroporation method. In an embodiment, the electroporation method is a pulsed electroporation method comprising the steps of: treating the TIL with a pulsed electric field to alter, manipulate or cause a defined and controlled, permanent or temporary TIL change, comprising the step of applying to the TIL at least three single operator-controlled, independently programmed DC electrical pulse trains having field strengths equal to or greater than 100V/cm, wherein the at least three DC electrical pulse trains have one, two or three of the following characteristics: (1) the pulse amplitudes of at least two of the at least three pulses are different from each other; (2) the pulse widths of at least two of the at least three pulses are different from each other; and (3) a first pulse interval of a first set of two pulses of the at least three pulses is different from a second pulse interval of a second set of two pulses of the at least three pulses. In an embodiment, the electroporation method is a pulsed electroporation method comprising the steps of: treating the TIL with a pulsed electric field to alter, manipulate or cause a defined and controlled, permanent or temporary TIL change, comprising the step of applying to the TIL at least three single operator-controlled, independently programmed DC electrical pulse trains having field strengths equal to or greater than 100V/cm, wherein pulse amplitudes of at least two of the at least three pulses are different from each other. In an embodiment, the electroporation method is a pulsed electroporation method comprising the steps of: treating the TIL with a pulsed electric field to alter, manipulate or cause a defined and controlled, permanent or temporary TIL change, comprising the step of applying to the TIL at least three single operator-controlled, independently programmed DC electrical pulse trains having field strengths equal to or greater than 100V/cm, wherein pulse widths of at least two of the at least three pulses are different from each other. In an embodiment, the electroporation method is a pulsed electroporation method comprising the steps of: treating the TIL with a pulsed electric field to alter, manipulate or cause a defined and controlled, permanent or temporary TIL change, comprising the step of applying to the TIL at least three single operator-controlled, independently programmed, DC electrical pulse trains having field strengths equal to or greater than 100V/cm, wherein a first pulse interval of a first set of two pulses of the at least three pulses is different from a second pulse interval of a second set of two pulses of the at least three pulses. In one embodiment, the electroporation method is a pulsed electroporation method comprising the steps of: treating the TIL with a pulsed electric field to induce pore formation in the TIL, comprising the step of applying to the TIL at least three sequences of DC electrical pulses having a field strength equal to or greater than 100V/cm, wherein the at least three sequences of DC electrical pulses have one, two or three of the following characteristics: (1) the pulse amplitudes of at least two of the at least three pulses are different from each other; (2) the pulse widths of at least two of the at least three pulses are different from each other; and (3) a first pulse interval of a first two pulses of the at least three pulses is different from a second pulse interval of a second two pulses of the at least three pulses such that the pores are induced for a relatively long period of time and such that viability of the TIL is maintained. In an embodiment, the method of genetically modifying a TIL population comprises the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating and endocytosis) are known in the art and are described in Graham and van der Eb, virology 1973,52, 456-; wigler et al, Proc. Natl. Acad. Sci. USA 1979,76, 1373-1376; and Chen and Okayarea, molecular cell biology 1987,7, 2745-; and U.S. patent No. 5,593,875, the disclosures of each of which are incorporated herein by reference. In an embodiment, the method of genetically modifying a TIL population comprises the step of lipofection. Lipofectin transfection methods, such as the method of using 1:1(w/w) liposome formulations of the cationic lipid N- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA) with Dioleoylphosphatidylethanolamine (DOPE) in filtered water, are known in the art and are described in Rose et al, Biotechnology 1991,10,520-525 and Felgner et al, Proc. Natl. Acad. Sci. USA, 1987,84,7413-7417 and U.S. Pat. No. 5,279,833; 5,908,635 No; 6,056,938 No; 6,110,490 No; 6,534,484 No; and 7,687,070, the disclosures of each of which are incorporated herein by reference. In embodiments, methods of genetically modifying TIL populations include the use of the compounds described in U.S. patent nos. 5,766,902; 6,025,337 No; 6,410,517 No; 6,475,994 No; and No. 7,189,705; the methods described in (a), the disclosure of each of which is incorporated herein by reference. The TILs may be a first population, a second population, and/or a third population of TILs as described herein.

According to an embodiment, the gene editing process may comprise the use of a programmable nuclease that mediates the generation of double-stranded or single-stranded breaks at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic sites, i.e., they rely on identifying specific DNA sequences within the genome that target the nuclease domain to this location and mediate the creation of double-strand breaks at the target sequence. Double-strand breaks in DNA subsequently recruit endogenous repair mechanisms to the band break sites to mediate genome editing by non-homologous end-joining (NHEJ) or homology-directed repair (HDR). Thus, repair of the break can result in the introduction of an insertion/deletion mutation that disrupts (e.g., silences, suppresses, or enhances) the target gene product.

Major classes of nucleases that achieve site-specific genome editing have been developed, including Zinc Finger Nucleases (ZFNs), transcription activator-like nucleases (TALENs), and CRISPR-associated nucleases (e.g., CRISPR/Cas 9). These nuclease systems can be broadly divided into two categories based on their DNA recognition patterns: ZFNs and TALENs achieve specific DNA binding through protein-DNA interactions, while CRISPR systems (e.g., Cas9) target specific DNA sequences through short RNA guides that base pair directly with the target DNA and through protein-DNA interactions. See, e.g., Cox et al, Nature Medicine, 2015, vol 21, vol 2.

Non-limiting examples of gene editing methods that can be used according to the TIL amplification methods of the present invention include CRISPR methods, TALE methods, and ZFN methods, which are described in more detail below. According to embodiments, a method for expanding TILs into a therapeutic population may be performed according to any embodiment of the methods described herein (e.g., GEN 3 process) or as described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, wherein the method further comprises genetically editing at least a portion of the TILs by one or more of a CRISPR method, a TALE method, or a ZFN method, so as to produce a TIL that can provide an enhanced therapeutic effect. According to an embodiment, the improved therapeutic effect of a gene-edited TIL may be assessed by comparing the gene-edited TIL to an unmodified TIL in vitro (e.g., by assessing in vitro effector function, cytokine profile, etc., as compared to the unmodified TIL). In certain embodiments, the methods comprise gene editing of a TIL population using CRISPR, TALE, and/or ZFN methods.

In some embodiments of the invention, electroporation is used to deliver gene editing systems, such as CRISPR, TALEN, and ZFN systems. In some embodiments of the invention, the electroporation system is a flow electroporation system. An example of a suitable flow electroporation system suitable for some embodiments of the present invention is the commercially available MaxCyte STX system. There are several alternative commercially available electroporation instruments that may be suitable for use in the present invention, such as the AgilePoulse System or ECM 830, Cellaxess Elektra (Cellecricon), Nuclear transfection Instrument (Lonza/Amaxa), GenePulser MXcell (BIORAD), iPromator-96 (Primax) or SiPORTer96(Ambion), available from BTX-Harvard Apparatus, BTX-Harvard Apparatus. In some embodiments of the invention, the electroporation system and the rest of the TIL amplification method form a closed sterile system. In some embodiments of the invention, the electroporation system is a pulsed electroporation system as described herein, and forms a closed sterile system with the rest of the TIL amplification method.

The method for expanding a TIL into a therapeutic population may be performed according to any of the embodiments of the methods described herein (e.g., process GEN 3) or as described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, wherein the method further comprises gene editing at least a portion of the TIL by a CRISPR method (e.g., CRISPR/Cas9 or CRISPR/Cpf 1). According to particular embodiments, the expression of one or more immune checkpoint genes is silenced or reduced in at least a portion of a therapeutic TIL population using CRISPR methods during a TIL amplification process. Alternatively, the use of CRISPR methods during a TIL amplification process allows for the expression of one or more immune checkpoint genes to be enhanced in at least a portion of a therapeutic TIL population.

CRISPR denotes "clustered regularly interspaced short palindromic repeats". Methods of gene editing using CRISPR systems are also referred to herein as CRISPR methods. There are three types of CRISPR systems that incorporate RNA and Cas proteins and that can be used according to the present invention: form I, form II and form III. Type II CRISPR (exemplified by Cas9) is one of the most well characterized systems.

CRISPR technology is adapted according to the natural defense mechanisms (domains of unicellular microorganisms) of bacteria and archaea. These organisms use CRISPR-derived RNA and various Cas proteins (including Cas9) to prevent attack by viruses and other foreign objects by chopping and destroying the DNA of foreign invaders. CRISPR is a DNA-specific region with two distinct properties: nucleotide repeats and spacers are present. Repeats of nucleotides are distributed throughout the CRISPR region, with short segments of foreign DNA (spacers) interspersed among the repeats. In type II CRISPR/Cas systems, the spacer is integrated within the CRISPR genomic site and is transcribed and processed into short CRISPR RNA (crRNA). These crrnas bind to trans-activating crRNA (tracrrna) and direct sequence-specific cleavage and silencing of pathogenic DNA by Cas proteins. Target recognition by Cas9 protein requires a "seed" sequence within the crRNA and a conserved dinucleotide-containing Protospacer Adjacent Motif (PAM) sequence upstream of the crRNA binding region. By redesigning the crRNA, the CRISPR/Cas system can then be retargeted to cleave almost any DNA sequence. crRNA and tracrRNA in natural systems can be reduced to single guide rna (sgrna) with about 100 nucleotides for genetic engineering. The CRISPR/Cas system is directly portable to human cells by co-delivering plasmids expressing the nuclease and essential crRNA components within Cas 9. Different variants of Cas proteins can be used to reduce targeting limitations (e.g., orthologs of Cas9, such as Cpf 1).

Non-limiting examples of genes that can permanently silence or inhibit TIL by editing by CRISPR methods include PD-1, CTLA-4, LAG-3, HAVCR (TIM-3), Cish, TGF β, PKA, CBL-2, PPP2, PTPN, PDCD, BTLA, CD160, TIGIT, CD, CRTAM, LAIR, SIGLEC, CD244, TNFRSF10, CASP, FADD, FAS, SMAD, SKI, SKIL, TGIF, IL10, HMOX, IL6, EIF2AK, CSK, PAG, SIT, FOBAT, PRXP, SMF, GUCY1A, GUCY1B, and GUCY 1B.

Non-limiting examples of genes that can be permanently enhanced by editing TIL by CRISPR methods include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15 and IL-21.

Examples of systems, methods, and compositions that alter expression of a target gene sequence by CRISPR methods and that can be used in accordance with embodiments of the present invention are described in U.S. patent nos. 8,697,359; 8,993,233 No; 8,795,965 No; 8,771,945 No; 8,889,356 No; 8,865,406 No; 8,999,641 No; 8,945,839 No; 8,932,814 No; 8,871,445 No; 8,906,616 No; and 8,895,308, which are incorporated herein by reference. Resources for performing CRISPR methods, such as plasmids expressing CRISPR/Cas9 and CRISPR/Cpf1, are commercially available from companies such as tassel (GenScript).

In embodiments, genetic modification of the TIL population can be performed using the CRISPR/Cpf1 system as described in U.S. patent No. US 9790490, the disclosure of which is incorporated herein by reference, as described herein.

The method for expanding a TIL into a therapeutic population may be performed according to any of the embodiments of the methods described herein (e.g., process 2A) or as described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, wherein the method further comprises gene editing at least a portion of the TIL by a TALE method. According to particular embodiments, the expression of one or more immune checkpoint genes is silenced or reduced in at least a portion of a therapeutic TIL population using a TALE method during a TIL amplification process. Alternatively, TALE methods are used during the TIL amplification process such that expression of one or more immune checkpoint genes is enhanced in at least a portion of the therapeutic TIL population.

TALE denotes a "transcription activator-like effector" protein, which comprises TALENs ("transcription activator-like effector nucleases"). Methods for gene editing using TALE systems may also be referred to herein as TALE methods. TALEs are naturally occurring proteins from the plant pathogenic genus Xanthomonas (Xanthomonas) and contain a DNA binding domain consisting of a series of 33-35 amino acid repeat domains, each of which recognizes a single base pair. TALE specificity was determined by two hypervariable amino acids called Repeat Variable Diresidues (RVDs). The modular TALE repeats join together to recognize adjacent DNA sequences. A particular RVD in a DNA-binding domain recognizes a base in the target locus, thereby providing structural features that assemble a predictable DNA-binding domain. The DNA binding domain of the TALE is fused to the catalytic domain of a type IIS FokI endonuclease to form a targetable TALE nuclease. To induce site-specific mutations, two separate TALEN arms separated by a 14-20 base pair spacer brought the fokl monomers very close to dimerize and create a targeted double-strand break.

Several large systematic studies using various assembly methods have shown that TALE repeats can be combined to identify almost any custom sequence. Specially designed TALE arrays are also commercially available from Cellectis Bioresearch (paris, france), Transposagen biopharmaceutics (liechstandon, kentucky, usa) and Life Technologies (Life Technologies) (glalander, new york, usa). TALEs and TALEN methods suitable for use in the present invention are described in U.S. patent application publication No. US 2011/0201118 a 1; US 2013/0117869 a 1; US 2013/0315884 a 1; the disclosures of said US patent applications are incorporated herein by reference, in US 2015/0203871 a1 and US 2016/0120906 a 1.

Non-limiting examples of genes that can permanently silence or suppress TIL by editing by TALE methods include PD-1, CTLA-4, LAG-3, HAVCR2(TIM-3), Cish, TGF β, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6, PAG 2, SAG 2, GUCY 2, GU 2, and GU 2.

Non-limiting examples of genes that can be permanently enhanced by gene editing of TIL by TALE methods include CCR2, CCR4, CCR5, CXCR2, CX3, CX3CR1, IL-2, IL12, IL-15 and IL-21.

Examples of systems, methods, and compositions that alter expression of a target gene sequence by TALE methods and that can be used according to embodiments of the present invention are described in U.S. patent No. 8,586,526, which is incorporated herein by reference.

The method for expanding TILs into a therapeutic population may be performed according to any of the examples of the methods described herein (e.g., process GEN 3) or as described in PCT/US2017/058610, PCT/US2018/012605, or PCT/US2018/012633, wherein the method further comprises gene editing at least a portion of the TILs by zinc finger or zinc finger nuclease methods. According to particular embodiments, zinc finger methods are used during the TIL amplification process such that expression of one or more immune checkpoint genes is silenced or reduced in at least a portion of the therapeutic TIL population. Alternatively, zinc finger approaches are used during the TIL amplification process such that expression of one or more immune checkpoint genes is enhanced in at least a portion of the therapeutic TIL population.

A single zinc finger contains approximately 30 amino acids in a conserved β β α configuration. Several amino acids on the surface of the alpha helix are typically exposed to 3bp in the major groove of DNA, with varying levels of selectivity. Zinc fingers have two protein domains. The first domain is a DNA binding domain that comprises a eukaryotic transcription factor and contains a zinc finger. The second domain is a nuclease domain, which comprises a fokl restriction enzyme and is responsible for catalytically cleaving DNA.

The DNA-binding domain of an individual ZFN typically contains three to six individual zinc finger repeats and each can recognize 9 to 18 base pairs. A pair of 3-finger ZFNs that recognize a total of 18 base pairs could theoretically target a single locus in the mammalian genome if the zinc finger domain was specific for its intended target site. One method of generating a new zinc finger array would be to combine smaller zinc finger "modules" of known specificity. Most common module assemblyThe process involves combining three individual zinc fingers, each of which can recognize a 3 base pair DNA sequence to produce a 3-finger array that can recognize 9 base pair target sites. Alternatively, selection-based approaches such as oligo library engineering (OPEN) can be used to select new zinc finger arrays from a random library that considers content-dependent interactions between neighboring fingers. Engineered zinc fingers are commercially available; the company Santamo Biosciences (Richmon, Calif., USA) collaborated with Sigma-Aldrich (Sigma-Aldrich) (St. Louis, Mo., USA) to develop a specialized platform for zinc finger construction

Non-limiting examples of genes that can permanently silence or suppress TIL by editing through a zinc finger approach include PD-1, CTLA-4, LAG-3, HAVCR (TIM-3), Cish, TGF β, PKA, CBL-2, PPP2, PTPN, PDCD, BTLA, CD160, TIGIT, CD, CRTAM, LAIR, SIGLEC, CD244, TNFRSF10, CASP, FADD, FAS, SMAD, SKI, SKIL, TGIF, IL10, HMOX, IL6, EIF2AK, CSK, PAG, SIT, FOBAT, PRXP, SMF, GUCY1A, GUCY1B, and GUCY 1B.

Non-limiting examples of genes that can be permanently enhanced by zinc finger methods for TIL gene editing include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15 and IL-21.

Examples of systems, methods, and compositions for altering expression of a target gene sequence by zinc finger methods that may be used according to embodiments of the present invention are described in U.S. Pat. nos. 6,534,261; 6,607,882 No; 6,746,838 No; 6,794,136 No; 6,824,978 No; 6,866,997 No; 6,933,113 No; 6,979,539 No; 7,013,219 No; 7,030,215 No; 7,220,719 No; 7,241,573 No; 7,241,574 No; 7,585,849 No; 7,595,376 No; 6,903,185 and 6,479,626, which are incorporated herein by reference.

Other examples of systems, methods and compositions for altering expression of a target gene sequence by the zinc finger method and which may be used according to embodiments of the invention are described in Beane et al, molecular therapy 2015, 231380-1390, the disclosures of which are incorporated herein by reference.

In some embodiments, the TIL is optionally genetically engineered to include additional functions, including but not limited to a high affinity T Cell Receptor (TCR), such as a TCR targeting a tumor-associated antigen (e.g., MAGE-1, HER2, or NY-ESO-1), or a Chimeric Antigen Receptor (CAR) that binds to a tumor-associated cell surface molecule (e.g., mesothelin) or lineage-restricted cell surface molecule (e.g., CD 19). In certain embodiments, the methods comprise genetically engineering the TIL population to comprise a high affinity T Cell Receptor (TCR), such as a TCR targeted to a tumor-associated antigen (e.g., MAGE-1, a TCR of HER2, or NY-ESO-1), or a Chimeric Antigen Receptor (CAR) that binds to a tumor-associated cell surface molecule (e.g., mesothelin) or a lineage-restricted cell surface molecule (e.g., CD 19). Suitably, the TIL population may be a first population, a second population and/or a third population as described herein.

K. Closed system for TIL manufacturing

The present invention provides for the use of a closed system during a TIL cultivation process. Such closed systems allow for avoiding and/or reducing microbial contamination, allow for the use of fewer flasks, and allow for cost reduction. In some embodiments, the closed system uses two containers.

Such containment systems are well known in the art and may be found, for example, in http:// www.fda.gov/cber/guidellines. htm and https:// www.fda.gov/biologics Blood lipids/Guidances company regulatory information/Guidances/Blood/ucm076779. htm.

A sterile connection device (STCD) produces a sterile weld between two compatible conduits. This procedure permits sterile connection of various container and catheter diameters. In some embodiments, the containment system includes a luer lock (luer lock) and a heat sealing system as described, for example, in example G. In some embodiments, the closed system is accessed by a syringe under sterile conditions to maintain the sterile and closed nature of the system. In some embodiments, a closed system as described in example G is employed. In some embodiments, the TIL is formulated into a final product formulation container according to the method described in example G, final formulation and fill section.

In some embodiments, the closed system uses one container from the time the tumor fragments are obtained until the TIL is ready for administration to a patient or cryopreservation. In some embodiments, when two containers are used, the first container is a closed G-container and the TIL population is centrifuged and transferred into an infusion bag without opening the first closed G-container. In some embodiments, when two containers are used, the infusion bag is a HypoThermosol containing infusion bag. The closed system or closed TIL cell culture system is characterized in that once the tumor sample and/or tumor debris has been added, the system is tightly sealed from the outside to form a closed environment free from bacterial, fungal invasion and/or any other microbial contamination.

In some embodiments, the reduction in microbial contamination is between about 5% and about 100%. In some embodiments, the reduction in microbial contamination is between about 5% and about 95%. In some embodiments, the reduction in microbial contamination is between about 5% and about 90%. In some embodiments, the reduction in microbial contamination is between about 10% and about 90%. In some embodiments, the reduction in microbial contamination is between about 15% and about 85%. In some embodiments, the reduction in microbial contamination is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100%.

The closed system allows for TIL growth in the absence of microbial contamination and/or with a significant reduction in microbial contamination.

In addition, the pH, partial pressure of carbon dioxide, and partial pressure of oxygen of the TIL cell culture environment each vary with the cells being cultured. Therefore, even if a medium suitable for cell culture is circulated, the closed environment needs to be constantly maintained as an optimal environment for proliferation of TIL. For this purpose, it is necessary to monitor physical factors of pH, partial pressure of carbon dioxide and partial pressure of oxygen in the culture liquid of the closed environment by means of sensors, signals of which are used to control a gas exchanger installed at an inlet of the culture environment, and adjust the partial pressure of gas of the closed environment in real time according to changes in the culture liquid so as to optimize the cell culture environment. In some embodiments, the present invention provides a closed cell culture system incorporating a gas exchanger equipped with a monitoring device at the inlet of the closed environment that measures the pH, carbon dioxide partial pressure, and oxygen partial pressure of the closed environment and optimizes the cell culture environment by automatically adjusting the gas concentration based on signals from the monitoring device.

In some embodiments, the pressure within the closed environment is controlled continuously or intermittently. That is, the pressure in the closed environment may be changed by means of, for example, a pressure maintaining device, thereby ensuring that the space is suitable for TIL growth in a positive pressure state, or promoting fluid exudation in a negative pressure state, and thus promoting cell proliferation. Furthermore, by intermittently applying the negative pressure, it is possible to uniformly and efficiently replace the circulating liquid in the closed environment by means of temporary contraction of the volume of the closed environment.

In some embodiments, optimal culture components for TIL propagation may be substituted or added, and factors including, for example, IL-2 and/or OKT3, and combinations may be added.

Optional cryopreservation of TIL

Ontological TIL populationOr an expanded population of TILsCryopreservation may optionally be performed. In some embodiments, the therapeutic TIL population is cryopreserved. In some embodiments, the TILs collected after the second amplification are cryopreserved. In some embodiments, in FIG. 1 (specifically, e.g., FIG. 1B and/or FIG. 1C)In exemplary step F, TIL is cryopreserved. In some embodiments, the TIL is cryopreserved in an infusion bag. In some embodiments, the TIL is cryogenically preserved prior to being placed in an infusion bag. In some embodiments, the TIL is cryopreserved and not placed in an infusion bag. In some embodiments, cryopreservation media is used for cryopreservation. In some embodiments, the cryopreservation media contains dimethyl sulfoxide (DMSO). This is typically accomplished by placing the TIL population in a freezing solution (e.g., 85% complement inactivated AB serum and 15% dimethyl sulfoxide (DMSO)). The cell-containing solution was placed in a cryovial and stored at-80 ℃ for 24 hours, with optional transfer to a gaseous nitrogen freezer for cryopreservation. See, Sadeghi et al, Acta Oncology, 2013,52, 978-.

When appropriate, cells were removed from the freezer and thawed in a 37 ℃ water bath until approximately 4/5 solution was thawed. The cells are typically resuspended in complete medium and optionally washed one or more times. In some embodiments, thawed TILs may be counted and viability assessed as known in the art.

In a preferred embodiment, the TIL population is cryopreserved using CS10 cryopreservation medium (CryoStor 10, bio life solutions). In a preferred embodiment, the TIL population is cryopreserved using a cryopreservation medium containing dimethyl sulfoxide (DMSO). In a preferred embodiment, the TIL population is cryopreserved using CS10 and cell culture medium in a 1:1 (volume: volume) ratio. In a preferred embodiment, the TIL population is cryopreserved using CS10 and cell culture medium in an approximately 1:1 (volume: volume) ratio further including additional IL-2.

As discussed above, and as illustrated in steps a through E provided in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C), cryopreservation may occur at multiple points throughout the TIL amplification process. In some embodiments, the amplified TIL population after the second amplification (as provided, for example, according to step D of fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) may be cryopreserved. Cryopreservation can generally be achieved by placing the TIL population in a freezing solution (e.g., 85% complement inactivated AB serum and 15% dimethyl sulfoxide (DMSO)). The cell-containing solution was placed in a cryovial and stored at-80 ℃ for 24 hours, with optional transfer to a gaseous nitrogen freezer for cryopreservation. See, Sadeghi et al, proceedings of oncology, 2013,52, 978-. In some embodiments, TIL is cryopreserved in 5% DMSO. In some embodiments, TILs are cryopreserved in cell culture medium plus 5% DMSO. In some embodiments, the TIL is cryopreserved according to the method provided in example D.

In some cases, the step B TIL population may be cryopreserved immediately using the protocol discussed below. Alternatively, the bulk TIL population may be subjected to steps C and D and then cryo-preserved after step D. Similarly, where a genetically modified TIL is to be used in therapy, the step B or step D TIL population may be genetically modified for appropriate treatment.

Phenotypic characterization of amplified TILs

In some embodiments, TILs are analyzed for expression of a plurality of phenotypic markers after amplification, including those described herein and in the examples. In embodiments, the expression of one or more phenotypic markers is detected. In some embodiments, the TIL is analyzed for phenotypic characteristics after the first amplification in step B. In some embodiments, the phenotypic characteristics of the TIL are analyzed during the transition in step C. In some embodiments, the phenotypic characteristics of TIL are analyzed during the transition according to step C and after cryopreservation. In some embodiments, the TIL is analyzed for phenotypic characteristics after the second amplification according to step D. In some embodiments, the phenotypic characteristics of the TIL are analyzed after two or more amplifications according to step D.

In some embodiments, the marker is selected from the group consisting of CD8 and CD 28. In some embodiments, expression of CD8 is detected. In some embodiments, expression of CD28 is detected. In some embodiments, the expression of CD8 and/or CD28 on the TIL produced according to the inventive process is higher compared to other processes (e.g., the Gen 3 process as provided, for example, in fig. 1 (specifically, for example, fig. 1B)) compared to the 2A process as provided, for example, in fig. 1 (specifically, for example, fig. 1B). In some embodiments, the expression of CD8 on the TIL produced according to the inventive process is higher compared to other processes (e.g., the Gen 3 process as provided, for example, in fig. 1 (specifically, for example, fig. 1B and/or fig. 1C)) compared to the 2A process as provided, for example, in fig. 1 (specifically, for example, fig. 1B and/or fig. 1C). In some embodiments, the expression of CD28 on the TILs produced according to the inventive process is higher compared to other processes (e.g., the Gen 3 process as provided, for example, in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)) compared to the 2A process as provided, for example, in fig. 1 (specifically, e.g., fig. 1A). In some embodiments, high CD28 expression indicates a younger, more durable TIL phenotype. In embodiments, the expression of one or more regulatory markers is measured.

In embodiments, the selection of the first TIL population, the second TIL population, the third TIL population, or the harvested TIL population based on CD8 and/or CD28 expression is not performed during any of the steps of the methods of expanding Tumor Infiltrating Lymphocytes (TILs) described herein.

In some embodiments, the percentage of central memory cells is higher on the TILs produced according to the inventive process compared to other processes (e.g., the Gen 3 process as provided, for example, in fig. 1 (specifically, e.g., fig. 1B)) compared to the 2A process as provided, for example, in fig. 1 (specifically, e.g., fig. 1A). In some embodiments, the memory marker of the central memory cell is selected from the group consisting of CCR7 and CD 62L.

In some embodiments, CD4+ and/or CD8+ TIL memory subpopulations may be divided into different memory subpopulations. In some embodiments, CD4+ and/or CD8+ TIL comprises naive (CD45RA + CD62L +) TIL. In some embodiments, CD4+ and/or CD8+ TIL comprise Central memory (CM; CD45RA-CD62L +) TIL. In some embodiments, CD4+ and/or CD8+ TIL comprises effector memory (EM; CD45RA-CD62L-) TIL. In some embodiments, CD4+ and/or CD8+ TIL comprise RA + effector memory/effector (TEMRA/TEFF; CD45RA + CD62L +) TIL.

In some embodiments, the TIL expresses more than one marker selected from the group consisting of granzyme B, perforin and granulysin. In some embodiments, the TIL expresses granzyme B. In some embodiments, the TIL expresses perforin. In some embodiments, the TIL expresses granulysin.

In embodiments, cytokine release from the re-stimulated TIL may also be assessed using a cytokine release assay. In some embodiments, the TIL may be evaluated for interferon-gamma (IFN- γ) secretion. In some embodiments, IFN- γ secretion is measured by an ELISA assay. In some embodiments, IFN- γ secretion is measured by an ELISA assay after the rapid second amplification step, after step D as provided, for example, in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C). In some embodiments, the TIL health is measured by IFN-gamma secretion. In some embodiments, IFN- γ secretion is indicative of active TIL. In some embodiments, a potency assay for IFN- γ production is employed. IFN- γ production is another measure of cytotoxic potential. IFN- γ production can be measured by determining the level of the cytokine IFN- γ in TIL medium stimulated with antibodies against CD3, CD28, and CD137/4-1 BB. IFN- γ levels in the media of these stimulated TILs can be determined by measuring IFN- γ release. In some embodiments, an increase in IFN- γ production in the Gen 3 process, e.g., in the step dtil, as provided in figure 1 (particularly, e.g., figure 1B and/or figure 1C), as compared to, e.g., step D, as provided in the 2A process, as provided in figure 1 (particularly, e.g., figure 1A), is indicative of an increase in the cytotoxic potential of the step dtil. In some embodiments, IFN- γ secretion is increased by one, two, three, four, or five times or more. In some embodiments, IFN- γ secretion is doubled. In some embodiments, IFN- γ secretion is increased by two-fold. In some embodiments, IFN- γ secretion is increased three-fold. In some embodiments, IFN- γ secretion is increased four-fold. In some embodiments, IFN- γ secretion is increased by five fold. In some embodiments, IFN- γ is measured using a Quantikine ELISA kit. In some embodiments, IFN- γ is measured in ex vivo TIL. In some embodiments, IFN- γ is measured in an ex vivo TIL, including TILs produced by the methods of the invention, including, for example, the methods of fig. 1B and/or fig. 1C.

In some embodiments, a TIL capable of secreting at least one, two, three, four, or five or more times IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the method of fig. 1B and/or fig. 1C). In some embodiments, a TIL capable of secreting at least one-fold more IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the method of fig. 1B and/or fig. 1C). In some embodiments, a TIL capable of secreting at least twice as much IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the method of fig. 1B and/or fig. 1C). In some embodiments, a TIL capable of secreting at least three times as much IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the method of fig. 1B and/or fig. 1C). In some embodiments, a TIL capable of secreting at least four times more IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the method of fig. 1B and/or fig. 1C). In some embodiments, a TIL capable of secreting at least five times more IFN- γ is a TIL produced by an amplification method of the invention (including, e.g., the method of fig. 1B and/or fig. 1C).

The diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited but large number of gene segments. These gene segments: v (variable), D (diverse), J (linked) and C (constant) determine the binding specificity and downstream applications of immunoglobulins to T Cell Receptors (TCRs). The present invention provides a method for producing TILs that exhibit and increase T cell bank diversity. In some embodiments, the TILs obtained by the methods of the invention exhibit an increase in diversity of the T cell pool. In some embodiments, TILs obtained by the methods of the invention exhibit an increase in T cell bank diversity as compared to freshly harvested TILs and/or TILs prepared using other methods in addition to those provided herein, including, for example, methods other than those practiced in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C). In some embodiments, the TILs obtained by the methods of the invention exhibit an increase in T cell bank diversity compared to freshly harvested TILs and/or TILs prepared using a method referred to as process 2A, as exemplified in fig. 1 (specifically, e.g., fig. 1A). In some embodiments, the TIL obtained in the first expansion exhibits an increase in T cell bank diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the diversity of immunoglobulins is in immunoglobulin heavy chains. In some embodiments, the immunoglobulin diversity is in an immunoglobulin light chain. In some embodiments, the diversity is in a T cell receptor. In some embodiments, the diversity is in one of the T cell receptors selected from the group consisting of: alpha receptors, beta receptors, gamma receptors, and delta receptors. In some embodiments, expression of T Cell Receptors (TCR) α and/or β is increased. In some embodiments, expression of T Cell Receptor (TCR) α is increased. In some embodiments, expression of T Cell Receptor (TCR) β is increased. In some embodiments, expression of TCRab (i.e., TCR α/β) is increased. In some embodiments, a process as described herein (e.g., the Gen 3 process) shows higher clonal diversity compared to other processes (e.g., the process referred to as Gen 2) based on the number of unique peptide CDRs within the sample (see, e.g., fig. 12-14).

In some embodiments, TILs prepared by the methods of the invention (including, for example, the method described in fig. 1) exhibit increased polyclonality as compared to TILs produced by other methods (including methods not exemplified in fig. 1, such as the method referred to as the process 1C method). In some embodiments, a significantly increased polyclonality and/or increased polyclonality is indicative of therapeutic efficacy and/or increased clinical efficacy of a cancer treatment. In some embodiments, polyclonality refers to T cell bank diversity. In some embodiments, an increase in polyclonality may be indicative of therapeutic efficacy with respect to administration of the TIL produced by the methods of the invention.

In some embodiments, the polyclonality is increased by one-fold, two-fold, ten-fold, 100-fold, 500-fold, or 1000-fold as compared to a TIL prepared using a method other than the methods provided herein (including, e.g., a method other than the method embodied in fig. 1). In some embodiments, the polyclonality is doubled compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein (including, e.g., a method other than that embodied in fig. 1). In some embodiments, the polyclonality is increased by two-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those embodied in fig. 1). In some embodiments, the polyclonality is increased ten-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein (including, e.g., a method other than the method embodied in fig. 1). In some embodiments, the polyclonality is increased 100-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein (including, e.g., a method other than the method embodied in fig. 1). In some embodiments, the polyclonality is increased 500-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein (including, e.g., a method other than the method embodied in fig. 1). In some embodiments, the polyclonality is increased 1000-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, e.g., methods other than those embodied in fig. 1).

In some embodiments, activation and depletion of TIL may be determined by examining one or more markers. In some embodiments, activation and depletion can be determined using multi-color flow cytometry. In some embodiments, the activation and depletion of the marker comprises, but is not limited to, one or more markers selected from the group consisting of: CD3, PD-1, 2B4/CD244, CD8, CD25, BTLA, KLRG, TIM-3, CD194/CCR4, CD4, TIGIT, CD183, CD69, CD95, CD127, CD103 and/or LAG-3. In some embodiments, the activation and depletion of the marker comprises, but is not limited to, one or more markers selected from the group consisting of: BTLA, CTLA-4, ICOS, Ki67, LAG-3, PD-1, TIGIT and/or TIM-3. In some embodiments, the activation and depletion of the marker comprises, but is not limited to, one or more markers selected from the group consisting of: BTLA, CTLA-4, ICOS, Ki67, LAG-3, CD103+/CD69+, CD103+/CD69-, PD-1, TIGIT, and/or TIM-3. In some embodiments, T cell markers (including activation and depletion markers) can be determined and/or analyzed to examine T cell activation, inhibition, or function. In some embodiments, the T cell marker may comprise, but is not limited to, one or more markers selected from the group consisting of: TIGIT, CD3, FoxP3, Tim-3, PD-1, CD103, CTLA-4, LAG-3, BTLA-4, ICOS, Ki67, CD8, CD25, CD45, CD4 and/or CD 59.

In some embodiments, the phenotypic characterization is detected after cryopreservation.

Further process examples

In some embodiments, the present invention provides a method for expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) priming a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and OKT-3, wherein the priming first expansion is performed for about 1 to 8 days to obtain a second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) performing a rapid second expansion by contacting the second TIL population with cell culture medium comprising IL-2, OKT-3, and exogenous Antigen Presenting Cells (APCs) to produce a third TIL population, wherein the rapid second expansion is performed for about 1 to 10 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and (d) collecting the therapeutic TIL population obtained from step (c). In some embodiments, the step of rapid second amplification is divided into multiple steps to achieve a scaled-up culture by: (1) performing the rapid second expansion by culturing the second TIL population in a small-scale culture in a first vessel (e.g., a G-REX100MCS vessel) for a period of about 2 to 4 days, and then (2) effecting transfer of the second TIL population from the small-scale culture to a second vessel larger than the first vessel (e.g., a G-REX500MCS vessel), wherein in the second vessel the second TIL population from the small-scale culture is cultured in a larger-scale culture for a period of about 4 to 8 days. In some embodiments, the step of rapid amplification is divided into multiple steps to achieve the outwardly expanding culture by: (1) performing the rapid second expansion by culturing the second TIL population in a first small-scale culture in a first vessel (e.g., a G-REX100MCS vessel) for a period of about 3 to 4 days, and then (2) effecting transfer and distribution of the second TIL population from the first small-scale culture into or among at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels of equal size to the first vessel, wherein in each second vessel the portion of the second TIL population transferred from the first small-scale culture to this second vessel is cultured in a second small-scale culture for a period of about 4 to 8 days. In some embodiments, the step of rapid amplification is divided into multiple steps to achieve the culture of scale-out and scale-up by: (1) performing the rapid second expansion by culturing the second TIL population in a small-scale culture in a first vessel (e.g., a G-REX100MCS vessel) for a period of about 2 to 4 days, and then (2) effecting a transfer and distribution of the second TIL population from the first small-scale culture into and among at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels that are larger in size than the first vessel (e.g., a G-REX500MCS vessel), wherein in each second vessel the portion of the second TIL population transferred from the small-scale culture to this second vessel is cultured in a larger-scale culture for a period of about 4 to 8 days. In some embodiments, the step of rapid amplification is divided into multiple steps to achieve the culture of scale-out and scale-up by: (1) performing the rapid second expansion by culturing the second TIL population in a small-scale culture in a first vessel (e.g., a G-REX100MCS vessel) for a period of about 3 to 4 days, and then (2) effecting the transfer and partitioning of the second TIL population from the first small-scale culture into and into 2, 3, or 4 second vessels of larger size than the first vessel (e.g., a G-REX500MCS vessel), wherein in each second vessel the portion of the second TIL population transferred from the small-scale culture to this second vessel is cultured in a larger-scale culture for a period of about 5 to 7 days.

In some embodiments, the present invention provides a method for expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) priming a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and OKT-3, wherein the priming first expansion is performed for about 1 to 8 days to obtain a second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) performing a rapid second expansion by contacting the second TIL population with cell culture medium comprising IL-2, OKT-3, and exogenous Antigen Presenting Cells (APCs) to produce a third TIL population, wherein the rapid second expansion is performed for about 1 to 8 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and (d) collecting the therapeutic TIL population obtained from step (c). In some embodiments, the step of rapid second amplification is divided into multiple steps to achieve a scaled-up culture by: (1) performing the rapid second expansion by culturing the second TIL population in a small-scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 2 to 4 days, and then (2) effecting transfer of the second TIL population from the small-scale culture to a second vessel larger than the first vessel (e.g., a G-REX 500MCS vessel), wherein in the second vessel the second TIL population from the small-scale culture is cultured in a larger-scale culture for a period of about 4 to 8 days. In some embodiments, the step of rapid amplification is divided into multiple steps to achieve the outwardly expanding culture by: (1) performing the rapid second expansion by culturing the second TIL population in a first small-scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 2 to 4 days, and then (2) effecting transfer and distribution of the second TIL population from the first small-scale culture into or among at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels of equal size to the first vessel, wherein in each second vessel the portion of the second TIL population transferred from the first small-scale culture to this second vessel is cultured in a second small-scale culture for a period of about 4 to 6 days. In some embodiments, the step of rapid amplification is divided into multiple steps to achieve the culture of scale-out and scale-up by: (1) performing the rapid second expansion by culturing the second TIL population in a small-scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 2 to 4 days, and then (2) effecting a transfer and distribution of the second TIL population from the first small-scale culture into and among at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels of a larger size than the first vessel (e.g., a G-REX 500MCS vessel), wherein in each second vessel the portion of the second TIL population transferred from the small-scale culture to this second vessel is cultured in a larger-scale culture for a period of about 4 to 6 days. In some embodiments, the step of rapid amplification is divided into multiple steps to achieve the culture of scale-out and scale-up by: (1) performing the rapid second expansion by culturing the second TIL population in a small-scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 3 to 4 days, and then (2) effecting the transfer and partitioning of the second TIL population from the first small-scale culture into and into 2, 3, or 4 second vessels of larger size than the first vessel (e.g., a G-REX 500MCS vessel), wherein in each second vessel the portion of the second TIL population transferred from the small-scale culture to this second vessel is cultured in a larger-scale culture for a period of about 4 to 5 days.

In some embodiments, the present invention provides a method for expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) obtaining a first TIL population from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments; (b) priming a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and OKT-3, wherein the priming first expansion is performed for about 1 to 7 days to obtain a second TIL population, wherein the second TIL population is greater in number than the first TIL population; (c) performing a rapid second expansion by contacting the second TIL population with cell culture medium comprising IL-2, OKT-3, and exogenous Antigen Presenting Cells (APCs) to produce a third TIL population, wherein the rapid second expansion is performed for about 1 to 11 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; and (d) collecting the therapeutic TIL population obtained from step (c). In some embodiments, the step of rapid second amplification is divided into multiple steps to achieve a scaled-up culture by: (1) performing the rapid second expansion by culturing the second TIL population in a small-scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 3 to 4 days, and then (2) effecting transfer of the second TIL population from the small-scale culture to a second vessel larger than the first vessel (e.g., a G-REX 500MCS vessel), wherein in the second vessel the second TIL population from the small-scale culture is cultured in a larger-scale culture for a period of about 4 to 7 days. In some embodiments, the step of rapid amplification is divided into multiple steps to achieve the outwardly expanding culture by: (1) performing the rapid second expansion by culturing the second TIL population in a first small-scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 3 to 4 days, and then (2) effecting transfer and distribution of the second TIL population from the first small-scale culture into or among at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels of equal size to the first vessel, wherein in each second vessel the portion of the second TIL population transferred from the first small-scale culture to this second vessel is cultured in a second small-scale culture for a period of about 4 to 7 days. In some embodiments, the step of rapid amplification is divided into multiple steps to achieve the culture of scale-out and scale-up by: (1) performing the rapid second expansion by culturing the second TIL population in a small-scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 3 to 4 days, and then (2) effecting transfer and distribution of the second TIL population from the first small-scale culture into and among at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels that are larger in size than the first vessel (e.g., a G-REX 500MCS vessel), wherein in each second vessel the portion of the second TIL population transferred from the small-scale culture to this second vessel is cultured in a larger-scale culture for a period of about 4 to 7 days. In some embodiments, the step of rapid amplification is divided into multiple steps to achieve the culture of scale-out and scale-up by: (1) performing the rapid second expansion by culturing the second TIL population in a small-scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 4 days, and then (2) effecting transfer and partitioning of the second TIL population from the first small-scale culture into and into 2, 3, or 4 second vessels of larger size than the first vessel (e.g., a G-REX 500MCS vessel), wherein in each second vessel the portion of the second TIL population transferred from the small-scale culture to this second vessel is cultured in a larger-scale culture for a period of about 5 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (b) the priming for the first expansion is performed by contacting the first TIL population with a culture medium further comprising exogenous Antigen Presenting Cells (APCs), wherein the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that in step (c) the medium is supplemented with additional exogenous APCs.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 20: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 10: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 9: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 8: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 7: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 6: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 5: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 4: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 3: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 2.9: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 2.8: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 2.7: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 2.6: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 2.5: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 2.4: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 2.3: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 2.2: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 2.1: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 1.1:1 to at or about 2: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of or about 2:1 to or about 10: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of or about 2:1 to or about 5: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of or about 2:1 to or about 4: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of or about 2:1 to or about 3: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 2:1 to at or about 2.9: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 2:1 to at or about 2.8: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of or about 2:1 to or about 2.7: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of or about 2:1 to or about 2.6: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 2:1 to at or about 2.5: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of or about 2:1 to or about 2.4: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of or about 2:1 to or about 2.3: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of at or about 2:1 to at or about 2.2: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is selected from the range of or about 2:1 to or about 2.1: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is at or about 2: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the ratio of the number of APCs added in the rapid second amplification to the number of APCs added in step (b) is at or about 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.1, 4:1, 4.6:1, 3.7:1, 3.8:1, 4:1, 4.4:1, 4:1, 4.6:1, or 4: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that the number of APCs added in priming the first amplification is at or about 1 x 10⁸、1.1×10⁸、1.2×10⁸、1.3×10⁸、1.4×10⁸、1.5×10⁸、1.6×10⁸、1.7×10⁸、1.8×10⁸、1.9×10⁸、2×10⁸、2.1×10⁸、2.2×10⁸、2.3×10⁸、2.4×10⁸、2.5×10⁸、2.6×10⁸、2.7×10⁸、2.8×10⁸、2.9×10⁸、3×10⁸、3.1×10⁸、3.2×10⁸、3.3×10⁸、3.4×10⁸Or 3.5X 10⁸APC, and the number of APC added in the rapid second amplification is made to be at or about 3.5X 10⁸、3.6×10⁸、3.7×10⁸、3.8×10⁸、3.9×10⁸、4×10⁸、4.1×10⁸、4.2×10⁸、4.3×10⁸、4.4×10⁸、4.5×10⁸、4.6×10⁸、4.7×10⁸、4.8×10⁸、4.9×10⁸、5×10⁸、5.1×10⁸、5.2×10⁸、5.3×10⁸、5.4×10⁸、5.5×10⁸、5.6×10⁸、5.7×10⁸、5.8×10⁸、5.9×10⁸、6×10⁸、6.1×10⁸、6.2×10⁸、6.3×10⁸、6.4×10⁸、6.5×10⁸、6.6×10⁸、6.7×10⁸、6.8×10⁸、6.9×10⁸、7×10⁸、7.1×10⁸、7.2×10⁸、7.3×10⁸、7.4×10⁸、7.5×10⁸、7.6×10⁸、7.7×10⁸、7.8×10⁸、7.9×10⁸、8×10⁸、8.1×10⁸、8.2×10⁸、8.3×10⁸、8.4×10⁸、8.5×10⁸、8.6×10⁸、8.7×10⁸、8.8×10⁸、8.9×10⁸、9×10⁸、9.1×10⁸、9.2×10⁸、9.3×10⁸、9.4×10⁸、9.5×10⁸、9.6×10⁸、9.7×10⁸、9.8×10⁸、9.9×10⁸Or 1X 10⁹And (4) APC.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the number of APCs added in priming the first amplification is selected from at or about 1 x 10⁸APC to at or about 3.5X 10⁸A range of APCs, and wherein at a fast secondThe number of APCs added during amplification is selected from the group consisting of at or about 3.5X 10⁸APC to at or about 1X 10⁹Range of individual APC.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the number of APCs added in priming the first amplification is selected from at or about 1.5 x 10⁸APC to at or about 3X 10⁸A range of APCs, and wherein the number of APCs added in the rapid second amplification is selected from at or about 4 × 10 ⁸APC to at or about 7.5X 10⁸Range of individual APC.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the number of APCs added in priming the first amplification is selected from at or about 2 x 10⁸APC to at or about 2.5X 10⁸A range of APCs, and wherein the number of APCs added in the rapid second amplification is selected from at or about 4.5X 10⁸APC to at or about 5.5X 10⁸Range of individual APC.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the first amplification addition to priming is at or about 2.5 x 10⁸APC, and added to the rapid second amplification at or about 5X 10⁸And (4) APC.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the antigen presenting cells are Peripheral Blood Mononuclear Cells (PBMCs).

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that a plurality of tumour fragments are dispensed into a plurality of separate containers, in each of which separate containers a first population of TILs is obtained in step (a), a second population of TILs is obtained in step (b), and a third population of TILs is obtained in step (c), and the therapeutic TILs from the plurality of containers in step (c) are combined to obtain the collected TIL population from step (d).

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that a plurality of tumours are evenly distributed into a plurality of individual containers.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, the method being modified such that the plurality of individual containers comprises at least two individual containers.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the plurality of individual containers comprises from two to twenty individual containers.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the plurality of individual containers comprises from two to fifteen individual containers.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the plurality of individual containers comprises between two and ten individual containers.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, the method being modified such that the plurality of individual containers comprises two to five individual containers.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, that is modified such that the plurality of individual containers comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 individual containers.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that for each vessel in which a first population of TILs is subjected to priming first amplification in step (b), a second population of TILs produced from such first population of TILs is subjected to rapid second amplification in step (c) in the same vessel.

In another embodiment, the present disclosure provides a method as described in any one of the preceding paragraphs, where applicable, modified such that each of the individual containers comprises a first gas-permeable surface area.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that a plurality of tumour fragments are dispensed into a single container.

In another embodiment, the present disclosure provides a method as described in any one of the preceding paragraphs, where applicable, modified such that a single container includes a first gas-permeable surface area.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming of the first expansion is performed by supplementing cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein in step (b) the APCs are layered on the first gas permeable surface region in an average thickness layer of from or about one cell layer to at or about three cell layers.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that in step (b) the APCs are layered on the first gas permeable surface region at an average thickness of from at or about 1.5 cell layers to at or about 2.5 cell layers.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that in step (b) the APCs are layered on the first gas-permeable surface region at an average thickness of at or about 2 cell layers.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that in step (b) the APCs are layered on the first gas permeable surface region at an average thickness of at or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 cell layers.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that in step (c) the APCs are layered on the first gas permeable surface region at an average thickness of from at or about 3 cell layers to at or about 10 cell layers.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that in step (c) the APCs are layered on the first gas permeable surface region at an average thickness of from at or about 4 cell layers to at or about 8 cell layers.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that in step (c) the APCs are layered on the first gas permeable surface region at an average thickness of at or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that in step (c) the APCs are stacked on the first gas permeable surface region at an average thickness of or about 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7.8, 7.8, 7.9, 7.8, or 7.8 cells.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that in step (b) the priming first amplification is performed in a first container comprising a first gas-permeable surface region, and in step (c) the rapid second amplification is performed in a second container comprising a second gas-permeable surface region.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the second container is larger than the first container.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable, the method being modified such that in step (b) the APCs are layered on the first gas permeable surface region in a layer of cells having an average thickness of at or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that in step (c) the APCs are layered on the second gas permeable surface region at an average thickness of from at or about 3 cell layers to at or about 10 cell layers.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that in step (c) the APCs are layered on the second gas permeable surface region at an average thickness of from at or about 4 cell layers to at or about 8 cell layers.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that in step (c) the APCs are layered on the second gas permeable surface region at an average thickness of at or about 3, 4, 5, 6, 7, 8, 9 or 10 cell layers.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that in step (c) the APCs are stacked on the second gas permeable surface region at an average thickness of or about 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7.8, 7.8, 7.9, or 8 cells.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that in step (b) the priming first amplification is performed in a first container comprising a first gas-permeable surface region, and in step (c) the rapid second amplification is performed in the first container.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.1 to at or about 1: 10.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.1 to at or about 1: 9.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.1 to at or about 1: 8.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.1 to at or about 1: 7.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.1 to at or about 1: 6.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.1 to at or about 1: 5.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.1 to at or about 1: 4.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.1 to at or about 1: 3.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.1 to at or about 1: 2.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.2 to at or about 1: 8.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.3 to at or about 1: 7.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.4 to at or about 1: 6.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.5 to at or about 1: 5.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.6 to at or about 1: 4.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.7 to at or about 1: 3.5.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.8 to at or about 1: 3.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, wherein the method is modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the range of at or about 1:1.9 to at or about 1: 2.5.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, the method being modified such that in step (b) the priming first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected to be or to be in the range of about 1:2.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, the method is modified such that in step (b) the priming of the first expansion is performed by supplementing the cell culture medium of the first TIL population with further Antigen Presenting Cells (APCs), wherein the number of APCs added in step (c) is greater than the number of APCs added in step (b), and wherein the ratio of the average number of layers of APCs stacked in step (b) to the average number of layers of APCs stacked in step (c) is selected from the group consisting of or about 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:3, 1.3, 1:2.3, 1:2.4, 1:2.5, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7.7, 1:8, 1:6.7, 1:7, 1:8.7, 1:6.7, 1:8, 1:6.7, 1.7, 1:8, 1:6.7, 1:8, 1.7, 1:6.7, 1:8, 1:6.7, 1.7, 1:8, 1.7, 1:6.7, 1:8, 1.7, 1.8, 1.7, 1:6.8, 1:8, 1:6.7, 1.7, 1:8, 1:6.7, 1.8, 1:6.7, 1:8, 1.7, 1.8, 1:8, 1.8, 1:6, 1.8, 1:8, 1:6, 1:6.7, 1.8, 1:8, 1.8, 1.7, 1.8, 1:6, 1.7, 1:6, 1:7, 1.7, 1:8, 1:6, 1.8, 1.7, 1:8, 1.8, 1:8, 1.7, 1:6, 1.8, 1.7, 1:6, 1.8, 1.7, 1.8, 1:8, 1.8, 1:6, 1.8, 1:8, 1.7, 1.8, 1:8, 1.7, 1:6, 1.7, 1.8, 1:6, 1.7, 1.8, 1:7, 1.8, 1:9.8, 1:9.9 or 1: 10.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is from or about 1.5:1 to or about 100: 1.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, that is modified such that the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is at or about 50: 1.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, that is modified such that the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is at or about 25: 1.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, that is modified such that the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is at or about 20: 1.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, that is modified such that the ratio of the number of TILs in the second TIL population to the number of TILs in the first TIL population is at or about 10: 1.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the second TIL population is at least or about 50 times greater in number than the first TIL population.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the second TIL population is at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 times greater in number than the first TIL population.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, which method is modified such that in step (c) the cell culture medium is supplemented with additional IL-2 at or about 2 days or at or about 3 days after the start of the second period of time.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified to further comprise the step of cryopreserving the collected TIL population in step (d) using a cryopreservation process.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified to include a further step (e) performed after step (d): transferring the collected TIL population from step (d) to an infusion bag optionally containing hypo thermosol.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified to include the step of cryopreserving the infusion bag including the collected TIL population in step (e) using a cryopreservation process.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the cryopreservation process is performed using a 1:1 ratio of the collected TIL population to cryopreservation media.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the PBMCs are irradiated and allogeneic.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that the total number of APCs added to the cell culture in step (b) is 2.5 x 10⁸。

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicableA method modified such that the total number of APCs added to the cell culture in step (c) is 5X 10⁸。

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the APCs are PBMCs.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the antigen presenting cells are artificial antigen presenting cells.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the collecting in step (d) is performed using a membrane-based cell processing system.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs applied above, modified such that the collecting in step (d) is performed using a LOVO cell processing system.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (b) the plurality of fragments comprises from at or about 5 to at or about 60 fragments per container.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (b) the plurality of fragments comprises from at or about 10 to at or about 60 fragments per container.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (b) the plurality of fragments comprises from at or about 15 to at or about 60 fragments per container.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (b) the plurality of fragments comprises from at or about 20 to at or about 60 fragments per container.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (b) the plurality of fragments comprises from at or about 25 to at or about 60 fragments per container.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (b) the plurality of fragments comprises from at or about 30 to at or about 60 fragments per container.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (b) the plurality of fragments comprises from at or about 35 to at or about 60 fragments per container.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (b) the plurality of fragments comprises from at or about 40 to at or about 60 fragments per container.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (b) the plurality of fragments comprises from at or about 45 to at or about 60 fragments per container.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (b) the plurality of fragments comprises from at or about 50 to at or about 60 fragments per container.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that in step (b) the plurality of fragments comprises at or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 fragments per container.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, the method being modified such that the volume of each fragment is at or about 27mm ³。

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, the method being modified such that the volume of each fragment is at or about 20mm³To or about 50mm³。

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, the method being modified such that the volume of each fragment is at or about 21mm³To or about 30mm³。

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, the method being modified such that the volume of each fragment is at or about 22mm³To or about 29.5mm³。

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, the method being modified such that the volume of each fragment is at or about 23mm³To or about 29mm³。

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, the method being modified such that the volume of each fragment is at or about 24mm³To or about 28.5mm³。

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, the method being modified such that the volume of each fragment is at or about 25mm ³To or about 28mm³。

In another embodiment, the present invention provides the aboveA method as in any preceding paragraph adapted such that the volume of each fragment is at or about 26.5mm³To or about 27.5mm³。

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the volume of each fragment is at or about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50mm³。

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that the plurality of fragments comprises from at or about 30 to at or about 60 fragments, wherein the total volume is at or about 1300mm³To or about 1500mm³。

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that the plurality of fragments comprises at or about 50 fragments, wherein the total volume is at or about 1350mm³。

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the plurality of fragments comprises at or about 50 fragments, wherein the total mass is at or about 1 gram to about 1.5 grams.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the cell culture medium is provided in a container, which is a G-container or Xuri cell bag.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the concentration of IL-2 in the cell culture medium is from about 10,000IU/mL to about 5,000 IU/mL.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the concentration of IL-2 in the cell culture medium is about 6,000 IU/mL.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the cryopreservation media comprises dimethyl sulfoxide (DMSO).

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the cryopreservation media comprises 7% to 10% DMSO.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the first period of time in step (b) is performed over a period of time of at or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the second period of time in step (c) is performed over a period of time of at or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the first period of time in step (b) and the second period of time in step (c) are each performed separately over a period of time of at or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the first period of time in step (b) and the second period of time in step (c) are each performed separately over a period of time of at or about 5 days, 6 days, or 7 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the first period of time in step (b) and the second period of time in step (c) are each performed separately over a period of time of at or about 7 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed for a total of from or about 14 days to or about 18 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed for a total of from or about 15 days to or about 18 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed for a total of from or about 16 days to or about 18 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed for a total of from or about 14 days to or about 17 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed for a total of from or about 15 days to or about 17 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed for a total of from or about 14 days to or about 16 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed for a total of from or about 15 days to or about 16 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed for a total of at or about 14 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed for a total of at or about 15 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed for a total of at or about 16 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed for a total of at or about 17 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed for a total of at or about 18 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed in a total of or about 14 days or less.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed in a total of or about 15 days or less.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed in a total of or about 16 days or less.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed in a total of or about 17 days or less.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that steps (a) to (d) are performed in a total of or about 18 days or less.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the population of therapeutic TILs collected in step (d) includes sufficient TILs for a therapeutically effective dose of TILs.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the amount of TIL sufficient for a therapeutically effective dose is at or about 2.3 x 10¹⁰To or about 13.7 x 10¹⁰。

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the third TIL population in step (c) provides increased efficacy, increased interferon-gamma production and/or increased polyclonality.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the third population of TILs in step (c) provides at least one to five times or more interferon-gamma production compared to TILs prepared by a process longer than 16 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the third population of TILs in step (c) provides at least one to five times or more interferon-gamma production compared to TILs prepared by a process longer than 17 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the third population of TILs in step (c) provides at least one to five times or more interferon-gamma production compared to TILs prepared by a process longer than 18 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs for use above, modified such that effector T cells and/or central memory T cells obtained from the third TIL population in step (c) exhibit increased expression of CD8 and CD28 relative to effector T cells and/or central memory T cells obtained from the second population of cells in step (b).

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that each container recited in the method is a closed container.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that each container recited in the method is a G-container.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that each container recited in the method is GREX-10.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that each container recited in the method is GREX-100.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that each container recited in the method is GREX-500.

In another embodiment, the invention provides a therapeutic Tumor Infiltrating Lymphocyte (TIL) population prepared by the method as described in any of the preceding paragraphs, where applicable, above.

In another embodiment, the invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from tumor tissue of a patient, wherein the therapeutic TIL population provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality as compared to TILs prepared by a process of first expansion of TILs without the addition of any Antigen Presenting Cells (APCs) or OKT 3.

In another embodiment, the present invention provides a therapeutic Tumor Infiltrating Lymphocyte (TIL) population prepared from tumor tissue of a patient, wherein the therapeutic TIL population provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality as compared to TILs prepared by a process of first expansion of TILs without the addition of any Antigen Presenting Cells (APCs).

In another embodiment, the present invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from tumor tissue of a patient, wherein the therapeutic TIL population provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality as compared to TILs prepared by a process of first expansion of TILs without the addition of any OKT 3.

In another embodiment, the invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population prepared from tumor tissue of a patient, wherein the therapeutic TIL population provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality as compared to TILs prepared by a process of first expansion of TILs without the addition of Antigen Presenting Cells (APCs) or without the addition of OKT 3.

In another embodiment, the invention provides a therapeutic Tumor Infiltrating Lymphocyte (TIL) population prepared from tumor tissue of a patient, wherein the therapeutic TIL population provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality compared to TILs prepared by a process longer than 16 days of the process.

In another embodiment, the invention provides a therapeutic Tumor Infiltrating Lymphocyte (TIL) population prepared from tumor tissue of a patient, wherein the therapeutic TIL population provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality compared to TILs prepared by a process longer than 17 days of the process.

In another embodiment, the invention provides a therapeutic Tumor Infiltrating Lymphocyte (TIL) population prepared from tumor tissue of a patient, wherein the therapeutic TIL population provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality compared to TILs prepared by a process longer than 18 days of the process.

In another embodiment, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs for use above that provides increased interferon- γ production.

In another embodiment, the present invention provides a therapeutic TIL population as described in any of the preceding paragraphs for use above that provides increased polyclonality.

In another embodiment, the present invention provides a therapeutic TIL population as described in any of the preceding paragraphs for use above that provides increased efficacy.

In another embodiment, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs for use above, modified such that the therapeutic TIL population is capable of producing at least one-fold more interferon- γ than a TIL prepared by a process longer than 16 days. In another embodiment, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs for use above, modified such that the therapeutic TIL population is capable of producing at least one-fold more interferon- γ than a TIL prepared by a process longer than 17 days. In another embodiment, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs for use above, modified such that the therapeutic TIL population is capable of producing at least one-fold more interferon- γ than a TIL prepared by a process longer than 18 days. In some embodiments, the TIL is enabled to produce at least one-fold more interferon- γ as a result of the amplification process described herein, e.g., as described in steps a-F above or according to steps a-F above (also as shown, e.g., in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)).

In another embodiment, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs for use above, modified such that the therapeutic TIL population is capable of producing at least two times more interferon- γ than a TIL prepared by a process longer than 16 days. In another embodiment, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs for use above, modified such that the therapeutic TIL population is capable of producing at least two times more interferon- γ than a TIL prepared by a process longer than 17 days. In another embodiment, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs for use above, modified such that the therapeutic TIL population is capable of producing at least twice as much interferon- γ as TILs prepared by a process longer than 18 days. In some embodiments, the TIL is capable of producing at least twice as much interferon- γ as a result of the amplification process described herein, e.g., as described in steps a-F above or according to steps a-F above (also as shown, e.g., in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)).

In another embodiment, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs for use above, modified such that the therapeutic TIL population is capable of producing at least three times more interferon- γ than a TIL prepared by a process longer than 16 days. In another embodiment, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs for use above, modified such that the therapeutic TIL population is capable of producing at least three times more interferon- γ than a TIL prepared by a process longer than 17 days. In another embodiment, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs for use above, modified such that the therapeutic TIL population is capable of producing at least three times more interferon- γ than a TIL prepared by a process longer than 18 days. In some embodiments, the TIL is capable of producing at least three times more interferon- γ as a result of the amplification process described herein, e.g., as described in steps a-F above or according to steps a-F above (also as shown, e.g., in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)).

In another embodiment, the invention provides a population of therapeutic Tumor Infiltrating Lymphocytes (TILs) capable of producing at least one-fold more interferon- γ than TILs prepared by a process of first expansion of TILs without the addition of any Antigen Presenting Cells (APCs). In some embodiments, the TIL is enabled to produce at least one-fold more interferon- γ as a result of the amplification process described herein, e.g., as described in steps a-F above or according to steps a-F above (also as shown, e.g., in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)).

In another embodiment, the invention provides a therapeutic tumor-infiltrating lymphocyte (TIL) population capable of producing at least one-fold more interferon- γ than TIL prepared by a process of first expansion of TIL without the addition of any OKT 3. In some embodiments, the TIL is enabled to produce at least one-fold more interferon- γ as a result of the amplification process described herein, e.g., as described in steps a-F above or according to steps a-F above (also as shown, e.g., in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)).

In another embodiment, the invention provides a population of therapeutic TILs capable of producing at least two-fold more interferon- γ than TILs prepared by a process of first amplification of TILs without addition of any APC.

In another embodiment, the invention provides a population of therapeutic TILs capable of producing at least two-fold more interferon- γ than TILs prepared by a process of first amplification of TILs without addition of any APC. In some embodiments, the TIL is capable of producing at least twice as much interferon- γ as a result of the amplification process described herein, e.g., as described in steps a-F above or according to steps a-F above (also as shown, e.g., in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)).

In another embodiment, the invention provides a therapeutic TIL population capable of producing at least two-fold more interferon- γ than TIL prepared by a process in which a first amplification of TIL is performed without the addition of any OKT 3. In some embodiments, the TIL is capable of producing at least twice as much interferon- γ as a result of the amplification process described herein, e.g., as described in steps a-F above or according to steps a-F above (also as shown, e.g., in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)).

In another embodiment, the invention provides a population of therapeutic TILs capable of producing at least three times more interferon- γ than TILs prepared by a process of performing a first amplification of TILs without the addition of any APC. In some embodiments, the TIL is enabled to produce at least one-fold more interferon- γ as a result of the amplification process described herein, e.g., as described in steps a-F above or according to steps a-F above (also as shown, e.g., in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)).

In another embodiment, the invention provides a therapeutic TIL population capable of producing at least three times more interferon- γ than TIL prepared by a process in which a first amplification of TIL is performed without the addition of any OKT 3. In some embodiments, the TIL is capable of producing at least three times more interferon- γ as a result of the amplification process described herein, e.g., as described in steps a-F above or according to steps a-F above (also as shown, e.g., in fig. 1 (specifically, e.g., fig. 1B and/or fig. 1C)).

In another embodiment, the present invention provides a method of expanding T cells, the method comprising: (a) priming a first expansion of a first population of T cells obtained from a donor by culturing the first population of T cells to effect growth and prime activation of the first population of T cells; (b) after the onset of decay of activation of the first population of T cells elicited in step (a), eliciting a rapid second expansion of the first population of T cells by culturing the first population of T cells to effect growth and enhance activation of the first population of T cells to obtain a second population of T cells; and (c) collecting the second population of T cells. In another embodiment, the step of rapid second amplification is divided into multiple steps to achieve a scaled-up culture by: (a) performing a rapid second expansion by culturing the first T cell population in a small-scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 3 to 4 days, and then (b) effecting transfer of the first T cell population from the small-scale culture to a second vessel larger than the first vessel (e.g., a G-REX 500MCS vessel), and culturing the first T cell population from the small-scale culture in a larger-scale culture in the second vessel for a period of about 4 to 7 days. In another embodiment, the step of rapid amplification is divided into multiple steps to achieve the outwardly expanding culture by: (a) performing a rapid second expansion by culturing the first population of T cells in a first small-scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 3 to 4 days, and then (b) effecting transfer and distribution of the first population of T cells from the first small-scale culture into or among at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels of equal size to the first vessel, wherein in each second vessel the portion of the first population of T cells transferred from the first small-scale culture to this second vessel is cultured in a second small-scale culture for a period of about 4 to 7 days. In another embodiment, the step of rapid amplification is divided into multiple steps to achieve the culture of scale-out and scale-up by: (a) performing a rapid second expansion by culturing the first T cell population in a small-scale culture in a first container (e.g., a G-REX 100MCS container) for a period of about 3 to 4 days, and then (b) effecting a transfer and distribution of the first T cell population from the small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers that are larger in size than the first container (e.g., a G-REX 500MCS container), wherein in each second container the portion of the first T cell population that is transferred from the small-scale culture to this second container is cultured in a larger-scale culture for a period of about 4 to 7 days. In another embodiment, the step of rapid amplification is divided into multiple steps to achieve the culture of scale-out and scale-up by: (a) performing a rapid second expansion by culturing the first T cell population in a small-scale culture in a first container (e.g., a G-REX 100MCS container) for a period of about 4 days, and then (b) effecting transfer and partitioning of the first T cell population from the small-scale culture into at least 2, 3, or 4 second containers of a larger size than the first container (e.g., a G-REX 500MCS container), wherein in each second container the portion of the first T cell population transferred from the small-scale culture to this second container is cultured in a larger-scale culture for a period of about 5 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that the step of rapid second amplification is divided into a plurality of steps to achieve scale-up culture by: (a) performing a rapid second expansion by culturing the first T cell population in a small-scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 2 to 4 days, and then (b) effecting transfer of the first T cell population from the small-scale culture to a second vessel larger than the first vessel (e.g., a G-REX 500MCS vessel), and culturing the first T cell population from the small-scale culture in a larger-scale culture in the second vessel for a period of about 5 to 7 days.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that the step of rapid amplification is divided into a plurality of steps to effect the culture of the outward expansion by: (a) performing a rapid second expansion by culturing the first population of T cells in a first small-scale culture in a first vessel (e.g., a G-REX 100MCS vessel) for a period of about 2 to 4 days, and then (b) effecting transfer and distribution of the first population of T cells from the first small-scale culture into or among at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second vessels of equal size to the first vessel, wherein in each second vessel the portion of the first population of T cells transferred from the first small-scale culture to this second vessel is cultured in a second small-scale culture for a period of about 5 to 7 days.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the step of rapid amplification is divided into a plurality of steps to achieve the outwardly expanding and scale-up culture by: (a) performing a rapid second expansion by culturing the first T cell population in a small-scale culture in a first container (e.g., a G-REX 100MCS container) for a time period of about 2 to 4 days, and then (b) effecting transfer and distribution of the first T cell population from the small-scale culture into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 second containers of a larger size than the first container (e.g., a G-REX 500MCS container), wherein in each second container the portion of the first T cell population transferred from the small-scale culture to this second container is cultured in a larger-scale culture for a time period of about 5 to 7 days.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the step of rapid amplification is divided into a plurality of steps to achieve the outwardly expanding and scale-up culture by: (a) performing a rapid second expansion by culturing the first T cell population in a small-scale culture in a first container (e.g., a G-REX 100MCS container) for a period of about 3 to 4 days, and then (b) effecting transfer and partitioning of the first T cell population from the small-scale culture into at least 2, 3, or 4 second containers of a larger size than the first container (e.g., a G-REX 500MCS container), wherein in each second container the portion of the first T cell population transferred from the small-scale culture to this second container is cultured in a larger-scale culture for a period of about 5 to 6 days.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the step of rapid amplification is divided into a plurality of steps to achieve the outwardly expanding and scale-up culture by: (a) performing a rapid second expansion by culturing the first T cell population in a small-scale culture in a first container (e.g., a G-REX 100MCS container) for a period of about 3 to 4 days, and then (b) effecting transfer and partitioning of the first T cell population from the small-scale culture into at least 2, 3, or 4 second containers of a larger size than the first container (e.g., a G-REX 500MCS container), wherein in each second container the portion of the first T cell population transferred from the small-scale culture to this second container is cultured in a larger-scale culture for a period of about 5 days.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the step of rapid amplification is divided into a plurality of steps to achieve the outwardly expanding and scale-up culture by: (a) performing a rapid second expansion by culturing the first T cell population in a small-scale culture in a first container (e.g., a G-REX 100MCS container) for a period of about 3 to 4 days, and then (b) effecting transfer and partitioning of the first T cell population from the small-scale culture into at least 2, 3, or 4 second containers of a larger size than the first container (e.g., a G-REX 500MCS container), wherein in each second container the portion of the first T cell population transferred from the small-scale culture to this second container is cultured in a larger-scale culture for a period of about 6 days.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the step of rapid amplification is divided into a plurality of steps to achieve the outwardly expanding and scale-up culture by: (a) performing a rapid second expansion by culturing the first T cell population in a small-scale culture in a first container (e.g., a G-REX 100MCS container) for a period of about 3 to 4 days, and then (b) effecting transfer and partitioning of the first T cell population from the small-scale culture into at least 2, 3, or 4 second containers of a larger size than the first container (e.g., a G-REX 500MCS container), wherein in each second container the portion of the first T cell population transferred from the small-scale culture to this second container is cultured in a larger-scale culture for a period of about 7 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that the priming first amplification of step (a) is performed during a period of up to 7 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that the rapid second amplification of step (b) is performed during a period of up to 8 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that the rapid second amplification of step (b) is performed during a period of up to 9 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that the rapid second amplification of step (b) is performed during a period of up to 10 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that the rapid second amplification of step (b) is performed during a period of up to 11 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the priming first amplification of step (a) is performed during a period of 7 days and the rapid second amplification of step (b) is performed during a period of up to 9 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the priming first amplification in step (a) is performed during a period of 7 days and the rapid second amplification of step (b) is performed during a period of up to 10 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the priming first amplification of step (a) is performed during a period of 7 or 8 days and the rapid second amplification of step (b) is performed during a period of up to 9 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the priming first amplification of step (a) is performed during a period of 7 or 8 days and the rapid second amplification of step (b) is performed during a period of up to 10 days.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the priming first amplification of step (a) is performed during a period of 8 days and the rapid second amplification of step (b) is performed during a period of up to 9 days.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs as applicable above, modified such that the priming first amplification in step (a) is performed during a period of 8 days and the rapid second amplification of step (b) is performed during a period of up to 8 days.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, which method is modified such that in step (a) the first population of T cells is cultured in a first medium comprising OKT-3 and IL-2.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the first medium comprises a 4-1BB agonist, OKT-3 and IL-2.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the first medium comprises OKT-3, IL-2 and Antigen Presenting Cells (APCs).

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the first culture medium comprises a 4-1BB agonist, OKT-3, IL-2, and Antigen Presenting Cells (APCs).

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, the method being modified such that in step (a) the first population of T cells is cultured in a first culture medium in a container comprising a first gas permeable surface, wherein the first culture medium comprises OKT-3, IL-2 and a first population of Antigen Presenting Cells (APCs), wherein the first population of APCs is exogenous to a donor of the first population of T cells and the first population of APCs is layered on the first gas permeable surface, wherein in step (b) the first population of T cells is cultured in a second culture medium in the container, wherein the second culture medium comprises OKT-3, IL-2 and a second population of APCs, wherein the second population of APCs is exogenous to a donor of the first population of T cells and the second population of APCs is layered on the first gas permeable surface, and wherein the second population of APCs is greater than the first population of APCs.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs for use above, the method modified such that in step (a), the first population of T cells is cultured in a first culture medium in a container comprising a first gas permeable surface, wherein the first culture medium comprises a 4-1BB agonist, OKT-3, IL-2, and a first population of Antigen Presenting Cells (APCs), wherein the first population of APCs is exogenous to a donor of the first population of T cells and is layered on the first gas permeable surface, wherein in step (b), the first population of T cells is cultured in a second culture medium in the container, wherein the second culture medium comprises OKT-3, IL-2, and a second population of APCs, wherein the second population of APCs is APCs to a donor of the first population of T cells and is layered on the first gas permeable exogenous population, and wherein the second population of APCs is greater than the first population of APCs.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, the method is modified such that in step (a) the first population of T cells is cultured in a first culture medium in a container comprising a first gas permeable surface, wherein the first culture medium comprises OKT-3, IL-2 and a first population of Antigen Presenting Cells (APCs), wherein the first population of APCs is exogenous to a donor of the first population of T cells and the first population of APCs is layered on the first gas permeable surface, wherein in step (b) the first population of T cells is cultured in a second culture medium in the container, wherein the second culture medium comprises a 4-1BB agonist, OKT-3, IL-2 and a second population of APCs, wherein the second population of APCs is APCs to a donor of the first population of T cells and the second population of APCs is layered on the first gas permeable surface, and wherein the second population of APCs is greater than the first population of APCs.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs for use above, the method modified such that in step (a), the first population of T cells is cultured in a first culture medium in a container comprising a first gas permeable surface, wherein the first culture medium comprises a 4-1BB agonist, OKT-3, IL-2, and a first population of Antigen Presenting Cells (APCs), wherein the first population of APCs is exogenous to a donor of the first population of T cells and is layered on the first gas permeable surface, wherein in step (b), the first population of T cells is cultured in a second culture medium in the container, wherein the second culture medium comprises a 4-1BB agonist, OKT-3, IL-2, and a second population of APCs, wherein the second population of APCs is exogenous to a donor of the first population of T cells and is layered on the first gas permeable surface And wherein the second population of APCs is greater than the first population of APCs.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, that is modified such that the ratio of the number of APCs in the second population of APCs to the number of APCs in the first population of APCs is about 2: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that the number of APCs in the first population of APCs is about 2.5 x 10⁸And the number of APCs in the second APC population is about 5X 10⁸。

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, that is modified such that in step (a) a first population of APCs is layered on the first gas-permeable surface at an average thickness of 2 layers of APCs.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, that is modified such that in step (b) a second population of APCs is layered on the first gas-permeable surface with an average thickness selected from a range of 4 to 8 layers of APCs.

In another embodiment, the present invention provides the method of any one of the preceding paragraphs as applicable above, modified such that the ratio of the average number of layers of APCs laminated on the first gas-permeable surface in step (b) to the average number of layers of APCs laminated on the first gas-permeable surface in step (a) is 2: 1.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (a) the first population of APCs is selected to be at or about 1.0 x 10 ⁶APC/cm²To or about 4.5 x 10⁶APC/cm²Is seeded on the first gas-permeable surface.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (a) the first population of APCs is selected to be at or about 1.5 x 10⁶APC/cm²To or about 3.5 x 10⁶APC/cm²Is seeded on the first gas-permeable surface.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (a) the first population of APCs is selected to be at or about 2.0 x 10⁶APC/cm²To or about 3.0 x 10⁶APC/cm²Is seeded on the first gas-permeable surface.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that in step (a) the first population of APCs is at or about 2.0 x 10⁶APC/cm²Is seeded on the first gas-permeable surface.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (b) the second population of APCs is selected to be at or about 2.5 x 10 ⁶APC/cm²To or about 7.5 x 10⁶APC/cm²Is seeded on the first gas-permeable surface.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that in step (b) the second population of APCs is treated with a second agentIs selected from the group consisting of at or about 3.5X 10⁶APC/cm²To or about 6.0 x 10⁶APC/cm²Is seeded on the first gas-permeable surface.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (b) the second population of APCs is selected to be at or about 4.0 x 10⁶APC/cm²To or about 5.5 x 10⁶APC/cm²Is seeded on the first gas-permeable surface.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that in step (b) the second population of APCs is at or about 4.0 x 10⁶APC/cm²Is seeded on the first gas-permeable surface.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (a) the first population of APCs is selected to be at or about 1.0 x 10 ⁶APC/cm²To or about 4.5 x 10⁶APC/cm²Is seeded on the first gas permeable surface, and the second population of APCs is seeded in step (b) at a density selected from at or about 2.5X 10⁶APC/cm²To or about 7.5 x 10⁶APC/cm²Is seeded on the first gas-permeable surface.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable, the method being modified such that in step (a) the first population of APCs is selected to be at or about 1.5 x 10⁶APC/cm²To or about 3.5 x 10⁶APC/cm²Is seeded on the first gas permeable surface, and the second population of APCs is seeded in step (b) at a density selected from at or about 3.5X 10⁶APC/cm²To or about 6.0 x 10⁶APC/cm²Is seeded on the first gas-permeable surface.

At another placeIn one embodiment, the present invention provides a method as described in any of the preceding paragraphs as applicable above, the method being modified such that in step (a) the first population of APCs is selected to be at or about 2.0 x 10⁶APC/cm²To or about 3.0 x 10⁶APC/cm²Is seeded on the first gas permeable surface, and the second population of APCs is seeded in step (b) at a density selected from at or about 4.0X 10 ⁶APC/cm²To or about 5.5 x 10⁶APC/cm²Is seeded on the first gas-permeable surface.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that in step (a) the first population of APCs is at or about 2.0 x 10⁶APC/cm²Is seeded on the first gas permeable surface, and in step (b) the second population of APCs is at or about 4.0X 10⁶APC/cm²Is seeded on the first gas-permeable surface.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the APCs are Peripheral Blood Mononuclear Cells (PBMCs).

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the PBMCs are irradiated and exogenous to the donor of the first T cell population.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that the T cells are Tumour Infiltrating Lymphocytes (TILs).

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the T cells are Marrow Infiltrating Lymphocytes (MILs).

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that the T cells are Peripheral Blood Lymphocytes (PBLs).

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the first population of T cells is obtained by separation from whole blood of a donor.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the first population of T cells is obtained by isolation from an apheresis product of a donor.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the first population of T cells is isolated from whole blood or apheresis products of a donor by positive or negative selection for a T cell phenotype.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that the T cell phenotype is CD3+ and CD45 +.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, which method is modified such that T cells are separated from NK cells prior to performing the priming first expansion of the first population of T cells. In another embodiment, the T cells are separated from NK cells in the first population of T cells by removing CD3-CD56+ cells from the first population of T cells. In another embodiment, CD3-CD56+ cells are removed from the first population of T cells by cell sorting the first population of T cells using a gating strategy that removes the CD3-CD56+ cell fraction and restores the negative fraction. In another embodiment, the foregoing method is used for T cell expansion in a first population of T cells characterized by a high percentage of NK cells. In another embodiment, the foregoing method is used for T cell expansion in a first population of T cells characterized by a high percentage of CD3-CD56+ cells. In another embodiment, the foregoing method is used for T cell expansion in tumor tissue characterized by the presence of large numbers of NK cells. In another embodiment, the foregoing methods are used for T cell expansion in tumor tissue characterized by a plurality of CD3-CD56+ cells. In another embodiment, the foregoing method is used for T cell expansion in tumor tissue obtained from a patient having a tumor characterized by the presence of a large number of NK cells. In another embodiment, the foregoing methods are used for T cell expansion in tumor tissue obtained from a patient having a tumor characterized by the presence of a plurality of CD3-CD56+ cells. In another embodiment, the foregoing methods are used for T cell expansion in tumor tissue obtained from a patient having ovarian cancer.

In another embodiment, the present invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that at or about 1 x 10 from the first population of T cells⁷Individual T cells were seeded in a vessel to initiate an initial first expansion culture in this vessel.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, modified such that the first population of T cells is distributed into a plurality of containers and in each container is seeded with at or about 1 x 10 cells from the first population of T cells⁷The individual T cells were cultured to initiate an initial first expansion in this vessel.

In another embodiment, the invention provides a method as described in any of the preceding paragraphs, where applicable, which is modified such that the second population of T cells collected in step (c) is a therapeutic TIL population.

Pharmaceutical compositions, dosages and dosing regimens

In embodiments, the TIL amplified using the methods of the present disclosure is administered to a patient in the form of a pharmaceutical composition. In the examples, the pharmaceutical composition is a suspension of TIL in sterile buffer. TILs amplified using PBMCs of the present disclosure may be administered by any suitable route as known in the art. In some embodiments, the T cells are administered as a single intra-arterial or intravenous infusion, preferably for about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.

Any suitable agent may be administeredAmount of TIL. In some embodiments, about 2.3 x 10 is administered¹⁰To about 13.7X 10¹⁰TILs of which average about 7.8X 10¹⁰TIL, especially where the cancer is melanoma. In some embodiments, about 1.2 x 10 is administered¹⁰To about 4.3X 10¹⁰And (4) TIL. In some embodiments, about 3 x 10 is administered¹⁰To about 12X 10¹⁰And (4) TIL. In some embodiments, about 4 x 10 is administered¹⁰To about 10X 10¹⁰And (4) TIL. In some embodiments, about 5 x 10 is administered¹⁰To about 8X 10¹⁰And (4) TIL. In some embodiments, about 6 x 10 is administered¹⁰To about 8X 10¹⁰And (4) TIL. In some embodiments, about 7 x 10 is administered¹⁰To about 8X 10¹⁰And (4) TIL. In some embodiments, the therapeutically effective dose is about 2.3 x 10¹⁰To about 13.7X 10¹⁰. In some embodiments, the therapeutically effective dose is about 7.8 x 10¹⁰TIL, especially where the cancer is melanoma. In some embodiments, the therapeutically effective dose is about 1.2 x 10¹⁰To about 4.3X 10¹⁰And (4) TIL. In some embodiments, the therapeutically effective dose is about 3 x 10¹⁰To about 12X 10¹⁰And (4) TIL. In some embodiments, the therapeutically effective dose is about 4 x 10¹⁰To about 10X 10¹⁰And (4) TIL. In some embodiments, the therapeutically effective dose is about 5 x 10¹⁰To about 8X 10 ¹⁰And (4) TIL. In some embodiments, the therapeutically effective dose is about 6 x 10¹⁰To about 8X 10¹⁰And (4) TIL. In some embodiments, the therapeutically effective dose is about 7 x 10¹⁰To about 8X 10¹⁰And (4) TIL.

In some embodiments, the concentration of TIL provided in a pharmaceutical composition of the invention is less than, e.g., 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001%, w/v/w/v% or w/v/w of the pharmaceutical composition.

In some embodiments, the concentration of TIL provided in a pharmaceutical composition of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50%, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75%, 7.50%, 7.25%, 6.25%, 6.5%, 6.75%, 3.5%, 4.75%, 3.75%, 3.25%, 3.75%, 4.25%, 3.25%, 4.25%, 3.25%, 3.75%, 3.25%, 4.25%, or more% of the pharmaceutical composition, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v.

In some embodiments, the TIL is provided in a pharmaceutical composition of the invention at a concentration in a range of about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/v, or w/v of the pharmaceutical composition.

In some embodiments, the TIL is provided in a pharmaceutical composition of the invention at a concentration in a range from about 0.001% to about 10%, from about 0.01% to about 5%, from about 0.02% to about 4.5%, from about 0.03% to about 4%, from about 0.04% to about 3.5%, from about 0.05% to about 3%, from about 0.06% to about 2.5%, from about 0.07% to about 2%, from about 0.08% to about 1.5%, from about 0.09% to about 1%, from about 0.1% to about 0.9% w/w, w/v, or v/v of the pharmaceutical composition.

In some embodiments, TIL is provided in the pharmaceutical compositions of the invention in an amount equal to or less than 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0.95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, 0.35g, 0.3g, 0.25g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.06g, 0.008g, 0.35g, 0000.3 g, 0.25g, 0.2g, 0.15g, 0.1g, 0.09g, 0.06g, 00006 g, 0.06g, 0.04g, 0.01g, 0.3g, 0.06g, 0.3g, 0.01g, 0.06g, 0.01g, 0.06g, 0.3g, 0.01g, 0.06g, 0.01g, 0.3g, 0.01g, 0.3g, 0.06g, 0.01g, 0.3g, 0.01g, 0.3 g.

In some embodiments, TIL is provided in a pharmaceutical composition of the invention in an amount greater than 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002g, 0.0025g, 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007g, 0.0075g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025g, 0.03g, 0.035g, 0.04g, 0.05g, 060.06 g, 0.06g, 0.15g, 0.7g, 0.06g, 0.5g, 0.15g, 0.7g, 0.15g, 0.7g, 0.15g, 0.6g, 0.7g, 0.6g, 0.15g, 0.7g, 0.6g, 0.15g, 0.7g, 0.15g, 0.7g, 0.6g, 0.15g, 0.7g, 0.15g, 0.6g, 0.15g, 0.7g, 0.6g, 0.7g, 0.15g, 0.7g, 0.6g, 0.15g, 0.6g, 0.7g, 0.15g, 0.6g, 0.7g, 4g, 0.15g, 4g, 0.6g, 0.7g, 4g, 0.7g, 4g, 0.6g, 0.15g, 4g, 0.7g, 1g, 4g, 0.15g, 0, 8.5g, 9g, 9.5g or 10 g.

The TILs provided in the pharmaceutical compositions of the present invention are effective over a wide dosage range. The exact dosage will depend upon the route of administration, the form of administration of the compound, the sex and age of the individual to be treated, the weight of the individual to be treated and the preferences and experience of the attending physician. Clinically determined TIL doses may also be used where appropriate. The amount of the pharmaceutical composition (e.g., the dose of TIL) administered using the methods herein will depend on the severity of the human or mammal, disorder or condition being treated, the rate of administration, the disposition of the active pharmaceutical ingredient, and the judgment of the prescribing physician.

In some embodiments, TIL may be administered in a single dose. Such administration may be by injection, for example intravenous injection. In some embodiments, the TIL may be administered in multiple dosage forms. The administration may be once, twice, three times, four times, five times, six times or more than six times per year. Administration may be monthly, biweekly, weekly, or every other day. Administration of TIL may be continued as long as necessary.

In some embodiments, an effective dose of TIL is about 1 × 10⁶、2×10⁶、3×10⁶、4×10⁶、5×10⁶、6×10⁶、7×10⁶、8×10⁶、9×10⁶、1×10⁷、2×10⁷、3×10⁷、4×10⁷、5×10⁷、6×10⁷、7×10⁷、8×10⁷、9×10⁷、1×10⁸、2×10⁸、3×10⁸、4×10⁸、5×10⁸、6×10⁸、7×10⁸、8×10⁸、9×10⁸、1×10⁹、2×10⁹、3×10⁹、4×10⁹、5×10⁹、6×10⁹、7×10⁹、8×10⁹、9×10⁹、1×10¹⁰、2×10¹⁰、3×10¹⁰、4×10¹⁰、5×10¹⁰、6×10¹⁰、7×10¹⁰、8×10¹⁰、9×10¹⁰、1×10¹¹、2×10¹¹、3×10¹¹、4×10¹¹、5×10¹¹、6×10¹¹、7×10¹¹、8×10¹¹、9×10¹¹、1×10¹²、2×10¹²、3×10¹²、4×10¹²、5×10¹²、6×10¹²、7×10¹²、8×10¹²、9×10¹²、1×10¹³、2×10¹³、3×10¹³、4×10¹³、5×10¹³、6×10¹³、7×10¹³、8×10¹³And 9X 10¹³. In some embodiments, an effective dose of TIL is 1X 10⁶To 5X 10⁶、5×10⁶To 1X 10⁷、1×10⁷To 5X 10⁷、5×10⁷To 1X 10⁸、1×10⁸To 5X 10⁸、5×10⁸To 1X 10⁹、1×10⁹To 5X 10⁹、5×10⁹To 1X 10¹⁰、1×10¹⁰To 5X 10¹⁰、5×10¹⁰To 1X 10¹¹、5×10¹¹To 1X 10¹²、1×10¹²To 5X 10¹²And 5X 10¹²To 1X 10¹³Within the range of (1).

In some embodiments, an effective dose of TIL is between about 0.01mg/kg to about 4.3mg/kg, about 0.15mg/kg to about 3.6mg/kg, about 0.3mg/kg to about 3.2mg/kg, about 0.35mg/kg to about 2.85mg/kg, about 0.15mg/kg to about 2.85mg/kg, about 0.3mg to about 2.15mg/kg, about 0.45mg/kg to about 1.7mg/kg, about 0.15mg/kg to about 1.3mg/kg, about 0.3mg/kg to about 1.15mg/kg, about 0.45mg/kg to about 1mg/kg, about 0.55mg/kg to about 0.85mg/kg, about 0.65mg/kg to about 0.8mg/kg, about 0.7mg/kg to about 0.75mg/kg, about 0.7mg/kg to about 2.85mg/kg, about 0.85mg/kg to about 1.85mg/kg, about 0.85mg/kg, About 1.15mg/kg to about 1.7mg/kg, about 1.3mg/kg to about 1.6mg/kg, about 1.35mg/kg to about 1.5mg/kg, about 2.15mg/kg to about 3.6mg/kg, about 2.3mg/kg to about 3.4mg/kg, about 2.4mg/kg to about 3.3mg/kg, about 2.6mg/kg to about 3.15mg/kg, about 2.7mg/kg to about 3mg/kg, about 2.8mg/kg to about 3mg/kg, or about 2.85mg/kg to about 2.95 mg/kg.

In some embodiments, an effective dose of the TIL is in the range of about 1mg to about 500mg, about 10mg to about 300mg, about 20mg to about 250mg, about 25mg to about 200mg, about 1mg to about 50mg, about 5mg to about 45mg, about 10mg to about 40mg, about 15mg to about 35mg, about 20mg to about 30mg, about 23mg to about 28mg, about 50mg to about 150mg, about 60mg to about 140mg, about 70mg to about 130mg, about 80mg to about 120mg, about 90mg to about 110mg, or about 95mg to about 105mg, about 98mg to about 102mg, about 150mg to about 250mg, about 160mg to about 240mg, about 170mg to about 230mg, about 180mg to about 220mg, about 190mg to about 210mg, about 195mg to about 205mg, or about 198 mg to about 207 mg.

An effective amount of TIL may be administered in a single or multiple dosage form by any of the recognized modes of agent administration with similar utilities, including intranasal and transdermal routes, by intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topical, by implantation, or by inhalation.

In another embodiment, the invention provides an infusion bag comprising a population of therapeutic TILs as described in any of the preceding paragraphs as applicable above.

In another embodiment, the invention provides a Tumor Infiltrating Lymphocyte (TIL) composition comprising a therapeutic TIL population as described in any of the preceding paragraphs for use above and a pharmaceutically acceptable carrier.

In another embodiment, the present invention provides an infusion bag comprising a TIL composition as described in any of the preceding paragraphs as applicable above.

In another embodiment, the invention provides a cryopreserved formulation of a therapeutic TIL population as described in any of the preceding paragraphs for use above.

In another embodiment, the invention provides a Tumor Infiltrating Lymphocyte (TIL) composition comprising a therapeutic TIL population as described in any of the preceding paragraphs for use above and a cryopreservation medium.

In another embodiment, the invention provides a TIL composition as described in any of the preceding paragraphs as applicable above, modified such that the cryopreservation media contains DMSO.

In another embodiment, the invention provides a TIL composition as described in any of the preceding paragraphs, where applicable, modified such that the cryopreservation media contains 7-10% DMSO.

In another embodiment, the present invention provides a cryopreserved formulation of a TIL composition as described in any of the preceding paragraphs for use as described above.

Methods of treating patients

The treatment method was started with initial TIL collection and TIL culture. Such methods are described in the art by, for example, Jin et al, journal of immunotherapy, 2012,35(3):283-292, which is incorporated herein by reference in its entirety. Examples of methods of treatment are described in the entire section below (including examples).

The amplified TILs produced according to the methods described herein, including, for example, as described above for steps a-F or as produced according to above steps a-F (as also shown, for example, in fig. 1 (specifically, e.g., fig. 1B)), have particular utility in treating patients with cancer (e.g., as described in Goff et al, J. Clin. Oncology, 2016,34(20): 2389-. In some embodiments, TIL is grown from excised metastatic melanoma deposits as previously described (see Dudley et al, J. Immunotherapy 2003,26: 332-. Fresh tumors can be dissected under sterile conditions. Representative samples can be collected for formal pathological analysis. Can use 2mm³To 3mm³Of the single fragment. In some embodiments, 5, 10, 15, 20, 25, or 30 samples per patient are obtained. In some embodiments, 20, 25, or 30 samples per patient are obtained. In some embodiments, 20, 22, 24, 26, or 28 samples per patient are obtained. In some embodiments, 24 samples per patient are obtained. Samples can be placed into individual wells of a 24-well plate, maintained in growth medium with high doses of IL-2(6,000IU/mL), and monitored for tumor destruction and/or proliferation of TILs. Any tumor remaining with viable cells after treatment can be enzymatically digested as described herein Single cell suspensions were formed and stored cryogenically.

In some embodiments, successfully grown TILs may be sampled for phenotypic analysis (CD3, CD4, CD8, and CD56) and tested for autologous tumors when available. TIL can be considered reactive if overnight co-culture produces interferon-gamma (IFN-. gamma.) levels of > 200pg/mL and twice the background. (Goff et al, J.Immunotherapy, 2010,33: 840-847; which is incorporated herein by reference in its entirety). In some embodiments, a culture with evidence of autoreactivity or sufficient growth pattern may be selected for a second amplification (e.g., as provided according to step D of fig. 1 (specifically, e.g., fig. 1B)), including a second amplification sometimes referred to as rapid amplification (REP). In some embodiments, amplified TILs with higher autoreactivity (e.g., high proliferation during the second amplification) are selected for additional second amplifications. In some embodiments, TILs with high auto-reactivity (e.g., high proliferation during the second amplification as provided in step D of fig. 1 (specifically, e.g., fig. 1B)) are selected for further second amplification according to step D of fig. 1 (specifically, e.g., fig. 1B).

In some embodiments, the patient does not move directly to ACT (adoptive cell transfer), e.g., in some embodiments, the cells are not utilized immediately after tumor acquisition and/or first expansion. In some embodiments, the TIL may be cryopreserved and thawed 2 days prior to administration to a patient. In some embodiments, the TIL may be cryopreserved and thawed 1 day prior to administration to a patient. In some embodiments, the TIL may be cryopreserved and thawed immediately prior to administration to a patient.

Cryopreserved samples of infusion bag TIL may be analyzed for cell phenotype by flow cytometry (e.g., FlowJo) against the surface markers CD3, CD4, CD8, CCR7, and CD45RA (BD biosciences), as well as by any of the methods described herein. Serum cytokines were measured by using standard enzyme-linked immunosorbent assay techniques. Serum IFN-g elevation was defined as > 100pg/mL and greater than 43 baseline levels.

In some embodiments, TILs produced by the methods provided herein, such as those illustrated in fig. 1 (specifically, e.g., fig. 1B), provide a surprising improvement in the clinical efficacy of TILs. In some embodiments, TILs produced by methods provided herein, such as those illustrated in fig. 1 (specifically, e.g., fig. 1B), exhibit increased clinical efficacy as compared to TILs produced by methods other than those described herein, including, for example, methods other than those illustrated in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, methods other than those described herein include methods referred to as process 1C and/or generation 1 (Gen 1). In some embodiments, increased efficacy is measured by DCR, ORR, and/or other clinical responses. In some embodiments, TILs produced by methods provided herein, such as those illustrated in fig. 1 (specifically, e.g., fig. 1B), exhibit similar response times and safety profiles as compared to TILs produced by methods other than those described herein, including, for example, methods other than those illustrated in fig. 1 (specifically, e.g., fig. 1B), such as the Gen 1 process.

In some embodiments, IFN- γ (IFN- γ) indicates therapeutic efficacy and/or increased clinical efficacy. In some embodiments, IFN- γ in the blood of a subject treated with TIL is indicative of active TIL. In some embodiments, a potency assay for IFN- γ production is employed. IFN- γ production is another measure of cytotoxic potential. IFN- γ production can be measured by determining the level of the cytokine IFN- γ in ex vivo blood, serum, or TIL of a subject treated with a TIL prepared by the methods of the invention, including as described, for example, in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, an increase in IFN- γ is indicative of the therapeutic efficacy of a patient treated with a TIL produced by the methods of the invention. In some embodiments, IFN- γ is increased by one, two, three, four, or five fold or more compared to an untreated patient and/or compared to a patient treated with a TIL prepared using methods other than those provided herein, including, for example, methods other than those embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, IFN- γ secretion is doubled compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, IFN- γ secretion is increased by two-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, IFN- γ secretion is increased three-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, IFN- γ secretion is increased four-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, IFN- γ secretion is increased by five-fold as compared to an untreated patient and/or as compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, IFN- γ is measured using a Quantikine ELISA kit. In some embodiments, IFN- γ is measured in ex vivo TILs of subjects treated with TILs prepared by the methods of the invention, including as described, for example, in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, IFN- γ is measured in the blood of a subject treated with a TIL prepared by the methods of the invention, including as described, for example, in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, IFN- γ is measured in TIL serum of a subject treated with TIL prepared by the methods of the invention, including as described, for example, in fig. 1 (specifically, e.g., fig. 1B).

In some embodiments, TILs prepared by the methods of the invention (including, for example, the methods described in fig. 1 (specifically, e.g., fig. 1B)) exhibit increased polyclonality as compared to TILs produced by other methods (including methods not exemplified in fig. 1 (specifically, e.g., fig. 1B), such as the method referred to as the process 1C method). In some embodiments, significantly increased polyclonality and/or increased polyclonality is indicative of therapeutic efficacy and/or increased clinical efficacy. In some embodiments, polyclonality refers to T cell bank diversity. In some embodiments, an increase in polyclonality may be indicative of therapeutic efficacy with respect to administration of the TIL produced by the methods of the invention. In some embodiments, the polyclonality is increased by one-fold, two-fold, ten-fold, 100-fold, 500-fold, or 1000-fold as compared to a TIL prepared using a method other than the methods provided herein, including, for example, a method other than the method embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, the polyclonality is doubled compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, the polyclonality is increased by two-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, the polyclonality is increased ten-fold as compared to an untreated patient and/or as compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, the polyclonality is increased 100-fold as compared to an untreated patient and/or as compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, the polyclonality is increased 500-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than the method embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, the polyclonality is increased 1000-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than the method embodied in fig. 1 (specifically, e.g., fig. 1B).

The measures of efficacy may include Disease Control Rate (DCR) and Overall Response Rate (ORR), as known in the art and described herein.

1. Methods of treating cancer and other diseases

The compositions and methods described herein may be used in methods of treating diseases. In embodiments, the compositions and methods are used to treat hyperproliferative disorders. The compositions and methods may also be used to treat other conditions as described herein and in the following paragraphs.

In some embodiments, the hyperproliferative disorder is cancer. In some embodiments, the hyperproliferative disorder is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of: glioblastoma (GBM), gastrointestinal cancer, melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma. In some embodiments, the hyperproliferative disorder is hematological malignancy. In some embodiments, the solid tumor cancer is selected from the group consisting of: chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-hodgkin's lymphoma, follicular lymphoma, and mantle cell lymphoma.

In some embodiments, the cancer is a highly mutated cancer phenotype. Highly mutated cancers are widely described in Campbell et al (cells, 171:1042-1056 (2017); which are incorporated herein by reference in their entirety for all purposes). In some embodiments, a highly mutated tumor comprises 9 to 10 mutations per megabase (Mb). In some embodiments, the pediatric hypermutant tumor comprises 9.91 mutations per megabase (Mb). In some embodiments, an adult hypermutant tumor comprises 9 mutations per megabase (Mb). In some embodiments, the enhanced hypermutated tumor comprises 10 to 100 mutations per megabase (Mb). In some embodiments, the enhanced pediatric hypermutant tumor comprises 10 to 100 mutations per megabase (Mb). In some embodiments, the enhanced adult hypermutant tumor comprises 10 to 100 mutations per megabase (Mb). In some embodiments, the hypermutation tumor comprises more than 100 mutations per megabase (Mb). In some embodiments, the pediatric hypermutation tumor comprises greater than 100 mutations per megabase (Mb). In some embodiments, an adult hypermutation tumor comprises greater than 100 mutations per megabase (Mb).

In some embodiments, the highly mutant tumor has a mutation in a replication repair pathway. In some embodiments, the highly mutant tumor has a mutation in a DNA polymerase associated with replication repair. In some embodiments, the highly mutated tumor has microsatellite instability. In some embodiments, the ultra-high mutant tumor has a mutation in a DNA polymerase associated with replication repair and microsatellite instability. In some embodiments, a high mutation in a tumor is associated with a response to an immune checkpoint inhibitor. In some embodiments, the highly mutated tumor is resistant to treatment with an immune checkpoint inhibitor. In some embodiments, the TILs of the invention may be used to treat highly mutated tumors. In some embodiments, the hypermutation in the tumor is caused by an environmental factor (exogenous exposure). For example, UV light can be the major cause of a number of mutations in malignant melanoma (see, e.g., Pfeifer, g.p., You, y.h., and Besaratinia, a. (2005): mutation research (mut. res.). 571, 19-31.; Sage, E. (1993): photochemical and photobiological (photochem. photobiological.). 57, 163-) 174.). In some embodiments, high mutations in tumors can be caused by more than 60 carcinogens in tobacco smoke of lung and throat tumors, as well as other tumors, due to direct mutagen exposure (see, e.g., Pleasance, e.d., Stephens, p.j., O' Meara, s., McBride, d.j., Meynert, a., Jones, d., Lin, m.l., Beare, d., Lau, k.w., Greenman, c., et al, (2010) & Nature 463, 184-. In some embodiments, high mutations in tumors are caused by dysregulation of apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like (APOBEC) family members, which have been shown to result in increased levels of C to T conversion in a wide range of cancers (see, e.g., Roberts, s.a., Lawrence, m.s., Klimczak, l.j., Grimm, s.a., Fargo, d., Stojanov, p., Kiezun, a., Kryukov, g.v., Carter, s.l., Saksena, g., 2013 et al (nat. genet.) -45, 970-) -976). In some embodiments, the high mutations in the tumor are caused by defective DNA replication repair due to lesion-corrected mutations by the primary replicase Pol3 and Pold 1. In some embodiments, the high mutations in the tumor are caused by a defect in DNA mismatch repair that is associated with high mutations in colorectal, endometrial, and other cancers (see, e.g., Kandoth, c., Schultz, n., Cherniack, a.d., Akbani, r., Liu, y., Shen, h., Robertson, a.g., Pashtan, i., Shen, r., Benz, c.c., et al; (2013): nature 497, 67-73.; Muzny, d.m., bainbrid, m.n., Chang, k., Dinh, h.h., drumond, j.a., Fowler, g., Kovar, c.l., Lewis, l.r., morg., mo, m.b., nham, nehams.r., 487., etc.;.330, nature 487). In some embodiments, DNA replication repair mutations are also found in cancer susceptibility syndromes, such as constitutive or biallelic mismatch repair deficiency (CMMRD), linch syndrome (Lynch syndrome), and polymerase corrected associated polyposis (PPAP).

In embodiments, the invention comprises a method of treating cancer with a TIL population, wherein the cancer is a highly mutated cancer. In embodiments, the invention comprises a method of treating cancer with a TIL population, wherein the cancer is an enhanced hypermutant cancer. In embodiments, the invention comprises a method of treating cancer with a TIL population, wherein the cancer is a hyper-mutated cancer.

In embodiments, the invention comprises a method of treating cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to infusion of a TIL according to the present disclosure. In the examples, the non-myeloablative chemotherapy was cyclophosphamide 60mg/kg/d for 2 days (day 27 and 26 before TIL infusion) and fludarabine 25mg/m²D lasted 5 days (day 27 to day 23 before TIL infusion). In the examples, following non-myeloablative chemotherapy and TIL infusion according to the present disclosure (day 0), patients received intravenous infusion of 720,000IU/kg IL-2 every 8 hours until physiological tolerance.

The compounds and compound combinations described herein can be tested for efficacy in treating, preventing, and/or managing the indicated disease or disorder using various models known in the art that provide guidance for treatment of human diseases. For example, models for determining the efficacy of treatment of ovarian cancer are described, for example, in Mulleny et al, Endocrinology 2012,153,1585-92; and Fong et al, journal of ovarian research (j. ovarian Res.) 2009,2, 12. Models for determining efficacy in treating pancreatic cancer are described in Herreros-Villanueva et al, J.world gastroenterology (World J.gastroenterol.) 2012,18, 1286-1294. Models for determining the efficacy of treatment for Breast Cancer are described, for example, in Fantozzi, Breast Cancer research (Breast Cancer Res.) 2006,8, 212. Models for determining efficacy in treating Melanoma are described in Damsky et al, Pigment Cell and Melanoma research (Pigment Cell & Melanoma Res.) 2010,23, 853-859. Models for determining efficacy in treating lung cancer are described, for example, in Meuwissen et al, Genes & Development 2005,19, 643-664. Models for determining efficacy in treating lung cancer are described, for example, in Kim, "clinical and experimental otolaryngology (clin. exp. otorhinolaryngol.) 2009,2, 55-60; and Sano, Head and Neck oncology (Head cock Oncol) 2009,1, 32.

In some embodiments, IFN- γ (IFN- γ) is indicative of therapeutic efficacy of treatment of a hyperproliferative disorder. In some embodiments, IFN- γ in the blood of a subject treated with TIL is indicative of active TIL. In some embodiments, a potency assay for IFN- γ production is employed. IFN- γ production is another measure of cytotoxic potential. IFN- γ production can be measured by determining the level of the cytokine IFN- γ in the blood of a subject treated with a TIL prepared by the methods of the invention, including as described, for example, in FIG. 1 (specifically, e.g., FIG. 1B). In some embodiments, the TIL obtained by the methods of the invention provides increased IFN- γ in the blood of a subject treated with a TIL of the methods of the invention as compared to a subject treated with a TIL prepared using a method referred to as the Gen 3 process (as exemplified in fig. 1 (specifically, e.g., fig. 1B) and throughout the present application). In some embodiments, an increase in IFN- γ is indicative of the therapeutic efficacy of a patient treated with a TIL produced by the methods of the invention. In some embodiments, IFN- γ is increased by one, two, three, four, or five fold or more compared to an untreated patient and/or compared to a patient treated with a TIL prepared using methods other than those provided herein, including, for example, methods other than those embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, IFN- γ secretion is doubled compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, IFN- γ secretion is increased by two-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, IFN- γ secretion is increased three-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, IFN- γ secretion is increased four-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, IFN- γ secretion is increased by five-fold as compared to an untreated patient and/or as compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, IFN- γ is measured using a Quantikine ELISA kit. In some embodiments, IFN- γ is measured using a Quantikine ELISA kit. In some embodiments, IFN- γ is measured in TIL ex vivo from a patient treated with TIL produced by the methods of the invention. In some embodiments, IFN- γ is measured in the blood of a patient treated with TIL produced by the methods of the invention. In some embodiments, IFN- γ is measured in the serum of a patient treated with TIL produced by the methods of the invention.

In some embodiments, TILs prepared by the methods of the invention (including, for example, the methods described in fig. 1 (specifically, e.g., fig. 1B)) exhibit increased polyclonality as compared to TILs produced by other methods (including methods not exemplified in fig. 1 (specifically, e.g., fig. 1B), such as the method referred to as the process 1C method). In some embodiments, a significantly increased polyclonality and/or increased polyclonality is indicative of therapeutic efficacy and/or increased clinical efficacy of a cancer treatment. In some embodiments, polyclonality refers to T cell bank diversity. In some embodiments, an increase in polyclonality may be indicative of therapeutic efficacy with respect to administration of the TIL produced by the methods of the invention. In some embodiments, the polyclonality is increased by one-fold, two-fold, ten-fold, 100-fold, 500-fold, or 1000-fold as compared to a TIL prepared using a method other than the methods provided herein, including, for example, a method other than the method embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, the polyclonality is doubled compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, the polyclonality is increased by two-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, the polyclonality is increased ten-fold as compared to an untreated patient and/or as compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, the polyclonality is increased 100-fold as compared to an untreated patient and/or as compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than that embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, the polyclonality is increased 500-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than the method embodied in fig. 1 (specifically, e.g., fig. 1B). In some embodiments, the polyclonality is increased 1000-fold compared to an untreated patient and/or compared to a patient treated with a TIL prepared using a method other than the methods provided herein, including, for example, a method other than the method embodied in fig. 1 (specifically, e.g., fig. 1B).

2. Method of co-administration

In some embodiments, a TIL produced as described herein (comprising, e.g., a TIL derived from the methods described in steps a through F of fig. 1 (specifically, e.g., fig. 1B)) can be administered in combination with one or more immune checkpoint modulator(s) (such as an antibody described below). For example, antibodies that target PD-1 and that can be co-administered with a TIL of the invention include, for example, but are not limited to, nivolumab (BMS-936558, hitemet & noble company;) Pembrolizumab (lambertilizumab, MK03475 or MK-3475, merck corporation;) H12.1, PD1.3.1, NAT 105, humanized anti-PD-1 antibody JS001 (Shanghai Junshi Co.), monoclonal anti-PD-1 antibody TSR-042(Tesaro Co.), pidilizumab (anti-PD-1 mAb CT-011, Madisison healthcare Co.), anti-PD-1 monoclonal antibody BGB-A317 (Baiji Shenzhou Co.) and/or anti-PD-1 antibody SHR-1210 (Shanghai Henry Co., Ltd.)) Human monoclonal antibody REGN2810 (regenerators), human monoclonal antibody MDX-1106 (BaishiGuibao Co.) and/or humanized anti-PD-1 IgG4 antibody PDR001 (Nowa Co.). In some embodiments, the PD-1 antibody is from clone: RMP1-14 (rat IgG) -BioXcell catalog number BP 0146. Other suitable antibodies suitable for use in the method of co-administration with TILs produced according to steps a through F as described herein are anti-PD-1 antibodies disclosed in U.S. patent No. 8,008,449, which is incorporated herein by reference. In some embodiments, the antibody or antigen-binding portion thereof specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity. Any antibody known in the art that binds to PD-L1 and disrupts the interaction between PD-1 and PD-L1 and stimulates an anti-tumor immune response is suitable for use in the method of co-administration with TIL produced according to steps a through F as described herein. For example, antibodies targeted to PD-L1 and in clinical trials included BMS-936559 (centella asiatica) and MPDL3280A (genethak). Other suitable antibodies targeting PD-L1 are disclosed in U.S. patent No. 7,943,743, which is incorporated herein by reference. It will be understood by those of ordinary skill in the art that any antibody that binds to PD-1 or PD-L1, disrupts the PD-1/PD-L1 interaction, and stimulates an anti-tumor immune response is suitable for use in the method of co-administration with TILs produced according to steps a through F as described herein. In some embodiments, a subject administered a combination of TILs produced according to steps a through F is co-administered with an anti-PD-1 antibody when the patient has a type of cancer that is refractory to administration of the anti-PD-1 antibody alone. In some embodiments, when the patient has refractory melanoma, the patient is administered a combination of TIL and anti-PD-1. In some embodiments, when the patient has non-small cell lung cancer (NSCLC), the patient is administered TIL in combination with anti-PD-1.

3. Optional pretreatment for depletion of lymphocytes in a patient

In embodiments, the invention comprises a method of treating cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to infusion of a TIL according to the present disclosure. In embodiments, the invention comprises a TIL for treating cancer in a patientA population, the patient having been pre-treated with non-myeloablative chemotherapy. In embodiments, the TIL population is administered by infusion. In the examples, the non-myeloablative chemotherapy was cyclophosphamide 60mg/kg/d for 2 days (day 27 and 26 before TIL infusion) and fludarabine 25mg/m²D lasted 5 days (day 27 to day 23 before TIL infusion). In the examples, following non-myeloablative chemotherapy and TIL infusion according to the present disclosure (day 0), patients received intravenous infusion of 720,000IU/kg IL-2 (aldesleukin, commercially available as PROLEUKIN) every 8 hours up to physiological tolerance. In certain embodiments, the TIL population is used in combination with IL-2 in the treatment of cancer, wherein the IL-2 is administered after the TIL population.

Experimental results show that lymphocyte depletion plays a key role in enhancing the efficacy of treatment by eliminating competing elements of the regulatory T cells and the immune system ("cytokine bank") before adoptive transfer of tumor-specific T lymphocytes. Thus, some embodiments of the invention employ a lymphocyte depletion step (sometimes also referred to as "immunosuppression modulation") on the patient prior to introducing the TILs of the invention.

Typically, lymphocyte depletion is achieved using fludarabine or cyclophosphamide (an active form known as macsfamide) and combinations thereof. Such methods are described in Gassner et al, Cancer immunology and immunotherapy 2011,60, 75-85; muranski et al, Nat. Clin. Pract. Oncol.) (2006, 3, 668-681; dudley et al, journal of clinical Oncology 2008,26, 5233-.

In some embodiments, the fludarabine is administered at a concentration of 0.5 μ g/mL to 10 μ g/mL fludarabine. In some embodiments, the fludarabine is administered at a concentration of 1 μ g/mL fludarabine. In some embodiments, the fludarabine is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 or more days. In some embodiments, the fludarabine is administered at a dose of 10mg/kg per day, 15mg/kg per day, 20mg/kg per day, 25mg/kg per day, 30mg/kg per day, 35mg/kg per day, 40mg/kg per day, or 45mg/kg per day. In some embodiments, the fludarabine is administered at 35mg/kg per day for 2-7 days. In some embodiments, the fludarabine is administered at 35mg/kg per day for 4-5 days. In some embodiments, the fludarabine is administered at 25mg/kg per day for 4-5 days.

In some embodiments, concentration of cyclophosphamide (the active form of cyclophosphamide) is between 0.5 μ g/mL and 10 μ g/mL is obtained by administration of cyclophosphamide. In some embodiments, concentration of 1 μ g/mL of cyclophosphamide (the active form of cyclophosphamide) is obtained by administration of cyclophosphamide. In some embodiments, cyclophosphamide is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 or more days. In some embodiments, 100 mg/m²150 mg/m/day²Daily 175 mg/m²200 mg/m/day²225 mg/m/day²Daily, 250 mg/m²275 mg/m/day²Daily or 300 mg/m²Cyclophosphamide was administered at a daily dose. In some embodiments, cyclophosphamide is administered intravenously (i.e., i.v.). In some embodiments, cyclophosphamide is administered at 35mg/kg daily for 2-7 days. In some embodiments, 250 mg/m per day²Cyclophosphamide was administered intravenously for 4-5 days. In some embodiments, 250 mg/m per day²Cyclophosphamide was administered intravenously for 4 days.

In some embodiments, lymphocyte depletion is performed by administering fludarabine and cyclophosphamide together to the patient. In some embodiments, 25 mg/m per day over 4 days ²Daily intravenous administration of fludarabine and at 250 mg/m per day²Cyclophosphamide was administered intravenously/day.

In the examples, the dosage is adjusted by 60 mg/m per day²Cyclophosphamide was administered in a daily dose for two days, followed by 25 mg/m daily²The dose per day was administered fludarabine for five days for lymphocyte depletion.

Scheme for IL-2

In embodiments, the IL-2 regimen comprises a high dose IL-2 regimen, wherein the high dose IL-2 regimen comprises starting intravenous administration of aldesleukin or a biological analog or variant thereof on the day following administration of the therapeutically effective portion of the therapeutic TIL population, wherein aldesleukin or a biological analog or variant thereof is administered at a dose of 0.037mg/kg or 0.044mg/kg IU/kg (patient body weight) using a 15 minute bolus intravenous infusion every eight hours until tolerated for up to 14 doses. After 9 days of rest, the regimen of 14 additional doses can be repeated, for a total of up to 28 doses.

In embodiments, the IL-2 regimen comprises a decreasing IL-2 regimen. Decreasing IL-2 regimens have been described in O' Day et al, J.Clin.Oncology 1999,17,2752-61 and Eton et al, Cancer (Cancer) 2000,88,1703-9, the disclosures of which are incorporated herein by reference. In the examples, the decreasing IL-2 regimen comprises intravenous administration of 18X 10 over 6 hours ⁶IU/m²Followed by intravenous administration of 18X 10 over 12 hours⁶IU/m²Followed by intravenous administration of 18X 10 over 24 hours⁶IU/m²Followed by intravenous administration of 4.5X 10 over 72 hours⁶IU/m². This treatment cycle can be repeated every 28 days for a maximum of four cycles. In the examples, the decreasing IL-2 regimen comprises 18,000,000IU/m on day 1²9,000,000IU/m on day 2²And 4,500,000IU/m on days 3 and 4²。

In embodiments, the IL-2 regimen comprises administering pegylated IL-2 at a dose of 0.10 to 50 mg/day every 1 day, every 2 days, every 4 days, every 6 days, every 7 days, every 14 days, or every 21 days.

5. Adoptive cell transfer

Adoptive Cell Transfer (ACT) is a very effective form of immunotherapy and involves the transfer of immune cells with anti-tumor activity into cancer patients. ACT is a therapeutic approach that involves identifying lymphocytes with anti-tumor activity in vitro, expanding these cells to large numbers in vitro and infusing them into a host carrying the cancer. Lymphocytes used for adoptive transfer can be derived from the stroma of the resected tumor (tumor infiltrating lymphocytes or TILs). TIL of ACT can be prepared as described herein. In some embodiments, the TIL is prepared, for example, according to the method described in fig. 1 (specifically, e.g., fig. 1B). They may also be derived or derived from blood if they are genetically engineered to express anti-tumor T Cell Receptors (TCRs) or Chimeric Antigen Receptors (CARs), enriched mixed lymphocyte tumor cell cultures (MLTCs), or cloned using autologous antigen presenting cells and tumor-derived peptides. ACT in which lymphocytes are derived from a cancer-bearing host to be infused is referred to as autologous ACT. U.S. publication No. 2011/0052530, which is incorporated herein by reference in its entirety, relates to a method for adoptive cell therapy to promote cancer regression, primarily for treating patients with metastatic melanoma. In some embodiments, the TIL may be administered as described herein. In some embodiments, TIL may be administered in a single dose. Such administration may be by injection, for example intravenous injection. In some embodiments, the TIL and/or cytotoxic lymphocytes may be administered in a multi-dose format. The administration may be once, twice, three times, four times, five times, six times or more than six times per year. Administration may be monthly, biweekly, weekly, or every other day. Administration of TIL and/or cytotoxic lymphocytes may continue as long as necessary.

6. Additional methods of treatment

In another embodiment, the invention provides a method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of a therapeutic TIL population as described in any of the preceding paragraphs applicable above.

In another embodiment, the invention provides a method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of the TIL composition as described in any of the preceding paragraphs as applicable above.

In another embodiment, the present invention provides a method for treating a subject having a cancer as described in any of the preceding paragraphs for use above, the method being modified such that a non-myeloablative lymphocyte depletion regimen has been administered to the subject prior to administering a therapeutically effective dose of a therapeutic TIL population and a TIL composition as described in any of the preceding paragraphs for use above, respectively.

In another embodiment, the present invention provides a method for treating a subject having a cancer as described in any of the preceding paragraphs to which it applies, the method being modified such that the non-myeloablative lymphocyte depletion regimen comprises the steps of: at 60 mg/m ²Cyclophosphamide was administered for two days at a dose of 25 mg/m²The dose of fludarabine was administered for five days.

In another embodiment, the invention provides a method for treating a subject having a cancer as described in any of the preceding paragraphs for which the above applies, the method being modified to further comprise the step of beginning treatment of the subject with the high dose IL-2 regimen the day after administering the TIL cells to the subject.

In another embodiment, the invention provides a method for treating a subject having a cancer as described in any of the preceding paragraphs for use above, the method being modified such that the high dose IL-2 regimen comprises administering 600,000 or 720,000IU/kg every eight hours in 15 minute bolus infusions until tolerated.

In another embodiment, the present invention provides a method for treating a subject having a cancer as described in any of the preceding paragraphs for which it is applicable, the method being modified such that the cancer is a solid tumor.

In another embodiment, the invention provides a method for treating a subject having a cancer as described in any of the preceding paragraphs for which the above applies, the method being modified such that the cancer is melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer or renal cell carcinoma.

In another embodiment, the invention provides a method for treating a subject having a cancer as described in any of the preceding paragraphs for which it is applicable, the method being modified such that the cancer is melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM) and gastrointestinal cancer.

In another embodiment, the present invention provides a method for treating a subject having a cancer as described in any of the preceding paragraphs for which it is applicable, the method being modified such that the cancer is melanoma.

In another embodiment, the present invention provides a method for treating a subject having a cancer as described in any of the preceding paragraphs for which the above applies, the method being modified such that the cancer is HNSCC.

In another embodiment, the invention provides a method for treating a subject having a cancer as described in any of the preceding paragraphs for which the above applies, the method being modified such that the cancer is cervical cancer.

In another embodiment, the present invention provides a method for treating a subject having a cancer as described in any of the preceding paragraphs for which it is applicable, the method being modified such that the cancer is NSCLC.

In another embodiment, the present invention provides a method for treating a subject having a cancer as described in any of the preceding paragraphs for which it is applicable, the method being modified such that the cancer is a glioblastoma (including GBM).

In another embodiment, the invention provides a method for treating a subject having a cancer as described in any of the preceding paragraphs for which the above applies, the method being modified such that the cancer is a gastrointestinal cancer.

In another embodiment, the present invention provides a method for treating a subject having a cancer as described in any of the preceding paragraphs for which it is applicable, the method being modified such that the cancer is a highly mutant cancer.

In another embodiment, the present invention provides a method for treating a subject having a cancer as described in any of the preceding paragraphs for which it is applicable, the method being modified such that the cancer is a pediatric high mutation cancer.

In another embodiment, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs for use above for use in a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of the therapeutic TIL population.

In another embodiment, the invention provides a TIL composition as described in any of the preceding paragraphs for use above in a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective dose of the TIL composition.

In another embodiment, the invention provides a therapeutic TIL population as described in any of the preceding paragraphs above applicable or a TIL composition as described in any of the preceding paragraphs above applicable, modified such that a non-myeloablative lymphocyte depletion regimen has been administered to the subject prior to administering to the subject a therapeutically effective dose of a therapeutic TIL population as described in any of the preceding paragraphs above applicable or a TIL composition as described in any of the preceding paragraphs above applicable.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the non-myeloablative lymphocyte depletion regimen comprises the steps of: at 60 mg/m²Cyclophosphamide was administered for two days at a dose of 25 mg/m²The dose of fludarabine was administered for five days.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified to further comprise the step of beginning treatment of the patient with a high dose IL-2 regimen the day after administering the TIL cells to the patient.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the high dose IL-2 regimen comprises administration of 600,000 or 720,000IU/kg as a 15 minute bolus intravenous infusion every eight hours until tolerated.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is a solid tumor.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer or renal cell carcinoma.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM) and gastrointestinal cancer.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is melanoma.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is HNSCC.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, adapted such that the cancer is cervical cancer.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is NSCLC.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is glioblastoma (including GBM).

In another embodiment, the invention provides a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is a gastrointestinal cancer.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is a highly mutated cancer.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is a pediatric high mutation cancer.

In another embodiment, the invention provides the use of a therapeutic TIL population as described in any of the preceding paragraphs for use above in a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective dose of the therapeutic TIL population.

In another embodiment, the invention provides the use of a TIL composition as described in any of the preceding paragraphs for use above in a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective dose of the TIL composition.

In another embodiment, the invention provides the use of a therapeutic TIL population as described in any one of the preceding paragraphs above applicable or a TIL composition as described in any one of the preceding paragraphs above applicable in a method of treating cancer in a subject, the method comprising administering to the subject a non-myeloablative lymphocyte depletion regimen, and then administering to the subject a therapeutically effective dose of a therapeutic TIL population as described in any one of the preceding paragraphs above applicable or a therapeutically effective dose of a TIL composition as described in any one of the preceding paragraphs above applicable.

In another embodiment, the invention provides the use of a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs as applicable above, modified such that the non-myeloablative lymphocyte depletion protocol comprises the steps of: at 60 mg/m²Cyclophosphamide was administered for two days at a dose of 25 mg/m²Dose administration per day fludarabineThe five days are continued.

In another embodiment, the invention provides the use of a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified to further comprise the step of beginning treatment of the patient with a high dose IL-2 regimen the day after administering the TIL cells to the patient.

In another embodiment, the invention provides the use of a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs as applicable above, modified such that the high dose IL-2 regimen comprises administration of 600,000 or 720,000IU/kg as a 15 minute bolus intravenous infusion every eight hours until tolerated.

In another embodiment, the invention provides the use of a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is a solid tumor.

In another embodiment, the invention provides the use of a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer or renal cell carcinoma.

In another embodiment, the invention provides the use of a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM) and gastrointestinal cancer.

In another embodiment, the invention provides the use of a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is melanoma.

In another embodiment, the invention provides the use of a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is HNSCC.

In another embodiment, the invention provides the use of a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, adapted such that the cancer is cervical cancer.

In another embodiment, the present invention provides the use of a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs, where applicable, modified such that the cancer is NSCLC.

In another embodiment, the invention provides the use of a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is glioblastoma (including GBM).

In another embodiment, the invention provides the use of a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is a gastrointestinal cancer.

In another embodiment, the invention provides the use of a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is a highly mutant cancer.

In another embodiment, the invention provides the use of a therapeutic TIL population or TIL composition as described in any of the preceding paragraphs for use above, modified such that the cancer is a pediatric high mutation cancer.

Examples of the invention

Embodiments encompassed herein are now described with reference to the following examples. These examples are provided for illustrative purposes only, and the disclosure contained herein should in no way be construed as limited to these examples, but rather should be construed to cover any and all variations which become evident as a result of the teachings provided herein.

Example 1: preparation of the Medium for the REP Pre-and REP Process

This example describes methods for preparing tissue culture media for protocols involving culturing Tumor Infiltrating Lymphocytes (TILs) derived from various tumor types, including but not limited to metastatic melanoma, Head and Neck Squamous Cell Carcinoma (HNSCC), ovarian cancer, triple negative breast cancer, and lung adenocarcinoma. This medium can be used to prepare any of the TILs described in the present application and examples.

Preparation of CM1

The following reagents were removed from refrigeration and were bathed at 37 ℃: (RPMI1640, human AB serum, 200mM L-glutamine). CM1 medium was prepared according to table 19 below by adding each component to the top portion of a 0.2 μm filter unit appropriate for the volume to be filtered. Store at 4 ℃.

Table 19: preparation of CM1

Composition (I)	Final concentration	Final volume 500ml	Final volume IL
				RPMI1640	NA	450ml	900m1
Human AB serum, heat inactivated 10%	50ml	100ml
				200mM L-Glutamine	2mM	5ml	10m1
55mM BME	55μM	0.5ml	1ml
				50mg/ml gentamicin sulfate	50μg/ml	0.5ml	1ml

On the day of use, the required amount of CM1 was prewarmed in a 37 ℃ water bath and 6000IU/m1 IL-2 was added.

Additional supplements are made as necessary according to table 20.

Table 20: CM1 is additionally supplemented as necessary.

Preparation of CM2

Prepared CM1 was removed from the refrigerator or fresh CM1 was prepared according to table 19 above. Removing from refrigeratorAnd by mixing prepared CM1 with equal volumeMix in sterile medium flasks to prepare the required amount of CM 2. On the day of use, 3000IU/ml IL-2 was added to CM2 medium. On the day of use, a sufficient amount of CM2 with 3000IU/ml IL-2 was made. Marking the culture medium bottle with the name of the CM2 medium bottle, the initials of the name of the producer, the date of filtration/production, the two week expiry dateSeeds and stored at 4 ℃ until required for tissue culture.

Preparation of CM3

CM3 was prepared on the day of use. CM3 andthe medium was the same, supplemented with 3000IU/ml IL-2 on the day of use. An amount of CM3 sufficient to meet the experimental needs was prepared by adding IL-2 stock solution directly into AIM-V bottles or bags. Mix well by gentle shaking. The bottles were marked immediately after addition to AIM-V with "3000 IU/ml IL-2". If there is an excess of CM3, it is stored in a bottle at 4 ℃ that is labeled with the media name, the initials of the name of the producer, the date the media was prepared, and its expiration date (7 days after preparation). After 7 days of storage at 4 ℃, the medium supplemented with IL-2 was discarded.

Preparation of CM4

CM4 is identical to CM3, but additionally supplemented with 2mM G1utaMAX^TM(final concentration). For every 1L of CM3, 10ml of 200mM G1utaMAX was added^TM. By mixing IL-2 stock solution with G1utaMAX^TMStock solutions were added directly to AIM-V bottles or bags to prepare an amount of CM4 sufficient to meet the needs of the experiment. Mix well by gentle shaking. The bottles were marked immediately after addition to AIM-V with "3000 IL/ml IL-2 and G1 utaMAX". If there is excess CM4, it is stored in bottles at 4 ℃ marked with the media name, "G1 utaMAX" and its expiration date (7 days after preparation). After 7 days of storage at 4 ℃, the medium supplemented with IL-2 was discarded.

Example 2: use of IL-2, IL-15 and IL-21 cytokine mixtures

This example describes the use of IL-2, IL-15 and IL-21 cytokines in combination with the TIL process of examples A through G, which act as additional T cell growth factors.

Using the process described herein, TIL was grown from colorectal, melanoma, cervical, triple negative breast, lung and kidney tumors in the presence of IL-2 in one group of experiments and in the replacement of IL-2 with a combination of IL-2, IL-15 and IL-21 in another group at the start of culture. Before REP was completed, cultures were evaluated for expansion, phenotype, function (CD107a + and IFN- γ), and TCR ν β repertoire. IL-15 and IL-21 are described elsewhere herein and Gruijl et al, IL-21 contributes to the expansion of CD27+ CD28+ tumor infiltrating lymphocytes with high cytotoxic potential and low collateral expansion of regulatory T cells; santegoes, S.J., J Transl Med., 2013,11:37(https:// www.ncbi.nlm.nih.gov/PMC/articles/PMC3626797 /).

The results show that CD4 under conditions of IL-2, IL-15, and IL-21 treatment was observed in various histologies relative to IL-2 only conditions⁺And CD8⁺Enhancement of TIL expansion in both cells: (>20%). The presence of major CD8 with biased TCR V.beta.repertoire in TILs obtained from IL-2, IL-15 and IL-21 treated cultures relative to IL-2 only cultures⁺And (4) a group. IFN-. gamma.and CD107a were elevated in IL-2, IL-15, and IL-21 treated TILs compared to IL-2 treated TILs alone.

Example 3: preparation of IL-2 stock solution (CELLGENIX)

This example describes a process of dissolving purified lyophilized recombinant human interleukin-2 into a stock sample suitable for use in further tissue culture protocols, including all those described in this application and examples, including those involving the use of rhIL-2.

Procedure

A 0.2% acetic acid solution (HAc) was prepared. 29mL of sterile water was transferred to a 50mL conical tube. 1mL of 1N acetic acid was added to a 50mL conical tube. Mix well by inverting the tube 2-3 times. The HAc solution was sterilized by filtration using a Steriflip filter.

PBS containing 1% HSA was prepared. 4mL of a stock solution of 25% HSA was added to 96mL of PBS in a 150mL sterile filter unit. The solution was filtered. Store at 4 ℃. For each vial of rhIL-2 prepared, a form was filled out.

Preparation of stock rhIL-2 solution (6X 10)⁶IU/mL final concentration). rhI of each batchL-2 all differ and require information found in the manufacturer's certificate of analysis (COA), such as: 1) mass of rhIL-2 per vial (mg), 2) specific activity of rhIL-2 (IU/mg) and 3) recommended 0.2% reconstituted volume of HAc (mL).

The volume of 1% HSA required for rhIL-2 batches was calculated by using the following equation:

for example, according to CellGenix's rhIL-2 batch 10200121 COA, the specific activity of 1mg vial was 25X 10⁶IU/mg。

It was suggested to reconstitute rhIL-2 in 2mL of 0.2% HAc.

The rubber stoppers of the IL-2 vials were wiped with ethanol wipes. The recommended 0.2% HAc volume was injected into the vial using a 16G needle attached to a 3mL syringe. Care was taken not to remove the stopper when withdrawing the needle. The vial was inverted 3 times and vortexed until all the powder was dissolved. Carefully remove the plug and rest on the ethanol wipe. The calculated volume of 1% HSA was added to the vial.

Storing the rhIL-2 solution. For short-term storage (<72 hours), the vials were stored at 4 ℃. For long-term storage (>72 hours), vials were aliquoted into smaller volumes and stored in cryovials at-20 ℃ until ready for use. Freeze/thaw cycles are avoided. The expiration date was recorded 6 months after the preparation date. The Rh-IL-2 label contains the supplier and catalog number, lot number, expiration date, initials of the operator's name, concentration and volume of the aliquot.

Example 4: low-temperature preservation process

This example describes the cryopreservation process using a CryoMed controlled rate freezer, model 7454 (Thermo Scientific) for TIL prepared using the simplified sealing procedure described in example G.

The equipment used was as follows: aluminum cassette holder (compatible with CS750 freezer bag), cryo-preservation cassette for 750mL bags, low pressure (22psi) liquid nitrogen tank, refrigerator, thermocouple sensor (tape type of bag) and CryoStore CS750 freezer bag (OriGen science).

The freezing process provided a 0.5 ℃ rate from nucleation to-20 ℃ and a 1 ℃ per minute cooling rate to the-80 ℃ end point temperature. The program parameters were as follows: step 1-wait at 4 ℃; step 2: 1.0 ℃/min (sample temperature) to-4 ℃; and step 3: 20.0 ℃/minute (chamber temperature), to-45 ℃; and 4, step 4: 10.0 ℃/minute (chamber temperature), to-10.0 ℃; and 5: 0.5 ℃/minute (chamber temperature), up to-20 ℃; and step 6: 1.0 ℃/min (sample temperature), to-80 ℃.

Example 5: GEN 3 exemplary Process

The examples provide a comparison between Gen 2 and Gen 3 processes. This example describes the development of a robust TIL amplification platform. Modifications to the Gen 2 process reduce risks and simplify manufacturing processes by reducing the number of operator interventions, reduce overall time of manufacture, optimize the use of reactants, and facilitate flexible semi-closed and semi-automated cell production processes suitable for high throughput manufacturing on a commercial scale.

The processes Gen 2 and Gen 3 TIL consist of autologous TIL derived from individual patients by surgical resection of the tumor and then ex vivo expansion.Is a cell culture in the presence of interleukin-2 (IL-2) and the monoclonal antibody OKT3, which OKT3 targets the T cell co-receptor CD3 on the backbone of irradiated Peripheral Blood Mononuclear Cells (PBMCs).

The manufacture of Gen 2 TIL products consists of two stages: 1) pre-rapid amplification (pre-REP) and 2) rapid amplification protocol (REP). During the pre-REP period, excised tumors were cut into ≦ 50 pieces of 2-3mm each size, which were cultured with serum-containing medium (RPMI 1640 medium containing supplemented 10% HuSAB) and 6,000IU/mL of interleukin-2 (IL-2) for a period of 11 days. On day 11TIL was harvested and introduced into a large-scale secondary REP amplification. REP consists of: in a 5X 10 load of 150ug monoclonal anti-CD 3 antibody (OKT3)⁹200X 10 from Pre-REP activation of irradiated allogeneic PBMC feeder cells in 5L volume of CM2 supplemented with 3000IU/mL rhIL-2⁶And (5) living cells for 5 days. On day 16, the volume of the culture was reduced by 90% and the cell fraction was increased to ≧ 1X 10 per flask ⁹Live lymphocytes were split into multiple G-REX-500 flasks and plated with CM4 QS to 5L. The TIL was incubated for another 6 days. REP is collected on day 22, washed, formulated and cryopreserved before transport to a clinical site for infusion at-150 ℃.

The manufacture of Gen 3 TIL products consists of three stages: 1) priming the first amplification protocol, 2) the rapid second amplification protocol (also referred to as rapid amplification phase or REP) and 3) subculture are separated. To achieve the initiation of first expansion TIL proliferation, the excised tumor was cut into 120 pieces each 2-3mm in size. On day 0 of priming the first amplification, in each of 3 100MCS containers, at about 100cm²On the surface area of (A) establishes about 2.5X 10⁸A feeder layer of allogeneic irradiated PBMC feeder cells. Tumor fragments were distributed and cultured in 3 100MCS containers for a period of 7 days, each container having 500mL of CM1 medium with serum and 6,000IU/mL of interleukin-2 (IL-2) and 15ug of OKT-3. On day 7, the composition was adjusted by mixing approximately 5X 10⁸An additional feeder cell layer of allogeneic irradiated PBMC feeder cells was incorporated into the tumor disruption culture phase in each of 3 100MCS containers and cultured with 500mL CM2 medium and 6,000IU/mL IL-2 and 30ug OKT-3 to initiate REP. REP priming is enhanced by activating the entire priming first expansion culture in a 100MCS container using closed system fluid transfer of feeder cells loaded with OKT3 into the same container. For Gen 3, TIL expansion or split involves a process step in which the entire cell culture is scaled up into larger vessels and transferred (from 100M flask to 500M flask) by closed system fluid transfer and an additional 4L of CM4 medium is added. REP cells were harvested on day 16, washed, conditioned and cryopreserved before being stored at-150 deg.C Delivered to a clinical site for infusion.

Overall, the Gen 3 process is a shorter, more scalable and easily modifiable amplification platform that will accommodate robust manufacturing and process comparability.

Table 21: comparison of exemplary Gen 2 and exemplary Gen 3 manufacturing processes.

On day 0, for both processes, the tumors were washed 3 times and the debris was randomized and divided into two pools; one tank for each process. For the Gen 2 process, the splits were transferred to a GREX100MCS flask with 1L of CM1 medium containing 6,000IU/mL rhIL-2. For the Gen 3 process, the splits were transferred to a GREX100MCS flask having 500mL containing 6,000IU/mL rhIL-2, 15ug OKT-3, and 2.5X 10⁸CM1 for individual feeder cells.

TIL inoculation on Rep priming days was performed on different days according to each process. For the Gen 2 process, in which the G-REX 100MCS flask was reduced by 90% volume, the collected cell suspension was transferred to new G-REX 500MCS to start REP priming on day 11 in CM2 medium containing IL-2(3000IU/mL) plus 5e9 feeder cells and OKT-3(30 ng/mL). Cells were expanded and split according to protocol at day 16 into multiple GREX 500MCS flasks with IL-2(3000IU/mL) in CM4 medium. Cultures were then harvested and cryopreserved on day 22 according to the protocol. For the Gen 3 process, a REP start occurs at day 7, where the REP start is performed using the same G-REX 100 MCS. Briefly, 500mL of CM2 medium containing IL-2(6000IU/mL) and 5X 10 with 30ug of OKT-3 were combined ⁸One feeder cell was added to each flask. On days 9-11, scale up cultures were performed. The entire volume (1L) of G-Rex100M was transferred to a G-REX 500MCS and 4L of CM4 containing IL-2(3000IU/mL) was added. The flask was incubated for 5 days. Cultures were harvested on day 16 and cryopreserved.

Three different tumors were included in the comparison, two lung tumors (L4054 and L4055) and one melanoma tumor (M1085T).

For L4054 and L4055, CM1 (medium 1), CM2 (medium 2), and CM4 (medium 4) media were prepared in advance and kept at 4 ℃. CM1 and CM2 media were prepared without filtration to compare cell growth with filtration and without filtration.

At REP initiation and scale-up, the culture medium of L4055 tumors was previously warmed up to 24 hours at 37 ℃.

Summary of results

For total viable cells obtained, Gen 3 fell to within 30% of Gen 2. Gen 3 end products exhibit higher INF- γ production after restimulation. The Gen 3 end product exhibited increased clonal diversity as measured by the presence of the total unique CDR3 sequence. Gen 3 end products exhibit longer mean telomere length.

The results obtained

Cell count and viability%

pre-REP and REP amplification for the Gen 2 and Gen 3 processes follow the details described above.

Table 22: REP pre-cell counts. For each tumor, both pools contained equal numbers of fragments. Due to the small size of the tumor, the maximum number of fragments per flask was not reached. At day 11 for the Gen 2 process and day 7 for the Gen 3 process, total REP pre-cells (TVCs) were collected and counted. To compare the two REP pre-groups, the cell count was divided by the number of debris provided in the culture in order to calculate the average number of viable cells per debris. As indicated in the table below, each fragment of the Gen 2 process consistently grew more cells than did the Gen 3 process. The extrapolated calculation of the predicted TVC number for day 11 of the Gen 3 process is calculated by dividing the REP pre-TVC by 7 and then multiplying by 11.

Table 22: REP Pre-cell count

L4055, unfiltered medium.

Table 23: total viable cell count and fold expansion for TIL end product: for the Gen 2 and Gen 3 processes, TVCs were counted according to process conditions and the percent viable cells for each day of the process was generated. At the time of collection, cells on day 22 (Gen 2) and day 16 (Gen 3) were collected and TVC counts were determined. The TVC was then divided by the number of debris provided on day 0 to calculate the average number of viable cells per debris. Fold expansion was calculated by dividing the harvested TVC by the REP starting TVC. As shown in the table, comparing Gen 2 to Gen 3, the fold amplification of L4054 was similar; in the case of L4055, the amplification fold of the Gen 2 process is higher. Specifically, in this case, the medium is warmed 24 before the REP initiation date. For M1085T, a higher fold amplification in Gen 3 was additionally observed. The extrapolated calculation of the predicted TVC number for Gen 3 process day 22 is calculated by dividing the REP TVC by 16 and then multiplying by 22.

Table 23: total viable cell count and fold expansion for the TIL end product

L4055, unfiltered medium.

Table 24: viability of TIL final product%: at the time of harvest, the final TIL REP product was compared to release standards to yield% viability. All conditions of Gen 2 and Gen 3 processes exceed 70% survival criteria and are comparable between processes and tumors.

Table 24: viability of REP%

Table 25: estimated cell counts for each additional flask of the Gen 3 process. Since the number of debris per flask was below the maximum required number, the estimated cell count on the day of acquisition was calculated for each tumor. The estimation is based on the expectation that clinical tumors are large enough to inoculate the 2 nd or 3 rd flasks on day 0.

Table 25: extrapolated estimated cell count calculations for full-scale (full scale) 2 nd and 3 rd flasks in the Gen 3 process

Immunophenotype:

comparison of phenotypic markers for the TIL end product:

in both Gen 2 and Gen 3 processes, three tumors, L4054, L4055 and M1085T, all underwent TIL amplification. At the time of harvest, flow cytometric analysis of the REP TIL end product was performed to test purity, differentiation and memory markers. For all conditions, the percentage of TCR a/b + cells exceeded 90%.

TILs collected from the Gen 3 process exhibited higher CD8 and CD28 expression compared to TILs collected from the Gen 2 process. The Gen 2 process exhibits a higher percentage of CD4 +. See fig. 3(A, B, C).

Memory marker comparison for TIL end product:

TILs collected from the Gen 3 process displayed higher expression on the central memory compartment than TILs collected from the Gen 2 process. See fig. 4(A, B, C).

Activation and depletion marker comparisons for TIL end products:

activation and depletion markers were analyzed in TILs from two tumors, L4054 and L4055, to compare the final TIL products of the Gen 2 and Gen 3TIL amplification processes. The activation and depletion markers are comparable between Gen 2 and Gen 3 processes. See fig. 5(A, B); fig. 6(A, B).

Interferon gamma secretion after restimulation:

on day 22 for Gen 2 and 16 for Gen 3 collected, TILs underwent overnight restimulation with coated anti-CD 3 plates for L4054 and L4055. M1085T was restimulated with anti-CD 3, CD28 and CD137 beads. After another 24 hours of stimulation under all conditions the supernatant was collected and frozen. Using the same ELISA plateThe supernatants from both processes were evaluated simultaneously by IFN γ analysis by ELISA. Higher production of IFN γ from the Gen 3 process was observed. See fig. 7(A, B, C).

Measurement of IL-2 levels in the culture media:

to compare IL-2 consumption between Gen 2 and Gen 3 processes, cell supernatants were collected for tumors L4054 and L4055 on REP initiation, scale-up and collection days. The amount of IL-2 in the cell culture supernatant was measured by the Quantitate ELISA kit from R & D. The overall trend indicates that the IL-2 concentration in the Gen 3 process is still higher when compared to the Gen 2 process. This is probably due to the higher IL-2 concentration at the start of REP for Gen 3 (6000IU/mL) combined with medium residues during the whole process. See fig. 8(A, B).

Metabolic substrate and metabolite analysis

The levels of metabolic substrates such as D-glucose and L-glutamine are measured as surrogate amounts of total media consumption. Their reciprocal metabolites, such as lactic acid and ammonia, were measured. Glucose is a monosaccharide in the culture medium that mitochondria use to produce energy in the form of ATP. When glucose is oxidized, lactic acid (lactate is an ester of lactic acid) is produced. Lactic acid is produced to a large extent during the exponential growth phase of the cells. High levels of lactic acid have a negative impact on the cell culture process. See fig. 9(A, B).

For both processes Gen 2 and Gen 3, the spent media of L4054 and L4055 were collected on REP start-up, scale-up and collection days. Spent media collections were performed on days 11, 16, and 22 for Gen 2; spent media collections for Gen 3 at days 7, 11 and 16The supernatants were analyzed on a CEDEX bioanalyzer for glucose, lactate, glutamine, glutamax and ammonia concentrations.

L-glutamine is an unstable essential amino acid required in cell culture media formulations. Glutamine contains an amine and this amide moiety can transport and deliver nitrogen to cells. When L-glutamine is oxidized, the cell produces toxic ammonia as a by-product. To counteract L-glutamine degradation, the media of the Gen 2 and Gen 3 processes are supplemented with Glutamax, which is more stable in aqueous solution and does not degrade spontaneously. In both tumor strains, Gen 3 group showed that L-glutamine and Glutamax decreased during the process and ammonia increased throughout REP. In the Gen 2 group, a constant concentration of L-glutamine and Glutamax was observed, and a slight increase in ammonia production. The ammonia of the Gen 2 and Gen 3 processes was comparable on the day of collection and showed slight differences in L-glutamine degradation. See fig. 10(A, B, C).

Telomeric repeats measured by Flow-FISH:

the Flow-FISH technique was used to measure the average length of telomeric repeats on L4054 and L4055 under Gen 2 and Gen3 processes. Determination of Relative Telomere Length (RTL) was calculated using telomere PNA kit/FITC from DAKO for flow cytometry analysis. Gen3 displays telomere length comparable to Gen 2.

CD3 analysis

To determine the clonal diversity of the cell products produced in each process, TIL end products collected against L4054 and L4055 were sampled and the clonal diversity analysis was examined by sequencing the CDR3 portion of the T cell receptor.

Table 26: gen 2 as a percentage of the shared unique CDR3 sequence on TIL collected cell products for L4054 was compared to Gen 3. The Gen3 and Gen 2 end products share 199 sequences between them, corresponding to 97.07% of the first 80% unique CDR3 sequences shared by Gen 2 and Gen3 end products.

Table 26: comparison of shared uCDR3 sequences between Gen 2 and Gen3 processes for L4054.

Table 27: gen 2 as a percentage of the shared unique CDR3 sequence on TIL collected cell products for L4055 was compared to Gen 3. 1833 sequences were shared between the Gen3 and Gen 2 end products, corresponding to 99.45% of the first 80% unique CDR3 sequences shared by Gen 2 and Gen3 end products.

Table 27: comparison of shared uCDR3 sequences between Gen 2 and Gen 3 processes for L4055.

CM1 and CM2 media were prepared in advance without filtration and kept at 4 ℃ until used for tumor L4055 for use on Gen 2 and Gen 3 processes.

On the day of REP initiation of the Gen 2 and Gen 3 processes, the tumor L4055 medium was warmed up 24 hours at 37 ℃ in advance.

In the supernatant collected from the process, no LDH was measured.

M1085T TIL cell counts were performed using a K2 cellometer cell counter.

With respect to tumor M1085T, no samples were available, such as supernatant for metabolic analysis, TIL product for activation and depletion marker analysis, telomere length and CD3-TCR analysis.

Conclusion

This example compares the functional quality attribute of 3 individual donor tumor tissues, as well as the extended phenotype characterization and media consumption during the Gen 2 and Gen 3 processes.

With respect to the viability of the total viable and total nucleated cell populations produced, Gen 2 was evaluated in comparison to Gen 3 pre-REP and REP amplifications. The TVC cell dose on the day of collection was not appropriate between Gen 2 (day 22) and Gen 3 (day 16). The Gen 3 cell dose was lower than Gen 2, about 40% of the total viable cells collected at the time of harvest.

For the Gen 3 process, the extrapolated cell numbers were calculated assuming pre-REP collection on day 11 instead of day 7 and REP collection on day 22 instead of day 16. The number of TVCs was shown to be closer in both cases compared to the Gen 2 process, indicating that early activation may allow overall better performance of TIL growth. Bottom row of tables 4 and 5.

For the extrapolated values for the extra flask (2 or 3) of the Gen 3 process, it was assumed that larger size tumors were treated and the maximum number of fragments required for each process as described was achieved. It was observed that for the Gen 3 process, at day 16 harvest, TVC could reach similar doses compared to day 22 of the Gen 2 process. This observation is very important and only early activation of the culture may allow better performance of TIL in less treatment time.

For phenotypic characterization, higher CD8+ and CD28+ expression was observed on three tumors in the Gen 3 process compared to the Gen 2 process. This data indicates that the Gen 3 process has improved final TIL product attributes compared to Gen 2.

The Gen 3 process exhibits a slightly higher central memory compartment than the Gen 2 process.

Despite the short duration of the Gen 3 process, Gen 2 and Gen 3 processes exhibit comparable activation and depletion markers.

Of the three tumors analyzed, IFN γ (IFN gamma) production on the Gen 3 final product was 3-fold higher compared to Gen 2. This data indicates that the Gen 3 process produces highly functional and more efficient TIL products than the Gen 2 process, probably due to higher CD8 and CD28 expression in Gen 3.

Telomere length on the TIL end product is comparable between Gen 2 and Gen 3.

Glucose and lactate levels were comparable between Gen 2 and Gen 3 end products, indicating that nutrient levels in the medium of the Gen 3 process were unaffected because no volume reduction removal was performed every day for the process and the overall culture volume was smaller in the process compared to Gen 2.

Overall, the Gen 3 process exhibits almost a two-fold reduction in processing time compared to the Gen 2 process, which will result in。

IL-2 consumption indicates a general trend for IL-2 consumption in the Gen 2 process, and in the Gen 3 process, IL-2 is higher due to the old medium not being removed.

The Gen 3 process shows higher clonal diversity as measured by CDR3 TCRab sequence analysis.

The addition of feeder cells and OKT3 on day 0 before REP allows for early activation of TIL and overall better TIL growth performance using the Gen 3 process.

Table 28 describes various examples and results of the Gen 3 process compared to the current Gen 2 process:

table 28: exemplary Gen 3 process.

Example 6: direct in vitro selection and expansion of PD-1+ cells: methods for enhancing tumor-reactive TIL for ACT therapy

Adoptive T cell therapy with autologous Tumor Infiltrating Lymphocytes (TILs) has demonstrated a sustained response rate in a cohort of patients with metastatic melanoma [1 ]. The TIL products used for therapy comprise heterogeneous T cells recognizing tumor-specific antigens, mutant patient-specific neo-antigens and non-tumor associated antigens [2, 3 ]. Studies have demonstrated that neoantigen-specific T cells contribute significantly to the anti-tumor activity of TIL [4 ]. Strategies to enrich TIL for tumor reactivity are expected to produce more effective therapeutic products, especially in epithelial cancers known to contain a high proportion of bystander T cells [5 ]. Several studies have demonstrated that expression of PD1 (a marker usually associated with T cell depletion) on TILs identifies autologous tumor-reactive T cells [6, 7, 8 ]. Presented herein is the development of a new protocol designed to select PD1+ cells and enrich for TIL product against autologous tumor-reactive T cells. This example provides a protocol for sorting and amplifying PD1+ TIL and characterizing the resulting product.

This protocol involves the use of a 2-REP protocol to amplify PD1+ TIL from ex vivo sorting of melanoma, lung, breast (triple negative and ER/PR tumors) and sarcomas. The amplified TILs were evaluated for growth, viability, phenotype, function (IFN γ secretion, CD107a mobilization), tumor killing (X-celgene), and TCR ν β repertoire (by flow cytometry and RNA sequencing). An exemplary method is depicted in the chart provided in fig. 7.

This example encompasses the PD1 selection procedure aimed at enriching the TIL product for TAA-specific TILs. Based on the following ideas: tumor/neoantigen specific T cells are responsible for the therapeutic activity of the TIL product, and the PD1+ subset of TILs includes tumor-reactive T cells.

Method-program

Preparation of tumor

Freshly excised tumor samples were obtained from the research consortium (UPMC, Moffitt) and tissue procurement suppliers (biotime and MTG group). Tumors were shipped overnight in HypoThermosol (BioLife solutions Inc., Washington, Cat. 101104) (with antibiotics) or RPMI 1640 (Semmel Feishel technologies, Pa., Cat. No. 11875-.

Tumors were removed from their primary and secondary packaging, vials with tumors and shipping media were weighed and the mass recorded. The tumor was removed from the vial, and the vial and transport medium were re-weighed. The mass of the tumor (vial mass + transport medium + tumor) - (vial + transport medium) was calculated.

Fragmenting the whole tumor to about 4-6-mm³To carry out tumor digestion. If the tumor is large enough, four 3mm are established³The fragments are processed.

Enzyme preparation for tumor digestion

The lyophilized enzymes were reconstituted in the amounts of sterile HBSS indicated for each of the following digestive enzymes. These enzymes were prepared as 10X. Ensuring that any residual powder is captured from the sides of the bottle and from the protective foil over the bottle opening. Pipette up and down several times and vortex to ensure complete reconstitution.

1-g of collagenase IV (Sigma, MO, C5138) was reconstituted in 10-ml HBSS (to make a 100-mg/ml stock). Mixing was performed by pipetting up and down to dissolve. If not dissolved after reconstitution, placed in 37 ℃ H₂O bath for 5 minutes. Aliquoted into 1-ml vials. This is a 100-mg/ml 10 × working stock of collagenase.

Stock solutions (10,000-IU/ml) of DNase (Sigma, MO, D5025) were prepared. The DNase units for each batch are provided in the accompanying data sheet. The appropriate volume of HBSS was calculated to reconstitute a 100-mg lyophilized DNase stock. For example, if the DNase stock solution is 2000-U/mg, the total DNase in the stock solution is 200,000-IU (2000-IU/mg X100-mg). To dilute to 10,000IU of working stock, 20-ml of HBSS was added to 100mg of dnase (200,000IU/20ml to 10,000U/ml). Aliquoted into 1-ml vials. This is a 10X working stock of 10,000IU/ml DNase.

Stock solutions of 10-mg/ml hyaluronidase (Sigma, MO, H2126) were prepared. 500-mg vials were reconstituted with 50-ml HBSS to prepare 10-mg/ml stock solutions. Aliquoted into 1-ml vials. This is a 10-mg/ml10X working stock of hyaluronidase.

Tumor treatment and digestion

Stock digests were diluted 1X. To prepare a 1 × working solution, 500- μ l of each of collagenase, DNase, and hyaluronidase was added to 3.5-ml of HBSS.

If GentleMeACS Octopdissociator is used, tumor fragments are transferred to GentleMeACS C-tubes (C-tubes) or 50-ml conical tubes in the 5-ml digestion mix described above (in HBSS). 2-3 pieces (4-6mm) were transferred to each C-tube.

Each C-tube (Miltenyi Biotec, Germany, 130-. Used according to the manufacturer's instructions. Note that each tumor histology had a suggested tumor dissociation procedure. The appropriate procedure was chosen for the corresponding tumor histology. Dissociation will take about one hour.

If GentleMeACS OctopDisociator is not applicable, a standard rotator is used. 2-3 tumor fragments were placed in 50-ml conical tubes (sealed with parafilm to avoid leakage) and fixed to a rotator. The rotator was placed at 37 ℃ in 5% CO ₂The rotation in the humidified incubator was continued for 1-2 hours. Alternatively, the tumor fragments may be digested overnight at room temperature, also with constant rotation.

After digestion, the C-tubes were removed from the Octodissociator or spinner. A0.22- μm sieve was attached to a sterile Falcon conical tube. Using a pipette, all contents from either the C-tube or the 50-ml conical tube (5ml) were passed through a 0.22- μm sieve into the 50-ml conical tube. The C tubes/50-ml conical tubes were washed with 10-ml HBSS and applied to a strainer. The blunt end of the sterile syringe plunger was used to dissociate any remaining undigested tumor through the filter. CM1 or HBSS was added up to 50-ml and the tubes were capped.

The samples were pelleted by centrifugation at 1500rpm for 5 minutes at room temperature. The liquid was carefully removed and the pellet was resuspended in 5-ml of CM1 for cell counting and viability assessment.

Whole tumor digests were set aside for the following: 1. cell culture (control for PD1+ and PD 1-); FMO flow cytometry control; 3. determining the digestive phenotype of the whole tumor before sorting; 4. freezing was used for tumor reactivity/cell killing assay. The number of cells left will depend on the total digestion yield and tumor histology.

Cell count and viability

Procedures for obtaining cell and viability counts using a Nexcelom cell meter K2(Nexcelom, massachusetts) have been described.

Staining of digested tumors for flow cytometry analysis and cell sorting

Tumor digests were stained with a mixture comprising incubation with nivolumab and staining with live/dead violet, anti-IgG 4 Fc-PE (secondary antibody against nivolumab) and CD3-FITC according to the following method.

After counting, the cells were resuspended in 10-ml HBSS. The cells were pelleted by centrifugation at 1500rpm for 5 minutes at room temperature (9 rpm). The pellet was resuspended in 5ml of HBSS. Add 5- μ L of live/dead blue dye (Sammerfell, Mass., Cat. L23105) to a final concentration of 1/1000. Incubate on ice for 20-30 minutes. Cells were pelleted by centrifugation at 1500rpm for 5 minutes at room temperature.

The pellet was resuspended in FACS buffer (1X HBSS, 1mM EDTA, 2% fetal bovine serum). The amount of FACS buffer added to the pellet is based on the size of the pellet. The staining volume should be about 3 times the size of the pellet. Thus, if 300- μ l of cells are present, the volume of buffer should be at least 900- μ l. Add 1. mu.g/ml of nivolumab (creative biological laboratories, New York, Cat. No. TAB-770). Dilution will depend on the antibody stock. Incubate at 4 ℃ for 30 minutes. The pellet was resuspended in 5ml of cold HBSS.

The cells were pelleted by centrifugation at 1500rpm for 5 minutes at room temperature (9 rpm). Wash 2X was repeated. The pellet was resuspended in 5ml of cold HBSS. Add 5- μ L of live/dead blue dye (Sammerfell, Mass., Cat. L23105) to a final concentration of 1/1000. Incubate at 4 ℃ for 20-30 minutes.

The cells were pelleted by centrifugation at 1500rpm for 5 minutes at room temperature (9 rpm). The pellet was resuspended in FACS buffer (1X HBSS, 1mM EDTA, 2% fetal bovine serum) as described above. The amount of FACS buffer added to the pellet is based on the size of the pellet. The staining volume should be about 3 times the size of the pellet. Thus, if 300- μ l of cells are present, the volume of buffer should be at least 900- μ l.

For antibody addition, each 100- μ l volume corresponds to one test (titer of antibody). That is, if a volume of 1-ml is present, a 10 × titration of the amount of antibody is required. 3- μ l of anti-CD 3-FITC (BD biosciences, N.J., Cat. No. 561807) was added per 100- μ l of the sample. anti-IgG 4 Fc-PE (Southern Biotech, Cat. 9200-09, Albama) was added at 1: 500. Thus, for every 500. mu.l of FACS buffer, 1. mu.l of anti-IgG 4 Fc-PE was added. Cells were incubated on ice for 30 minutes. Light was protected during incubation. During the incubation period, stirring was performed several times. The cells were resuspended in 20-ml FACS buffer. The solution was passed through a 70- μm cell strainer into a new 50-ml conical tube. Centrifuge at 1500rpm for 5 minutes at room temperature (9 rpm). And (6) pumping. Resuspend cells in FACS buffer up to 10e ⁶TOTAL (alive + dead) of (c). The minimum volume is 300- μ l.

Transferred to sterile polypropylene FACS tubes. 3 ml/tube for FACS sorting.

FACS sorting (FX500 Start)

At the time of setting up the system and waiting for the calibration to complete, the following are prepared:

five sterile 15-ml conical tubes were prepared with 10-ml sterile deionized water.

Five sterile 5-ml FACS tubes were prepared with 4-ml sterile deionized water.

Five sterile 15-ml conical tubes were prepared with 12-ml of 70% EtOH.

Five sterile 15-ml conical tubes were prepared with 12-ml of 10% sodium hypochlorite.

Sample collection

Confirm that the sample chamber and collection chamber are at 5 ℃ and choose vortexing as the stirring sample. The PD1 gate was adjusted as needed.

When the door is satisfactory, as many events as possible (or up to 20,000 CD3 events) are recorded. The sample pressure can be set to 10 to speed up this collection. The collection was stopped and the tube removed.

The sample chamber door was opened and a 15-ml collection chamber block was loaded into the chamber. Loading a collection tube containing a collection buffer into a chamberIn the chamber block. Sample pressure was adjusted to maintain a sorting efficiency of at least 85%. 50,000 CD3 events were recorded. If the cells collected in any of the fractions exceed 4.5X 10⁶One or more collection tubes will need to be replaced. Sorting was continued until all samples were removed from the sample tubes.

REP1 Start (Start priming first amplification step)

The conditions with the least number of cells (PD1+ or PD1-) were used to determine the number of CD3+ cells for REP1 priming. The% (determined during sorting) of CD3 cells will be used to calculate the total number of cells in the whole digest required to initiate REP1 with the same number of CD3 cells as the PD1+ and PD 1-samples. Total REP 1-initiated total digested cells equals the number of sorted cells seeded in REP 1/% of CD3 cells.

Approximately 1000 cells of CD3+ cells were placed in G-Rex 24 or G-Rex10 and treated with 7-ml or 40-ml of CM2 (50% RPMI 1640+ 10% human serum, glutamax, gentamicin and 50% AimV), and 3000-IU/ml of IL-2, respectively, for 11 days. At least one G-Rex flask was initiated for PD1+ and PD 1-sorted populations and whole tumor digests. At the start of the culture, anti-CD 3 (clone: OKT3) (30-ng/ml) and feeder cells (ratio 1:100(TIL: feeder cells)) were added to each flask.

Cells were incubated in plates/flasks for 11 days without medium change (REP 1).

Upon completion of REP1, approximately 5-ml of media was removed for G-Rex 24 and 30-ml of media was removed for G-Rex 10. The cells were resuspended in the remaining medium by pipetting up and down. Cells were placed in 50-ml conical tubes and centrifuged at 1500rpm for 5 minutes.

The medium was aspirated and the cells were resuspended in 10-20-ml of CM2 for counting and viability assessment.

REP2 Start (second Rapid amplification Start)

For mini-REP2 priming, 1e5 cells were plated in G-Rex10 with 40-ml of CM2 medium and 3000-IU/ml of IL-2. At the start of the culture, anti-CD 3 (clone: OKT3) (30-ng/ml) and feeder cells (ratio 1:100, TIL: feeder cells) were added.

For "full scale runs", 2e6-30e6 cells were expanded in 1-L of CM2 medium and 3000-IU/m of G-Rex100M in IL-2. At the start of the culture, anti-CD 3 (clone: OKT3) (30-ng/ml) and feeder cells (ratio 1:100, TIL: feeder cells) were added.

Medium change (for small scale) or medium change + split ("full scale run") was performed on day 5 of REP2 (day 16 of the process). The volume of the flask was reduced to about 10-ml (G-Rex 10) or 100-ml (G-Rex 100M) and supplemented to 40-ml (G-Rex 10) or 1-L (G-Rex 100M) with CM2 or AimV +3000-IU/ml IL-2. For "full scale runs", the flasks were split 1: 2.

On day 11 of REP2 (or day 22 of the process), the flask was reduced in volume and centrifuged at 1500rpm for 5 minutes at room temperature.

The final products were evaluated for cell count, viability, phenotype (TIL1, TIL2(TIL surface antigen stained TIL2 group), TIL3 and function (CD107a (TIL function assessed by CD107a mobilization and IFN γ assay)). according to the manufacturer's instructions, the V.beta.pool was evaluated by FACS (Beckman Coulter, Calif., catalog No. IM3497), the FACS assesses 24 specificities (70% of total V.beta.family), additional cells (1e6-5e6 cells) were pelleted and frozen for RNA sequencing and analysis. And tumor reactivity was assessed by co-culture and/or killing (% cytolysis) using the xcelligene system (ACEA biosciences, inc., ca).

Materials and methods

PD1 positive (PD1+) cells were sorted directly from fresh tumor digests by flow cytometry and expanded in vitro.

Samples from six melanomas, three sarcomas, six breast cancers and eight lung cancers were evaluated.

3 populations were studied:

·PD1⁺sorted TIL;

PD-sorted TIL;

bulk TIL (whole tumor unsorted digest).

TIL productivity (cell count), phenotype (flow cytometry), TCR ν β pool (RNA sequencing), non-specific function (anti-CD 3 and PMA), and tumor reactivity and killing (co-culture assay) were evaluated.

A protocol has been developed for expanding TILs selected by PD1 from melanoma, lung, breast and sarcoma to clinically relevant numbers.

In vitro amplification of PD 1-selected TILs resulted in products that were phenotypically comparable to the bulk TIL.

T cell markers were upregulated relative to pre-sorted TIL, indicating high activation levels. Relative to pre-sorted TIL, T cell markers modulated at the surface of expanded PD1+ TIL comprise PD1 and CD25 and indicate high activation levels. Importantly, in vitro amplification of PD1+ TIL resulted in a product that was phenotypically comparable to the bulk TIL, suggesting a powerful therapeutic potential. The functionality of amplified PD1+ TIL was confirmed by robust IFN γ and CD107a expression in response to non-specific stimulation. Compared to PD 1-derived and bulk TILs, amplified PD1+ TILs showed oligoclonality, which is evidence of antigen-driven clonal expansion at the tumor site. Preliminary data demonstrated that autologous tumor cells were killed by PD1+ instead of PD 1-derived TIL.

PD1+ derived TILs showed oligoclonality compared to PD 1-derived TILs and bulk TILs.

All TIL products were functional as assessed by non-specific stimulation.

Autologous melanoma cell killing was observed in PD1+ -derived TIL, but not in PD 1-derived TIL and in autologous TIL.

Results and acceptance criteria

PD1+ sorted cells will show a defect in proliferative capacity. The yield of the final product will be > or ═ 1e 9. PD1+ cells will be oligoclonal compared to PD 1-. Based on the premise that PD1+ cells are more likely to be antigen-specific, PD1+ cells will likely exhibit enhanced tumor-specific killing compared to their PD 1-counterpart.

Reference to the literature

Rosenberg, S.A. et al, complete responses Durable using T cell metastatic immunotherapy in severely pretreated patients with metastatic melanoma (viral complex responses in fatty pretreated patients with viral metabolic tissue using T-cell transfer immunotherapy) & clinical Cancer research (Clin Cancer Res., 2011.17(13): pages 4550-7.

Kvistborg, P. et al, TIL therapy broadens the tumor-reactive CD8(+) T cell compartment (TIL therapy vaccines the tumor-reactive CD8(+) T cell compartment in melanoma patients) tumor immunology (Oncoumunology), 2012.1(4), page 409 and 418.

Simoni, Y, et al, Bystander CD8(+) T cells are abundant in human tumor infiltration and phenotypically different (Bystander CD8(+) T cell array and phenotypicaly distintine in human tumor inflilters.) Nature (Nature), 2018.557(7706) at page 575-579.

Schumacher, t.n. and r.d. schreiber, Neoantigens in cancer immunotherapy (Neoantigens in cancer immunotherapy), Science (Science), 2015.348(6230), pages 69-74.

Turcotte, S. et al, phenotype and function of T cells infiltrating visceral metastases from gastrointestinal cancer and melanoma: adoptive cell transfer therapy (pharmaceuticals and functions of T cells encapsulating viral vectors from diverse sites and media: pharmaceuticals for adaptive cell transfer therapy) J immunological J2013.191 (5) p 2217-25.

Inozume, T. et al, selected CD8+ PD-1+ lymphocytes (Selection of CD8+ PD-1+ lymphocytes in fresh human melanomas enriched for tumor-reactive T cells) J.Immunotherapy (J.Immunoth), 2010.33(9) pp.956-64.

Gross, A. et al, PD-1 identified a patient-specific CD8(+) tumor-reactive repertoire infiltrating human tumors (PD-1 identities the patient-specific CD8(+) tumor-reactive permeability tumors), journal of clinical research (J Clin Invest), 2014.124(5): pages 2246-59.

Thommen, D.S. et al, a transcriptionally and functionally diverse PD-1(+) CD8(+) T cell pool (A transcriptional and functional differentiation PD-1(+) CD8(+) T cell with predictive positional in non-small cell lung cancer treated with PD-1 blocking.) Nature medicine (Nat Med), 2018.

Cohen et al, Isolation of neo-antigen specific T cells from tumor and peripheral lymphocytes (Isolation of neo-specific T cells from tumor and peripheral lymphocytes) journal of clinical research 2015; 125(10):3981-3991.

Example 7: further examples for direct in vitro selection and expansion of PD-1+ cells: methods of enhancing tumor-reactive TIL for ACT therapy

Study: detection of PD1 Using M1H4 and EH12.2H7 clones

Based on previous data, anti-PD 1 MIH4 mAb was used for early research work. Cells sorted using M1H4 demonstrated feasibility of amplification and product functionality for PD 1-selected TIL:

A staining protocol was prepared to:

ensure staining of all PD1+ TIL.

Incorporation of conjugated anti-IgG 4 for detection of pre-bound receptor.

Protocol using anti-PD 1 EH12.2H7 mAb.

Further characterization of the PD1 sorting protocol in order to confirm selection of the appropriate PD1+ population, a new sorting strategy has been initiated to use the following selection PD1^{Height of}TIL。

Strategy:

a) 16 tumors of H & N (N ═ 4), melanoma (N ═ 4) and lung (N ═ 7)

Omicron 5 experiments were direct comparisons (i.e., PD 1)⁺Relative to PD1^{Height of})。

b) Analysis (including functional and TCRv β library)

Table 29: protocol comparison

Preliminary results and summary:

the differences in phenotype and functionality of the amplified TIL (after REP1 and REP 2) relative to the sorted high population of PD1 provide a novel selection/sorting strategy.

Studies have demonstrated PD1 from lung and melanoma⁺The TILs selected are antigen specific and have greater effector function.

Cold tumors that do not have PD1 activity (PD1/PD-L1 axis) "are not suitable for PD1 selection.

Due to PD1^{Height of}Association of cells with neoantigen/tumor specificity, PD1^{Height of}TIL tumors are ideal for selection.

Further analysis will include phenotypic, functional, physiological, clonal, and analysis for impurities.

Phenotype:

t cell subsets (memory/α - β vs γ - δ)

Functionality

IFNg and granzyme (bead stimulation)

CD107a (PMA/IO stimulation)

Tumour reactivity of IFNg or CD107a/TNF or granzyme B

Omicron- (tumor digest or HLA matched or HLA mismatched or other cancer antigens)

Tumor killing assay (Xcelligence)

Versatility (IsoLight)?

Physiology of human body

Telomere length

Cell expansion fold

Cell cycle analysis, mitotic index

Depletion/senescence/activation markers

Clonality of

Diversity (number of unique clones)

Identity overlap of unique clones iREP

Shannon entropy index

Impurities

Contaminating tumor cells, NK cells, other cells, process residues

Example 8: tumor expansion process with defined medium.

The processes disclosed in examples 1 to 7 were carried out by using defined media (e.g., CTS) according to the invention^TMOpTmizer^TMT cell expansion SFM, sermer feishel, containing, for example, DM1 and DM2) was performed in place of CM1 and CM2 medium.

Example 9: selection and amplification of PD-1+ TIL for full-scale manufacturing

Purpose(s) to

This example describes the results from the selection and amplification of PD-1+ TIL in small-scale and full-scale manufacturing experiments as described in this example.

Information

Several studies have demonstrated that surface expression of PD-1, a marker often associated with T cell depletion, identifies autologous tumor-reactive T cells in the tumor microenvironment. The protocol was designed to select PD-1 positive (PD-1+) cells from the tumor digest to enrich for TIL products from autologous tumor-reactive T cells. The scheme is adapted and modified for both small-scale and full-scale manufacturing.

Range

PD-1+ selected TIL was amplified from one lung tumor digest and one melanoma tumor digest using the small scale PD-1+ selected Gen2 process (PD-1+ Gen 2). The TIL final product was characterized according to protocol TP-19-004.

PD-1+ selected TIL was amplified from two head and neck tumor digests and one melanoma tumor digest using the full scale PD-1+ selected Gen2 process (PD-1+ Gen 2). The TIL final product was characterized according to protocol TP-19-004.

Design of experiments

Descriptions of small-scale and full-scale manufacturing processes are shown in tables 30, 31 below.

The small scale study (phase 1) is a feasibility study that scaled up and optimized the TIL amplification process to clinical scale. Additional conditions were tested to explore the use of defined media and early REP (short REP 1) in PD-1+ TIL amplification.

TABLE 30 summary of the Small Scale Process for PD-1+ TIL culture (study/PD-1 + Gen 2/defined composition culture) Base/early REP)

^*PD-1^{Negative of}Gen 2 and PD-1 bulk TIL Gen 2 the same conditions for small scale cultures as PD-1+ Gen 2 were used.

Defined media conditions CTS OpTmizer with 3% CTS immune cell serum replacement was used.

TABLE 31 overview of the full-Scale Process for PD-1+ TIL culture (PD-1+ Gen 2)

Table 31 below lists the tumors and associated histologies used in this study.

Table 31: tumors used in the study

Acceptance criteria

The characterization tests of these batches were carried out for the parameters listed in tables 3 and 4 below, for reference only.

Table 33 below specifies the acceptance criteria for evaluating the performance of the phase 2/full-scale batch.

TABLE 33 collected product testing and acceptance criteria

Table 34 below specifies additional final product characterization tests for the phase 2 full-scale batch for reference only.

TABLE 34 characterization of the final product (for reference only)

Results

Phase 1 small Scale feasibility results

Lung (L4093) and melanoma (M1135) tumors were used in phase 1 studies. Briefly, each tumor was enzymatically digested, PD-1+ cells were sorted by FACS, and the following cultures were determined as test conditions on day 0:

(1) Study of PD-1+

(2)PD-1+Gen 2

(3) PD-1+ defined medium

(4) PD-1+ early REP.

Two additional cultures were also initiated from each tumor as controls. These two conditions were compared to the PD-1+ conditions for amplification kinetics and TCR-V.beta.clonotypes.

(5)PD-1^{Negative of}Gen 2

(6) Ontology TIL Gen 2

PD-1+ early REP sections were collected on day 17, while all other cultures were collected on day 22 (see Table-1 a).

Cell sorting output

FACS outputs for two tumors used in phase 1 studies are summarized in table 35 below.

Table 35: purity of PD-1+ TIL before and after sorting by flow cytometry.

Properties	Lung (L4093)	Melanoma (M1135)
			CD3+％	8.17％	1.77％
PD-1 +% (of CD3 +)	79％	65％
			Estimation of before TVC sorting (CD3+ PD-1 +%)	5.6e5(6.5％)	4.88e4(1.2％)
Sorted TVC (% yield)	4.5e5(80.7％)	4.8e4(98.4)
			Purity after sorting (PD-1 +%). multidot.	94％	88％

^*The purity was based on FSC/CD3+/PD-1+ and not on live cells, since no viability dye was added during flow sorting of cells for subsequent culture.

The% yield and post-sort purity of PD-1+ cells were high for both tumors. These results show that the experimental parameters for the small scale feasibility study are satisfactory.

REP1 and REP2 outputs

Total Viable Cells (TVCs) were measured using NC 200 after REP-1 and REP-2, and are shown in Table 36 below. The numbers represented in the table for TVC are extrapolated to full scale processes using factors.

Table 36: Small-Scale manufacturing and summary of product attributes

Fold expansion versus harvested TVC/vaccinated TVC

CD 107A% calculated from CD3+ CD4+ or CD3+ CD8+ gated population

The process yield is as follows: the experimental PD-1+ Gen2 group produced more than 200e6 at REP-1 collection and more than 90e9 TIL at REP-2 collection (with viability)>98% and 94% CD45+ CD3+ cells), indicating that PD1+ Gen2 is a viable process to generate enough cells for full-scale manufacturing. When it is reacted with PD-1^{Negative of}Or bulk TIL conditions, REP-1 collected from PD-1+ Gen2 conditions showed lower fold amplification. This finding is consistent with previous findings.

The functions are as follows: the TIL amplified from the PD-1+ Gen2 process released IFN γ and granzyme B at levels similar to that produced using the research process in response to stimulation with anti-CD 3/CD28/CD137 beads (Table 34). In all parameters tested, the REP-1 and REP-2 products from PD-1+ defined medium conditions were similar to the corresponding products for PD-1+ Gen2 conditions. Interestingly, PD-1+ early REP conditions (22 days compared to the PD-1+ Gen2 process) with a total culture duration of 17 days resulted in corresponding PD-1+ Gen2 conditions of 57% (Rep-1) and 37% (REP-2). Further, PD-1+ early REP TIL produced IFN γ and granzyme B levels that were more than 2-fold higher when compared to other conditions, suggesting that PD-1+ early REP TIL may grow and metabolize more actively than TIL produced using other conditions. Given that the doubling time of TILs in culture is typically <1 day, this together suggests that comparable cell output (relative to cell number) and higher functionality (relative to IFN γ and granzyme B release) can be achieved by slightly increasing the duration of PD-1+ early REP culture to 18 or 19 days relative to 22 days of PD-1+ Gen 2. CD107A expression on the surface of activated T cells is a measure of T lymphocyte function. All PD1+ TILs expressed high levels of CD107A when stimulated with PMA/IO (both CD4+ and CD8+ TIL).

The TIL life: table 37 describes TIL telomere lengths for REP-2 collection as determined by fluorescence in situ hybridization Flow cytometry (FISH Flow).

Table 37: summary of Relative Telomere Length (RTL) compared to control (1301) cell lines

Telomere length in samples of L4093 and M1135 was compared to a control cell line (1301 leukemia). The control was a tetraploid cell line with long stable telomeres that allowed calculation of relative telomere length. When compared to the bulk TIL, telomeres of PD1+ TIL were longer in one case and slightly shorter in the other, indicating that PD-1+ TIL maintained its lifetime relative to the bulk TIL.

TIL clonality: table 38 describes the clonality of TILs from REP 2 collections as measured by the TCR V β repertoire.

Table 38: summary of TCR V.beta.libraries for L4093 and M1135

The number of unique CDR3 sequences for PD-1+ Gen2 conditions was comparable for both lung and melanoma TILs. In addition, the TCR V.beta.repertoire of PD-1+ Gen2 conditions showed more than 10% overlap with the corresponding repertoire of bulk TILs. The diversity index (shannon entropy) of PD1+ Gen2 conditions was less than that of the bulk TIL, indicating that the TCR V β repertoire of PD1+ Gen2 conditions was less and oligoclonal compared to the corresponding repertoire of bulk TIL.

Expanded phenotype: tables 39-41 describe the results of the expanded phenotypic analysis from TILs. Multicolor flow cytometry was used to characterize the TIL purity, identity, memory subpopulation, activation and depletion status of REP-2 TIL.

Table 39: TIL purity, identity and memory phenotype characterization

An NK cell; natural killer cells, B cells, gated on live/CD 14-/CD3-/CD19-/CD56+ and/or CD16 +; (ii) gating of viable/CD 14-/CD3-/CD19+, monocytes; live/CD 14+, TCR α/β; gating of live/CD 14-/CD3+/TCR γ/δ -memory cells; (ii) gating live/CD 14-/CD3+/TCR γ/δ -, T-EM; an effector, T-CM; central memory, T-EFF/TEMRA; effector/RA + effector memory.

Table 40: activation and depletion states of CD4+ TIL

Percentages are calculated from (CXCR3+ CCR4+ and CXCR3-CCR4 +).

Percentages are calculated from (CXCR3-CCR 4-and CXCR3+ CCR 4-).

Table 41: activation and depletion states of CD8+ TIL

Percentages are calculated from (CXCR3+ CCR4+ and CXCR3-CCR4 +).

Percentages are calculated from (CXCR3-CCR 4-and CXCR3+ CCR 4-).

PD-1+ Gen 2 TIL comprises predominantly TCR α β and less than 0.2% TCR γ δ cells. non-T cell populations comprising B cells, monocytes and NK cells are each < 0.3%. All conditions comprising PD-1+ Gen 2 TIL were predominantly effector memory phenotype and were less differentiated with high levels of CD28+, BTLA +, CD95+ expression.

The activation (CD69+) and depletion (KLRG1+) status of TIL for PD-1+ conditions is comparable to the historical results for melanoma TIL produced using Gen 2 manufacturing process.

Based on process yield, function, phenotype, PD-1+ Gen 2 showed promising quality attributes when compared to PD-1+ defined medium and PD-1+ early REP.

Results of phase 2 full Scale experiments

One melanoma (M1137) and two head and neck (H3032 and H3034) tumors were used in phase 2 studies. Briefly, each tumor was subjected to enzymatic digestion, PD-1+ cells were flow sorted, and culture was initiated at full scale on day 0 using the PD-1+ Gen 2 process described in table 31.

Streaming sort output

The output of flow sorting of the three tumors used in the phase 2 study is summarized in table 42 below.

TABLE-42: purity of PD-1+ TIL before and after sorting by flow cytometry.

Purity was based on FSC/CD3+/PD1+ and not on live cells, as no viability dye was added during flow sorting of cells for subsequent culture.

Purity after sorting (PD-1 +%) for all three tumors met the > 80% criterion. A slightly lower purity of melanoma tumors was observed relative to head and neck tumors, most likely due to lower expression of PD-1+ cells upon sorting (appendix-1).

REP-1 and REP-2 outputs

Table 43 summarizes the total viable cell count and product attributes from three full-scale experiments.

Table 43: full-Scale manufacturing and summary of product attributes

Range of TVC inoculated at REP-2 based on currently established range of Gen 2REP Process, and not formal acceptance criteria in protocols

Fold expansion versus harvested TVC/vaccinated TVC

CD 107A% calculated from CD3+ CD4+ or CD3+ CD8+ gated population

The process yield is as follows: at the end of REP-1, all three PD-1+ TILs were amplified to > 80e6(> 1500-fold amplification), with sufficient yield to initiate REP-2 culture. The range of 5-200e6 TVC inoculated at REP-2 is based on the current Gen 2REP process. At REP-2 collection, all cultures produced >26e9 TVC with > 90% recovery after Lovo.

Dosage: final product dose >26e9 TVC (with viability > 94% and 93% CD45+ CD3+ cells), i.e., a highly enriched TIL population.

The functions are as follows: the function of TIL was characterized based on overnight restimulation of PD-1+ TIL with Dynabeads. After another 24 hours of stimulation, the supernatant was collected and frozen. IFN gamma and granzyme B ELISA was performed on the supernatant. IFN γ release met the acceptance criteria and all three TIL cultures secreted high levels of granzyme B upon stimulation. Similar to the TIL product produced in phase 1 studies, all three PD1+ TILs expressed profound CD107A when stimulated with PMA/IO (both CD4+ and CD8+ TILs).

The TIL life: the opposite end particle lengths of PD-1+ TIL for H3032, M1137, H3034 were 7, 4.1 and 5.2, respectively, and were comparable to Gen 2(QP-17-011R 01).

TIL clonality: the data is pending.

Expanded phenotype: tables 44 and 45 describe the expanded phenotypic analysis of TIL. Multicolor flow cytometry was used to characterize the TIL purity, identity, memory subpopulation, activation and depletion status of REP-2 TIL.

Table 44:TIL purity, identity and memory phenotype characterization

Characterization (%)	H3032	M1137	H3034
				NK cells (CD3-CD56+)	0.1	0	0.1
B cell (CD3-CD19+)	0	0	0
				Mononuclear cells (CD14+)	0	0	0
TCRαβ	92.2	97	97.8
				TCRγδ	0.5	0.3	0.3
TCRαβ+CD4+	93.7	89.1	92.9
				TCRαβ+CD8+	5.7	10.1	2.1
In the beginning: CCR7+ CD45RA +	0	0	0
				T-EM：CCR7-CD45RA-	96.4	97.3	99.3
T-CM：CCR7+CD45RA-	2.5	2.6	0.6
				T-EFF/TEMRA：CCR7-CD45RA+	1.1	0.1	0.1
T-CM：CD62L+CD45RA-	7.6	11.7	3.5

Table 45: activation and depletion states of TIL

Percentages are calculated from (CXCR3+ CCR4+ and CXCR3-CCR4 +). Percentages are calculated from (CXCR3-CCR 4-and CXCR3+ CCR 4-).

No detectable B cells, monocytes or NK cells were present in the final collected TILs (Table-14). REP TIL is composed primarily of TCR α β and effector memory differentiation.

All three PD-1+ TILs appear to be the CD4 dominant phenotype, with effector memory phenotype and high CD27 expression (Table 44).

The depletion marker KLRG1 was less than 13% except for M1137 (table 45). CD57, BTLA4, LAG3, PD1+, TIGIT levels were similar to the historical results of melanoma TIL produced using Gen 2 manufacturing process.

Metabolite analysis: for all conditions, spent media was collected on the last day of collection. The supernatants were analyzed on a CEDEX bioanalyzer for glucose, lactate, ammonia, glutamine, Glutamax and cholesterol levels (appendix 3). Glucose, glutamine and cholesterol levels for PD-1+ Gen 2 conditions were comparable to bulk TIL conditions. The Glutamax levels of PD-1+ Gen 2 conditions were slightly higher than bulk TIL, indicating that the availability of nutrients does not limit the growth of the culture. Byproducts such as lactate and ammonia were comparable to bulk TIL.

Difference and deviation

A sample of the PD-1+ TIL end product from the full scale experiment was sent to iRepertore for TCR V.beta.sequencing. Once the data is available, the report is modified.

Due to material limitations, small scale runs on L4093 and M1135 were run on the 1/50 th scale rather than the 1/100 th scale. Instead of transferring 10% volume, max 2e6 cells into a G-Rex 5M flask, 20% volume, max 4e6 cells were transferred. Upscaling is affected by changing the upscaling formula from TVC/10e6 (round up, max. 5) to TVC/20e6 (round up, max. 5). These changes allow the final extrapolation to 50X instead of 100X to full scale. The details of the previous section of this report reflect this change.

Conclusion

The PD-1+ Gen2 process was developed at full scale to amplify PD-1+ TIL to >25e9 in 22 days. All quality attributes, such as phenotypic characterization (including purity, memory, activation, depletion marker and function of TIL) generated using the PD-1+ Gen2 process were comparable to melanoma Gen 2.

Summary table 46:

test parameters	Acceptance criteria	H&N 3032	M1137	H&N 3034
					Appearance of the product	The bag was intact with no evidence of clumping	Qualified	Qualified	Qualified
Cell viability	≥70％	Qualified	Qualified	Qualified
					Total viable cell count	1 × 10e9 to 150 × 10e9	Qualified	Qualified	Qualified
Identity (CD 3/% CD45 +%)	>90% CD3+ CD45+ cells	Qualified	Qualified	Qualified
					IFN gamma (stimulated-unstimulated)	≥500pg/mL	Qualified	Qualified	Qualified

The PD-1+ Gen2 process was chosen for further development of PD-1-selected TIL products.

Based on the results obtained at the small scale, additional experiments were performed using PD-1+ early REP conditions to characterize the PD-1+ amplification process as a shorter duration of 17-19 days without compromising dose or product function. See fig. 156.

Reference to example 10:

rosenberg, S.A. et al, complete response sustained in severely pretreated patients with metastatic melanoma using T cell metastatic immunotherapy clinical cancer research 2011.17(13): pages 4550-7.

Kvistborg, P. et al, TIL therapy broadens the tumor-reactive CD8(+) T-cell compartment in melanoma patients, tumor immunology 2012.1(4) p.409-.

Simoni, Y, et al, bystander CD8(+) T cells are abundant in human tumor infiltration and are phenotypically different, Nature 2018.557(7706) p. 575-579.

Schumacher, T.N. and R.D.Schreiber, neoantigens in cancer immunotherapy, science 2015.348(6230): pages 69-74.

Turcotte, S. et al, phenotype and function of T cells infiltrating visceral metastases from gastrointestinal cancer and melanoma: adoptive cell transfer therapy has been suggested in journal of immunology 2013.191(5), pages 2217-25.

Inozume, T. et al, selected CD8+ PD-1+ lymphocytes in fresh human melanoma enriched for tumour-reactive T cells, J.Immunotherapy, 2010.33(9): pages 956-64.

Gros, A. et al, PD-1 identified a patient-specific CD8(+) tumor-reactive pool infiltrating human tumors, J.Clin. Res. 2014.124(5): pp.2246-59.

A pool of transcriptionally and functionally distinct PD-1(+) CD8(+) T cells with predictive potential in non-small cell lung cancer treated with PD-1 blockade by Thommen, d.s. et al.

Table 47: summary of TIL amplification (extrapolation to full Scale) of L4093

Table 48: summary of TIL amplification (extrapolated to full-Scale) by M1135

Table 49: summary of the bulk TIL amplifications (extrapolated to full-Scale) of H3032, M1137 and H3034

NA-not applicable

Table 50: summary of metabolite levels in spent media

Watch 51

Example 10: direct in vitro selection and amplification of PD1^{Height of}Cell: methods of enhancing tumor-reactive TIL for ACT therapy

Introduction to

Adoptive T cell therapy with autologous Tumor Infiltrating Lymphocytes (TILs) has demonstrated a sustained response rate in a cohort of patients with metastatic melanoma [1]. TIL products for therapy comprise heterogeneous T cells that recognize tumor-specific antigens, mutant patient-specific neo-antigens and non-tumor associated antigens [2, 3 ]]. Studies have demonstrated that neoantigen-specific T cells contribute significantly to the anti-tumor activity of TIL [4]. Strategies to enrich TIL for tumor reactivity are expected to produce more effective therapeutic products, especially in epithelial cancers known to contain a high proportion of bystander T cells [5]. Several studies have demonstrated that expression of PD1 (a marker usually associated with T cell depletion) on TIL identifies autologous tumor-reactive T cells [6, 7, 8 ]. Presented in this example is the development of a new scheme designed to select PD1^{Height of}Cells and enriched for TIL product against autologous tumor-reactive T cells.

Purpose(s) to

This example provides methods for sorting and amplifying PD1^{Height of}TIL and protocol for characterization of the resulting product.

Range

This study involved the expanded ex vivo sorting of PD1 from melanoma, lung and head and neck cancers using a 2-REP protocol^{Height of}And (7) TIL. Targeting growth, viability, phenotype, function (IFN γ secretion, CD107a mobilization), tumor killingThe amplified TIL was evaluated for wound reactivity (X-CELLigence) and TCRv β pool (by flow cytometry and RNA sequencing). A schematic method overview is provided in fig. 131.

Material

Tumor tissue

Tumors of various histologies were obtained from the UPMC, Moffitt, Biotheme and MT groups.

A standard reagent for TIL growth comprising: G-Rex 24 well plates, and 10 and 100M flasks (Wilson wolff, Wilson Wolf, Minn., catalog No. P/N80192M; catalog No. 80040S; catalog No. P/N80500); CM2 medium; RPMI 1640 medium (life technologies, ca, catalog No. 11875093); AIMV medium (Gibco, Mass., Cat. No. 0870112-DK); glutamate (Gibco, Mass., catalog No. 30050-; beta-mercaptoethanol (Gibco, Mass., Cat. 21985-023); human AB serum (Gemini, Cat. 100- & 512, Calif.); 0.5mg/ml gentamicin (Gibco, Mass., catalog number 15750-; and GMP recombinant IL-2(Cell-Genix, Germany, catalog No. 1020-1000).

Analytical reagent

Flow cytometry compensation beads: amine reactivity compensation bead kit (ARC) (life technologies, ca, cat # a10346) and VersaComp antibody capture kit (beckmann coulter, ca, cat # B22804).

Flow cytometry antibodies (TIL1, TIL2 (TIL 2 group for surface antigen staining of TIL, v1 and v 2)), TIL3 and (CD107a (TIL function assessed by CD107a mobilization)); the ArC amine reactivity compensation bead kit (Fisher Scientific, massachusetts, catalog No. a 10346); phorbol 12-myristate 13-acetate (PMA) (sigma, catalog No. P1586, missouri); corning Bio-Coat T cell activation plate anti-CD 3 (feishell technologies, inc., ma, catalog No. NC 9937781); corning Bio-Coat T cell activation control plate anti-CD 3 (feishell technologies, inc., ma, catalog No. NC 1108453); human IFN γ Quantikine kit by R & D systems (R & D systems, mn, catalog No. SIF 50); debris removal solution (Gentle and whirly Biotech company, Germany, catalog No. 130-; and R & D systems human IFN γ Quantikine kit (R & D systems, mn, catalog No. SIF 50).

Procedure

Preparation of tumor

Freshly excised tumor samples were obtained from the research consortium (UPMC, Moffitt) and tissue procurement suppliers (biotime and MTG group). Tumors were shipped overnight in HypoThermosol (BioLifecol solutions, Washington, Cat. 101104) (containing antibiotics).

Fragmenting the entire tumor to approximately 4-6-mm³To carry out tumor digestion. If the tumor is large enough, four 3mm are established for Gen2³Of the chip (a). The tumor can be digested with any of the protons described herein.

Enzyme preparation for tumor digestion (using research grade dnase, collagenase and hyaluronidase)

The lyophilized enzymes were reconstituted in the amounts of sterile HBSS indicated for each of the following digestive enzymes. These enzymes were prepared as 10X. Pipette up and down several times and vortex to ensure complete reconstitution.

1-g collagenase IV (Sigma, MO, C5138) was reconstituted in 10-ml HBSS (to make a 100-mg/ml stock). Mixing was performed by pipetting up and down to dissolve. If not dissolved after reconstitution, placed in 37 ℃ H ₂0 bath for 5 minutes. Aliquoted into 1-ml vials. This is a 100-mg/ml 10 × working stock of collagenase.

Stock digests were diluted 1X. To prepare a 1 × working solution, 500-ml of each of collagenase, DNase, and hyaluronidase was added to 3.5-ml of HBSS. The digestion mixture was added directly to the C-tube.

Enzyme preparation for tumor digestion (using GMP collagenase and neutral protease)

The lyophilized enzymes were reconstituted in the amounts of sterile HBSS indicated for each of the following digestive enzymes. Pipette up and down several times and vortex to ensure complete reconstitution.

Collagenase AF-1(Nordmark, Sweden, N0003554) was reconstituted in 10-ml sterile HBSS. The concentration of lyophilized stock enzyme was 2892PZ U/vial. Thus, after reconstitution, the collagenase stock was 289.2PZ U/ml. Note that the stock solution of enzyme can be varied to confirm the concentration of the lyophilized stock solution and modify the final amount of enzyme added to the digestion mixture accordingly.

Neutral protease (Nordmark, Sweden, N0003553) was reconstituted in 1-ml sterile HBSS. The lyophilized stock enzyme concentration was 175DMC U/vial. Thus, after reconstitution, the neutral protease stock solution was 175 DMC/ml. Note that the stock solution of enzyme can be varied to confirm the concentration of the lyophilized stock solution and modify the final amount of enzyme added to the digestion mixture accordingly.

DNase I was reconstituted in 1-ml sterile HBSS (Roche, Switzerland, 03724751). The concentration of lyophilized stock enzyme was 4 KU/vial. Thus, after reconstitution, the DNase stock was 4 KU/vial. Note that the stock solution of enzyme can be varied to confirm the concentration of the lyophilized stock solution and modify the final amount of enzyme added to the digestion mixture accordingly.

The working GMP digestion mixture was prepared. 10.2- μ l of neutral protease (0.36DMC U/ml), 21.3- μ l of collagenase AF-1(1.2PZ/ml) and 250- μ l of DNase I (200U/ml) were added to 4.7-ml of sterile HBSS. The digestion mixture was placed directly in the C-tube.

Tumor treatment and digestion

Each C-tube (America whirlpool Biotechnology, Germany, 130-. Used according to the manufacturer's instructions. Note that each tumor histology had a suggested tumor dissociation procedure. The appropriate procedure was chosen for the corresponding tumor histology. Dissociation took about one hour.

If GentleMeACS OctopDisociator is not applicable, a standard rotator is used. 2-3 tumor fragments were placed in 50-ml conical tubes (sealed with parafilm to avoid leakage) and fixed to a rotator. The rotator was placed at 37 ℃ in 5% CO₂The rotation in the humidified incubator was continued for 1-2 hours. Alternatively, the tumor fragments may be digested overnight at room temperature, also with constant rotation.

After digestion, the C-tubes were removed from the Octodissociator or spinner. A0.22- μm sieve was attached to a sterile Falcon conical tube. All contents from the C-tube or 50ml conical tube (5ml) were passed through a 0.22- μm sieve into a 50ml conical tube using a pipette. The C tubes/50-ml conical tubes were washed with 10-ml HBSS and applied to a strainer. The blunt end of the sterile syringe plunger was used to dissociate any remaining undigested tumor through the filter. CM1 or HBSS was added up to 50-ml and the tubes were capped.

The sample was pelleted by centrifugation at 1500rpm for 5 minutes at room temperature (with an acceleration and deceleration of 9).

The liquid was carefully removed and the pellet was resuspended in 5-ml of CM1 for cell counting and viability assessment.

Whole tumor digests were set aside for the following: 1. cell culture (unselected TIL control); FMO flow cytometry control; 3. determining the digestive phenotype of the whole tumor before sorting; 4. freezing was used for tumor reactivity/cell killing assay. The number of cells left depends on the total digestion yield and tumor histology.

Cleaning digesta Using debris removal kit

Debris was removed from the tumor digests using a debris removal solution (America whirlwind Biotech, Germany, catalog No. 130-. The tumor cell suspension was centrifuged at 300Xg for 10 min at 4 ℃ and the supernatant was aspirated completely. The cell suspension was carefully resuspended with the appropriate volume of cold buffer according to the table below and transferred to a 15ml conical tube. There was no vortex.

Table 52: solutions of

	Resuspension (PBS)	Debris removal solution	Cover layer (PBS)
				0.5-1g tissue	6200-ul	1800-ul	4-ml
，>0.5g of tissue	3100-ul	900-ul	4-ml

An appropriate volume of cold debris removal solution was added and mixed well by pipetting up and down slowly 10-20 times using a 5-ml pipette. Very gentle coverage with 4-ml of cold buffer. The tubes were tilted and pipetted very slowly to ensure that the PBS/D-PBS phase covered the cell suspension and that the phases did not mix. The tumor cell suspension was centrifuged at 3000Xg for 10 min at 4 ℃ with complete acceleration and complete interruption. Three stages are formed. The two top phases are completely sucked and discarded. The bottom phase contains debris removal solution and cells. Leaving at least as much bottom volume as the added debris removal solution. (i.e., if 1ml of solution is added, at least 1-ml of solution remains at the bottom of the tube). Incubate up to 15-ml with cold buffer and invert the tube at least three times. There was no vortex. Centrifugation was carried out at 4 ℃ and 1000Xg for 10 minutes with complete acceleration and complete interruption. Cells were resuspended in HBSS or media for cell counting.

Staining of digested tumors for flow cytometry analysis and cell sorting

Tumor digests were stained with a mixture comprising stained PD1-PE, anti-IgG 4 Fc-PE (secondary antibodies against nivolumab and pembrolizumab), and CD3-FITC according to the following protocol. After counting, the cells were resuspended in 10-ml HBSS.

The pellet was resuspended in FACS buffer (1X HBSS, 1mM EDTA, 2% fetal bovine serum). The amount of FACS buffer added to the pellet is based on the size of the pellet. The staining volume should be about 3 times the size of the pellet (300- μ l of cells, volume of buffer should be at least 900- μ l).

For antibody addition, each 100- μ l volume corresponds to one test (titer of antibody). That is, if a volume of 1-ml is present, a 10 × titration of the amount of antibody is required.

3- μ l of anti-CD 3-FITC (BD biosciences, Cat. No. 561807), 2.5- μ l of anti-PD 1-PE (Biolegend, Cat. No. 329906) were added per 100- μ l volume. anti-IgG 4Fc-PE (southern Biotechnology, Inc., Albama, Cat. No. 9200-09) was also added at 1: 500. For each 500. mu.l FACS buffer, 1. mu.l of anti-IgG 4Fc-PE was added.

Cells were incubated on ice for 30 minutes. Light was protected during incubation. During the incubation period, stirring was performed several times. The cells were resuspended in 20-ml FACS buffer. The solution was passed through a 70- μm cell strainer into a new 50-ml conical tube. Centrifuge at 400Xg for 5 minutes at room temperature (acceleration and deceleration 9). And (6) pumping. Resuspend cells in FACS buffer up to 10e ⁶In/ml TOTAL (live + dead). The minimum volume is 300- μ l. Transfer to sterile polypropylene FACS tubes or 15-ml conical tubes. 3 ml/tube for FACS sorting. A15-ml collection tube was prepared for the sorted population. 2ml of FACS buffer was placed in the tube.

Cell count and viability

Cells were obtained and viability counts were taken using a Nexcelom cell meter K2(Nexcelom, ma).

FACS sorting (FX500 Start)

The machine was turned on and the cell sorter software was run. An automatic calibration is run.

Five sterile 15-ml conical tubes were prepared with 10-ml sterile deionized water. Five sterile 5-ml FACS tubes were prepared with 4-ml sterile deionized water. Five sterile 15-ml conical tubes were prepared with 12-ml of 70% EtOH. Five sterile 15-ml conical tubes were prepared with 12-ml of 10% sodium hypochlorite.

Sample collection

Confirm the sample chamber and collection chamber are at 5 ℃ and select stir sample icon. Using a sample collection software program.

Tubes containing PBMC controls were placed on the sample collection platform.

A 100,000 cell set was selected for the two drop down menus seen above. Confirming that the cell population was correctly gated. The three populations were differentiated by using PBMC, FMO control and the sample itself, gates were set at high, medium (also called intermediate) and low (also called negative). See fig. 132.

It may be necessary to adjust BSC or FSC settings. The voltage of any other channel is not adjusted. PE FMO control tubes were loaded and samples run. The PD1 gate was adjusted as needed. See fig. 133.

When the door is satisfactory, as many events as possible (or up to 20,000 CD3 events) are recorded. The sample pressure can be set to 10 to speed up this collection. The collection was stopped and the tube removed. The previously prepared 15-ml sterile dH20 cone was loaded onto the sample platform. 10 was selected as the sample pressure. The software is run. The samples were collected for one minute. The event is repeated until the CD3 gate is empty. The dH20 sample tube was removed and discarded. At the bottom and middle point of the meniscus, a line is drawn on the tube to be collected with a permanent marker. The sample to be collected is added to the loading platform. Note that: there were a total of four fractions to be collected-negative, medium, high and CD 3. The PD-1 fraction was collected first. The CD3 fractions were then finally collected.

4 was selected as the sample pressure. The software is run. Wait for the cells to appear on the screen. About 15 seconds. Pause was pressed when 3 PD-1 fractions were visible. The lowest 2 of the 3 fractions were collected first.

The sample chamber door was opened and a 15-ml collection chamber block was loaded into the chamber. The collection tube containing the collection buffer was loaded into the chamber block. The capped tube was inverted several times to coat the top of the tube with the collection buffer. The tube was tapped on the surface of the BSC to remove excess buffer from the top of the tube and cap. Two tubes are labeled with sample name and neg, medium or high. The fraction with the lowest percentage of PD-1 cells was selected. The lid is removed and the tube is placed in the sample chamber block. The correct right/left orientation is selected to match the tube position. And continuing to carry out loading collection. The sample pressure was adjusted so that the total events per second was below 5,000. Sample pressure was adjusted to maintain a sorting efficiency of at least 85%. 50,000 CD3 events were recorded.

Sorting was stopped when the sample reached a null value of about 2/3. The collected sample containing the most events was removed. Recapped and placed on ice or at 4 ℃. Leaving the sample with the lower amount in the collection chamber allows more cells to be collected during the collection of the highest percentage of PD-1 sample. The collection tube is labeled and the cap removed. It is placed in a collection chamber. The appropriate left/right orientation of the sorted collection is selected. And loading the collecting pipe. Press play, record and start sort. When the sample reached approximately one-third of the empty value. And stopping sorting. The collected fractions were removed. Recapped and placed on ice or at 4 ℃, and the CD3 collection tube placed to the left of the holder. Let the left side be CD3 and the right side be sort blank. Sorting was continued until all samples were removed from the sample tubes. It is also possible if the tube runs "dry". The sample tube is removed from the sample chamber. And (4) discarding. The sorted fraction is removed from the collection chamber. The tube was capped and inverted several times gently to incorporate a droplet near the top of the tube into the solution. Gently tap the tube onto the surface of the BSC to remove excess solution from the top of the tube and cap. The tubes were placed on ice. The purity percentage of the PD1 fraction was confirmed. A14-ml sterile dH2O conical tube was placed over the sample chamber. And (6) washing. And (6) repeating. The dH2O tube was removed and a positive fraction tube was added. The sample pressure value was changed to 10. 75 CD3 positive events were recorded. The tube is immediately stopped and unloaded from the sample chamber. Repeat for the remaining samples. Data was exported and the instrument was turned off.

REP1 Start

The conditions with the least number of cells (high PD1, negative PD1 intermediate or PD 1) were used to determine the number of cells for REP1 priming. The% (determined during sorting) of CD3 cells was used to calculate the total number of cells in the whole digest required to initiate REP1 under unselected TIL conditions with the same number of CD3 cells as PD1 high, PD1 intermediate, or PD1 negative samples. Total REP 1-initiated total digested cells equals the number of sorted cells seeded in REP 1/% of CD3 cells.

Approximately 1000-cell 100,000 CD3+ cells were placed in G-Rex10 and treated with 7-ml or 40-ml CM2 (50% RPMI 1640+ 10% human serum, glutamax, gentamicin and 50% AimV), and 3000-IU/ml IL-2 for 11 days, respectively. At least one G-Rex flask was started for PD1 high, PD1 intermediate and PD1 negatively sorted populations and unselected TILs. At the start of the culture, anti-CD 3 (clone: OKT3) (30-ng/ml) and feeder cells (ratio 1:100(TIL: feeder cells)) were added to each flask.

Upon completion of REP1, approximately 30-ml of medium was removed for G-Rex 10. The cells were resuspended in the remaining medium by pipetting up and down. Cells were placed in 50-ml conical tubes and centrifuged at 1500rpm for 5 minutes (9 rpm).

REP2 Start

On day 11 of REP2 (or day 22 of the process), the flask was reduced in volume and centrifuged at 1500rpm for 5 minutes at room temperature (9 rpm ramp).

The final products were evaluated for cell count, viability, phenotype (TIL1, DIG1) and function (IFN γ and CD107 a). For V.beta.pool analysis, 1e6-5e6 cells were pelleted and frozen. RNA sequencing was performed by irpertoire. Tumor reactivity of the final product was also assessed in a co-culture assay and IFN γ was assessed. Thawed whole tumor digests were co-cultured with TIL and evaluated for tumor reactivity (secretion by IFN γ) and killing (% cytolysis) using the xcelligene system (ACEA biosciences, ltd, ca).

Results

Cells highly sorted by PD1 showed a defect in proliferative capacity. The expected final product yield expected > or ═ 1e 9. The expanded PD1 high cells were also oligoclonal compared to PD1 intermediate, PD1 negative, and unselected TIL. Based on the premise that PD1+/PD1 high cells are more likely to be antigen-specific, PD1 high cells exhibit enhanced tumor-specific killing capacity compared to their unselected TIL counterparts.

Reference to example 10

Example 11: analysis of TCR libraries in PD 1-selected TILs

Purpose(s) to

To determine the polyclonality and diversity of Tumor Infiltrating Lymphocytes (TILs) selected for programmed cell death protein 1(PD1) and to compare it to unselected TILs.

Range

Seven pairs of matched PD 1-selected and unselected TIL T Cell Receptor (TCR) pools generated from human Head and Neck Squamous Cell Carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) samples were analyzed.

Information

Cancer immunotherapy utilizes the immune system to recognize and destroy tumor cells. The success achieved by immune checkpoint inhibitors (CPI) targeting cytotoxic T lymphocyte antigen 4 and PD1 has transformed cancer treatment and established immunotherapy, along with surgery, chemotherapy, and radiation therapy, as one of the standard treatment approaches. CPI therapy results in a significantly long-lasting clinical response, but only in a subset of patients with certain types of cancer, and often at the cost of severe side effects [1, 2 ].

Adoptive Cell Therapy (ACT) with autologous Tumor Infiltrating Lymphocytes (TIL) has emerged as a powerful and potentially curative therapy for several cancers (Geukes Foppen et al, molecular oncology (Mol Oncol) 2015). The TIL product used in ACT is an unselected, nongenomic preparation of polyclonal T cells that recover directly from tumor tissue and expand extensively ex vivo [3 ]. This process ensures the restoration of a potentially diverse pool of patient tumor-specific memory T cells without prior knowledge of the nature or identity of the antigen [4 ]. In summary, ACT is a simpler, less biased, safer and potentially more effective approach than other cell therapies, such as Chimeric Antigen Receptors (CARs) and TCR T cells that target single tissue-specific or tumor-specific antigens and require the insertion of transgenes. However, the current TIL process may also allow recovery and expansion of variable fractions of T cells that are not associated with cancer (so-called bystander TIL) and recognize antigens, such as antigens from Epstein-Barr virus (EBV), human Cytomegalovirus (CMV) or influenza viruses [5 ].

Multisystem evidence supports neoantigen recognition followed by tumor cell killing as the primary mechanism of action for TIL therapy [6 ]. TILs enriched for tumor neoantigen-specific T cells while remaining unbiased to maintain a degree of diversity and avoid the need for antigen identification represents an attractive means for optimizing products.

As an activation-induced T cell regulator, PD1 has been shown to be specifically expressed in response to recent antigen encounters, and specifically labels neoantigen-specific cells in the case of T cells infiltrating cancer tissues [7, 8 ]. Thus a method is performed by which TIL is selected for PD1 expression prior to ex vivo amplification to enrich for relevant TIL relative to bystander TIL.

In the present example, the T cell clonal composition of a PD 1-selected TIL was compared to that of a matching unselected TIL to confirm that the selection process produced patient-specific TIL products having different compositions and corresponding to a subset of the unselected bulk TIL population.

Design of experiments

The clonal composition of 7 pairs of PD 1-selected and unselected TILs was established by RNA sequencing of the complementarity determining region 3(CDR3) of the TCR β subunit variable region (v β). Each T cell clone in the TIL product expresses a unique TCR identifiable by its CDR3v β. Thus, the unique CDR3v β sequence provides clonal identity by which T cell repertoires of TIL products can be defined and studied.

Material

Tumor samples and TIL products used in this work are described in table 53.

Table 53: description of PD 1-selected and unselected TILs used in the study

TIL selected by PD1 was obtained from 2 HNSCC and 5 NSCLC samples according to procedure example 10. Briefly, whole tumor biopsies were digested with a mixture of dnase, hyaluronidase, and collagenase IV. A portion of the resulting single cell suspension was stained for PD1 and sorted on FX500 instrument (sony, headquarters, new york). PD1 sorted cells and unselected whole tumor digests were subjected to two 11-day Rapid Expansion Phases (REP) to obtain PD1 selected TIL and unselected TIL, respectively. The TIL product was stored frozen and thawed prior to use according to the following procedure.

Method

RNA extraction

According to the manufacturer's protocol (QIAGEN, Higemann, Maryland), use was made ofMini kit, total RNA was extracted from 1-2e6 TIL.

RNA sequencing

CDR3v β was amplified in a semi-quantitative manner using the repertore proprietary arm PCR (amplicon-rescued multiplex PCR) technique with its HTBIvc assay (repertore, henckville, alabama). The HTBIvc assay is a nested reverse transcription multiplex PCR assay that captures VDJ rearrangements from leukocytes, particularly β -chain VDJ rearrangements from T cells. The resulting libraries generated from input RNA were sequenced using the knominal Next Generation Sequencing (NGS) platform (lonminal (Illumina), san diego, ca) at a standard read depth of approximately 1 million reads per library. The final data covers the variable gene region from within frame 3 to the constant gene origin as shown in FIG. 134. The CDR3 portion of the variable gene region corresponds to the "DJ" rearrangement site at the genomic level. The MiSeq platform of enomie was used for all samples. All experiments were performed with iprerture.

Sequencing data analysis

Preliminary analysis of sequencing results was performed by iprerture using a pipeline to filter sequencing and amplification errors and identify the CDR3v β sequence and its frequency for each sample (iprerture, huntville, ala). The data were normalized using a custom script written in Python and additional analysis of the unique CDR3v β profile was performed, including the identification of overlapping CDR3 clones.

The number of unique CDR3v β was defined as the number of unique peptide CDR3 within the sample. The frequency of each individual clonotype was calculated by counting the number of sequencing reads containing the unique clonotype that passed through the demultiplexing and filter within the library. The Shannon diversity index (H) is a formulaCalculated, where S is the total number of clones in the colony (abundance), and p_iIs the proportion of S constituted by clone i. The overlap between PD 1-selected and unselected TILs from the same tumor sample was determined by identifying clones found in both samples and reported in two ways: reporting the percentage of clones shared by dividing the number of clones shared by the total number of unique clones reported in each type of product; the percentage of total TCR population shared was determined by normalizing the frequencies between samples and summing the frequencies of shared clones for each TIL product.

Expected result

Comparative analysis of TCR libraries expected to pair PD 1-selected and unselected TILs revealed that the selected TILs represented a portion of the unselected TILs and were oligoclonal. TIL clones shared between PD 1-selected and unselected products were expected to show different frequencies among the 2 products, reflecting altered competition kinetics.

The results obtained

Number and diversity of unique CDR3v β in PD 1-selected and unselected TILs

In vivo TILs comprise T cells that are not only specific for tumor-specific antigens (e.g., neoantigens), but also recognize a wide range of epitopes not associated with cancer (e.g., epitopes from EBV, CMV, or influenza viruses) [5 ]. These non-cancer-associated or bystander TILs can overgrow tumor-specific cells during a broad in vitro culture period that is required to produce a sufficient number of T cells for patient treatment and may result in the production of TIL products with a low frequency of tumor-reactive T cells [9 ].

To test whether sorting TILs expressing PD1 prior to in vitro expansion allowed the recovery of products containing T cell pools different from those of non-pre-sorted TILs, the TCR compositions of the products of the two methods were compared. Sequencing of CDR3v β was performed on 14 samples as described in the examples. The data were analyzed according to the described method to generate the number and diversity index of the unique CDR3v β for each sample. The results are shown in fig. 134 and table 54.

The number of unique CDRs 3v β varied from 1,027 to 2,778 and 648 to 1,975 in PD 1-selected and unselected TILs, respectively (fig. 134A). No specific pattern of HNSCC relative to HNSCC was noted. 4 of the 7 PD 1-selected samples exhibited fewer unique CDR3v β clones than their matched unselected samples, indicating a reduced tendency for polyclonality in the PD 1-selected TIL relative to the unselected TIL, which would require testing of additional samples to be confirmed. Similar to the number of unique CDR3v β clones, the indices representing clonal diversity of PD 1-selected and unselected TILs were not significantly different (fig. 134B).

The oligoclonality of PD1+ TIL in vivo was reported for melanoma and NSCLC and is thought to reflect the selective expansion of neoantigen-specific TIL within the tumor microenvironment [7, 8 ]. These results are consistent with those reported, perhaps because the amplification phase to which the TILs from this study were subjected prior to sequencing allowed the appearance of low frequency clones that would not be detectable prior to amplification. None of the TILs analyzed in the published reports were amplified, so the least frequent clones may not be considered. A potential implication of the observations in this example is that there may be more PD1+ or tumor specific T cells in the TME than originally assumed. The relatively high (mean 38.4%, range 21 to 78%) PD1+ TIL detected in the original 7 tumor digests was consistent with this hypothesis (SR-19-009-. Alternatively, the apparent polyclonality of the TIL product selected by PD1 may result from amplification of contaminating PD 1-TIL. Sorting purity was about 93% (study data) and few PD1-TIL may have proliferated to detectable levels during amplification in view of initial proliferation advantage [8, 10, 11 ].

Since the number and frequency of unique CDR3v β clones in PD1 selected and unselected TILs may have reached a level during in vitro culture, it was proposed to compare the identity of T cell clonotypes comprising PD1 selected and unselected TILs.

Number and percentage of overlapping T cell clones between PD 1-selected and unselected TILs

In TME, PD1 was shown to specifically identify TILs that recognize tumor antigens, which represent a fraction of T cells infiltrating tissues [7, 8 ]. As noted above, at any given time, there may also be a wide range of non-cancer related T cells in the TME that are not expected to recently upregulate PD1 expression and for which the PD1 sorting strategy is aimed at selecting. Thus, it was desired to determine which part of the TIL clonotypes present in the unselected product comprised the PD 1-selected product. To this end, the degree of clonal overlap between PD 1-selected and unselected products was evaluated for each pair of TIL samples. Results expressed as number, percentage and fraction of overlapping clones are shown in the analysis.

The average number of ucrd 3v β clones shared by 5.4% and 5.36% of the total CDR3v β reads, in both PD 1-selected and unselected TILs, was numbered (fig. 2). These numbers indicate that the pool of PD 1-selected clones only partially overlaps with the pool of unselected clones, and that there is a significant population of clonotypes identified in the PD 1-selected TIL that were not detected in the matching unselected TIL. Since PD 1-selected and unselected TILs were originally from the same tumor digest, the results of the overlap analysis indicated that: 1) presumably a substantial portion of bystander TILs did not make it a PD 1-selected TIL product, and 2) a variable portion of TILs that may contain tumor-specific cells was recovered in a PD 1-selected product, which was lost during the expansion phase of unselected TILs. Bystander TILs appearing at the start of culture may be able to exceed the low proliferation PD1+ TILs in unselected cell pools, while these same PD1+ TILs are given the opportunity to expand when cultured in the absence of bystanders under conditions selected via PD 1. Significant differences were noted between the 2 histologies studied here. Although the fraction of clonotypes shared among the PD 1-selected products was increased relative to the unselected products for all 5 NSCLC samples, the opposite was observed for the 2 HNSCC samples. In addition, the unselected formulations corresponding to these 2 samples consisted of a relatively high proportion of shared TIL. Given the limited sample size, this difference can be anecdotal; the differences may also reflect differences in the types of tumor antigens present in those underlying HPV-associated cancers. Overall, the results are consistent with the profound effect of the selection step on the composition of the amplified TIL product, and indicate that the resulting PD 1-selected TIL can be greatly enriched for tumor-specific TILs that would otherwise be reduced during the amplification phase. See fig. 135.

Comparative frequency of top PD 1-selected TIL clones in PD 1-selected and unselected products

Both results from previous evaluations indicate that tumor-specific TILs expressing PD1 are susceptible to competition by non-tumor-associated bystander T cell clones and have the benefit of isolating tumor-associated TILs from the pool. To further assess the difference in tumor-specific T cells in PD 1-selected and unselected TILs indicates that the ranking of the top 10 highest frequency PD 1-selected TIL clones was determined in unselected TILs. The results are shown in fig. 136 and table 56.

Of all paired TIL products, the majority of highly represented PD 1-selected TIL clones were represented to a low or absent extent in the unselected products, confirming the significant effect of the selection step on the final composition of the amplification product and the potential enrichment of tumor-specific T cells in the PD 1-selected TIL.

Conclusions and suggestions

The number and diversity index of unique CDR3v β sequences of PD 1-selected TILs evaluated were comparable to matching unselected TILs, indicating that polyclonal and highly diverse products can be amplified after PD1 sorting.

The pool of PD 1-selected TIL clones partially overlapped the pool of unselected TILs, indicating that a greater number of tumor-specific TILs could be recovered when using the selection process.

The high frequency of TIL clones selected by PD1 appeared less frequently in the unselected TIL product, again supporting the enrichment of tumor-specific TILs in the new product.

Overall, this study showed that amplification of the PD 1-sorted TIL produced TIL products that differed in their composition from the unselected TILs. This difference probably reflects the modest performance of tumor-specific TILs and outgrowth of bystander TILs, which occurred in the absence of PD1 selection.

Reference to example 11

Sharma, P.and J.P.Allison, future of immune checkpoint therapy (The future of immune checkpoint therapy) science 2015.348(6230) pages 56-61.

Michot, j.m. et al, immune-related adverse events with blockade of immune checkpoints: a comprehensive review (Immune-related events with an Immune checkpoint block: a comprehensive review) of the European journal of Cancer (Eur J Cancer), 2016.54: page 139, 148.

Wardell, S. et al, cryo-preserved TIL Product LN-144(A Cryopressed TIL Product, LN-144, produced with an Abbreviated Method Suitable For High-Throughput Commercial Manufacturing of exhibiting Favorable Quality Attributes For Adoptive Cell Transfer.) Journal of Cancer ImmunoTherapy (Journal For immunological therapy of Cancer), 2017.5 ((Journal 2)).

Gontcharova, V. et al, C-144-01 study of advanced metastatic melanoma the Persistence of the cryopreserved tumor-infiltrating lymphocyte product, lifilurel (LN-144) (Persistence of secreted tumor-infiltrating lymphocyte product, lifilurel (LN-144) in C-144-01 study of advanced metastatic melanoma.) Cancer study (Cancer Res), 2019.79 (supplement 13).

A pool of transcriptionally and functionally distinct PD-1(+) CD8(+) T cells with predictive potential in non-small cell lung cancer treated with PD-1 blockade by Thommen, d.s. et al. 24(7) page 994 and 1004.

Yossef, r. et al, Enhanced detection of neoantigen-reactive T cells targeting distinct and shared oncogenes for personalized cancer immunotherapy (Enhanced detection of neo-active-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy), journal of clinical investigation (JCI Insight), 2018.3 (19).

Zhang, y, et al, Programmed death 1 upregulation has been associated with dysfunction of tumor-infiltrating CD8+ T lymphocytes in human non-small Cell lung cancer (Programmed death-1 alignment is corraded with a dynamic function of tumor-infiltrating CD8+ T lymphocytes in human non-small Cell lung cancer) Cell and molecular immunology (Cell Mol Immunol), 2010.7(5), pp 389-95.

Fernandez-Poma, S.M. et al, Expansion of PD-1 Expressing Tumor Infiltrating CD8(+) T cells improved the Efficacy of Adoptive T cell Therapy (Expansion of Tumor-Infiltrating CD8(+) T cells Expressing PD-1 improvements the Efficacy of adjuvant T-cell Therapy), cancer research 2017.77(13), p. 3672-3684.

Table 54: counts of unique CDR3 sequences of unselected and PD 1-selected TIL products and aromatic diversity index.

Table 55: clonal overlap between PD 1-selected and unselected TILs

Table 56: frequency of the first 10 most frequently detected clones in PD 1-sorted TIL products among PD 1-sorted and unsorted TIL products.

Example 12: PD1 expression cells in tumor digests

Purpose(s) to

This example evaluated the expression of programmed cell death protein 1(PD1) in the whole tumor digest.

Range

PD1 assessing whole tumor digest following tumor histology; melanoma, non-small lung cancer (NSCLC), Head and Neck Squamous Cell Carcinoma (HNSCC), Ovarian Cancer (OC), Triple Negative Breast Cancer (TNBC), Prostate Cancer (PC), and colorectal cancer (CRC).

Information

PD1 is a multidimensional phenotypic marker associated with activation, antigen specificity and depletion. It is rapidly induced upon activation and maintained on cells undergoing antigens in a chronic disease setting, including cancer [1, 2 ]. Molecularly, PD1 is a member of the CD28 family that regulates cell surface receptors and is expressed on chronically activated T cells, NKT cells, B cells and monocytes [3-5 ]. Engagement with its ligands PD-L1 and PD-L2 induces a signaling cascade leading to a reduction in T cell activation, proliferation, survival and cytokine production [6 ].

Despite the immunosuppressive role of PD1, the presence of Tumor Infiltrating Lymphocytes (TILs) expressing PD1 has been associated with favorable clinical outcomes in the head as well as HNSCC and NSCLC, suggesting that these TILs may be involved in controlling tumor progression [7] [8, 9 ].

Studies in melanoma and NSCLC have demonstrated that most tumor-reactive TILs are included in the PD1+ T cell subset [4, 8, 10 ].

Based on the concept that PD1+ TIL is a neoantigen/tumor-specific lymphocyte, Iovance is developing a novel PD 1-selected TIL product LN-145-S1 enriched in PD1+ TIL sorted directly from whole tumor digests.

Although PD1 expression is essential for response to anti-PD 1 therapy, PD1 expression alone does not predict responsiveness to therapy. As an example, PD1 is present on TILs in OCs and its expression is correlated with survival [11 ]. However, recent clinical trials in OC demonstrated that anti-PDL 1 drug aviluzumab (Avelumab) in combination with chemotherapy did not enhance progression-free survival [12 ]. This study, together with the large number of patients resistant to anti-PD 1 therapy expressing PD1+ in the tumor microenvironment, showed that in vivo blockade of the PD1/PDL1 axis was insufficient to control most cancers.

Adoptive T cell therapy using lifileucel has demonstrated significant efficacy in melanoma patients refractory to anti-PD 1, suggesting that the TIL process expands the T cell population that is not restored by in vivo PD1 blockade [13 ].

In all PD1+ cancer histologies, sorting PD1+ TIL prior to ex vivo expansion could further improve the response rate to TIL therapy.

The purpose of this example was to investigate various tumor histologies for the presence of PD1+ TIL to support its clinical targeting with these TILs.

Design of experiments

Tumor digests from various tumor histologies were evaluated by flow cytometry for PD1 expression.

Material

The tumor digest used in this example is depicted in fig. 137.

Method

Tumor treatment

Tissue sample fractions weighing 0.2g to 1.5g were cut into 4-6mm pieces and digested into single cell suspensions containing tumor, stroma and immune cells. The tissue was digested with a triple enzyme mixture comprising DNase (500IU/ml), hyaluronidase (1mg/ml) and collagenase IV (10ng/ml) at 37 ℃ for 1 hour with gentle agitation.

PD1 staining

Staining of whole tumor digests was performed according to the following table. Cells were stained in 100. mu.l/1 e6 cells.

Table 57: PD1 flow cytometry staining plate

Antibody/staining	Cloning	Fluorescent dyes	Manufacturer(s)	Amount (μ l/1e6 cells)
					7-AAD	N/A	N/A	BD bioscience Co.	20
CD3	UCHT1	FITC	BD bioscience Co.	3
					CD4	OKT4	PE/Cy	BioLegend Co	1
PD1	EH12.2H7	PE	BioLegend Co	2.5

PD1 selection and gating strategy

Stained cells were placed on either an FX500 cell sorter (sony, new york) or ZE5 cell analyzer (berle corporation (BioRad), ca) and analyzed based on the following gating strategy. First, single cells were identified based on forward and backward or side scatter. Next, live cells were gated based on negative/low 7-AAD or live-dead blue fluorescence. TIL was identified using CD 3. PD1 cells were identified using normal donor peripheral blood (ND-PBL) as a control. The selection gate for PD1 was placed above the baseline for PD1 expression in ND-PBL.

Data analysis was performed using FlowJo v8.1 software (FlowJo ltd (FlowJo LLC), oregon). Results were plotted using GraphPad v8.

Expected result

PD1 was expected to be expressed in most of the tumor digests assayed. The percentage of PD1 is expected to be variable within each tumor subtype and between different tumor histologies.

The results obtained

Expression of PD1 in tumor digests

To identify which histologies were candidates for PD1 selection, expression of PD1 was assessed in multiple tumor samples from several cancer histologies using flow cytometry. A total of 4 melanomas, 7 NSCLCs, 5 HNSCCs, 3 OCs, 5 TNBCs, 2 PCs and 8 CRCs were tested according to the procedure TMP-18-015 (abbreviated section 5.2). CRC consists of microsatellite stabilised (MSS) (n ═ 6) and microsatellite unstable (MSI) (n ═ 2) tumors. After digestion, a portion of the resulting single cell suspension was stained for PD1, analyzed by flow, and sorted to obtain PD1+ cells when >5e6 cells were available. PD1 sorted cells were subjected to two 11 day Rapid Expansion Phases (REP) to obtain PD1 selected TILs. Tumor ID, histology, and experimental fate are listed in fig. 137. The results of the streaming analysis are shown in fig. 138.

All tumor digests assayed expressed the percentage of PD1+ cells within the CD3 population. PD 1% was variable and ranged from 11% to 78%, with an average of 35% in the histology determined. Melanoma (n ═ 4) and PC (n ═ 2) produced the lowest average expression of PD1 of 27% and 21%, respectively. The average percentage of PD 1expression was not correlated with the clinical response rates observed for those histologies. Histology responsive to anti-PD 1 blockade, such as melanoma and NSCLC, did not have higher PD1 levels/expression compared to histology not responsive to anti-PD 1 blockade (i.e., OC and PC).

Importantly, in all cases in which culture could be initiated, PD 1-selected products could be obtained following in vitro expansion by PD1+ cells (fig. 138). Therefore, all histologies determined were potential candidates for PD1 selection based on the expression of PD 1.

Conclusion

In all tumor digests assayed, PD1 was expressed on CD3 cells.

There is a wide range of intratumoral and intratumoral variability in PD1 expression.

PD 1expression was not associated with histology that has been shown to be responsive to anti-PD 1 therapy.

Reference to example 12

Simon, s. and n.labarrire, PD-1expression on tumor-specific T cells: friends or enemies of immunotherapy? (PD-1expression on tumor-specific T cells: Friend or for immunology) tumor immunology 2017.7(1): p.e1364828.

Simon, S. et al, PD-1expression T cell avidity with an anti-specific reagent within the conditional antigen specificity library (PD-1expression conditions T cell avidity) tumor immunology 2016.5(1): p.e1104448.

Ahmadzadeh, m. et al, Tumor antigen-specific CD 8T cells infiltrating tumors express high levels of PD-1and are functionally impaired (Tumor antigen-specific CD 8T cells encapsulating the Tumor expression high levels of PD-1and are functionally impaired) & Blood (Blood), 2009.114(8) & pages 1537-44.

Formation and phenotypic characterization of the CD 8T cell population expressing CD49a, CD49b and CD103 in Melssen, M.M. et al, human metastatic melanoma (formatting and phenotypic characterization of CD49a, CD49b and CD103 expression CD 8T cell publications in human metastatic melanoma.) tumor immunology 2018.7(10): p.e1490855.

Lee, J. et al, restoration of depleted T Cells by blocking the PD-1Pathway (Reinforming Exhausted T Cells by Block of the PD-1Pathway) For immunopathological disease therapy (For Immunopathol disease therapy), 2015.6(1-2) p.7-17.

Badoual, C. et al, PD-1 expressing tumor-infiltrating T cells are a favorable prognostic biomarker in HPV-associated head and neck cancers (PD-1-expressing tumor-encapsulating T cell areas a volatile protective biomaker in HPV-associated head and neck cancer.) cancer research 2013.73(1): pages 128-38.

Kansy, B.A. et al, PD-1Status in CD8(+) T Cells was associated with Survival in Head and Neck Cancer and Anti-PD-1 treatment Outcomes (PD-1Status in CD8(+) T Cells Associates with Survival and Anti-PD-1 Therapeutic Outcome in Head and cock Cancer). Cancer research 2017.77(22) at page 6353-.

Gross, A. et al, PD-1 identified patient-specific CD8(+) tumor-reactive pools infiltrating human tumors, J.Clin. Res. 2014.124(5): pp 2246-59.

Webb, J.R., K.Milne and B.H.Nelson, PD-1and CD103 Are Widely co-expressed in Human Ovarian Cancer on Intraepithelial CD 8T Cells with Favorable prognosis (PD-1and CD103 Are Widel Coexpressed on viral connective Intra CD 8T Cells in Human Ocarian Cancer) & Cancer immunology research (Cancer Immunol Res., 2015.3(8): pages 926-35).

Columbus, G. Abelmuzumab missed the Primary endpoint in the stage III Ovarian Cancer test (Avelumab Misss Primary endpoint in Phase III Ovarian Cancer Trial). 2018; available from the following websites: https:// www.onclive.com/web-exclusive/avelumab-messes-primary-end-in-phase-iii-overview-cane r-tertiary.

Sarniak, a. phase 2 multiple center study for assessing the efficacy and safety of autologous tumor infiltrating lymphocytes (LN-144) for treating patients with metastatic melanoma (a phase 2, multicenter study to access the efficacy and safety of autogous tumor organizing lymphocytes (LN-144) for the treatment of patients with metastatic melanoma 2018; available from the following websites: https:// ascopubs.org/doi/abs/10.1200/JCO.2018.36.15_ Suppl.TPS9595.

Example 13: amplification of PD 1-selected TIL

Purpose(s) to

This example evaluated the expansion of Tumor Infiltrating Lymphocytes (TILs) sorted by programmed cell death protein 1(PD1) compared to matched unselected TILs.

Range

Both PD 1-selected and unselected TILs from melanoma (n-4), non-small cell lung cancer (NSCLC) (n-7) and Head and Neck Squamous Cell Carcinoma (HNSCC) (n-2) were amplified using a two-cycle Iovance rapid amplification protocol (REP). The amplification of selected and unselected TILs was evaluated at the completion of REP1 (day 11) and REP2 (day 22).

Information

PD1 is a member of the CD28 family and is expressed on chronically activated T cells, NKT cells, B cells and monocytes [1, 2 ]. Expression of PD1 in T cells has been best characterized in that it is induced upon TCR stimulation [2,3] and maintained on antigen-specific cells in a chronic disease setting [4, 5 ].

Upon conjugation to its ligands PD-L1 and PD-L2, signaling through PD1 results in inhibition of T cell proliferation, survival and cytokine production. Several studies in chronic disease models (including HIV, hepatitis c, and cancer) have demonstrated a significant reduction in fold expansion of PD1+ cells compared to PD1-TIL [1, 6, 7 ]. In murine multiple myeloma models, TIL selected by PD1 was less efficient in proliferation when compared to its PD1 counterpart, as evidenced by a 10-fold lower amplification rate.

Despite the reduced proliferative capacity of TIL selected by PD1, PD1+ cells have been shown to proliferate in vitro in the presence of anti-CD 3 and allogeneic feeder cells with IL-2 [5-7 ]. Furthermore, PD1+ TIL kills autologous tumors in vitro and generates an anti-tumor response in vivo in mice [8 ].

PD1+ -sorted TIL (selected by PD 1) and TIL derived from whole tumor digests (unselected TIL) were amplified using two consecutive 11-day REPs. Fold amplifications of TILs were calculated to determine whether or not TILs selected by PD1 could be amplified and how this compared to matching unselected TILs. Fold expansion was calculated at the completion of REP1 (day 11) and REP2 (day 22) based on the initial CD3 seeding count and the number of cells at harvest.

Design of experiments

Both PD 1-selected and unselected TILs were amplified during 22 days using a two-step amplification procedure comprising an 11-day activation step followed by 11-day REP. Fold amplification was calculated to obtain the proliferative capacity of both TIL products.

Material

Tumor samples and TIL products used in this work are depicted in fig. 139.

According to example 10, PD 1-selected and unselected TIL products were obtained from 4 melanomas, 7 NSCLCs and 2 HNSCCs. Briefly, whole tumor biopsies were digested with a mixture of dnase, hyaluronidase, and collagenase IV. A portion of the resulting single cell suspension was stained for PD1 and sorted on FX500 instrument (sony, headquarters, new york). PD1 sorted cells and unselected whole tumor digests were subjected to a 22 day expansion process to obtain PD1 selected TIL and unselected TIL, respectively.

Method

Tumor treatment

Tissue sample fractions weighing 0.2g to 1.5g were cut into 4-6mm pieces and digested into single cell suspensions containing tumor, stroma and immune cells. The tissue was digested with a triple enzyme mixture comprising DNase (500IU/ml), hyaluronidase (1mg/ml) and collagenase IV (10ng/ml) at 37 ℃ for 1 hour with gentle agitation. To ensure capture of the in situ phenotype, PD1 cells were selected directly after digestion [2 ].

PD1 staining

Table 58: PD1 flow cytometry staining plate

PD1 selection and gating strategy

Stained cells were placed on an FX500 cell sorter (sony, new york) and analyzed based on the following gating strategy. First, single cells were gated based on forward and back scatter, then on negative or low 7-AAD fluorescence viable cells, followed by CD3 and PD1 expression. PD1 cells were identified using normal donor peripheral blood (ND-PBL) as a control. The selection gate for PD1 was placed above the baseline for PD1 expression in ND-PBL.

TIL Rapid amplification protocol by PD1 selection

PD 1-selected and unselected TILs were amplified using a two-step process comprising an 11-day activation step followed by 11-days REP for a total of 22 days. TIL was expanded using OKT3(30ng/ml, Gentle Biotech) and allogeneic irradiated peripheral blood mononuclear cells (TIL: feeder ratio of 1: 100). The number of TILs inoculated ranged between 5,000-100,000 CD3+, and was dependent on the number of pre-sorted cells, CD3 infiltration, and PD1 expression.

Calculating the amplification fold of TIL

On day 11 (activation collection) and day 22 (REP collection), TILs were collected and counted using a Cellometer K2 fluorescence viability cell counter (Nexcelom, massachusetts). Fold expansion of PD 1-selected and unselected populations was calculated based on seeded CD3 counts and collected cell counts (i.e., fold activation expansion ═ day 11 cell count/day 0 cell count and fold REP expansion ═ day 22 cell count/day 11 seed count). The number of seeded cells used for the activation step under unselected TIL conditions was normalized to the number of CD3 cells in PD1 selection on day 0. Data were plotted using GraphPad Prism v 8.

Results

The PD 1-selected TILs expanded in the presence of anti-CD 3 and feeder cells, but to a lesser extent than the matched unselected TILs.

The results obtained

Fold amplification in PD 1-selected and unselected TILs

Classically, PD1+ cells have shown impaired cytokine production and reduced proliferation [3, 4 ]. Blocking PD1 or its ligand PD-L1 in situ has been shown to partially reverse proliferative dysfunction in TIL [2, 9 ]. In vitro, PD1+ cells can proliferate but not to the extent of PD1-TIL when stimulated with anti-CD 3 and allogeneic feeder cells in the presence of IL2 [1 ].

To determine whether the PD 1-selected TIL was able to proliferate in vitro and produce therapeutically appropriate amounts of TIL for infusion, PD 1-selected and unselected TIL from 4 melanomas, 7 NSCLC and 2 HNSCC were expanded using a two-step procedure with an 11-day activation step, followed by 11-day REP, and fold expansion was assessed.

During the activation step, the amplification level of the PD 1-selected TIL was reduced compared to the unselected TIL. The mean fold amplification for the activation step was 833, while the fold amplification for unselected TILs was 2650. Interestingly, for the REP step, the PD 1-selected TIL overcome the initial proliferation defect in the activation step, as the fold expansion of the PD 1-selected TIL (1308) was similar to that of the unselected TIL (1418). Reduced proliferation of R in the activation step EP1 was observed in both melanoma and NSCLC, but not in HNSCC. However, the number of HNSCC tumors determined was low (n ═ 2). Furthermore, the proliferative capacity in REP across three histologies is similar between TIL populations.

Conclusion

TILs selected by PD1 were successfully amplified from tumor digests of melanoma, NSCLC and HNSCC. See fig. 141.

Amplification in REP1 was significantly reduced for the PD 1-selected TIL compared to the unselected TIL.

There was no reduction in proliferation in TILs selected with PD1 during REP 2.

Despite being derived from sorted digests, the TIL selected by PD1 amplified well within the REP fold range (54-28, 214) of the generation 2 product, lifileucel, Iovance.

Although the proliferative capacity of TIL selected via PD1 was reduced during REP1, the 13/13 TIL selected via PD1 generated using the 2-REP process exceeded the lifileucel threshold of infusion (i.e., >1e 9).

Since PD1+ TIL enriches tumor/neoantigen-specific cells, it is essential that it is present in considerable amounts in the final product [10, 11 ]. The lower proliferative capacity of PD 1-selected TIL suggests that it will be competitive in unselected TIL preparations, further strengthening the rationale for selection prior to amplification.

Reference to example 13

Ahmadzadeh, m. et al, tumor antigen-specific CD 8T cells infiltrating tumors express high levels of PD-1 and are functionally impaired blood, 2009.114(8): pages 1537-44.

Lee, J, et al, for the reconstitution of depleted T cells by blocking the PD-1 pathway for immunopathological disease therapy 2015.6(1-2): pages 7-17.

Virgin, h.w., e.j.where and r.ahmed, reduction of chronic viral infection Cell infection 2009.138(1): pages 30-50.

Simon, s. and n.labarrire, PD-1 expression on tumor-specific T cells: friends or enemies of immunotherapy? Tumor immunology 2017.7(1) p.e1364828.

Simon, S. et al, PD-1 expresses T cell avidity within a conditional antigen specificity repertoire (tumor immunology 2016.5(1): p.e1104448).

Biochemical signaling of PD-1 on T cells and functional implications thereof in Boussiositis, V.A., P.Chatterjee and L.Li, PD-1, J.Cancer J. 2014.20(4), pp.265-71.

Petrelli, A. et al, PD-1+ CD8+ T cells are clonal expansion effectors in human chronic inflammation (PD-1+ CD8+ T cell cyclic expansion effectors in human chronic inflammation), J.Clin.Res. 2018.128(10), p.4669-4681.

Amplification of PD-1 expressing tumor-infiltrating CD8(+) T cells improves the efficacy of adoptive T cell therapy in Fernandez-Poma, S.M. et al, cancer research, 2017.77(13): 3672-3684.

Tumeh, P.C. et al, PD-1blockade induced responses by inhibition of adoptive immune resistance (PD-1 blocked responses by inhibiting adaptive immune responses) Nature 2014.515(7528) pp 568-71.

Thommen, D.S. et al, a transcriptionally and functionally diverse pool of PD-1(+) CD8(+) T cells with predictive potential in non-small cell lung cancer treated with PD-1blockade, Nature medicine, 2018.

Example 14: functional assessment of TIL selected by PD1

Purpose(s) to

This example evaluates the effector function of amplified apoptosis protein 1(PD1) selected TIL and compared to unselected TIL.

Range

IFN γ secretion, granzyme B release and CD107a mobilization of PD 1-selected and unselected TILs from melanoma and non-small cell lung cancer (NSCLC) and Head and Neck Squamous Cell Carcinoma (HNSCC) were assessed in response to non-specific stimulation.

Information

PD1 is a multidimensional phenotypic marker associated with activation, antigen specificity and depletion. It is rapidly induced upon activation and maintained on antigen-specific cells in a chronic disease setting, including cancer [1, 2 ]. Molecularly, PD1 is a member of the CD28 family that regulates cell surface receptors and is expressed on chronically activated T cells, NKT cells, B cells and monocytes [3-5 ]. Engagement with its ligands PD-L1 and PD-L2 induces a signaling cascade leading to a reduction in T cell activation, proliferation, survival and cytokine production [6 ].

Despite the immunosuppressive role of PD1, the presence of TIL expressing PD1 has been associated with favorable clinical outcomes in HNSCC [7], NSCLC [8] and ovarian cancer [9 ]. The encounter of antigens in the tumor microenvironment leads to up-regulation of PD 1. Several studies have demonstrated that neoantigen/tumor-reactive TILs are predominantly included in the PD1+ T cell subset [3, 10 ]. Therefore, the TIL selected for expression of PD1 is expected to enrich the TIL product of tumor/neoantigen specific T cells.

Selected PD1+ TIL recovered from tumor lesions have been evaluated for functionality in response to non-specific stimulation. The uncultured sorted PD1+ TIL showed a significant reduction in IFN γ production relative to its PD1 counterpart [3, 8, 11 ]. However, the effector function of amplified PD1+ TIL was restored upon in vitro culture [4, 8 ].

Based on PD1 expression (selected by PD 1), TIL products were developed to enrich for tumor-specific T cells. Both PD 1-selected TIL and TIL derived from whole tumor digests (unselected TIL) were amplified using a two-step procedure, employing an 11-day activation step followed by an 11-day rapid amplification protocol (REP). To determine whether the resulting amplified PD 1-selected TIL exhibited effector function, TILs were evaluated in a series of in vitro functional assays and compared to matching unselected TILs.

Design of experiments

Both PD 1-selected and unselected TILs were amplified during 22 days, using a two-step process with an 11-day activation step followed by 11-day REP. The functionality of the final TIL product to non-specific stimuli was evaluated in terms of IFN γ and granzyme B secretion.

Material

Tumor samples and TIL products used in this work are depicted in fig. 14.

Method

Tumor treatment

PD1 staining

Staining of whole tumor digests was performed according to the following table. Cells were stained in 100. mu.l/1 e6 cells. The PD-1 flow cytometry staining set is provided in table 58 in example 13 above.

PD1 selection and amplification

PD1+ cells were selected using FX500 cells (sony, new york). Both PD 1-selected and unselected TILs were amplified using a two-step procedure with an 11-day activation step followed by 11-day REP. TIL was expanded using OKT3(30ng/ml, Gentle Biotech) and allogeneic irradiated peripheral blood mononuclear cells (TIL: feeder ratio of 1: 100).

IFN gamma and granzyme B secretion

TIL was seeded at 5e5 cells/well in 1ml 48-well plates +300IU/ml IL2(CellGenix, N.J.). TIL stimulated +/-100. mu.l/well of alpha CD 3/alpha CD 28/alpha 41BB beads (Semmer Feishell science, Mass.) for 12-18 hours. Supernatants were collected and evaluated by ELISA for IFN γ (R & D systems, mn) and granzyme B (life technologies, ca). ELISA plates were read on an american pentium microplate reader (BioTek, buddont, usa) and evaluated using Gen5 data analysis software. Data were plotted using GraphPad Prism v 8.

CD107 mobilization

TIL selected with PD1 and unselected with PMA/ionomycin (Biolegend, ca) were stimulated for 2 hours (to prevent protein secretion) in the presence of monensin (monensin). TIL was then stained with live/dead dye and antibodies against CD3 and CD107 a. Stained cells were detected by flow cytometry. Expression of CD107a in CD3+ cells was analyzed using FlowJo software (beckmann dickinson). The gating strategy is as follows: single cell (singlet) (FSC and SSC), live cells, CD3 and CD 107. All data were plotted using GraphPad Prism v 8.

Results

In response to non-specific stimulation, amplified TILs selected by PD1 produced IFN γ and granzyme B.

Secretion of IFN γ and granzyme B in PD 1-selected and unselected TILs

Previous reports have demonstrated that PD1+/PD1 high cells either reduced or failed to produce IFN γ at all in response to PMA and ionomycin [4] or anti-CD 3/anti-CD 28 stimulation [8, 11 ]. These studies were performed with uncultured PD1+ TIL, which expressed high levels of the co-inhibitory receptors LAG3 and Tim3 in addition to PD1 [8, 10, 12 ]. Pre-cultured PD1+ is considered to be "dysfunctional" due to its "depleted" phenotype (i.e. high expression of inhibitory receptors) and its inability to produce effector functions. However, once the PD1+/PD1 high cells expand in vitro (by anti-CD 3 and allogeneic feeder cells), TILs restore their ability to produce IFN γ [3, 4, 8 ]. Enhanced effector function is also associated with a significant reduction in PD1 expression [3, 4, 8, 13 ]. These studies indicate that the observed anergy in uncultured PD1+/PD1 high TIL can be reversed with in vitro culture.

To assess whether PD1+ TIL was functional in cytokine production after expansion, PD 1-selected and matched unselected TILs from 13 tumors were non-specifically stimulated with α CD3/α CD28/α 41BB activated beads, and IFN γ and granzyme B secretion were assessed.

The TIL selected by PD1 secreted considerable levels of IFN γ and granzyme B in response to non-specific stimuli (α CD3/α CD28/α CD137 beads), indicating that these did function after amplification. See fig. 143.

Although it was able to produce IFN γ after amplification, the TILs selected with PD1 secreted significantly less IFN γ than the unselected TILs. Thus, in response to non-specific stimulation, TILs selected via PD1 have a reduced ability to produce IFN γ. However, TILs selected with PD1 have been shown to produce significantly higher levels of IFN γ when co-cultured with autologous tumors compared to PD1-TIL [3, 8, 13 ]. Expanded PD 1-selected and unselected TILs were co-cultured with autologous tumors and IFN γ was evaluated. Compared to unselected TILs, the TILs selected with PD1 secreted higher levels of IFN γ, indicating not only its ability to secrete IFN γ, but also secretion in a tumor-specific manner.

The PD 1-selected TIL produced similar levels when compared to the unselected TIL, but the granzyme B levels were slightly elevated, consistent with previous studies in HNSCC [11 ]. Since granzyme B is considered an activation marker, these results further indicate that the amplified PD 1-selected TIL is not in a depleted or non-reactive state after amplification.

Stimulation of CD107a mobilization in PD 1-selected and unselected TILs by PMA/ionomycin

CD107a cell surface expression is considered a reliable marker of TIL effector function. CD107a (LAMP1) was mobilized to the cell surface upon stimulation and was used as a measure of cell degranulation capacity. Degranulation is a prerequisite for perforin granzyme mediated killing and is required for immediate lytic function mediated by responding to antigen-specific CD8+ T cells [14, 15 ].

To further evaluate the functional capacity of the PD 1-selected TILs, 10 post-amplification TILs were mobilizated against CD107a and compared to matching unselected TILs.

CD107 expression was similar in the TILs selected with PD1 when compared to unselected TILs. See fig. 144. These results further support the following concept: expansion of the PD 1-selected TIL was highly functional and did not indicate a depleted cell population.

Conclusion

In response to non-specific stimulation, TILs selected via PD1 produced IFN γ and granzyme B and mobilized CD107 a.

In contrast to what has been demonstrated for uncultured PD1+/PD1 high cells, expanded PD 1-selected TIL is highly active and functional and therefore capable of producing anti-tumor effects once in vivo.

Reference to example 14

Formation and phenotypic characterization of a population of CD 8T cells expressing CD49a, CD49b and CD103 in Melssen, m.m. et al, human metastatic melanoma.

Lee, J, et al, restoration of depleted T cells by blocking the PD-1 pathway, for immunopathological disease therapy, 2015.6(1-2): pages 7-17.

Badoual, C.et al, tumor-infiltrating T cells expressing PD-1 are a favorable prognostic biomarker in HPV-associated head and neck cancer [ cancer research, 2013.73(1): pages 128-38 ].

Webb, J.R., K.Milne and B.H.Nelson, PD-1 and CD103 are widely co-expressed on intraepithelial CD 8T cells with favorable prognosis in human ovarian Cancer in Cancer immunology Res, 2015.3(8) pages 926-35.

The PD-1 status in CD8(+) T cells was correlated with survival in head and neck cancer and the outcome of anti-PD-1 therapy in Kansy, B.A. et al, cancer research, 2017.77(22): page 6353-6364.

Adoptive cell therapy with PD-1(+) myeloma-reactive T cells eliminates established myelomas in mice (Adoptive cell therapy using PD-1(+) myelomas-reactive T cells) (J.Immunotherapy cancer, 2017.5, page 51.

Amplification of PD-1 expressing tumor-infiltrating CD8(+) T cells improves the efficacy of adoptive T cell therapy in Fernandez-Poma, S.M. et al, cancer research, 2017.77(13): page 3672-3684.

Betts, m.r. and r.a.koup, detection of T cell degranulation: CD107a and b (Detection of T-Cell differentiation: CD107a and b.) Methods in Cell biology (Methods Cell Biol.), 2004.75: page 497-512.

Rubio, v. et al, Ex vivo identification, isolation and analysis of tumor cell-lytic T cells (Ex vivo identification, isolation and analysis of tumor-cytolytic T cells), naturel medicine, 2003.9(11), pages 1377-82.

Example 15: autologous tumor reactivity in PD 1-selected TIL

Purpose(s) to

This example evaluated autologous tumor reactivity/killing of expanded programmed cell death protein 1(PD1) sorted Tumor Infiltrating Lymphocytes (TILs) compared to matched unselected TILs.

Range

Thirteen matched TILs selected by PD1 and unselected TILs from melanoma, non-small cell lung cancer (NSCLC), and Head and Neck Squamous Cell Carcinoma (HNSCC) were evaluated for responsiveness and killing ability in response to autologous tumor stimulation. Reactivity and cytotoxicity were measured as IFN γ secretion and tumor cell death (% cytotoxicity), respectively.

Information

Adoptive T cell therapy (ACT) with autologous Tumor Infiltrating Lymphocytes (TIL) is considered as an effective treatment for metastatic melanoma and other solid tumors, eliciting a long-lasting and complete response even in heavily pretreated patients [1-6 ]. During tumorigenesis, malignant tumors acquire nonsynonymous mutations, denoted neoantigens [7, 8 ]. Recent studies have highlighted the importance of tumor neoantigens in tumor recognition, as well as potent anti-tumor T cell responses in vivo [8, 9 ]. The presence of tumor-specific T cells correlates with tumor regression and clinical efficacy on TIL therapy [10 ]. In particular, selected ACT of neoantigen-reactive T cells has mediated substantial objective clinical regression in patients with colon [8] and breast [11] cancers.

Until recently, knowledge of all pools and frequencies of tumor-specific TILs in tumors was limited [12, 13 ]. The key goal of ACT is to obtain a polyclonal TIL product enriched for tumor-reactive T cell clones. Recent studies have demonstrated that PD1 expression in TIL can be used as a marker and selection tool for the identification of neoantigen-specific lymphocytes. PD1 is expressed when encountered by an antigen and upregulated in T cells responsive to tumor antigens and undergoes clonal expansion at the tumor site [13-20 ].

Based on the concept that PD1+ TIL is a neoantigen-specific lymphocyte, the ability of PD1+ TIL to recognize autologous tumor lines ex vivo has been evaluated. To determine tumor reactivity, TILs were co-cultured with autologous tumor cell lines and IFN γ was determined. IFN γ is an essential effector cytokine in the Tumor Microenvironment (TME) and is considered as a surrogate marker for identifying antigen-specific T cells. Interestingly, PD1+ TIL secreted higher levels of IFN γ when co-cultured with autodigesta [14, 17] compared to its PD1 counterpart in both NSCLC and melanoma.

Tumor cell lysis/killing has also been used to identify antigen-specific T cells, however, these assays are not performed frequently due to the problems of deriving and maintaining autologous viable tumor cell lines. One melanoma study evaluated the killing of sorted PD1+ TIL and demonstrated a higher capacity of PD1+ TIL to lyse autologous tumor lines compared to PD1-TIL [13 ].

Based on the evidence discussed above, the TILs selected for expression of PD1 expression are expected to enrich for tumor/neoantigen specific T cells, which demonstrate greater autoreactivity in vitro. To capture tumor specific cells, PD1+ TIL was sorted from freshly digested tumors (selected by PD 1) using Fluorescence Activated Cell Sorting (FACS) prior to expansion. Both PD 1-selected TIL and unselected TIL were amplified using two consecutive 11-day REPs.

Tumor reactivity and cell lysis were evaluated to determine whether selection for PD1+ would enrich for antigen-specific T cells. TILs selected by PD1 were co-cultured with autologous tumor cells and evaluated for IFN γ production and tumor cell death. The results were compared to matching unselected TIL formulations.

Design of experiments

Co-cultures of TILs and autologous tumors were used to evaluate tumor cell killing and reactivity in 13 paired PD 1-selected and unselected TILs. Tumor lysis and IFN γ secretion were used to measure antigen specificity in the TIL product.

Material

Tumor samples and TIL products used in this work are depicted in fig. 145.

Both PD 1-selected and unselected TIL products were obtained from 4 melanomas, 7 NSCLC and 2 HNSCC according to the procedure TMP-18-015. Briefly, whole tumor biopsies were digested with a mixture of dnase, hyaluronidase, and collagenase IV. A portion of the resulting single cell suspension was stained for PD1 and sorted on FX500 instrument (sony, headquarters, new york). The remaining digesta was frozen and thawed before use in the assays indicated below. PD1 sorted cells and unselected whole tumor digests were subjected to two 11-day Rapid Expansion Phases (REP) to obtain PD1 selected TIL and unselected TIL, respectively.

Method

Tumor treatment and electroplating

Autologous whole tumor digests were treated using a dead cell removal kit (meitian whirlpool, germany). 1e5 live cells were plated in each well of a 96-well plate and allowed to adhere in an xCEELLigene instrument (ACEA biosciences, Inc., Calif.) for 18 hours at 37 ℃.

Co-cultivation set-up

1e5 PD 1-selected TIL and unselected TIL-derived autologous TIL were added to their respective wells to generate a 1:1(TIL: target) cell ratio and incubated for 48 hours.

Tumor cell lysis quantification

Killing of autologous target cells was recorded as an increase in impedance caused by cell detachment. Cell killing (% cytolysis) was calculated using the formula,% cytolysis [1- (NCIst)/(avgcntirt) ] × 100, where NCIst is the normalized cell index of the sample and NCIRt is the average of the normalized cell indices of the matched reference wells (digested alone). The% cell lysis was calculated using the RTCA software Pro (ACEA biosciences, ltd., ca).

Secretion of IFN gamma

Supernatants were collected 24 hours after TIL addition and IFN γ release was assessed by ELISA (R & D systems). ELISA plates were read on an american pentium microplate reader (american pentium, budd) and evaluated using Gen5 data analysis software. Data were plotted using GraphPad Prism v 8.

Results

This example examined whether the PD 1-selected TIL has greater killing and IFN γ -secreting ability than the unselected TIL when co-cultured with autologous tumor digests.

The results obtained

Tumor reactivity and killing in PD 1-selected TILs

Preclinical data in both mice and humans have demonstrated that expression of PD1 on T cells within tumors can identify pools of neoantigen-specific lymphocytes [13, 14, 17-20 ]. Several studies have demonstrated that purified PD1+ TIL amplified in vitro secretes significantly greater amounts of IFN γ than PD1-TIL when co-cultured with autologous tumors [14, 17 ]. Based on these studies, TILs selected for expression of PD1 expression are expected to enrich for tumor/neoantigen specific T cells, which will exhibit greater autoreactivity when evaluated in vitro.

Thirteen matched TILs selected with PD1 and unselected were evaluated for autologous tumor reactivity and killing. Tumor cell lysis was measured using the tumor cell index. The cell index is a measure of cell attachment calculated from the cell surface impedance of the well. As tumor cells adhere, the impedance increases, as does the cellular index. When tumor cells die and detach, the cellular index decreases due to the decrease in impedance. Thus, if TIL lyses tumor cells, the cell index will decrease and the calculated percentage of cell lysis will increase. However, if at any time during co-cultivation the cell index drops below zero, the cell lysis of the sample cannot be calculated.

Of the 13 tumors evaluated, tumor cell lysis was evaluated for only one melanoma tumor, due to poor tumor cell viability and insufficient adhesion of tumor cells to the plate. The cell index of the melanoma and% cell lysis of the tumor cells that can be assessed are shown in figures 146A and 146B below, respectively.

Supernatants from the above co-culture cell lysis assay were assayed for IFN γ. Among the 13 tumors evaluated, IFN γ secretion was detected in 3 melanomas and 2 NSCLCs (fig. 146C).

Due to technical difficulties, tumor cell lysis% was only assessed in 1/13 co-cultured tumors. In evaluable tumors with appropriate unselected TIL controls, the TIL selected with PD1 exhibited greater ability to kill autologous tumors as determined by a greater decrease in cellular index (fig. 1A) indicating more cell detachment and tumor cell death and a higher percentage of cytolysis (fig. 1B) compared to unselected TIL. These results are supported by melanoma studies that also demonstrate enhanced cytolysis in subpopulations selected by PD1+ using alternative assays of autologous tumor cell lines rather than whole tumor digests [13 ]. Despite the low number of tumors that can be assessed, results, as well as other results, have demonstrated that the TIL selected with PD1 has a greater ability to kill autologous tumors than its unselected or PD1 counterpart.

Among the 13 co-cultured tumors tested, IFN γ secretion could be detected in 5 tumors. Of the 5/5 tumors tested, the PD 1-selected TIL secreted higher levels of IFN γ than the unselected TIL when co-cultured with autologous tumor digests. Secretion of IFN γ was tumor specific, as blockade with anti-HLA-A, -B and-C reduced the amount of secreted IFN γ (FIG. 1C). The higher level of IFN γ production in the presence of autologous tumors indicates that the PD 1-selected TIL has a higher proportion of antigen-specific TIL than the unselected TIL.

Conclusion

TILs selected with PD1 showed enhanced killing of autologous tumor cells relative to unselected TILs.

In response to autologous tumors, IFN γ secretion in PD 1-selected TILs was significantly greater than unselected TILs.

These results demonstrate that the TIL selected with PD1 has excellent reactivity against autologous tumors in vitro compared to the unselected TIL.

Clinical efficacy in ACT is directly related to the presence of tumor-specific TIL. Therefore, enrichment of tumor-specific TIL by PD1 selection and amplification can enhance the ability of TIL to initiate effective and highly potent anti-tumor effects in vivo.

Reference to example 15

Stevanovic, S. et al, Complete regression of metastatic cervical cancer after treatment with human papillomavirus-targeted tumor-infiltrating T-cells (Complete regression of metastatic cervical cancer with human papulomyavirus-targeted tumor-inducing T-cells.) J.Clin Oncology 2015.33(14): pages 1543-50.

Stevanovic, S. et al, phase II study of tumor-infiltrating lymphocyte therapy for human papillomavirus-associated epithelial cancer (A phase II study of tumor-infiltrating lymphocyte therapy for human papillomavir-associated epithelial cancers) [ clinical cancer study ], 2018.

Tumor-infiltrating lymphocyte therapy (Tumor-infiltrating lymphocyte therapy for ovarian cancer and renal cell carcinoma) in Andersen, R. et al, human vaccine immunotherapy (Hum vaccine immunotherapy), 2015.11(12), pages 2790-5.

Andersen, R. et al, T Cell response in the Microenvironment of Primary Renal Cell Carcinoma-the significance of Adoptive Cell Therapy (T-Cell Responses in the Microenvironmental of Primary Renal Cell Carcinoma-indications for Adoptive Cell Therapy) -cancer immunology research 2018.6(2): page 222-235.

Westergaard, m.c.w. et al, tumor-reactive T cell subsets in the microenvironment of ovarian Cancer @ (tumor-reactive T cell subsets of the Cancer), journal of british Cancer (Br J Cancer), 2019.

Yossef, r. et al, enhanced detection of neoantigen-reactive T cells targeting distinct and shared oncogenes for personalized cancer immunotherapy, J.Clin. investigational insights, 2018.3 (19).

Tran, E.et al, Cancer immunotherapy based on mutation-specific CD4+ T cells of patients with epithelial Cancer (Cancer immunological based on mutation-specific CD4+ T cells in a patient with epithelial Cancer) science 2014.344(6184) page 641-5.

McGranahan, N.et al, cloning of neo-antigens to elicit T cell immune reactivity and sensitivity to immune checkpoint blockade (Clnal neoantigens Elicit cell immunoreactivity and sensitivity to immune checkpoint blockade) science 2016.351(6280) pages 1463-9.

Zacharakis, N.et al, Immune recognition of somatic mutations that lead to complete durable regression of metastatic breast cancer (Immune recognition of pathological lesions in metastatic breast cancer) Nature medicine 2018.24(6) page 724-730.

Prospective identification of neoantigen-specific lymphocytes in peripheral blood of melanoma patients (Selective identification of neoantigen-specific lymphocytes in the peripherical blood of melanomas), Nature medicine, 2016.22(4), pp 433-8.

Simon, S. et al, PD-1 expresses T cell avidity within a conditional antigen-specific repertoire (tumor immunology 2016.5(1): p.e1104448).

Thommen, D.S. et al, a transcriptionally and functionally diverse pool of PD-1(+) CD8(+) T cells with predictive potential in non-small cell lung cancer treated with PD-1 blockade, Nature medicine, 2018.

Amplification of PD-1 expressing tumor infiltrating CD8(+) T cells improves the efficacy of adoptive T cell therapy in Fernandez-Poma, S.M. et al, cancer research, 2017.77(13): 3672, 3684. f.

Adoptive cell therapy with PD-1(+) myeloma-reactive T cells abolished established myeloma in mice, J.Immunotherapy cancer 2017.5, p 51.

Donia, M. et al, PD-1(+) multifunctional T Cells in Cancer Tumor Infiltrating Lymphocyte Therapy with peripheral (PD-1(+) multifunctional T Cells Dominate the peripheral after the Cancer Therapy with infusion Therapy Heat Cancer research for Cancer 2017. 23(19), page 5779 and 5788.

Example 16: phenotypic characterization of TILs selected by PD1

Purpose(s) to

TILs selected for phenotypic characterization of programmed cell death protein 1(PD 1).

Range

This example relates to the expression of cell surface markers characterizing the PD 1-selected TIL that are characteristic of various T cell states and whose phenotype is compared to that of a matching unselected TIL.

Information

Cancer immunotherapy utilizes the immune system to recognize and destroy tumor cells. The success achieved by immune checkpoint inhibitors (CPI) targeting cytotoxic T lymphocyte antigen 4 and PD-1 has changed cancer therapy and established immunotherapy, along with surgery, chemotherapy and radiation therapy, as one of the standard treatment approaches. CPI therapy results in a significantly long-lasting clinical response, but only in a subset of patients with certain types of cancer, and often at the cost of severe side effects [1, 2 ].

Adoptive Cell Therapy (ACT) with autologous Tumor Infiltrating Lymphocytes (TIL) has emerged as a powerful and potentially curative therapy for several cancers [3 ]. The TIL product used in ACT is an unselected, non-genetically engineered preparation of polyclonal T cells that recover directly from tumor tissue and expand extensively ex vivo [4 ]. This process ensures the restoration of a potentially diverse pool of patient tumor-specific memory T cells without prior knowledge of the nature or identity of the antigen [5 ]. In summary, ACT is a simpler, less biased, safer and potentially more effective approach than other cell therapies, such as Chimeric Antigen Receptors (CARs) and TCR T cells that target single tissue-specific or tumor-specific antigens and require the insertion of transgenes. However, the current TIL process may also allow recovery and expansion of variable fractions of T cells that are not associated with cancer (so-called bystander TIL) and recognize antigens, such as antigens from Epstein Barr Virus (EBV), human Cytomegalovirus (CMV) or influenza viruses [6 ].

Multisystem evidence supports neoantigen recognition followed by tumor cell killing as the primary mechanism of action for TIL therapy [7 ]. TILs enriched for tumor neoantigen-specific T cells while remaining unbiased to maintain a degree of diversity and avoid the need for antigen identification represents an attractive means for optimizing products.

As an activation-induced T cell regulator, PD-1 has been shown to be specifically expressed in response to recent antigen encounter and, in the case of T cells infiltrating cancer tissues, to specifically label neo-antigen specific cells [8, 9 ]. Thus a method is implemented by which TILs are selected for PD-1 expression prior to ex vivo amplification to enrich for relevant TILs relative to bystander TILs. The protocol involves sorting PD-1+ TIL directly from freshly digested tumours using Fluorescence Activated Cell Sorting (FACS) and subjecting it to a two-step process comprising an 11 day activation step followed by an 11 day Rapid Expansion Protocol (REP) to obtain a therapeutically appropriate amount of PD-1 selected TIL.

In the current study, PD-1-selected TILs were phenotypically characterized to confirm that 1) the new product met LN-145 release specifications and 2) was comparable to the unselected IL product. Expression of cell surface markers of lineage, differentiation, memory, activation, depletion and resident memory of PD-1-selected TILs was assessed by flow cytometry.

Design of experiments

PD-1 selected and unselected TILs were amplified in a two-step process over a 22-day course, comprising an 11-day activation step followed by 11-day REP. The final TIL product was phenotypically characterized using flow cytometry. Notably, the unselected TIL product was obtained from the same whole tumor digest as the PD-1 selected TIL. The limited tumor tissue prevented the derivation of the unselected TIL control from the tumor fragments used to derive the LN-145 TIL product from Iovance. In addition, to amplify a small number of sorted PD-1 populations, unselected TILs are subjected to a 2-REP process, as opposed to a pre-REP and a single REP used to generate LN-145. Thus, while the unselected TIL represents a true control of PD-1 sorted TIL, it does not reflect LN-145 TIL.

Material

Tumor samples and TIL products used in this work are depicted in fig. 147.

Method

PD1 selection and amplification

PD-1-selected and unselected TIL products were obtained from 4 melanomas, 7 NSCLCs and 2 HNSCCs according to the procedure TMP-18-015. Briefly, whole tumor biopsies were digested with a mixture of dnase, hyaluronidase, and collagenase IV. A portion of the resulting single cell suspension was stained for PD-1 and sorted on FX500 instrument (sony, headquarters, new york). PD-1 selected and unselected TILs were subjected to an 11 day activation step in the presence of OKT3(30ng/ml, America whirlpool Biotech) and allogeneic irradiated peripheral blood mononuclear cells (TIL: feeder ratio of 1: 100), followed by 11 days REP.

Antibody staining

TILs were stained with live/dead markers and used for expression of CD3 and phenotypic markers defining T cell lineage, memory, differentiation, activation and depletion (figure 147). Two flow cytometry panels designated 1 and 2 were used to overlay the markers of interest. The antibodies and conjugated fluorophores are listed in table 59 below, where they are arranged by phenotypic parameters. The numbers in brackets indicate their respective panels.

Table 59: TIL-characterized phenotype Panel

FACS analysis

Stained cells were run on a ZE5 cell analyzer (burle, ca) following standard laboratory procedures. Briefly, by comparing the results in live (live/dead dye negative or low) and CD3⁺Identification of evaluable events by gating on single cells (using forward scatter and side scatter parameters). Single phenotypic markers were gated based on FMO (fluorescence minus one) and control normal donor peripheral blood mononuclear T cells.

Data analysis was performed using FlowJo v8.1 software (FlowJo, inc., oregon). Results were plotted using GraphPad v8.

Expected result

For most phenotypic markers, PD-1-selected TILs were expected to be comparable to unselected TILs and met LN-145 phenotypic release criteria. Based on published reports, PD-1 expression of PD-1-selected TILs is expected to decrease with in vitro amplification steps [10-12 ]. It was not known whether PD-1 levels of PD-1-selected TIL remained higher than levels of unselected TIL.

Results

CD4 and CD8 expression in PD 1-selected TILs

PD-1 is expressed in CD3+ T cells, but most have been characterized in CD8+ T cells, despite its expression in both the CD4+ and CD8+ lineages [10, 13 ]. To determine whether sorting of PD-1+ altered the ratio of CD4+ and CD8+ T cell lineages in amplified PD-1-selected TIL relative to unselected TIL, 13 paired samples were compared for expression of 2 markers. The results are shown in graph 148.

The average percentage of CD4+ and CD8+ cells in the amplified TIL was similar in PD-1 selected and unselected products. The percentage of CD4+ T cells was higher in both PD-1 selected and unselected TIL products than CD8+ T cells.

These results indicate that the ratio of CD4+ and CD8+ TIL does not differ significantly in the PD-1 selected T cell population relative to the unselected TIL product. These results indicate that the choice of PD-1 does not alter the T cell lineage of the final amplification product.

Markers of youth/differentiation in PD 1-selected TILs

Response to ACT requires a balance of effector function and persistence that is typical in differentiated T cells, which is associated with T cell adolescence and central memory phenotype [14, 15 ]. Classically, high CD27 and CD28 expression was associated with T cell adolescence, while CD56, CD57 and KLRG1 expression identified terminally differentiated cells. Thirteen paired PD-1-selected and unselected TIL products were stained for these markers and analyzed by flow cytometry. The results are shown in fig. 149.

PD-1-selected and unselected TILs expressed similar differentiation phenotypes as indicated by low levels of CD27, CD56, and KLRG1, and moderate levels of CD28 and CD 57. However, PD-1 selected TILs had significantly higher levels of CD27 and reduced levels of KLRG1 compared to unselected TILs, which may translate into a less differentiated phenotype in PD-1 selected TILs. These results are consistent with the report of selected PD-1 high TILs from NSCLC, where TIL is CD27+ and KLRG1-, compared to its PD-1 counterpart [2 ]. CD27+ TIL is also associated with anti-tumor activity in vivo, and KLRG1+ T is associated with decreased persistence in vivo of T cells [16 ]. These results indicate that TILs selected by PD-1 may be able to support the sustained antitumor activity required for a sustained response in vivo [7 ].

Memory T cell population in TIL selected by PD1

T cell memory subsets can be identified based on the differential expression of the 2 cell surface markers CD45RA and CCR 7. Effector memory T cells (TEM) are defined as CD45 RA-and CCR7-, central memory T Cells (TCM) as CD45 RA-and CCR7+, stem cell memory T cells (TSCM) as CD45RA + and CCR7+, and CD45RA + effector memory T cells or terminally differentiated T cells (TEMRA) as CD45RA + and CCR7- [17 ].

Published studies have shown that PD-1+ TIL comprises mainly effector memory T cells (TEM) [11, 18 ]. Furthermore, these TEMs have been shown to represent a major population of unselected TIL products demonstrating clinical activity [19 ]. To determine the proportion of each memory T cell subset in the TILs selected by PD-1, 13 products were evaluated for CD45RA and CCR7 expression by flow cytometry. The results are shown in graph 150.

As with the current TIL products of Iovance, lifileucel and LN-145, both PD-1-selected TILs and unselected TILs comprise predominantly TEM [20], as other TIL products demonstrated to have clinical efficacy. Selection of PD-1 did not appear to alter the memory pool of amplified TIL.

Activation State of TIL selected by PD1

Upon T cell activation, several cell surface markers are upregulated, each at a different stage of the activation process. One of the earliest markers of activation was CD69, an inducible cell surface glycoprotein expressed after activation by TCR [21 ]. CD25, the alpha subunit of the IL-2 receptor, is up-regulated slightly later than CD69 and plays a crucial role in regulating T cell proliferation [21 ]. In addition, co-stimulatory receptors such as CD134 and CD137 are also considered markers of T cell activation and are commonly used to identify antigen-specific T cells in infiltrating tumors [21, 22 ].

Based on the expression profile of these markers, TIL has been shown to exhibit an activated phenotype after REP, consistent with the ability of TIL products to initiate an effective anti-tumor T cell response after infusion [3 ].

Extensive studies have evaluated the activation state of PD-1+ and PD-1-TIL in both mice and humans. Data in mice show that PD-1+ TIL expresses a higher percentage of CD134 and CD137 than PD-1- [11, 23 ]. Similar results were obtained in human studies in which CD137 was found to be higher in PD-1+/PD-1 high TIL in patients with melanoma and NSCLC [8, 12 ].

Additional studies have evaluated the expression of CD69 and CD25 in PD-1+ TIL. A significant fraction of PD-1+ TIL has been shown to co-express CD69[24], whereas most PD-1+ lack expression of CD25[13 ].

To confirm that PD-1-selected TILs expressed an activated phenotype after amplification, the expression of CD25, CD69, CD134, and CD137 of the 13 TIL products was analyzed by flow cytometry and compared to unselected TILs. The results are shown in fig. 151.

On average 3.34% to 22.28% of 4 activation markers were detected in PD-1 selected TILs, indicating that a fraction of TILs expressed at least one marker indicative of activation in all products tested. The percentages of CD25+, CD69+, and CD134+ were comparable to those in unselected TILs, indicating that the PD-1 selection step did not alter the activation state of the in vitro expanded cells. However, unselected TILs exhibited significantly lower levels of CD137+ T cells than PD-1 selected TILs, which may reflect a slightly higher activation state of PD-1 selected TILs. Taken together, these results show that REP uniformly activates PD-1-selected and unselected TILs and indicates that PD-1+ TILs express an activated phenotype after in vitro culture [10 ].

Depletion markers in PD-1 selected TILs

Extensive studies have evaluated the co-expression of PD-1 with other co-suppression/depletion markers. Subsets of PD-1+ TIL consistently co-expressed TIM3, LAG3, TIGIT, BTLA and CTLA4[8, 11, 12, 18, 23 ]. However, these markers were evaluated in freshly isolated PD-1+ TIL and less information was available about their status in amplified PD-1+ TIL.

Interestingly, PD-1 expression in amplified PD-1+ TIL showed a decrease with culture and amplification and was interpreted as evidence of ex vivo restoration of TIL [10, 12 ].

To better understand the depletion/inhibition status of PD-1 selected TILs, the expression of four depletion/inhibition markers LAG3, PD-1, TIM3 and CD101 for 13 matched unselected and PD-1 selected TIL products were analyzed by flow cytometry. CD101 has been associated with advanced TIL dysfunction and is added to a standard list of depletion markers [25 ]. The results are shown in graph 152.

The PD-1 selected TIL expressed all 4 of the depletion/inhibition markers determined. The content of LAG3 was found to be 1.75 to 37.8%, the content of PD-1 was found to be 9.06 to 53.8%, the content of TIM3 was found to be 8.65-54.9%, and the content of CD101 was found to be 9.16-91.1%. Unselected TILs expressed similar LAG3, TIM3, and CD101 levels relative to the selected products, again indicating that sorting of PD-1+ TILs did not significantly skew the phenotype of the final products upon in vitro amplification. Only PD-1 levels differed significantly between 2 products, with the PD-1-selected product expressing a higher percentage of PD-1+ cells than the unselected cells. However, in the TIL selected by PD-1, the number of PD-1+ cells decreased significantly from 92.8% after sorting to an average of 27.1% after REP. This is consistent with data reported by others for melanoma and NSCLC TIL and indicates in vitro reconstitution [10, 12 ].

To compare the extension of PD-1 down-regulation induced by in vitro amplification between PD-1-selected and unselected TILs, the pre-amplification and post-amplification percentages of PD-1+ TILs were evaluated for both products. The results are shown in fig. 153.

The expression of PD-1 was significantly reduced in both PD-1-selected TIL (average 27.1%, range 9.06 to 43.6) and unselected TIL products (average 10.6%, range 4.93 to 29.3) relative to the initial average PD-1 level of 92.8% and 37.3%, respectively. Thus, the process of amplifying TIL also affected two TIL preparations in which the expression of PD-1 was reduced by > 3-fold.

Resident memory T cell markers in PD 1-selected TILs

Integrins mediate the retention of lymphocytes in peripheral tissues. Some of these integrins are expressed on subsets of T cells (called resident memory T cells). These cells, which are phenotypically very similar to effector memory T cells, do not circulate and reside within the tissue.

Several integrins such as α E β 7(CD103), α 1 β 1(CD49a) are expressed on variable fractions of freshly isolated TIL [7, 8 ]. Together with CD39, a T cell surface molecule involved in the adenosine pathway and associated with inhibitory signals, CD49 and CD103 were identified as tumor-reactive on PD-1+ TIL [2, 8, 10 ]. Furthermore, PD-1 and CD103 co-expression have been associated with favorable clinical outcomes in ovarian cancer [9 ].

To determine whether the PD 1-selected and unselected TIL products expressed markers associated with resident memory T cells, expression of CD39, CD49a, and CD103 expression was analyzed for 13 tumors. See fig. 153.

No difference was observed in the percentage of CD49a + and CD103+ cells in PD-1 selected TILs relative to unselected TILs, whereas CD39 expression was significantly higher in PD-1 selected TILs than unselected TILs. The difference may be associated with higher levels of CD39 in the unamplified PD-1+ TIL, as indicated by the association of this marker with neoantigen specificity [6 ]. Overall, these 3 markers were differentially expressed in the TIL product.

Conclusion

The observed differences in phenotypic expression of the determined cell surface markers are shown in table 60 below. In addition to KLRG1, the phenotypic markers listed in the PD 1-selected TIL were significantly upregulated compared to the unselected TIL.

Table 60: phenotypic markers differentially expressed in PD1+ -selected and unselected TILs

PD-1 selected TILs appear to differentiate to a lesser extent than unselected TILs, as evidenced by higher expression of CD27 and lower levels of KLRG 1. The efficacy and healing potential of TIL depends on its ability to kill all malignant cells in a tumor and persist long enough to eradicate all malignant cells in a tumor [14, 24 ]. Thus, a moderately differentiated phenotype may be a positive feature of TIL selected by PD-1.

The PD-1 selected TIL expressed a higher percentage of CD137 and CD39 when compared to the unselected TIL. These findings indicate that the PD-1-selected TIL is in an activated state, which may have the potential to enhance its effector function once transferred in vivo.

Overall, the results indicate that the amplified PD-1-selected TIL is composed mainly of non-differentiated TEMs with low depletion marker expression, indicating that these cells are restored after in vitro amplification.

The phenotypic characteristics of PD-1 selected TIL may be comparable to Iovance's unselected TIL products, lifileucel and LN-145, which have shown clinical efficacy in metastatic melanoma and cervical cancer, respectively.

Reference to example 16

Crompton, j.g., m.sukumar and n.p.restifo, Uncoupling T-cell expansion from effector differentiation in cell-based immunotherapy, immunological review (immunological Rev), 2014.257(1), page 264-.

Westergaard, m.c.w. et al, subpopulation of tumor-reactive T cells in the microenvironment of ovarian cancer, uk journal of cancer, 2019.

Bally, A.P., J.W.Austin and J.M.Boss, Gene and Epigenetic Regulation of PD-1 Expression (Genetic and Epigenetic Regulation of PD-1 Expression) J.Immunol 2016.196(6): pages 2431-7.

Goluovskaya, V. and L.Wu, Different subgroups of T Cells, Memory, Effector Functions and CAR-T Immunotherapy (differential Subsets of T Cells, Memory, Effector Functions, and CAR-T Immunotherapy) & Cancers (Basel), 2016.8 (3).

Radvanyi, L.G. et al, predicting response to adoptive cell therapy in metastatic melanoma patients using Specific lymphocyte subsets of expanded autologous tumor-infiltrating lymphocytes (Specific lymphocyte subsets predicting response to adoptive cell therapy using ex-panded autologous cancer tumor-infiltrating lymphocytes in metabolic tumor patients.) clinical cancer research 2012.18(24) pages 6758-70.

Wolfl, M. et al, Activation-induced expression of CD137 allows the detection, isolation and expansion of a full pool of CD8+ T cells in response to antigen without the need for knowledge of epitope specificity (Activation-induced expression of CD137 receptors detection, isolation, and expansion of the full response of CD8+ T cells suppression to antigen with out differentiation of epitopes). blood, 2007.110(1): pages 201-10.

Co-expression of CD39 and CD103 identified tumor-reactive CD 8T cells in human solid tumors (Co-expression of CD39 and CD103 analytes tumor-reactive CD 8T cells in human solid tumors), Nature letters, 2018.9(1): page 2724.

Rosenberg, S.A. et al, complete response sustained using T cell metastatic immunotherapy in severely pretreated patients with metastatic melanoma clinical cancer research 2011.17(13): pages 4550-7.

Example 17: selection of PD1 TIL for clinical manufacturing by flow cytometry sorting and full-scale amplification Using Nwaruzumab

Introduction to

This example is directed to the development of a protocol designed to select PD1 TIL from tumor digests to enrich TIL products from autologous tumor-reactive T cells. This example provides a protocol for obtaining PD 1-selected TIL using nivolumab as a PD1 staining antibody instead of PE-conjugated clone No. EH12.2H7.

Purpose(s) to

The objective of this protocol was to develop a method for sorting PD1 TIL using nivolumab as a selection agent and expanding the material for manufacturing clinical trials.

Range

The working range was to amplify sorted PD1 TIL from melanoma or lung or head and neck or ovarian tumors using a 2-REP protocol designed for full-scale clinical manufacturing (figure 154).

Two small scale experiments and one full scale experiment were performed.

On day 0, tumor digests were evenly distributed to purify PD1 TIL using a new staining method (using nivolumab) and a staining method (using anti-PD 1(EH12.2H7)), and PD1 TIL was flow sorted.

For the small scale process (1/100 scale), REP-1 was initiated on day 0 by calculating 10% of the PD1 TIL with the lowest sorting outcome and transferring that amount of TIL from each sort to the corresponding G-Rex-10M flask with feeder cells and OKT-3 with IL-2 medium. REP-2 starts as in example 9. A brief explanation of the relevant points in time is outlined in the method section below (fig. 155).

For the full scale process, REP-1 was initiated on day 0 using sorted PD1 TIL with 100e6 allogeneic feeder cells and 30ng/mL OKT3 for 11 days. REP-2 will be initiated on day 11 using the collected REP-1 product. The REP-2 (day 11) and subsequent day 16 and day 22 procedures were performed according to the IOVA manufacturing lot records. A brief explanation of the relevant points in time is outlined in the method section below (fig. 154).

Day 22 collection was initiated by volume reduction followed by cell counting on NC-200 for all conditions.

The expanded TIL and final product were evaluated for cell growth, viability, phenotype, telomere length and function (IFN γ and granzyme B secretion, CD107a mobilization).

4. Method of producing a composite material

Overview of digested PD1 Gen-2 Small-Scale and full-Scale Processes

Material

Tumor tissue

Various histological tumors were obtained from research unions and tissue procurement suppliers. A standard reagent for TIL growth comprising: G-Rex 100MCS and 500MCS flasks (Wilson Walf, Cat. Nos. 81100-CS, 85500S-CS, respectively); GMP recombinant IL-2(Cell-Genix, Germany, catalog No. 1020-1000); GlutaMAX 100X (Saimer Feishale, catalog number 35050061); and gentamicin 50mg/mL (seimer feishel, catalog No. 15750060).

Flow cytometry staining and analytical reagents

Flow cytometry antibodies

anti-PD 1 PE, clone EH12.2H7, Biolegend, Cat. No. 329906

anti-CD 3 FITC, clone OKT3, Biolegend, Cat. No. 317306

anti-IgG 4 Fc-PE, clone HP6025, southern Biotechnology Inc., Cat. No. 9200-09

Nivolumab [ trade name: opdivo 10mg/mL (Beziese noble Co., New York)

PE anti-human IgG4, clone HP6023, 0.5mg/mL (Biolegend, san Diego, Cat. No. 98155)

Sorting buffer

HBSS with 2% FBS.

Collecting the buffer

HBSS containing 50% hAB serum

Procedure

Preparation of tumor tissue

Freshly resected tumor samples were obtained from research consortia and tissue procurement suppliers. Tumors were transported overnight at 2-8 ℃ in HypoThermosol (BioLife solutions Co., Washington, Cat. 101104) (with gentamicin (10mg/mL) and amphotericin B (250. mu.g/mL)).

Images of the vial/tube tumors were taken. Tumors were removed from the package and washed 3X each time in tumor wash buffer (filtered HBSS with 50 μ g/mL gentamicin) for 2 minutes.

The entire tumor was fragmented into 4-6-mm3 pieces in preparation for tumor digestion. Mixing 4-6-mm³The fragments were held in wells of a 6-well plate containing 10mL of tumor wash buffer per well.

Enzyme preparation for tumor digestion

The tumors were digested with GMP collagenase, neutral protease and dnase I as described herein.

The lyophilized enzymes were reconstituted in the amounts of sterile HBSS indicated for each of the following digestive enzymes. Ensuring that any residual powder is captured from the sides of the bottle and from the protective foil over the bottle opening. Pipette up and down several times and vortex to ensure complete reconstitution.

Collagenase AF-1(Nordmark, Sweden, N0003554) was reconstituted in 10ml of sterile HBSS. The concentration of lyophilized stock enzyme was 2892PZ U/vial. Thus, after reconstitution, the collagenase stock was 289.2PZ U/ml. Note that the stock of enzymes can be varied, confirming the concentration of the lyophilized stock and modifying the final amount of enzymes added to the digestion mixture accordingly. Aliquots were divided into 100. mu.l aliquots and stored at-20 ℃.

Neutral protease (Nordmark, Sweden, N0003553) was reconstituted in 1ml sterile HBSS. The lyophilized stock enzyme concentration was 175DMC U/vial. Thus, after reconstitution, the neutral protease stock solution was 175 DMC/ml. Note that the stock of enzymes can be varied, confirming the concentration of the lyophilized stock and modifying the final amount of enzymes added to the digestion mixture accordingly. Aliquots were aliquoted into 20. mu.l aliquots and stored at-20 ℃.

DNase I (Roche, Switzerland, 03724751) was reconstituted in 1ml sterile HBSS. The concentration of lyophilized stock enzyme was 4 KU/vial. Thus, after reconstitution, the DNase stock was 4 KU/ml. Note that the stock of enzymes can be varied, confirming the concentration of the lyophilized stock and modifying the final amount of enzymes added to the digestion mixture accordingly. Aliquots were divided into 250 μ l aliquots and stored at-20 ℃.

The 3 components of the GMP digestion mixture were thawed and a working GMP digestion mixture was prepared as follows: mu.l of neutral protease (0.36DMC U/ml), 21.3. mu.l of collagenase AF-1(1.2PZ/ml) and 250. mu.l of DNase I (200U/ml) were added to 4.7ml of sterile HBSS. The digestion mixture was placed directly in the C-tube.

Tumor treatment and digestion

For GentleMeACS Octopdissociator, up to 4-6mm of tumor fragments were transferred to each GentleMeACS C tube (C tube) in 5ml of digestion mix described above. Additional tumor fragments were treated using additional gentlemecs acs C tubes.

Each C-tube was transferred to a GentleMACS octodispatcher. Digestion was performed by setting the dissociator to the appropriate procedure for the corresponding tumor histology listed in table 61 below. Dissociation took about one hour.

Table 61: american and whirlwind octodispatcher programs based on tumor tissue type.

Tumor tissue type	Name (R)	Procedure
			Melanoma, ovary, colon, hypopharynx and kidney	Is soft	37C_h_TDK_1
Lung and prostate gland	Medium and high grade	37C_h_TDK_2
			Mammary gland, pancreas, liver cell, head and neck squamous cell (HNSCC)	Hard and stiff	37C_h_TDK_3

After digestion, one or more C-tubes are removed from the octodis or spinner and placed in the BSC. Digests were removed from each C tube with a 25-mL serum pipette and bulk digests were passed through a 70- μm cell strainer into a 50-mL conical tube.

Note that: the digestate is not allowed to splash due to pressure from the pipette. The solution was poured gently into a 70- μm cell strainer. Avoiding the pipette tip from contacting the filter.

The undigested portion of the tumor may not pass through the screen, wash one or more C-tubes with an additional 10mL of HBSS, and pass the wash through the cell screen. 50-mL conical tubes QS were brought to 50mL with HBSS.

Digests were centrifuged at 400x G for 5 minutes at room temperature (full acceleration and full braking).

The conical tube was transferred to BSC and the supernatant was aspirated or decanted. The pellet was resuspended in 5mL of warmed CM1+6000IU/mL IL-2 and pipetted 5-6 times up and down. Without dilution, 2 cell counts were performed on NC-200.

Bulk controls were performed by placing 0.5-1mL of digest aside, and aliquots of 2X 500. mu.l of digest were cryopreserved for tumor reactivity assays. Digestion was maintained on ice.

Note that: once an ice-water slurry was observed, it was ensured that it was immediately replaced with crushed or pelletised ice.

The remaining cells were evenly distributed and subjected to anti-PD 1-PE (clone No. EH12.2H7) and nivolumab staining procedures.

Tumor digest flow cytometry staining using anti-PD 1-PE (clone No. EH12.2H7) and cell sorting

The first half of the tumor digest was stained with anti-PD 1-PE.

Tumor digest flow cytometry staining using nivolumab and cell sorting

For the second fraction, approximately 1e5 cells of unstained negative control, PE, and FITC monochrome compensation controls were removed into labeled 15-mL conical tubes. The remaining tumor digest will be stained with nivolumab and anti-IgG 4-PE (secondary antibody against nivolumab).

Preparation of sorting buffer (2% FBS): 2mL of HBSS was aspirated from a fresh 500mL HBSS bottle and 10mL of FBS was added. The sort buffer was kept in ice until further use.

Preparation of a solution of securabumab: to prepare the working solution, a 1:100 dilution was performed by adding 10- μ l of nivolumab [10mg/mL ] to 990- μ l of the sorting buffer.

Preparation of intermediate 1:50 IgG4 dilution:

10-uL of anti-IgG 4-PE was added to 490uL of sort buffer in a microcentrifuge tube and vortexed gently for 5 seconds to mix well. The intermediate dilutions were placed on ice until further use.

Preparation of tumor digest samples for flow sorting:

using the cell count data from above, the number of cells remaining in the tumor digestive tube was calculated.

Digestion was performed by adding 10mL of HBSS and centrifugation at 400x G for 5 minutes at room temperature (full acceleration and full braking).

The conical tube was transferred to BSC and the supernatant was decanted. The calculated volume of cells was resuspended at 10e6 cells/ml with sort buffer (resuspend sort buffer volume, nivolumab volume reference table number for TVC concentration).

Add 10- μ l L of working nivolumab per 1ml of cells.

Table 62: the suggested resuspension sort buffer and volume of nivolumab to be added.

The digests were gently mixed with a 1-mL micropipette and the cells were incubated on ice for 30 minutes. Light was protected during incubation. Agitation was performed by gently flicking every 10 minutes during incubation to ensure thorough staining.

After incubation, 10mL of sorting buffer was added to the sample digest, the monochrome compensation and the unstained negative control.

Centrifuge at 400x G for 5 minutes at room temperature (full acceleration and full braking).

The sample was decanted gently.

The pellet was resuspended in 400- μ L of sorting buffer and the total volume of the sample was measured using a serological pipette. mu.L of anti-CD 3-FITC was added per 100. mu.l, and 50. mu.L of intermediate-diluted anti-IgG 4-PE was added per 500. mu.l (see section: 9.5.3).

After incubation, 10-mL of sorting buffer was added to the sample digest.

The sample digest was filtered through a 70- μm cell strainer into a labeled 50-mL conical tube.

Cells were resuspended at up to 10e6 cells/ml in sorting buffer. The minimum volume was 300- μ l and transferred to a new 15mL conical tube.

The tubes were stored on ice, covered with aluminum foil until further use.

Preparation of monochrome compensation:

PE compensation controls were stained with nivolumab plus anti-IgG 4-PE secondary, and FITC compensation controls were stained with anti-CD 3-FITC.

10mL of HBSS was added to unstained PE and FITC compensation tubes and centrifuged at 400x G for 5 minutes at room temperature (full acceleration and full braking).

Undyed tube:

cells were resuspended in 500- μ L of sorting buffer and stored in ice until other samples were ready for sorting.

FITC Compensation tube:

cells were resuspended in 100- μ L of sorting buffer.

3- μ L of anti-CD 3-FITC was added per 100- μ L.

The digests were gently mixed with a 1-mL micropipette and the cells were incubated on ice for 30 minutes. Light was protected during incubation by covering the ice bucket with aluminum foil.

Cells were resuspended in 500 μ l of sorting buffer and stored in ice until other samples were ready for sorting, covered with aluminum foil until further use.

PE compensation pipe:

the conical tube was transferred to BSC and the supernatant was decanted. Cells were resuspended in 1mL of sorting buffer.

Add 10- μ L of working nivolumab per 1ml of cells.

After incubation, 10mL of sorting buffer was added and centrifuged at 400x G for 5 minutes at room temperature (full acceleration and full braking).

The sample was decanted gently and the pellet was resuspended in 500 μ Ι _ of sorting buffer and the total volume of the sample was measured using a serological pipette. 50 μ L of intermediate diluted anti-IgG 4-PE (see section: 9.5.3) anti-IgG 4-PE was added per 500 μ L of cells.

The digests were gently mixed with a 1-mL micropipette and the cells were incubated on ice for 30 minutes. Light was protected during incubation.

Preparation of a collection tube:

A15-mL collection tube was prepared for the sorted population. 2-mL of collection buffer (50% HBSS with 50% hAB serum) was placed in the tube. The collection tubes were stored on ice until further use.

Cell count and viability assessment

Procedures for obtaining cell and viability counts using a Chemometec NC-200 cell counter as described herein.

FACS sorting Using Sony FX500

TILs selected by PD1 were flow cytometrically sorted from tumor digests for sorting procedures and maintenance.

PD1 Rapid amplification protocol-full Scale REP day 0 (REP-1)

Preparation of the Medium

1L of CM1+6000IU/mL IL-2 was prepared in an incubator at 37 ℃ for at least 24 hours.

PBMC feeder cell preparation and TIL inoculation with TIL for REP-1

Example 9 provides instructions for initiating full-scale day 0 (REP-1) with the following exceptions:

the minimum number of PD 1-selected TILs resulting from both sorts will be used as the number of PD 1-selected TILs to add to both PD1-a and PD1-B conditions. The respective sorted volumes were calculated to achieve the numbers under both PD1-a and PD1-B conditions. The TIL volume was transferred to its corresponding G-Rex 100M flask.

PD1 Rapid amplification protocol-Small Scale REP day 0 (REP-1)

Preparation of the Medium

1L of CM1+6000IU/mL IL-2 was prepared and preheated in a 37 ℃ incubator for at least 24 hours.

PBMC feeder cell preparation

Thawing the appropriate number of vials (10 e6 per flask would be required; 60e6-80e6 PBMC per 1mL vial is assumed) for REP-1

40mL of warmed CM1+6000IU/mL IL-2 was placed in a 50mL conical tube and a 1mL PBMC feeder cell vial was pipetted into the conical tube.

Thawed PBMC feeder cells were pipetted up and down for thorough mixing and 2 cell counts on NC-200.

The volume necessary for transfer of 10e6 PBMC to G-Rex 10M was calculated and transferred.

To G-Rex 10M was added 3- μ L of aCD3 (OKT-3). The flask was placed in an incubator.

Inoculation of TIL for REP-1

Calculate 10% of the lowest PD1 sort results and calculate the volume of the respective sort to achieve the number under both PD1-a and PD1-B conditions. The TIL volume was transferred to its corresponding G-Rex 10M flask.

An equal number of CD3+ cells as PD1 cells were added to the bulk TIL control conditions. To obtain a suitable volume of digest, the following steps are followed:

CD3+ TVC/mL in the digest was calculated by multiplying the digest TVC obtained by the CD3 +% of live cells obtained from the lowest sort report. (i.e., 10e6 × 10% ═ 1e 6).

After this number was obtained, the number of PD1 cells used was divided by this number. (i.e., 1e5/1e6 ═ 0.1 mL).

This volume (0.1mL) of digest was added to the bulk TIL flask and filled to 100mL with CM1+6000IU/mL IL-2.

All flasks were placed in a 37 ℃ 5% CO2 incubator 9.10. PD1 Rapid amplification protocol-full Scale days 11, 16 and 22

Each manufacturing batch record follows a full scale process. The ontology TIL condition is processed similarly to the steps described in example 9.

Acceptance criteria

Table 63 below specifies the acceptance criteria for evaluating the performance of the small scale (extrapolated TVC) and full scale experiments.

Table 63: product release test and acceptance criteria in Process and Collection

Table 64 below specifies additional final product characterization tests performed.

Table 64: characterization of the final product (for reference only)

Example 17 reference

Examples 6 and 7: direct ex vivo selection and expansion of PD1+ cells: a process for enhancing tumor-reactive TIL for ACT therapy.

Example 9: PD1 for full-scale manufacturing⁺Selection and amplification of TIL.

Example 10: PD1 for full-scale manufacturing^{Height of}Selection and amplification of TIL.

Example 18: cells expressing PD-1 in tumor digests

Purpose(s) to

Expression of programmed cell death protein 1(PD-1) in the whole tumor digest was evaluated.

Range

Assessing expression of PD-1 in whole tumor digests from the following tumor histologies: melanoma, non-small lung cancer (NSCLC), Head and Neck Squamous Cell Carcinoma (HNSCC), Ovarian Cancer (OC), Triple Negative Breast Cancer (TNBC), Prostate Cancer (PC), and colorectal cancer (CRC).

Background information

PD-1 is a multidimensional phenotypic marker associated with activation, antigen specificity and depletion. It is rapidly induced upon activation and maintained on cells undergoing antigens in a chronic disease setting, including cancer [1, 2 ]. Molecularly, PD1 is a member of the CD28 family that regulates cell surface receptors and is expressed on chronically activated T cells, NKT cells, B cells and monocytes [3-5 ]. Engagement with its ligands PD-L1 and PD-L2 induces a signaling cascade leading to a reduction in T cell activation, proliferation, survival and cytokine production [6 ].

Despite the immunosuppressive role of PD-1, the presence of PD-1 expressing Tumor Infiltrating Lymphocytes (TILs) has been associated with favorable clinical outcomes in HNSCC and NSCLC, suggesting that these TILs may be involved in controlling tumor progression [7-9 ].

Studies in melanoma and NSCLC have demonstrated that most tumor-reactive TILs are included in PD-1⁺Within T cell subsets [4、8、10]。

Based on PD-1⁺TIL is a concept of neoantigen/tumor-specific lymphocytes and Iovance is developing a novel PD-1-selected TIL product LN-145-S1 enriched in PD-1 sorted directly from the whole tumor digest⁺TIL。

Although PD-1 expression is essential for response to anti-PD-1 therapy, PD-1 expression alone does not predict responsiveness to therapy. As an example, PD-1 is present on TIL in OC and its expression is correlated with survival [11 ]. However, recent clinical trials in OC demonstrated that anti-PD-L1 drug aviluzumab in combination with chemotherapy did not enhance progression-free survival [12]. This study together with the expression of PD-1 in the tumor microenvironment⁺Together with a large number of patients resistant to anti-PD-1 therapy, showed that in vivo blockade of the PD-1/PD-L1 axis was insufficient to control most cancers.

Adoptive T cell therapy using TIL has demonstrated significant efficacy in melanoma patients refractory to anti-PD-1, suggesting that protocols used to expand TIL ex vivo are able to restore TIL, as opposed to PD-1 blockade in vivo [13 ].

In this example, sorting PD-1 prior to ex vivo amplification was examined⁺TIL about all PD-1⁺Further improving the response rate to TIL therapy in cancer histology.

The aim of this study was to target PD-1⁺The presence of TIL investigates various tumor histologies to support clinical targeting with amplified TIL products.

Design of experiments

Tumor digests from various tumor histologies were evaluated for PD-1 expression by flow cytometry.

Material

Tumor digests used in this work are described in table 65. Abbreviations: CRC (colorectal cancer), HNSCC (head and neck squamous cell carcinoma), MSI (microsatellite instability), MSS (microsatellite stability), ND-PBL (normal donor peripheral blood lymphocytes), NSCLC (non-small cell lung cancer), OC (ovarian cancer), PD-1 (programmed cell death protein 1), REP (rapid expansion protocol), TIL (tumor infiltrating T-cells), and TNBC (triple negative breast cancer).

Table 65: description of tumor digests for these studies

Method

Tumor treatment

PD-1 staining

Table 66: PD-1 flow cytometry staining plate

Antibody/staining	Cloning	Fluorescent dyes	Manufacturer(s)	Amount (μ l/1e6 cells)
					7-AAD	N/A	N/A	BD bioscience Co.	20
CD3	UCHT1	FITC	BD bioscience Co.	3
					CD4	OKT4	PE/Cy	BioLegend Co	1
PD-1	EH12.2H7	PE	BioLegend Co	2.5

PD-1 selection and gating strategies

Stained cells were placed on either an FX500 cell sorter (sony, new york) or ZE5 cell analyzer (berle, ca) and analyzed based on the following gating strategy. First, single cells were identified based on forward and backward or side scatter. Next, live cells were gated based on negative/low 7-AAD or live-dead blue fluorescence. TIL was identified using CD 3. PD-1 cells were identified using normal donor peripheral blood (ND-PBL) as a control. The selection gate for PD-1 was placed above the baseline for PD-1 expression in ND-PBL. Data analysis was performed using FlowJo v8.1 software (FlowJo, inc., oregon). Results were plotted using GraphPad v8.

Results

Expression of PD-1 in tumor digests

To identify which histologies are candidates for PD-1 selection, flow cytometry was used on multiple tumors from several cancer histologiesExpression of PD-1 was assessed in tumor samples. A total of 4 melanomas, 7 NSCLCs, 5 HNSCCs, 3 OCs, 5 TNBCs, 2 PCs and 8 CRCs were tested according to the procedure TMP-18-015 (abbreviated section 5.2). CRC consists of microsatellite stabilised (MSS) (n ═ 6) and microsatellite unstable (MSI) (n ═ 2) tumors. After digestion, a portion of the resulting single cell suspension was stained for PD-1, analyzed by flow, and if so>5e6 cells were available, and sorting was performed to obtain PD-1⁺A cell. PD-1 sorted cells were subjected to a two-step process comprising an 11-day activation step followed by an 11-day Rapid Expansion Protocol (REP) to obtain PD-1 selected TILs. Tumor ID, histology and experimental fate are listed in table 65. The results of the streaming analysis are shown in fig. 157.

All tumor digests assayed expressed PD-1 in the CD3 population⁺Percentage of cells. PD-1% is variable and ranges from 11% to 78%, with an average of 35% in the histology determined. Melanoma (n ═ 4) and PC (n ═ 2) produced the lowest mean values for PD-1 expression of 30.1% and 25.8%, respectively. The average percentage of PD-1 expression was not correlated with the clinical response rates observed for those histologies. Histology that is responsive to anti-PD-1 blockade, such as melanoma and NSCLC, does not have higher PD-1 levels/expression than histology that is not responsive to anti-PD-1 blockade (i.e., OC and PC).

In all cases culture can be initiated by PD-1⁺The PD-1-selected products were obtained after in vitro expansion of the cells (Table 1). The results of this study are reported in literature example 13. Thus, all histologies determined are potential candidates for PD-1 selection based on PD-1 expression.

Conclusion

In all tumor digests assayed, PD-1 was expressed on CD3 cells.

There is a wide range of intratumoral and intratumoral variability in PD-1 expression.

PD1 expression was not associated with histology that has demonstrated responsiveness to anti-PD 1 therapy.

Example 18 reference

Webb, J.R., K.Milne and B.H.Nelson, PD-1 and CD103 are widely co-expressed on intraepithelial CD 8T cells with favorable prognosis in human ovarian cancer [ immunological studies on cancer ], 2015.3(8): pages 926-35.

12, Columbus, g, avillumab missed the primary endpoint in the stage III ovarian cancer test 2018; available from the following websites: https:// www.onclive.com/web-exclusive/avelumab-messes-primary-endings-in-phase-iii-auxiliary-cancer-tertiary.

Sarniak, a. phase 2 multicenter study for assessing the efficacy and safety of autologous tumor infiltrating lymphocytes (LN-144) for treating patients with metastatic melanoma 2018; available from the following websites: https:// ascopubs.org/doi/abs/10.1200/JCO.2018.36.15_ Suppl.TPS9595.

Example 19: selection of PD-1 by flow cytometry sorting and full-scale amplification Using Nitumumab⁺Use of TIL for clinical manufacture

Purpose(s) to

This report describes the results from amplification of PD-1 selected TIL using nivolumab for selection in the full scale manufacturing experiments described in this example.

Range

The working range was to amplify TIL selected by PD-1 from melanoma or lung or head and neck or ovarian tumors.

On day 0, tumor digests were equally distributed to two groups and stained for tumor digests in each group of the experiment using nivolumab or anti-PD 1 clone No. EH12.2H7 (research grade) as the primary antibody and FITC conjugated anti-IgG 4 secondary antibody. PD-1 expressing TILs were then selected from the stained population by flow sorting. PD-1-selected TILs were amplified using a two-step amplification procedure for full-scale clinical manufacturing. The first step of amplification ("activation") was performed from day 0 to day 11. The second step of the amplification process is performed from day 11 to day 22 ("rapid amplification phase" or "REP", including split at day 16). The final product was collected on day 22.

For the small scale process (1/100 scale), 10% of PD-1 selected TILs with the lowest sorting results were used on day 0 and the amount of TILs was transferred from each sort to the corresponding G-Rex-10M flask with feeder cells and OKT-3 with IL-2 medium to initiate activation. REP was initiated according to TP-19-004, split and collected. A brief explanation of the relevant time points is outlined in the method section below (table 67).

For the full scale process, activation was initiated on day 0 using PD-1 selected TIL with similar cell numbers, with 100e6 allogeneic feeder cells and 30ng/mL OKT3 for 11 days. REP was initiated on day 11 starting from the collected product. The REP (day 11) and subsequent day 16 (split) and day 22 (harvest) procedures were performed according to IOVA manufacturing lot records. A brief explanation of the relevant time points is outlined in the experimental design below (table 2).

The amplified final product TIL was evaluated for cell growth, viability, phenotype and function (IFN- γ and granzyme B secretion, post-stimulation CD107a mobilization).

Additional analysis was performed on the amplified characterization data to establish the equivalence of EH12.2H7 and nivolumab.

Background information

Previously developed protocols designed to select TILs expressing PD-1 from tumor digests using PE-conjugated anti-PD-1 antibodies (clone number EH12.2H7) to enrich TIL products from autologous tumor-reactive T cells are provided in examples 9 and 21.

In the current study, using nivolumab as an anti-PD 1 antibody instead of PE-conjugated clone No. EH12.2H7 and FITC-conjugated anti-IgG 4 antibody as a secondary staining antibody, examples 9 and 21 were suitable for obtaining TIL selected via PD 1.

Design of experiments

Two pilot experiments and bulk control conditions were performed per TP-19-004.

One full scale experiment was performed for each example 19.

A summary of small and full scale is provided in tables 67 and 68.

Table 67: 1/100 overview of small-Scale PD-1-selected TIL Process

Table 68: overview of full-Scale PD-1-selected TIL Process

Results

Table 69 below specifies the acceptance criteria for evaluating the performance of the small scale (extrapolated TVC) and full scale experiments according to example 19.

Table 69: product release test and acceptance criteria in Process and Collection

Applicable only to full scale experiments.

The results obtained

Table 70 below is a list of tumors and related histologies used in this study.

Table 70: tumors used in this study

Streaming sort output

Table 71: purity of TILs selected by PD-1 before and after sorting by flow cytometry.

^*Purity was based on PD-1 +% (gated at FSC/BSC/CD 3)

Purity after sorting (PD-1 +%) for all three tumors met the > 80% criterion.

Activation and REP Collection output

Table 72 below summarizes the total viable cell count and product attributes from two small-scale and one full-scale experiments, along with their ontological counterparts (indicated in parentheses).

Table 72: summary of product attributes from activation and REP

¹The bulk condition TVC shown above was extrapolated to full-scale control of nivolumab and EH12.2H7

²The currently established range based on the Gen 2 REP Process the range of 5-200 e6 TVC inoculated at REP, and is not a formal acceptance criteria in this protocol

³Fold expansion versus harvested TVC/vaccinated TVC

⁴Cell doubling was calculated based on the formula ═ LOG (TVC on day 22/TVC on day 11)/LOG (2)

⁵Batches were small-scale, no LOVO was performed

⁶A single LOVO operation is available. The conditions of nivolumab were selected for LOVO treatment, which represents the clinical manufacturing of the TIL process selected by PD-1.

⁷The NC-200 cytometer problem was identified during the post-LOVO counting process. The post-thaw recovery counts from the stability study (SP-19-003) were used to calculate% recovery.

The process yield is as follows: at the end of activation, staining of selected TILs with nivolumab or EH12 produced cells greater than 100e6(>1200 fold expansion, average 9.1 cell doublings) with sufficient yield to initiate REP culture.

At REP harvest, all cultures produced >80e9 TVC. An average of 9 cell doublings were observed between day 11 and day 22. The number of cell doublings was very similar to that observed in previous preclinical experiments (TP-19-004R and example 21R).

Dosage: from the full-scale run (H3046), the final product dose using nivolumab staining was 88.5e9 TVC, with 85% viability and 99.7% CD45+ CD3+ cells. The final product is a highly enriched TIL product.

The functions are as follows: the functionality of TIL was characterized based on stimulating the final product with aCD3/aCD28/aCD137 Dynabeads (LAB-016) overnight. Supernatants were collected and frozen after 24 hours of stimulation. ELISA was performed to determine the concentration of IFN γ and granzyme B released into the supernatant. IFN γ release met the acceptance criteria and all TIL cultures secreted high levels of granzyme B upon stimulation. Similar to the TIL product generated in the previous study (TP-19-004R, example 21), a high fraction of TILs from the final product expressed CD107A when stimulated with PMA/IO (both CD4+ and CD8+ TIL).

TIL telomere length and telomerase activity: the data is pending. When a report is available, the report will be modified to include this data.

TIL clonality: the data is pending. When a report is available, the report will be modified to include this data.

Expanded phenotype: tables 73, 74 and 75 describe the expanded phenotypic analysis of TILs. Multicolor flow cytometry was used to characterize TIL purity, identity, memory subpopulations, activation and depletion status of REP TIL. There were < 1% detectable B cells, monocytes or NK cells in the final collected TILs (table 7). REP TIL is composed primarily of TCR α/β and primarily effector memory differentiation. With the exception of ovarian tumors, the CD8/CD4 ratio between nivolumab and EH12.2H7 was comparable. The skewness of the CD8/CD4 ratio may be due to heterogeneity of ovarian tumor types and the lack of selectable markers for CD4 and CD8 during the selection procedure.

Table 73: TIL purity, identity and memory phenotype characterization

Note that: the gating algorithm for TIL purity is as follows:

monocytes: live CD14 +%)

NK (natural killer) cells: live CD14-, CD3-, CD56+ CD16 +%

B cell: % CD14-, CD3-, CD19 +%

All PD-1-selected TIL conditions showed both up-regulation of CD28 expression and down-regulation of CD27 expression due to TCR-stimulated proliferation of TIL. In addition, all PD-1 selected TILs showed a less differentiated phenotype with lower expression of KLRG 1.

CD27, CD28, CD56, CD57, BTLA, CD25, and CD69 levels were similar to the results for melanoma TIL produced using the Gen 2 manufacturing process (table 74).

There were no significant differences between the nivolumab and EH12.2H7 selection procedures in differentiation, activation, and depletion states.

Table 74: activation and depletion states of CD4+ TIL

Table 75: activation and depletion states of CD8+ TIL

Table 76: expression of CD27, CD28, CD56, CD57, BTLA, CD25 and CD69 at CD3+

Additional analyses were performed on the phenotypic characterization data to establish the equivalence of EH12.2H7 and nivolumab.

Evaluation of PD-1 Using EH12.2H7 and nivolumab by flow cytometry⁺TIL-produced PD-1-selected TIL expression of CD4, CD8, CCR7, CD45RA, and PD-1. No significant difference was observed in the expression of PD-1 selected CD4 and CD8 derived using nivolumab and EH12.2H7. Relative to CD4 for the three tumors tested⁺T cells, both TIL products produced a higher proportion of CD8⁺T cells (fig. 1). The similarity of CD4 and CD8 expression in the three PD-1 selected TIL products indicates that the ratio of CD4/CD8 was not altered by the use of nivolumab selection for PD-1+ compared to EH12.2H7. See fig. 159.

Like the T cell lineage, the memory status of TIL was similar in PD-1 selected TIL generated using EH12.2H7 and nivolumab. The TIL population consisted primarily of PD-1-selected TILs using effector memory T cells generated by nivolumab and EH12.2H7 using LN-145 study products similar to Iovance, indicating that selection of PD-1 using either anti-PD-1 clone did not skew the memory phenotype of TILs (fig. 160).

To assess whether PD-1 expression decreased similarly after culture, PD-1-selected TIL generated using nivolumab and EH12.2H7 was assessed before and after amplification. After sorting, the percentage of PD-1+ TIL in the two freshly sorted TIL preparations was close to 100% (table 71). In the PD-1 selection generated using EH12.2H7 and nivolumab, the expression of PD-1 was significantly and relatively reduced after amplification (fig. 161). As predicted, the reduction in PD-1 expression following amplification indicates that the previously high PD-1 expressor reverted to predominantly PD-1-with amplification in PD-1+ -sorted TIL using EH12.2H7 and nivolumab.

Functional characterization of PD-1-selected TIL generated from EH12.2H7 and the PD-1+ TIL sorted with Nwaruzumab.

To assess whether amplified PD-1+ TIL derived using nivolumab has similar function to TIL derived using EH12.2H7 clone, PD-1 selected TILs from 3 tumors were non-specifically stimulated with α CD3/α CD28/α 41BB activated beads and evaluated for IFN γ and granzyme B secretion. Nivolumab and EH12.2H7-derived PD-1-selected TIL produced similar levels of IFN γ and granzyme B in response to stimulation (figure 161). PD-1-selected TILs generated using nivolumab and EH12.2H7 secreted appreciable levels of IFN γ and granzyme B in response to non-specific stimulation (α CD3/α CD28/α CD137 beads), indicating that the selected TILs were highly functional after amplification.

Information

On day 0, the example did not receive fresh tumor due to logistic problems. All experiments were performed using frozen tumor digests instead of fresh tumors. Data from research studies showed no difference in PD-1 expression when testing fresh or frozen tumors.

Conclusions and suggestions

The PD-1-selected TIL process was developed at full scale to amplify PD-1+ TIL to >80e9 in 22 days. All six batches produced at the development scale (both nivolumab and EH12 staining methods, 2 full-scale and 4 mini-scale) met the acceptance criteria for release parameters.

Table 77: summary table:

NA, not applicable, cell Collection on a Small Scale

In general, this example demonstrates that PD-1-selected TIL generated from PD-1-sorted TIL using nivolumab is comparable to TIL generated using EH12.2H7 clone, supporting PD-1 selection using nivolumab in clinical manufacturing. See also fig. 162, 163 and 164.

Example 20: overview of PD-1 non-clinical Studies

Non-clinical overview

Introduction to

The TILs described in this example are preparations of autologous Tumor Infiltrating Lymphocytes (TILs) that have been selected based on expression of programmed cell death protein 1(PD-1) biomarkers. Thus, TIL is a subset in which TIL cells with higher PD-1 expression are selected for ex vivo expansion. The manufacturing process described throughout the examples provides a manufacturing method in which PD-1 positive (PD-1) is selected from the bulk TIL population using flow cytometry prior to its ex vivo expansion ⁺) T cells. The resulting TILs have been characterized and demonstrated activity in the ex vivo studies summarized below. Such TILs may be administered to a patient using a TIL protocol of Adoptive Cell Transfer (ACT) as described in the examples and this application.

This example summarizes non-clinical data supporting a phase 2 clinical trial that will investigate the safety and primary efficacy of TIL in patients with Head and Neck Squamous Cell Carcinoma (HNSCC). TIL products are patient-specific and do not act across species and therefore cannot be tested in traditional non-clinical pharmacological, pharmacokinetic and toxicological studies. The non-clinical and clinical safety of other agents (IL-2, cyclophosphamide and fludarabine) to be included in a TIL treatment regimen is well characterized.

Table 78 lists non-clinical studies performed by Iovance to support clinical studies of amplified TIL products. Reports of these studies are provided in the examples above.

Table 78: list of non-clinical studies

Non-clinical pharmacology

Selection of PD-1 in the TIL manufacturing Process prior to ex vivo amplification⁺TILs should enrich for neoantigen-specific T cells while retaining TIL diversity and thus exhibit the potential to recognize tumor neoantigen arrays. This strategy represents an attractive means to further optimize the TIL manufacturing process used in therapy. Based on the non-clinical studies summarized below, manufacturing processes have been developed that reliably produce highly functional PD-1-selected TIL products.

PD-1-selected TIL products will be examined for treatment of relapsed/refractory HNSCC, a refractory malignancy, for which PD-1 has been implicated⁺Correlations between cellular levels and clinical outcome were studied (Badoual et al, 2013).

Non-clinical studies

The PD-1-selected TIL product has been extensively characterized with respect to its composition and ex vivo anti-tumor activity. These analyses were preceded by infiltration of PD-1⁺Investigation of multiple tumor types of T cells and sorting of PD-1⁺Testing of the amplification process of TIL. All non-clinical studies were performed using a research-grade PD-1 specific monoclonal antibody (clone EH12.2H7) for detection and selection of PD-1⁺And (7) TIL. Therefore, a bridging study was performed to establish the comparability of EH12.2H7 with nivolumab, an anti-PD-1 monoclonal antibody used to produce TIL products (see, example 19). Overall, this work demonstrated that the PD-1-selected TIL was prepared histologically from a variety of tumors; it is on the watchSimilar in shape to the unselected TIL; and it exhibits many traits that are specifically associated with the neoantigen, including an initial reduced proliferative capacity and, importantly, tumor reactivity.

PD-1⁺The prevalence of TIL across multiple cancer types (report No.: example 18).

Assessment of PD-1 in multiple tumor samples from several cancer histologies⁺The presence of lymphocytes. A total of 34 tumors were evaluated in the following histology: melanoma (n ═ 4), non-small cell carcinoma (NSCLC) (n ═ 7), head and HNSCC (n ═ 5), OC (n ═ 3), TNBC (n ═ 4), PC (n ═ 2), and CRC (n ═ 8). Samples of CRC included microsatellite stabilised (MSS) tumours (n ═ 6) and tumours with microsatellite instability (MSI) (n ═ 2).

Tumor samples were dissociated using enzymatic digestion, and a portion of the resulting single cell suspension was stained for PD-1 and analyzed by flow cytometry. The results of the flow analysis study are summarized in fig. 165.

All tumor digests assayed were in CD3⁺The cell population contains a substantial fraction of PD-1⁺A cell. PD-1⁺The percentage of cells was variable and ranged from 11% to 78%, with an average of 35% in the histology determined. Melanoma (n ═ 4) and PC (n ═ 2) produced the lowest mean values for PD-1 expression of 27% and 21%, respectively. Histologies that have been shown to respond clinically to anti-PD-1 blockade (such as melanoma and NSCLC) do not have higher PD-1 levels/expression compared to other histologies (i.e., OC and PC).

Importantly, PD-1-selected products can be present at greater than 2X 10 prior to sorting⁶In all cases of individual cells in PD-1⁺Obtained after ex vivo expansion of the cells, this was achieved in 12 out of 13 tumor samples in the histology examined. The results of this study are discussed in example 13. Thus, all histologies examined were potential candidates for the production of TIL products based on the expression of PD-1 by TIL.

Proliferative Capacity of PD-1-selected TIL (example 13)

PD-1⁺The cells have shown a cellular causeImpaired seed production and reduced proliferation [3, 4]. In vivo blockade of PD-1 or its ligand, PD-L1, can restore the functionality of those cells to trigger an anti-tumor response (Schmacher et al, 2015; Shang et al, 2018). Tested in PD-1⁺Whether the defects observed in the cells can be reversed by displacing the cells from the immunosuppressive microenvironment and expanding the cells ex vivo in the presence of anti-CD 3 and allogeneic feeder cells, as described in several publications (Inozume et al, 2010; Thommen et al, 2018).

To determine whether PD-1-selected TILs can be expanded ex vivo to high numbers, PD-1-sorted TILs from 4 melanoma, 7 NSCLC and 2 HNSCC tumor samples were subjected to a process consisting of a two-step protocol consisting of an 11-day activation step followed by an 11-day rapid amplification protocol (REP), and fold-amplifications were evaluated. Matched unselected TILs amplified from whole tumor digests under similar conditions were used as controls.

The proliferative capacity of PD-1-selected TILs was initially reduced compared to unselected TILs, resulting in a lower level of amplification during the activation step. The mean fold amplification for PD-1 selected TIL in the activation step was 833, while the mean fold amplification for unselected TIL was 2650. However, in the REP step, comparable mean fold amplifications were calculated 1308 and 1418 for PD-1 selected and unselected TILs, respectively (fig. 166).

The delayed expansion in the activation step in PD-1 selected TILs resulted in a lower total viable cell yield in 9 of 13 paired samples (Table 79). Since REP is performed on a small scale, the achievable cell count on a manufacturing scale is estimated based on the fold expansion of REP and the total cell yield in the activation step (i.e. the extrapolated cell count ═ fold expansion of REP × total viable cell yield of activated cells). Notably, these extrapolated numbers may underestimate potential overall yield, as only a portion of the cell digest is used for PD-1 sorting.

The extrapolated cell yield of TIL selected by PD-1 ranged from 2.32X 10⁷To 209X 10⁹Wherein the average cell yield is 47.46X 10⁹And (4) cells. Importantly, the total cell counts generated after REP from 12 of the 13 cultures expanded with PD-1 selected TIL were within the range specified for the LN-145 study product (1X 10) ⁹To 150X 10⁹Total viable cells).

Table 79: final product yield in PD-1 selected TIL

Legend: PD-1 sorted and unselected TILs from 4 melanoma, 7 NSCLC and 2 HNSCC tumor samples were amplified using a 22-day procedure consisting of an 11-day activation step followed by 11-day REP. Shows inoculated CD3⁺Number of cells, fold expansion and extrapolated cell count.

In summary, PD-1 is present during the activation step⁺The reduced proliferative capacity of TIL did not prevent the PD-1-selected TIL product from reaching high cell counts in the final product. In fact, 12 of the 13 preparations produced using the expected manufacturing process for the amplified TIL product>1×10⁹And (4) total living cells. These yields were well within those specified for release of the standard LN-145 study product.

Phenotypic characterization of PD-1-selected TILs (example 16)

Phenotypic analysis was performed by flow cytometry to characterize the expression of cell surface markers of T cell lineages and memory subpopulations. In addition, PD-1 levels were evaluated in PD-1-selected TILs. The same sample set of 13 matched PD-1-selected and unselected TIL products from 4 melanoma, 7 NSCLC and 2 HNSCC tumor samples was stained with live/dead markers followed by antibody staining for multiple markers. The results for CD4, CD8, CCR7, CD45RA, and PD-1 are presented in figures 167, 168, and 169. More detailed results on additional activation, depletion, differentiation and tissue retention markers can be found in the complete report example 16.

CD4 and CD8 expression in PD-1 selected TILs

PD-1 has been largely described in CD8⁺T cells, although characterized in CD4⁺And CD8⁺Expression is found in both lineages (Ahmadzadeh et al, 2009; Inozume et al, 2010). To determine the selection and amplification of PD-1⁺Whether cells alter CD4⁺And CD8⁺Cell ratio, cell surface expression of CD4 and CD8 of the final PD-1 selected and unselected TIL products was evaluated.

There was no significant difference in the expression of CD4 and CD8 in PD-1 selected and unselected TIL products (167). CD4⁺And CD8⁺The proportion of T cells was variable in samples and histology (report example 16, but in general, relative to CD8⁺T cells, both TIL products produced a higher proportion of CD4⁺T cells).

The similarity of CD4 and CD8 expression across various PD-1 selected and matched unselected TIL products indicates that sorting of PD-1 does not alter the T cell lineage of the final amplification product.

Memory T cell populations in PD-1-selected TILs and unselected TILs

Published studies have shown that PD-1⁺TIL comprises mainly effector memory T cells (TEM) (Fernandez-Poma et al, 2017; Kansy et al, 2017). Furthermore, these TEMs have been shown to represent a major population of unselected TIL products demonstrating clinical activity (gross et al, 2014). The following markers are typically used to differentiate T cell memory subpopulations:

Effector memory T cells (TEM): CD45RA^-And CCR7^-；

Central memory T Cells (TCM): CD45RA^-And CCR7⁺；

Naive/stem cell memory T cells (TSCM): CD45RA⁺And CCR7⁺(ii) a And

effector T cells (TEMRA): CD45RA⁺And CCR7^-(Golubroovskaya and Wu, 2016).

To determine whether PD-1 selected TILs predominantly comprise TEM, PD-1 selected and matched unselected TIL products derived from 12 unique tumor samples were evaluated for CD45RA and CCR7 expression to define the individual memory subpopulations indicated above.

PD-1 selected and unselected TIL products were composed of similar ratios of various subsets of memory T cells, with TEM cells representing the majority of cells within each product (fig. 168).

Given the similar expression of memory-associated phenotypic markers, the selection of PD-1 prior to expansion does not appear to alter the relative proportion of memory T cell subsets in the TIL product. Notably, the profile of memory T cell subsets of TILs selected by PD-1 closely resembles that of LN-145.

Expression of PD-1 in PD-1-selected and unselected TILs

Amplified PD-1 has been shown⁺PD-1 expression in TIL decreases with ex vivo culture and amplification, which is considered to be evidence of TIL reversion (Inozume et al, 2010; Thommen et al, 2018).

To determine whether PD-1 expression changes with amplification, PD-1 expression was analyzed before and after amplification for PD-1 derived from PD-1-selected and matched unselected TIL products of 12 unique tumor samples.

PD-1 in unselected TIL before amplification⁺The percentage of cells represents the population of cells expressing PD-1 in the whole tumor digest (37.3% on average). Sorting PD-1 as expected⁺The cells result in high purity PD-1⁺A population wherein the average sort purity is 92.8%. After amplification, PD-1 expression was significantly reduced in PD-1-selected and unselected TIL products relative to the PD-1 level prior to amplification. PD-1 was observed in both PD-1-selected and unselected TIL formulations⁺The proportion of cells was reduced by more than 3-fold (FIG. 169).

The expression of additional co-inhibitory receptors associated with depletion of PD-1 selected and unselected TILs was also assessed. PD-1-selected and unselected TILs expressed similar TIM3, LAG3, and CD101 levels (example 16).

This is indicated by a significant reduction in PD-1 expression in TILs expressing high levels of PD-1 in situSome cells returned to PD-1 following expansion^-T cells, and thus are less likely to be inhibited by the PD-1/PD-L1 axis upon infusion.

In summary, phenotypic analysis of PD-1 selected TILs revealed products composed primarily of TEM cells with low PD-1 expression, indicating that these cells are restored after ex vivo expansion.

TCR library of PD-1-selected TIL (example 11)

Study in NSCLC investigated designation as PD-1^T(A "tumor-associated PD-1", i.e., PD-1 levels above those observed on PBMCs of healthy donors) whether a TIL clone expressing PD-1 is associated with PD-1^-The TIL is shared. Although some overlap of clones could be observed, PD-1^TThe major TCR in TIL is not present in PD-1^-Subgroup (Thommen et al, 2018). TCRv beta was cloned in PD-1^TAnd PD-1^-A low degree of clonotype sharing in TILs indicates that the ex vivo generated product contains TCRs with different antigen specificities.

It is expected that a PD-1 selection step performed prior to the ex vivo expansion phase of TIL will result in TIL product enrichment of tumor-specific T cells. To determine whether amplified PD-1 sorted TILs produced different products, PD-1 selected and unselected TILs were compared for their TCRv β compositions. To this end, the representation of the first 10 TCRv β clones present in PD-1 selected TIL products within the corresponding matched unselected TIL products was evaluated.

Of all paired TIL products, most highly representative PD-1-selected TIL clones were present at significantly reduced levels, or were not detected in matching unselected products (fig. 170).

The PD-1-selected TIL and unselected TIL products contained different high frequency TCRs. Thus, these two products are expected to exhibit a measurable difference in ex vivo testing of T cell reactivity.

In summary, the results demonstrate how the PD-1 selection step alters the composition of the amplified TIL product and indicate that the resulting PD-1 selected TIL can greatly enrich the specific TCRv β pool of potential tumor-reactivity.

Increased tumor reactivity of PD-1-selected TIL (example 15)

Published data for both mice and humans has demonstrated that expression of PD-1 on T cells within a tumor can identify a pool of tumor-reactive lymphocytes (containing tumor neoantigen-specific lymphocytes) (Donia et al, 2017; Fernandez-Poma et al, 2017; gross et al, 2014; Inozume et al, 2010; lacing et al, 2017; Thommen et al, 2018). In these studies, ex vivo amplified purified PD-1 was tested in co-culture with autologous tumor cells⁺Tumor reactivity of TIL and with PD-1 ^-TIL comparison, showed PD1⁺TILs secrete significantly greater amounts of IFN γ.

Based on these studies, TILs selected for expression of PD-1 expression are expected to enrich tumor/neoantigen specific T cells, which should exhibit greater autologous tumor reactivity when evaluated ex vivo.

Tumor reactivity in PD-1-selected TIL

To assess TIL tumor reactivity, IFN γ release was measured by ELISA upon co-culture with autologous tumor digests.

Of the 10 pairs of PD-1-selected and unselected TIL products evaluated (5 out of 13 tumor samples corresponding to the foci of this summary, supplemented with 5 recently obtained samples), 7 produced detectable amounts of IFN γ when co-cultured with autologous tumor digests. Upon co-culture, three pairs (2 ovarian pairs and 1 TNBC pair) produced no IFN γ under any conditions. In 7 evaluable co-cultures, PD-1-selected TIL produced significantly higher levels of IFN γ than its unselected counterpart, on average 4.57-fold higher (1.22 to 11.65) (fig. 171). This increased reactivity was antigen specific for 5 of the 7 responding PD-1 selected TILs, as evidenced by the reduction in IFN γ production following HLAI class blockade. As shown, positive values reflect HLA-specific anti-tumor responses, while negative or negative values reflect non-specific responses.

Therefore, selection of TILs that express PD-1 results in enrichment of TIL products in tumor-reactive T cells.

The enhancement of IFN γ production in the presence of autologous tumors suggests that TILs selected with PD1 may have greater potential for anti-tumor effects when administered to patients in an ACT background relative to unselected TILs. Clinical efficacy in ACT is directly related to the presence of tumor-specific TIL. Thus, enrichment of tumor-specific TILs by PD-1 selection and amplification may enhance the ability of TILs to initiate effective and efficient anti-tumor effects when administered to a patient.

Autologous tumor cell killing

Ten matched PD-1-selected TILs and unselected TILs were evaluated for autologous tumor killing. Tumor cell lysis was quantified by xcelligene real-time cellular analysis, which monitored tumor cell isolation as a measure of tumor cell death (Peper et al, 2014).

Of the 10 tumors evaluated, tumor cell lysis of only 1 melanoma could be evaluated due to poor tumor cell adhesion and low viability. Tumor cell lysis is estimated using the tumor cell index, which is a measure of plate impedance when cells attach or detach from it. If at any time during co-cultivation the cell index drops below zero, the cell lysis of the sample cannot be calculated. In 9 out of 10 tumors tested, the cellular index dropped below zero, resulting in an inability to properly assess tumor cell lysis.

Among evaluable tumors, the PD-1 selected TIL exhibited greater ability to kill autologous tumors compared to unselected TIL, as determined by a greater decrease in cell index (a parameter reflecting cell proliferation when increased and cell detachment/death when decreased) and a higher percentage of cytolysis (fig. 172).

The results of this analysis show that the TIL selected with PD-1 has a greater ability to kill autologous tumors when compared to the unselected counterpart. This result is in contrast to freshly isolated and ex vivo amplified PD-1⁺The antitumor activity of both TILs is superior to PD-1^-Published reports of antitumor activity of TIL are consistent (gross et al, 2014; Inozume et al, 2010). For PD-1 isolated from NSCLC⁺TIL has been performed similarlyObservations indicate that this finding is not melanoma specific (Thommen et al, 2018).

EH12.2H7 and nivolumab for selecting PD-1⁺Equivalence of TIL (example 19)

To establish equivalence, sorting of PD-1 derived from the use of study (EH12.2H7) and GMP (nivolumab) anti-PD-1 monoclonal antibodies was compared⁺TIL selected by PD-1. Three tumor digests (1 ovary, 1 melanoma, and 1 HNSCC) were stained with EH12.2H7 or nivolumab to identify PD-1 ⁺And (4) a group. Amplification of nivolumab and EH12.2H7 sorted PD-1 Using a two-step procedure⁺A cell, the two-step process comprising an 11-day activation step and 11-day REP for a total of 22 days.

In general, this work demonstrated that PD-1-selected TILs generated from PD-1-sorted TILs using nivolumab were comparable to TILs generated using EH12.2H7 clone, supporting PD-1 selection in the manufacture of amplified TIL product studies using nivolumab.

PD-1 sorted TIL ex vivo amplification Using nivolumab and EH12.2H7

To determine whether PD-1-selected TILs generated using nivolumab and EH12.2H7 proliferated similarly, PD-1-sorted TILs from 1 ovary, 1 melanoma, and 1 HNSCC were subjected to an 11-day activation step followed by 11-day REP, and yield and amplification were assessed.

Tumor digests stained with EH12.2H7 and nivolumab expressed similar levels of CD3⁺PD-1⁺The cells were also represented as indistinguishable dot patterns on the flow cytometer (fig. 172 and table 79). When comparing the two TIL populations, the fold expansion in the activation step and REP and the total extrapolated/actual cell yield are similar.

In conclusion, EH12.2H7 and nivolumab identified a similar percentage of PD-1 in tumor digests ⁺A cell. Fold expansion of TIL and total extrapolated cell counts in three nivolumab and EH12.2H7 stained tumor samples were comparable.

Use for PD-1⁺Phenotypic characterization of sorted EH12.2H7 and PD-1-selected TILs produced by nivolumab

Evaluation of PD-1 Using EH12.2H7 and nivolumab by flow cytometry⁺TIL-produced PD-1-selected TIL expression of CD4, CD8, CCR7, CD45RA, and PD-1. A broader phenotypic evaluation can be seen in example 19.

No significant difference was observed in the expression of PD-1 selected CD4 and CD8 derived using nivolumab and EH12.2H7. Relative to CD4 for the three tumors tested⁺T cells, both TIL products produced a higher proportion of CD8⁺T cells (fig. 174).

The similarity of CD4 and CD8 expression in the three PD-1-selected TIL products indicates that PD-1 was selected using nivolumab as compared to EH12.2H7⁺The ratio of CD4/CD8 was not changed.

Like the T cell lineage, the memory status of TIL was similar in PD-1 selected TIL generated using EH12.2H7 and nivolumab. The TIL population is composed mainly of effector memory T cells (fig. 175).

PD-1 selected TIL generated using nivolumab and EH12.2H7 was similar to the LN-145 study product, indicating that selection of PD-1 using either anti-PD-1 clone did not skew the memory phenotype of TIL.

To assess whether PD-1 expression decreased similarly after culture, PD-1-selected TIL generated using nivolumab and EH12.2H7 was assessed before and after amplification. After sorting, PD-1 in two freshly sorted TIL preparations⁺The percentage of TIL was close to 100% (table 79). In the PD-1 selection generated using EH12.2H7 and nivolumab, the expression of PD-1 was significantly and relatively reduced after amplification (fig. 176).

As discussed in FIG. 169, the reduction in PD-1 expression following amplification confirmed PD-1 with EH12.2H7 and nivolumab⁺Sorted TILs revert primarily to PD-1 with amplification^-. PD-1 sorted from EH12.2H7 and nivolumab⁺Functional characterization of TIL-generated PD-1-selected TIL.

To evaluate amplified PD-1 derivatized with nivolumab⁺Whether TIL has similar function to TIL derived using EH12.2H7 clone, the trans-3 tumors from whichPD-1-selected TILs were non-specifically stimulated with α CD3/α CD28/α 41 BB-activated beads and evaluated for IFN γ and granzyme B secretion.

Nivolumab and EH12.2H7-derived PD-1-selected TIL produced similar levels of IFN γ and granzyme B in response to stimulation (fig. 177).

PD-1-selected TILs generated using nivolumab and EH12.2H7 secreted appreciable levels of IFN γ and granzyme B in response to non-specific stimulation (α CD3/α CD28/α CD137 beads), indicating that the selected TILs were highly functional after amplification.

Conclusion

In summary, PD-1 was obtained from all 34 tumor samples tested⁺TIL, the tumor sample comprising samples of CRC, NSCLC, HNSCC, TNBC, melanoma, OC, and PC. The percentage of TILs expressing high levels of PD-1 is variable within a given tumor type, and is not correlated across tumor types with known clinical responsiveness to anti-PD-1 therapy.

Importantly, the predicted yield of PD-1-selected TILs following ex vivo amplification was well within the clinical dose range specified for the LN-145 study product. In addition, the phenotype of the amplified PD-1-selected TIL was similar to the matched unselected TIL product, as well as the phenotype exhibited by the LN-145 product, but the PD-1-selected TIL product maintained low to moderate levels of PD-1 expression.

Finally, the PD-1 selected TIL products showed excellent autologous tumor reactivity and tumor cell killing when compared to the matched unselected TILs. This observation is consistent with the significant enrichment of the most prevalent TCRv β sequences found in PD-1 selected products relative to the level of these sequences in the matched unselected TIL products. Since published studies indicate that neoantigen-reactive T cells obtained from tumors express PD-1(6, 8), enhanced tumor reactivity is expected.

Summary non-clinical studies directed to PD-1-selected TILs strongly support clinical development of amplified TIL products of ACT of solid tumors.

Reference to example 20

Ahmadzadeh M, Johnson LA, Heemskerk B, Wunderlich JR, Dudley ME, White DE and Rosenberg SA (2009), tumor antigen-specific CD 8T cells infiltrating tumors express high levels of PD-1 and are functionally impaired (blood 114: 1537-1544).

Badoual C, Hans S, Melillon N, Van Ryswick C, Ravel P, Benhamouda N, Leviononis E, Nizard M, Si-Mohamed A, Besnier N, Gey A, Rotem-Yehudar R, Pere H, Tran T, Guerin CL, Chauvat A, Dransart E, Alanio C, Albert S, Barry B, Sandoral F, Quintin-Colonna F, Bruneval P, Fridman WH, Lemoine FM, Oudard S, Johannes L, Olive D, Brasnud and Tartour E (2013), tumor-infiltrating T cells expressing PD-1 being advantageous biomarkers in HPV-related head and neck cancer 128: 73: 138-.

Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ and Ahmed R (2006), restoration function in depleted CD 8T cells during chronic viral infection (restoration function in oxygenated CD 8T cell reduction viral infection), Nature 439: 682-.

Chikuma S, Terawaki S, Hayashi T, Nabeshima R, Yoshida T, Shibayama S, Okazaki T and Honjo T (2009), PD-1-mediated inhibition of IL-2production induces CD8+ T cell anergy in vivo (PD-1-mediated depression of IL-2production inductors CD8+ T cell indexing in vivo) & J Immunol & 182:6682 and 6689.

Donia M, Kjeldsen JW, Andersen R, Westergaard MCW, Bianchi V, Legut M, Attaf M, Szomolay B, Ott S, Dolton G, Lyngaa R, Hadrup SR, Sewell AK and Svine IM (2017), PD-1(+) multifunctional T cells in cancer tumor infiltrating lymphocyte therapy after peripheral [ clinical cancer research 23:5779- ]5788.

Fernandez-Poma SM, Salas-Benito D, Lozano T, Casares N, Riezu-Boj JI, Mancheno U, Elizalde E, Alignani D, Zubeldia N, Otano I, Conde E, Sarobe P, Lasarte JJ and Hervas-Stubbs S (2017), expansion of tumor-infiltrating CD8(+) T cells expressing PD-1 improved the efficacy of adoptive T cell therapy [ cancer research 77: 3672-3684- ].

Geukes Foppen MH, Donia M, Svine IM and Haanen JB (2015), Tumor-infiltrating lymphocytes for the treatment of metastatic cancer (Tumor-infiltrating lymphocytes for the treatment of the metastatic cancer).: molecular oncology 9: 1918-.

Golubrookaya V and Wu L (2016), different subsets of T cells, memory, effector functions and CAR-T immunotherapy, cancer (Basel) 8.

Gros A, Robbins PF, Yao X, Li YF, Turcotte S, Tran E, Wunderlich JR, Mixon A, Farid S, Dudley ME, Handda K, Almeida JR, Darko S, Douek DC, Yang JC, and Rosenberg SA (2014), PD-1 identifies patient-specific CD8(+) tumor reactive pools infiltrating human tumors, J.Clin. Res.124: 2246-.

Inozume T, Hanada K, Wang QJ, Ahmadzadeh M, Wunderlich JR, Rosenberg SA and Yang JC (2010), selecting CD8+ PD-1+ lymphocytes in fresh human melanoma enriched for tumour-reactive T cells J.Immunotherapy 33: 956-964.

Jung W, Gershan JA, Blitzer GC, Palen K, Weber J, McOlash L, Riese M and Johnson BD (2017), adoptive cell therapy using PD-1(+) myeloma-reactive T cells abolished established myeloma in mice < J.Immunotherapy cancer 5: 51.

Kansy BA, Concha-Benavente F, Srivastava RM, Jie HB, Shayan G, Lei Y, Moskovitz J, Moy J, Li J, Brandau S, Lang S, Schmitt NC, Freeman GJ, Gooding WE, Clump DA and Ferris RL (2017), and the PD-1 status in CD8(+) T cells correlates with survival and anti-PD-1 treatment in head and neck cancer & cancer research 77: 6353-.

McGranahan N, furnessAJ, Rosenthal R, Ramskov S, Lyngaa R, Saini SK, Jamal-Hanjani M, Wilson GA, Birkbak NJ, Hiley CT, Watkins TB, Shafi S, Murugaseu N, Mitter R, Akarca AU, Linares J, Marafioti T, Henry JY, Van Allen EM, Miao D, Schilling B, Schendedorf D, Garraway LA, Makarov V, Rizvi NA, Snalder A, Hellmann MD, Merghoub T, Wolchok JD, Shugla SA, Wu CJ, Peggs KS, Chan TA, Hadrup SR, Quezada SA and Swanton C (2016T) and the scientific blocking sensitivity of immune response to immune response of cells to 1469.

Peper JK, Schuster H, Loffler MW, Schmid-Horch B, Rammensee HG and Stevanovic S (2014), impedance-based cytotoxicity assays for real-time and label-free assessment of T-cell-mediated killing of adherent cells (An impedance-based cytotoxicity assay for real-time and label-free assays of T-cell-mediated killing of additive cells) & J Immunol Methods 405: 192. 198.

Schumacher TN and Schreiber RD (2015), a novel antigen in cancer immunotherapy, science 348: 69-74.

Shang J, Song Q, Yang Z, Sun X, Xue M, Chen W, Yang J and Wang S (2018), Analysis of PD-1related immune transcriptional profiles in different Cancer types (Analysis of PD-1related immune transcriptional profile in differential Cancer types), "Cancer Cell International (Cancer Cell Int) 18: 218.

Thommen DS, Koelzer VH, Herzig P, Roller A, Trefny M, Dimeloe S, Kiialainen A, Hanhart J, Schill C, Hess C, Savic Prince S, Wiese M, Lardinois D, Ho PC, Klein C, Karanikas V, Mertz KD, Schumacher TN and Zippelius A (2018), transcriptional and functionally different PD-1(+) CD8(+) T cell pools with predictive potential in non-small cell lung cancer treated with PD-1 blocking.

Example 21: high PD-1 selection and amplification for manufacturing

Introduction to

Several studies have demonstrated that surface expression of high levels of PD-1, a marker often associated with T cell depletion, identifies autologous tumor-reactive T cells in the tumor microenvironment (section 11.10). This example provides a protocol designed to select PD-1 positive (PD-1+) cells from tumor digests to enrich TIL products from autologous tumor-reactive T cells (example 15). This scheme is suitable for selectively obtaining TILs with high levels of PD-1.

Purpose(s) to

The purpose of this example was to develop a method for sorting andamplification of PD-1^{Height of}TIL to a method of manufacturing clinical trial materials.

Range

The examples provide expanded sorted PD-1 from melanoma, lung and head and neck tumors using a 2-REP protocol designed for full-scale clinical manufacturing^{Height of}TIL。

Amplification of three full-Scale PD-1 s as described below^{Height of}Selected Gen 2 process cultures.

On day 0, tumor digests were isolated using a GMP digestion mix containing neutral protease, dnase I and collagenase. Digests were washed, stained and sorted by FACS to purify PD-1^{Height of}TIL。

Sorted PD-1 with 100e6 allogeneic feeder cells and 30ng/mL OKT3 on day 0^{Height of}TIL initiated REP-1 for 11 days.

REP-2 was initiated on day 11 using the collected REP-1 product. The REP-2 (day 11) and subsequent day 16 and day 22 procedures were performed according to IOVA manufacturing lot records (see chapter 12 annex). A brief explanation of the relevant time points is outlined below.

The expanded TILs were evaluated for cell growth, viability, phenotype, telomere length and function (IFN γ and granzyme B secretion, CD107a mobilization).

Method

Material

Tumor tissue

Various histological tumors will be obtained from research unions and tissue procurement suppliers.

A standard reagent for TIL growth comprising: G-Rex 100MCS and 500MCS flasks (Wilson Walf, Cat. Nos. 81100-CS, 85500S-CS, respectively); GMP recombinant IL-2(Cell-Genix, Germany, catalog No. 1020-1000); all media reagents for CM1, CM2, and CM4 can be found in manufacturing lot records, as seen in annex 5-7; GlutaMAX 100X (Saimer Feishale, catalog number 35050061); gentamicin 50mg/mL (Saimei Feishale, catalog number 15750060)

Flow cytometry staining and analytical reagents

Flow cytometry antibody: anti-PD-1 PE, clone EH12.2H7, Biolegend, Cat No. 329906; anti-CD 3 FITC, clone OKT3, Biolegend, catalog No. 317306; and anti-IgG 4 Fc-PE, clone HP6025, southern Biotechnology Inc., catalog No. 9200-09.

Sorting buffer solution: HBSS with 2% FBS, 1mM EDTA and sterile Gemini filtration.

Collecting a buffer solution: HBSS with 50% hAB serum.

Procedure

Preparation of tumor tissue

Freshly resected tumor samples will be obtained from research consortia and tissue procurement suppliers. Tumors were shipped overnight in HypoThermosol (BioLifecol solutions, Washington, Cat. 101104) (containing antibiotics).

Images of the vial/tube tumors were taken. Tumors were removed from the package and washed 3X each time in tumor wash buffer (filtered HBSS with 50ug/mL gentamicin) for 2 minutes.

The whole tumor was fragmented into 4-6-mm pieces in preparation for tumor digestion. The 6-mm fragments were held in the wells of a 6-well plate containing 10mL of tumor wash buffer.

Enzyme preparation for tumor digestion

Tumors were digested with GMP collagenase and neutral protease as described below.

Collagenase AF-1(Nordmark, Sweden, N0003554) was reconstituted in 10ml of sterile HBSS. The concentration of lyophilized stock enzyme was 2892PZ U/vial. Thus, after reconstitution, the collagenase stock was 289.2PZ U/ml. Note that the stock of enzymes can be varied, confirming the concentration of the lyophilized stock and modifying the final amount of enzymes added to the digestion mixture accordingly. Aliquots were divided into 100uL aliquots and stored at-20 ℃.

Neutral protease (Nordmark, Sweden, N0003553) was reconstituted in 1ml sterile HBSS. The lyophilized stock enzyme concentration was 175DMC U/vial. Thus, after reconstitution, the neutral protease stock solution was 175 DMC/ml. Note that the stock of enzymes can be varied, confirming the concentration of the lyophilized stock and modifying the final amount of enzymes added to the digestion mixture accordingly. Aliquots were divided into 20uL aliquots and stored at-20 ℃.

DNase I (Roche, Switzerland, 03724751) was reconstituted in 1ml sterile HBSS. The concentration of lyophilized stock enzyme was 4 KU/vial. Thus, after reconstitution, the DNase stock was 4 KU/ml. Note that the stock of enzymes can be varied, confirming the concentration of the lyophilized stock and modifying the final amount of enzymes added to the digestion mixture accordingly. Aliquots were divided into 250uL aliquots and stored at-20 ℃.

Tumor treatment and digestion

Each C-tube was transferred to a GentleMACS octodispatcher. Digestion was performed by setting the dissociator to the appropriate procedure for the corresponding tumor histology listed in table 80 below. Dissociation will take about one hour.

Table 80: american and whirlwind octodispatcher programs based on tumor tissue type.

After digestion, one or more C-tubes are removed from the octodis or spinner and placed in the BSC. Digests were removed from each C tube with a 25-mL serum pipette and bulk digests were passed through a 70- μm cell strainer into a 50-mL conical tube. Undigested portions of the tumor may not pass through the screen, not allowing splattering of digesta due to pressure from the pipette. Wash one or more C-tubes with an additional 10mL of HBSS and pass the wash through the cell strainer. 50-mL conical tubes QS were brought to 50mL with HBSS.

The conical tube was transferred to BSC and the supernatant was aspirated or decanted. The pellet was resuspended in 5mL of warmed CM-1+6000IU/mL IL-2 and pipetted 5-6 times up and down. 2 cell counts were performed on NC-200 without dilution per WRK LAB-056.

1mL of digest was placed aside for CD3+ bulk control and aliquots of 2X 500uL of digest were cryopreserved for tumor reactivity assays. Digestion was maintained on ice.

Staining of digested tumors for flow cytometry analysis and cell sorting

Small samples (approximately 1e5 cells) for PE and FITC monochrome compensation controls were set aside in 15-mL conical tubes.

The remaining tumor digests were stained with a mixture comprising anti-PD-1-PE, anti-IgG 4 Fc-PE (secondary antibodies against nivolumab and pembrolizumab), and anti-CD 3-FITC according to the following protocol. PE monochrome compensation control was stained with secondary anti-PD-1-PE plus IgG4, and FITC color compensation control was stained with anti-CD 3-FITC only.

After cell counting, 10mL of HBSS was added for digestion and centrifuged at 400x G for 5 minutes at room temperature (full acceleration and full braking).

The conical tube was transferred to BSC and the supernatant was decanted. A micropipette was used to obtain the volume of digest remaining after decantation. Add 3x this volume of sort buffer to the tube. That is, if the obtained volume is 150uL, 450uL of sorting buffer is added, and the total volume is 600 uL.

anti-CD 3-FITC was added in 3 μ L per 100 μ L (i.e. 6 × 3 ═ 18uL of antibody if the volume was 600 uL). (added to both samples).

2.5 μ L of anti-PD-1-PE was added per 100 μ L (i.e. if the volume was 600uL, 6 × 2.5 ═ 15.0uL of antibody was added). (not to be added to FMO).

anti-IgG 4-Fc-PE was added at a dilution of 1:500 (i.e., 1uL of antibody was added for each 500uL volume).

The fully stained cells were resuspended in 10mL of sorting buffer and 10mL of sorting buffer was added to the FMO.

The fully stained solution was passed through a 30- μm cell strainer into a 15-mL conical tube and the FMO was passed through a 30- μm cell strainer into a 15-mL conical tube as well.

Cells were resuspended in up to 10e6/mL total cells (live and dead) in sorting buffer. The minimum volume was 300. mu.l.

Transfer to a 15-mL conical tube. The tubes were stored on ice, covered with aluminum foil until further use.

A15-mL collection tube was prepared for the sorted population. 2mL of collection buffer (D-PBS with 2% hAB serum) was placed in the tube. The collection tubes were stored on ice until further use.

Cell count and viability assessment

Using Chemometec NC-200 cytometer, the procedure used to obtain cell and viability counts was used

FACS sorting (FX500 Start)

The BSC is turned on. The JUN-AIR vacuum pump was turned on. The FX500 is turned on by pressing the power/Standby button on the front of the instrument. By double-clicking the icon on the desktop, the cell sorter software is turned on, and the program is logged in and run.

Run auto-calibration

When prompted to load calibration beads, 15 drops of the auto-set beads were added to a 5-mL sterile FACS tube. And then the operation is carried out according to the prompt. When prompted to select settings for auto-calibration, the "standard" radio button is selected. While waiting for the calibration to complete, the following were prepared: preparing five sterile 15-mL conical tubes by using 10mL sterile deionized water; five sterile 5-mL FACS tubes were prepared with 4mL sterile deionized water; five sterile 15-mL conical tubes were prepared with 12mL of 70% EtOH; and five sterile 15-mL conical tubes were prepared with 12mL of 10% sodium hypochlorite.

Sample collection

Confirm the sample chamber and collection chamber are at 5 ℃ and select stir sample icon. Click the cytometer tab at the top of the screen. The 5 ℃ icon and the sample 5 ℃ icon are collected in a single click. Click the stir icon.

Confirm that the sample has been compensated. Click the compensation tab at the top of the screen. The compensation icon should be light blue. Tubes containing PBMC controls (5-mL FACS tubes or 15-mL conical tubes) were placed on the sample collection platform. The sample collection pressure was set to 6. Single click play, start sample collection. The gate and statistics are clicked to display the following at the top of the screen.

100,000 is selected for the two drop down menus seen above. Confirming that the cell population was correctly gated. See the examples below. It may be necessary to adjust BSC or FSC settings. The voltage of any other channel is not adjusted. PD-1 gates were not adjusted. Recording as many events as possible (or up to 20,000 CD3 events) can set the sample pressure to 10 to speed up this collection. The collection was stopped and the tube removed. The previously prepared 15-mL sterile dH20 cone was loaded onto the sample platform. 10 was selected as the sample pressure. A run icon is clicked. The samples were collected for one minute. Click the restart icon. The event is repeated until the CD3 gate is empty. The dH20 sample tube was removed and discarded. The sample to be collected is added to the loading platform. The confirmation setup is shown in the following figure:

The sample chamber door was opened and a 15-mL collection chamber block was loaded into the chamber. The collection tube containing the collection buffer was loaded into the chamber block. The capped tube was inverted several times to coat the top of the tube with the collection buffer. The tube was tapped on the surface of the BSC to remove excess buffer from the top of the tube and cap. One tube is labeled with the sample name and plus sign. The lid is removed and placed into the left chamber. The second tube is labeled with the sample name and minus sign. The cap is removed and placed into the right chamber.

Click on the load gather icon shown in the figure. 4 was selected as the sample pressure. A run icon is clicked. Wait for the cells to appear on the screen. About 15 seconds. The sample pressure was adjusted so that the total events per second was below 5,000. Click start sort icon. Sample pressure was adjusted to maintain a sorting efficiency of at least 85%. 50,000 CD3 events were recorded. See the figure below. The recording will automatically stop. Upon the appearance of the dialog box, the ok button is clicked.

The cells collected in either fraction exceeded 4.5X 10⁶In either case, one or more collection tubes would need to be replaced. Click-stop sort icon. A pause icon is clicked. Opening the collection chamber A door for the chamber, and replacing the original collection tube with a new one; the door was closed and the raw collection tube was placed on ice. Click the next tube icon. Click load collect icon. A play icon is clicked. Click start sort icon.

Sorting was continued until all samples were removed from the sample tubes. It is also possible if the tube runs "dry". The sample tube is removed from the sample chamber. And (4) discarding. The sorted fraction is removed from the collection chamber. The tube was capped and inverted several times gently to incorporate a droplet near the top of the tube into the solution. Gently tap the tube onto the surface of the BSC to remove excess solution from the top of the tube and cap. The tubes were placed on ice. The percent selectivity of the PD-1 fraction was confirmed. A14-mL EtOH cone was placed on the sample chamber. Click the probe wash icon. And (6) repeating. The EtOH tube was removed and then the positive fraction tube was added. The sample pressure value was changed to 7. Click the next tube icon. Tubes were named with sample name and "positive selection". A single click plays and records 75 CD3 positive events. The tube is immediately stopped and unloaded from the sample chamber. The steps were repeated for negatively selected samples.

And exporting the data. PD-1 FMO tubes were selected by double click. Select file/print. Printed in PDF format rather than a printer. This would provide a complete 6-page sample report. The above operation is repeated for each tube collected. The instrument was turned off.

PD-1^{Height of}Rapid amplification protocol

Day 0-REP 1

Preparing a culture medium: 1L of CM-1+6000IU/mL IL-2 was prepared or warmed.

PBMC feeder cell preparation: thawing an appropriate number of vials (100 e6 PBMC for full scale and 10e6 for bulk CD3+ control would be required, assuming 60e6-80e6 PBMC per 1mL vial) for REP-1. 40mL of warmed CM1+ IL-2 was placed in a 50mL conical tube and a 1mL PBMC feeder cell vial was pipetted into the conical tube. Thawed PBMC feeder cells were pipetted up and down for thorough mixing and 2 cell counts on NC-200.

Appropriate volumes were calculated for transfer to G-Rex 100M and G-Rex 10M to transfer 100e6 and 10e6 PBMCs, respectively.

30uL of aCD3(OKT-3) was added to G-Rex 100M and 3uL was added to G-Rex 10M. The flask was placed in an incubator

Inoculation of TIL for REP-1

All PD-1 is put^{Height of}Sorting was performed in G-Rex 100M. CD3+ bulk TIL control conditions will be all-scale to PD-1 at a ratio of 1/10^{Height of}The cells were supplemented with equal numbers of CD3+ cells. To obtain a suitable volume of digest, the following steps are followed: 1) CD3+ TVC/mL in the digest was calculated by multiplying the digest TVC obtained in step 9.3.5 by the CD3 +% of live cells obtained from the sort report. (i.e., 10e6 × 10% ═ 1e6), 2) after this number was obtained, PD-1 inoculated into full scale conditions ^{Height of}The number of cells is divided by this number. (i.e., 1e5/1e6 ═ 0.1mL), and 3) this volume (0.1mL) of digest was added to a bulk CD3+ TIL flask and filled to 100mL with CM1+ IL-2. All flasks were placed at 37 ℃ in 5% CO₂An incubator.

Day 11, day 16, day 22

Followed by a full scale process. The ontology CD3+ TIL condition was processed similarly to the steps described in example 9.

Acceptance criteria

Table 81 below specifies the acceptance criteria for evaluating the performance of three full-scale batches.

Table 81: collecting product test and acceptance criteria

Additional final product characterization tests performed are specified in table 82 below for reference only.

Table 82: characterization of the final product (for reference only)

Reference to example 21

Rosenberg, S.A. et al, using T cell metastatic immunotherapy, sustained complete response in severely pretreated patients with metastatic melanoma clinical cancer research 2011.17(13): 4550-7.

Kvistborg, P. et al, TIL therapy broadens the tumor-reactive CD8(+) T-cell compartment in melanoma patients tumor immunology, 2012.1(4): 409-.

Simoni, Y, et al, bystander CD8(+) T cells are abundant in human tumor infiltration and are phenotypically different (Nature, 2018.557(7706): 575) 579.

Schumacher, t.n. and r.d. schreiber, neoantigens in cancer immunotherapy, science 2015.348(6230): 69-74.

Turcote, s. et al, phenotype and function of T cells infiltrating visceral metastases from gastrointestinal cancer and melanoma: adoptive cell transfer therapy has been suggested in J.Immunol. 2013.191(5): 2217-25.

Gross, A. et al, PD-1 identified patient-specific CD8(+) tumor-reactive repertoire infiltrating human tumors, J.Clin. Res. 2014.124(5): pp 2246-59.

The examples set forth above are provided to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the compositions, systems, and methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains.

All headings and section designations are used for clarity and reference only and should not be considered limiting in any way. For example, those skilled in the art will recognize the usefulness of various aspects from different headings and sections combined as appropriate in accordance with the spirit and scope of the present invention as described herein.

All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent application was specifically or individually indicated to be incorporated by reference in its entirety for all purposes.

Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Sequence listing

<110> Aowenware Biotherapeutics, Inc. (Iovance Biotherapeutics, Inc.)

<120> improved selection of tumor-reactive T cells

<130> 116983-5020

<150> US 62/756,006

<151> 2018-11-05

<150> US 62/826,831

<151> 2019-03-29

<150> US 62/903,629

<151> 2019-09-20

<150> US 62/924,602

<151> 2019-10-22

<160> 126

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Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

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Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

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Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

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Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

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Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr

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Pro Leu Ala Pro Val Cys Gly Gly Thr Thr Gly Ser Ser Val Thr Leu

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Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp

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Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

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Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser

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Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser

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Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Pro Lys Ser Cys Asp

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Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

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Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

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Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

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Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

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Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

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Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

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Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

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Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

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Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

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Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

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Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

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Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

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Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

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Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser

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Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu

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Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

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Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Ala Asp Thr Ala Pro

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Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly

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Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn

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Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn

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Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser

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Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu

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His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr

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Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro

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Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu

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Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His

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Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu

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Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr

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Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser

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Ile Ile Ser Thr Leu Thr

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Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

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Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

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Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

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Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

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<213> Artificial Sequence (Artificial Sequence)

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Met His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn

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Ser Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr Asp

20 25 30

Ile Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg

35 40 45

Ala Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr

50 55 60

Arg Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu

65 70 75 80

Ile Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly

85 90 95

Leu Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn

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Phe Leu Glu Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys

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Ser Ser

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Met Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val

1 5 10 15

Leu Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly

20 25 30

Ser Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys

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Asp Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu

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Arg Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu

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Val Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys

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Ser Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp

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Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn

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Met Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

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Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

20 25 30

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

35 40 45

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

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Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

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Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

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Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

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Asn Thr Ser

115

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Met Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val

1 5 10 15

Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro

20 25 30

Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys

35 40 45

Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg

50 55 60

Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr

65 70 75 80

Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp

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Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser

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Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly

115 120 125

Ser Glu Asp Ser

130

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<213> Artificial Sequence (Artificial Sequence)

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<223> human 4-1BB, tumor necrosis factor receptor superfamily, member 9 (homo sapiens)

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Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu

180 185 190

Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu

195 200 205

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

210 215 220

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

225 230 235 240

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

245 250 255

<210> 10

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<223> murine 4-1BB, tumor necrosis factor receptor superfamily, member 9 (mus musculus)

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Met Gly Asn Asn Cys Tyr Asn Val Val Val Ile Val Leu Leu Leu Val

1 5 10 15

Gly Cys Glu Lys Val Gly Ala Val Gln Asn Ser Cys Asp Asn Cys Gln

20 25 30

Pro Gly Thr Phe Cys Arg Lys Tyr Asn Pro Val Cys Lys Ser Cys Pro

35 40 45

Pro Ser Thr Phe Ser Ser Ile Gly Gly Gln Pro Asn Cys Asn Ile Cys

50 55 60

Arg Val Cys Ala Gly Tyr Phe Arg Phe Lys Lys Phe Cys Ser Ser Thr

65 70 75 80

His Asn Ala Glu Cys Glu Cys Ile Glu Gly Phe His Cys Leu Gly Pro

85 90 95

Gln Cys Thr Arg Cys Glu Lys Asp Cys Arg Pro Gly Gln Glu Leu Thr

100 105 110

Lys Gln Gly Cys Lys Thr Cys Ser Leu Gly Thr Phe Asn Asp Gln Asn

115 120 125

Gly Thr Gly Val Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg

130 135 140

Ser Val Leu Lys Thr Gly Thr Thr Glu Lys Asp Val Val Cys Gly Pro

145 150 155 160

Pro Val Val Ser Phe Ser Pro Ser Thr Thr Ile Ser Val Thr Pro Glu

165 170 175

Gly Gly Pro Gly Gly His Ser Leu Gln Val Leu Thr Leu Phe Leu Ala

180 185 190

Leu Thr Ser Ala Leu Leu Leu Ala Leu Ile Phe Ile Thr Leu Leu Phe

195 200 205

Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln

210 215 220

Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser

225 230 235 240

Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu

245 250 255

<210> 11

<211> 441

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain of Utomimilu monoclonal antibody

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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Thr Tyr

20 25 30

Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Lys Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Ile Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe

180 185 190

Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro

210 215 220

Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210> 12

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain of Utomimilu monoclonal antibody

<400> 12

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Ile Gly Asp Gln Tyr Ala

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Lys Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Tyr Thr Gly Phe Gly Ser Leu

85 90 95

Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

115 120 125

Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

130 135 140

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

145 150 155 160

Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

165 170 175

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

180 185 190

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Thr Glu Cys Ser

210

<210> 13

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<223> heavy chain variable region of Utomimilu monoclonal antibody

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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Thr Tyr

20 25 30

Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Lys Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Gly Ile Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 14

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<223> light chain variable region of Utomimilu monoclonal antibody

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Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Ile Gly Asp Gln Tyr Ala

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Lys Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Tyr Thr Gly Phe Gly Ser Leu

85 90 95

Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 15

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR1 of Utomimilu monoclonal antibody

<400> 15

Ser Thr Tyr Trp Ile Ser

1 5

<210> 16

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR2 of Utomimilu monoclonal antibody

<400> 16

Lys Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 17

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR3 of Utomimilu monoclonal antibody

<400> 17

Arg Gly Tyr Gly Ile Phe Asp Tyr

1 5

<210> 18

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR1 of Utomimilu monoclonal antibody

<400> 18

Ser Gly Asp Asn Ile Gly Asp Gln Tyr Ala His

1 5 10

<210> 19

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR2 of Utomimilu monoclonal antibody

<400> 19

Gln Asp Lys Asn Arg Pro Ser

1 5

<210> 20

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR3 of Utomimilu monoclonal antibody

<400> 20

Ala Thr Tyr Thr Gly Phe Gly Ser Leu Ala Val

1 5 10

<210> 21

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain of Urru mab

<400> 21

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly

210 215 220

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 22

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain of Urelumab

<400> 22

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Ala Leu Thr Phe Cys Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 23

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> variable heavy chain of Urru mab

<400> 23

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys

20 25 30

Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe

35 40 45

Ser Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu

50 55 60

Glu Trp Ile Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro

65 70 75 80

Ser Leu Glu Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln

85 90 95

Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

100 105 110

Tyr Cys Ala Arg Asp Tyr Gly Pro

115 120

<210> 24

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> variable light chain of Urru mab

<400> 24

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

50 55 60

Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln

100 105 110

<210> 25

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR1 of Uriluzumab

<400> 25

Gly Tyr Tyr Trp Ser

1 5

<210> 26

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR2 of Uriluzumab

<400> 26

Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser

1 5 10 15

<210> 27

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR3 of Uriluzumab

<400> 27

Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu

1 5 10

<210> 28

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR1 of Uriluzumab

<400> 28

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210> 29

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR2 of Uriluzumab

<400> 29

Asp Ala Ser Asn Arg Ala Thr

1 5

<210> 30

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR3 of Uriluzumab

<400> 30

Gln Gln Arg Ser Asp Trp Pro Pro Ala Leu Thr

1 5 10

<210> 31

<211> 230

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fc Domain

<400> 31

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

1 5 10 15

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

20 25 30

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

35 40 45

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

50 55 60

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

65 70 75 80

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

85 90 95

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

100 105 110

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

115 120 125

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

130 135 140

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

145 150 155 160

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

165 170 175

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

180 185 190

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

195 200 205

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

210 215 220

Ser Leu Ser Pro Gly Lys

225 230

<210> 32

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> linker

<400> 32

Gly Gly Pro Gly Ser Ser Lys Ser Cys Asp Lys Thr His Thr Cys Pro

1 5 10 15

Pro Cys Pro Ala Pro Glu

20

<210> 33

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> linker

<400> 33

Gly Gly Ser Gly Ser Ser Lys Ser Cys Asp Lys Thr His Thr Cys Pro

1 5 10 15

Pro Cys Pro Ala Pro Glu

20

<210> 34

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> linker

<400> 34

Gly Gly Pro Gly Ser Ser Ser Ser Ser Ser Ser Lys Ser Cys Asp Lys

1 5 10 15

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

20 25

<210> 35

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> linker

<400> 35

Gly Gly Ser Gly Ser Ser Ser Ser Ser Ser Ser Lys Ser Cys Asp Lys

1 5 10 15

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

20 25

<210> 36

<211> 29

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> linker

<400> 36

Gly Gly Pro Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Lys Ser Cys

1 5 10 15

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

20 25

<210> 37

<211> 29

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> linker

<400> 37

Gly Gly Ser Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Lys Ser Cys

1 5 10 15

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

20 25

<210> 38

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> linker

<400> 38

Gly Gly Pro Gly Ser Ser Gly Ser Gly Ser Ser Asp Lys Thr His Thr

1 5 10 15

Cys Pro Pro Cys Pro Ala Pro Glu

20

<210> 39

<211> 23

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> linker

<400> 39

Gly Gly Pro Gly Ser Ser Gly Ser Gly Ser Asp Lys Thr His Thr Cys

1 5 10 15

Pro Pro Cys Pro Ala Pro Glu

20

<210> 40

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> linker

<400> 40

Gly Gly Pro Ser Ser Ser Gly Ser Asp Lys Thr His Thr Cys Pro Pro

1 5 10 15

Cys Pro Ala Pro Glu

20

<210> 41

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> linker

<400> 41

Gly Gly Ser Ser Ser Ser Ser Ser Ser Ser Gly Ser Asp Lys Thr His

1 5 10 15

Thr Cys Pro Pro Cys Pro Ala Pro Glu

20 25

<210> 42

<211> 246

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Fc Domain

<400> 42

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Ala Gly Asn Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

20 25 30

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

35 40 45

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

50 55 60

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

65 70 75 80

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

85 90 95

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

100 105 110

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

115 120 125

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

130 135 140

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

145 150 155 160

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

165 170 175

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

180 185 190

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

195 200 205

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

210 215 220

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

225 230 235 240

Leu Ser Leu Ser Pro Gly

245

<210> 43

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> linker

<400> 43

Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

1 5 10

<210> 44

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> linker

<400> 44

Ser Ser Ser Ser Ser Ser Gly Ser Gly Ser Gly Ser

1 5 10

<210> 45

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> linker

<400> 45

Ser Ser Ser Ser Ser Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

1 5 10 15

<210> 46

<211> 254

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 4-1BBL

<400> 46

Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro

1 5 10 15

Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val

20 25 30

Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe

35 40 45

Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser

50 55 60

Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp

65 70 75 80

Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val

85 90 95

Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp

100 105 110

Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu

115 120 125

Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe

130 135 140

Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser

145 150 155 160

Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala

165 170 175

Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala

180 185 190

Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala

195 200 205

Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His

210 215 220

Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val

225 230 235 240

Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

245 250

<210> 47

<211> 168

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 4-1BBL soluble Domain

<400> 47

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

1 5 10 15

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

20 25 30

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

35 40 45

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

50 55 60

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

65 70 75 80

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

85 90 95

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

100 105 110

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

115 120 125

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

130 135 140

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

145 150 155 160

Gly Leu Pro Ser Pro Arg Ser Glu

165

<210> 48

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 4B4-1-1 version 1 variable heavy chain

<400> 48

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Val Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Gly Asn Gly His Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Phe Thr Thr Ala Arg Gly Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser

115

<210> 49

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 4B4-1-1 version 1 variable light chain

<400> 49

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Gln Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asp Gly His Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 50

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 4B4-1-1 version 2 variable heavy chain

<400> 50

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Val Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn Pro Gly Asn Gly His Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Phe Thr Thr Ala Arg Gly Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 51

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 4B4-1-1 version 2 variable light chain

<400> 51

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Gln Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asp Gly His Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 52

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> variable heavy chain of H39E3-2

<400> 52

Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly

1 5 10 15

Ala His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asp Tyr Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Asp Ile Lys Asn Asp Gly Ser Tyr Thr Asn Tyr Ala

65 70 75 80

Pro Ser Leu Thr Asn Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Glu Leu Thr

115 120

<210> 53

<211> 109

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> variable light chain of H39E3-2

<400> 53

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala

20 25 30

Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser

35 40 45

Leu Leu Ser Ser Gly Asn Gln Lys Asn Tyr Leu Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Thr Arg Gln

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala

100 105

<210> 54

<211> 277

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> human OX40 (wisdom people)

<400> 54

Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu

1 5 10 15

Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val

20 25 30

Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro

35 40 45

Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys

50 55 60

Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro

65 70 75 80

Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys

85 90 95

Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly

100 105 110

Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys

115 120 125

Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp

130 135 140

Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn

145 150 155 160

Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro

165 170 175

Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr

180 185 190

Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu

195 200 205

Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val

210 215 220

Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu

225 230 235 240

Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly

245 250 255

Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser

260 265 270

Thr Leu Ala Lys Ile

275

<210> 55

<211> 272

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> murine OX40 (mouse)

<400> 55

Met Tyr Val Trp Val Gln Gln Pro Thr Ala Leu Leu Leu Leu Gly Leu

1 5 10 15

Thr Leu Gly Val Thr Ala Arg Arg Leu Asn Cys Val Lys His Thr Tyr

20 25 30

Pro Ser Gly His Lys Cys Cys Arg Glu Cys Gln Pro Gly His Gly Met

35 40 45

Val Ser Arg Cys Asp His Thr Arg Asp Thr Leu Cys His Pro Cys Glu

50 55 60

Thr Gly Phe Tyr Asn Glu Ala Val Asn Tyr Asp Thr Cys Lys Gln Cys

65 70 75 80

Thr Gln Cys Asn His Arg Ser Gly Ser Glu Leu Lys Gln Asn Cys Thr

85 90 95

Pro Thr Gln Asp Thr Val Cys Arg Cys Arg Pro Gly Thr Gln Pro Arg

100 105 110

Gln Asp Ser Gly Tyr Lys Leu Gly Val Asp Cys Val Pro Cys Pro Pro

115 120 125

Gly His Phe Ser Pro Gly Asn Asn Gln Ala Cys Lys Pro Trp Thr Asn

130 135 140

Cys Thr Leu Ser Gly Lys Gln Thr Arg His Pro Ala Ser Asp Ser Leu

145 150 155 160

Asp Ala Val Cys Glu Asp Arg Ser Leu Leu Ala Thr Leu Leu Trp Glu

165 170 175

Thr Gln Arg Pro Thr Phe Arg Pro Thr Thr Val Gln Ser Thr Thr Val

180 185 190

Trp Pro Arg Thr Ser Glu Leu Pro Ser Pro Pro Thr Leu Val Thr Pro

195 200 205

Glu Gly Pro Ala Phe Ala Val Leu Leu Gly Leu Gly Leu Gly Leu Leu

210 215 220

Ala Pro Leu Thr Val Leu Leu Ala Leu Tyr Leu Leu Arg Lys Ala Trp

225 230 235 240

Arg Leu Pro Asn Thr Pro Lys Pro Cys Trp Gly Asn Ser Phe Arg Thr

245 250 255

Pro Ile Gln Glu Glu His Thr Asp Ala His Phe Thr Leu Ala Lys Ile

260 265 270

<210> 56

<211> 451

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain of tavollizumab

<400> 56

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser Gly

20 25 30

Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu

65 70 75 80

Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 57

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain of tavollizumab

<400> 57

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ala Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 58

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain variable region of tavollizumab

<400> 58

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ser Gly

20 25 30

Tyr Trp Asn Trp Ile Arg Lys His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Thr Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu

65 70 75 80

Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr

115

<210> 59

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain variable region of tavollizumab

<400> 59

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ala Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 60

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR1 of tavollizumab

<400> 60

Gly Ser Phe Ser Ser Gly Tyr Trp Asn

1 5

<210> 61

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR2 of tavollizumab

<400> 61

Tyr Ile Gly Tyr Ile Ser Tyr Asn Gly Ile Thr Tyr His

1 5 10

<210> 62

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR3 of tavollizumab

<400> 62

Arg Tyr Lys Tyr Asp Tyr Asp Gly Gly His Ala Met Asp Tyr

1 5 10

<210> 63

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR1 of tavollizumab

<400> 63

Gln Asp Ile Ser Asn Tyr Leu Asn

1 5

<210> 64

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR2 of tavollizumab

<400> 64

Leu Leu Ile Tyr Tyr Thr Ser Lys Leu His Ser

1 5 10

<210> 65

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR3 of tavollizumab

<400> 65

Gln Gln Gly Ser Ala Leu Pro Trp

1 5

<210> 66

<211> 444

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain of 11D4

<400> 66

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ser Gly Trp Tyr Leu Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys

210 215 220

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr

290 295 300

Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 67

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain of 11D4

<400> 67

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 68

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 11D4 heavy chain variable region

<400> 68

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ser Gly Trp Tyr Leu Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 69

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 11D4 variable light chain region

<400> 69

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 70

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR1 of 11D4

<400> 70

Ser Tyr Ser Met Asn

1 5

<210> 71

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR2 of 11D4

<400> 71

Tyr Ile Ser Ser Ser Ser Ser Thr Ile Asp Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 72

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR3 of 11D4

<400> 72

Glu Ser Gly Trp Tyr Leu Phe Asp Tyr

1 5

<210> 73

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR1 of 11D4

<400> 73

Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala

1 5 10

<210> 74

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR2 of 11D4

<400> 74

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 75

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR3 of 11D4

<400> 75

Gln Gln Tyr Asn Ser Tyr Pro Pro Thr

1 5

<210> 76

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain of 18D8

<400> 76

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Gln Ser Thr Ala Asp Tyr Tyr Phe Tyr Tyr Gly Met Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg

210 215 220

Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg

290 295 300

Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 77

<211> 213

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain of 18D8

<400> 77

Glu Ile Val Val Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 78

<211> 124

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 18D8 heavy chain variable region

<400> 78

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Gln Ser Thr Ala Asp Tyr Tyr Phe Tyr Tyr Gly Met Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 79

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 18D8 light chain variable region

<400> 79

Glu Ile Val Val Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 80

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR1 of 18D8

<400> 80

Asp Tyr Ala Met His

1 5

<210> 81

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR2 of 18D8

<400> 81

Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 82

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR3 of 18D8

<400> 82

Asp Gln Ser Thr Ala Asp Tyr Tyr Phe Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 83

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR1 of 18D8

<400> 83

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210> 84

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR2 of 18D8

<400> 84

Asp Ala Ser Asn Arg Ala Thr

1 5

<210> 85

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR3 of 18D8

<400> 85

Gln Gln Arg Ser Asn Trp Pro Thr

1 5

<210> 86

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain variable region of Hu119-122

<400> 86

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Tyr Glu Phe Pro Ser His

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val

35 40 45

Ala Ala Ile Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met

50 55 60

Glu Arg Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Tyr Asp Asp Tyr Tyr Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 87

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain variable region of Hu119-122

<400> 87

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 88

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR1 of Hu119-122

<400> 88

Ser His Asp Met Ser

1 5

<210> 89

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR2 of Hu119-122

<400> 89

Ala Ile Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met Glu

1 5 10 15

Arg

<210> 90

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR3 of Hu119-122

<400> 90

His Tyr Asp Asp Tyr Tyr Ala Trp Phe Ala Tyr

1 5 10

<210> 91

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR1 of Hu119-122

<400> 91

Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His

1 5 10 15

<210> 92

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR2 of Hu119-122

<400> 92

Leu Ala Ser Asn Leu Glu Ser

1 5

<210> 93

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR3 of Hu119-122

<400> 93

Gln His Ser Arg Glu Leu Pro Leu Thr

1 5

<210> 94

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain variable region of Hu106-222

<400> 94

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 95

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain variable region of Hu106-222

<400> 95

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 96

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR1 of Hu106-222

<400> 96

Asp Tyr Ser Met His

1 5

<210> 97

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR2 of Hu106-222

<400> 97

Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys

1 5 10 15

Gly

<210> 98

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain CDR3 of Hu106-222

<400> 98

Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr

1 5 10

<210> 99

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR1 of Hu106-222

<400> 99

Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210> 100

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR2 of Hu106-222

<400> 100

Ser Ala Ser Tyr Leu Tyr Thr

1 5

<210> 101

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain CDR3 of Hu106-222

<400> 101

Gln Gln His Tyr Ser Thr Pro Arg Thr

1 5

<210> 102

<211> 183

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OX40L

<400> 102

Met Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg

1 5 10 15

Pro Arg Phe Glu Arg Asn Lys Leu Leu Leu Val Ala Ser Val Ile Gln

20 25 30

Gly Leu Gly Leu Leu Leu Cys Phe Thr Tyr Ile Cys Leu His Phe Ser

35 40 45

Ala Leu Gln Val Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val

50 55 60

Gln Phe Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln

65 70 75 80

Lys Glu Asp Glu Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn

85 90 95

Cys Asp Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu

100 105 110

Val Asn Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln

115 120 125

Leu Lys Lys Val Arg Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr

130 135 140

Tyr Lys Asp Lys Val Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser Leu

145 150 155 160

Asp Asp Phe His Val Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn

165 170 175

Pro Gly Glu Phe Cys Val Leu

180

<210> 103

<211> 131

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OX40L soluble Domain

<400> 103

Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln Phe Thr Glu

1 5 10 15

Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln Lys Glu Asp Glu

20 25 30

Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn Cys Asp Gly Phe

35 40 45

Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu Val Asn Ile Ser

50 55 60

Leu His Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln Leu Lys Lys Val

65 70 75 80

Arg Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr Tyr Lys Asp Lys

85 90 95

Val Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser Leu Asp Asp Phe His

100 105 110

Val Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn Pro Gly Glu Phe

115 120 125

Cys Val Leu

130

<210> 104

<211> 128

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OX40L soluble Domain (alternative)

<400> 104

Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln Phe Thr Glu Tyr Lys Lys

1 5 10 15

Glu Lys Gly Phe Ile Leu Thr Ser Gln Lys Glu Asp Glu Ile Met Lys

20 25 30

Val Gln Asn Asn Ser Val Ile Ile Asn Cys Asp Gly Phe Tyr Leu Ile

35 40 45

Ser Leu Lys Gly Tyr Phe Ser Gln Glu Val Asn Ile Ser Leu His Tyr

50 55 60

Gln Lys Asp Glu Glu Pro Leu Phe Gln Leu Lys Lys Val Arg Ser Val

65 70 75 80

Asn Ser Leu Met Val Ala Ser Leu Thr Tyr Lys Asp Lys Val Tyr Leu

85 90 95

Asn Val Thr Thr Asp Asn Thr Ser Leu Asp Asp Phe His Val Asn Gly

100 105 110

Gly Glu Leu Ile Leu Ile His Gln Asn Pro Gly Glu Phe Cys Val Leu

115 120 125

<210> 105

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 008 variable heavy chain

<400> 105

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Tyr Ser Gln Val His Tyr Ala Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser

115 120

<210> 106

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 008 variable light chain

<400> 106

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Ala Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Tyr

85 90 95

Tyr Asn His Pro Thr Thr Phe Gly Gln Gly Thr Lys

100 105

<210> 107

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 011 variable heavy chain

<400> 107

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Arg Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

65 70 75 80

Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Asp Arg Tyr Phe Arg Gln Gln Asn Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 108

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 011 variable light chain

<400> 108

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Ala Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Tyr

85 90 95

Tyr Asn His Pro Thr Thr Phe Gly Gln Gly Thr Lys

100 105

<210> 109

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 021 variable heavy chain

<400> 109

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Arg Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Tyr Ile Thr Leu Pro Asn Ala Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser

115 120

<210> 110

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 021 variable light chain

<400> 110

Asp Ile Gln Met Thr Gln Ser Pro Val Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Tyr

85 90 95

Lys Ser Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys

100 105

<210> 111

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 023 variable heavy chain

<400> 111

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val His Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Met

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Tyr Asp Asn Val Met Gly Leu Tyr Trp Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 112

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 023 variable light chain

<400> 112

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 113

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain variable region

<400> 113

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Tyr Tyr Gly Ser Ser Leu Ser Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 114

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain variable region

<400> 114

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 115

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain variable region

<400> 115

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Lys Asp Tyr

20 25 30

Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Gly Ile Tyr Pro Asn Asn Gly Gly Ser Thr Tyr Asn Gln Asn Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Gly Tyr His Gly Pro His Leu Asp Phe Asp Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Pro

115 120

<210> 116

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain variable region

<400> 116

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Leu Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Ala Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Thr Asp Tyr Phe Cys Gln Gln Tyr Ile Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 117

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain variable region of humanized antibody

<400> 117

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Asn Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr Trp

100 105 110

Gly His Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 118

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain variable region of humanized antibody

<400> 118

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 119

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain variable region of humanized antibody

<400> 119

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Arg

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 120

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain variable region of humanized antibody

<400> 120

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Arg

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Leu Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 121

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain variable region of humanized antibody

<400> 121

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu

1 5 10 15

Ser Leu Lys Leu Ser Cys Glu Ser Asn Glu Tyr Glu Phe Pro Ser His

20 25 30

Asp Met Ser Trp Val Arg Lys Thr Pro Glu Lys Arg Leu Glu Leu Val

35 40 45

Ala Ala Ile Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met

50 55 60

Glu Arg Arg Phe Ile Ile Ser Arg Asp Asn Thr Lys Lys Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg His Tyr Asp Asp Tyr Tyr Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 122

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain variable region of humanized antibody

<400> 122

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Tyr Glu Phe Pro Ser His

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val

35 40 45

Ala Ala Ile Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met

50 55 60

Glu Arg Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Tyr Asp Asp Tyr Tyr Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 123

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain variable region of humanized antibody

<400> 123

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 124

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain variable region of humanized antibody

<400> 124

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 125

<211> 138

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain variable region

<400> 125

Met Tyr Leu Gly Leu Asn Tyr Val Phe Ile Val Phe Leu Leu Asn Gly

1 5 10 15

Val Gln Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asp Ala Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu

50 55 60

Glu Trp Val Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr

65 70 75 80

Tyr Ala Glu Ser Val Asn Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser

85 90 95

Lys Ser Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Gly Ile Tyr Tyr Cys Thr Trp Gly Glu Val Phe Tyr Phe Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

130 135

<210> 126

<211> 126

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain variable region

<400> 126

Met Arg Pro Ser Ile Gln Phe Leu Gly Leu Leu Leu Phe Trp Leu His

1 5 10 15

Gly Ala Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

Ala Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ser Ser Gln Asp

35 40 45

Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro

50 55 60

Arg Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser

85 90 95

Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp

100 105 110

Asn Leu Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

115 120 125

Claims

1. A method for expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(e) Collecting the therapeutic TIL population obtained from step (d); and

(f) transferring the collected TIL population from step (e) to an infusion bag.

2. A method for expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

e) Collecting the therapeutic TIL population obtained from step (d).

3. The method of claim 2, wherein in step (b), the cell culture medium further comprises Antigen Presenting Cells (APCs), and wherein the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).

4. The method of claim 2, wherein in step (b), the cell culture medium further comprises Antigen Presenting Cells (APCs), and wherein the number of APCs in the culture medium in step (c) is equal to the number of APCs in the culture medium in step (b).

5. The method of claim 1 or 2, wherein the PD-1 positive TIL is PD-1 high TIL.

6. A method for expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(c) collecting the therapeutic TIL population obtained from step (b).

7. A method for expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(c) collecting the therapeutic TIL population obtained from step (b).

8. The method of claim 6, wherein in step (b), the cell culture medium further comprises Antigen Presenting Cells (APCs), and wherein the number of APCs in the medium in step (c) is greater than the number of APCs in the medium in step (b).

9. The method of claim 6, wherein in step (b), the cell culture medium further comprises Antigen Presenting Cells (APCs), and wherein the number of APCs in the medium in step (c) is equal to the number of APCs in the medium in step (b).

10. The method of claim 6 or 7, wherein the PD-1 positive TIL is PD-1 high TIL.

11. The method of claim 1 or 2 or 6 or 7, wherein the selecting of step (b) comprises the steps of: (i) exposing the first TIL population to an excess of monoclonal anti-PD-1 IgG4 antibody that binds to PD-1 through the N-terminal loop outside the IgV domain of PD-1; (ii) adding an excess of anti-IgG 4 antibody conjugated to a fluorophore; and (iii) performing flow-based cell sorting based on the fluorophore to obtain a population of PD-1 enriched TILs.

12. The method of claim 11, wherein the monoclonal anti-PD-1 IgG4 antibody is nivolumab (nivolumab), or a variant, fragment, or conjugate thereof.

13. The method of claim 12, wherein the anti-IgG 4 antibody is clone anti-human IgG4, clone HP 6023.

14. The method of claim 1 or 2 or 6 or 7, wherein the ratio of the number of APCs in the rapid second amplification to the number of APCs in the priming first amplification is selected from the range of about 1.5:1 to about 20: 1.

15. The method of claim 1 or 2 or 6 or 7, wherein the ratio is selected from the range of about 1.5:1 to about 10: 1.

16. The method of claim 1 or 2 or 6 or 7, wherein the ratio is selected from the range of about 2:1 to about 5: 1.

17. The method of claim 1 or 2 or 6 or 7, wherein the ratio is selected from the range of about 2:1 to about 3: 1.

18. The method of claim 1 or 2 or 6 or 7, wherein the ratio is about 2: 1.

19. The method of claim 1 or 2 or 6 or 7, wherein the number of APCs in the priming first amplification is selected from about 1 x 10⁸APC to about 3.5X 10⁸A range of APCs, and wherein the number of APCs in the rapid second amplification is selected from about 3.5 x 10 ⁸APC to about 1X 10⁹Range of individual APC.

20. The method of claim 1 or 2 or 6 or 7, wherein the number of APCs in the priming first amplification is selected from about 1.5 x 10⁸APC to about 3X 10⁸A range of APCs, and wherein the number of APCs in the rapid second amplification is selected from about 4 x 10⁸APC to about 7.5X 10⁸Range of individual APC.

21. The method of claim 1 or 2 or 6 or 7, wherein the number of APCs in the priming first amplification is selected from about 2 x 10⁸APC to about 2.5X 10⁸A range of APCs, and wherein the number of APCs in the rapid second amplification is selected from about 4.5 x 10⁸APC to about 5.5X 10⁸Range of individual APC.

22. According to claim 1 or 2 or 6 or7, wherein about 2.5 x 10⁸Addition of individual APCs to the priming first amplification, and 5X 10⁸Individual APCs were added to the rapid second amplification.

23. The method of any one of claims 1-22, wherein a ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is about 1.5:1 to about 100: 1.

24. The method of any one of claims 1-22, wherein a ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is about 50: 1.

25. The method of any one of claims 1-22, wherein a ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is about 25: 1.

26. The method of any one of claims 1-22, wherein a ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is about 20: 1.

27. The method of any one of claims 1-22, wherein a ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is about 10: 1.

28. The method of any one of claims 1-22, wherein the second TIL population is at least 50-fold greater in number than the first TIL population.

29. The method of any one of claims 2-28, wherein the method comprises performing the following additional steps after the step of collecting the therapeutic TIL population:

the collected therapeutic TIL populations were transferred to infusion bags.

30. The method of any one of claims 1-28, wherein the plurality of tumor fragments are dispensed into a plurality of separate containers, in each of the separate containers, the second TIL population is obtained from the first TIL population in the step of priming a first expansion and the third TIL population is obtained from the second TIL population in the step of rapid second expansion, and wherein the therapeutic TIL populations obtained from the third TIL population are collected from each of the plurality of containers and combined to produce the collected TIL population.

31. The method of claim 30, wherein the plurality of individual containers comprises at least two individual containers.

32. The method of claim 30, wherein the plurality of individual containers comprises two to twenty individual containers.

33. The method of claim 30, wherein the plurality of individual containers comprises two to ten individual containers.

34. The method of claim 30, wherein the plurality of individual containers comprises two to five individual containers.

35. The method of any one of claims 30-34, wherein each of the individual containers includes a first gas-permeable surface region.

36. The method of any one of claims 1-29, wherein the plurality of tumor fragments are dispensed in a single container.

37. The method of claim 36, wherein the single container includes a first gas-permeable surface region.

38. The method of claim 33 or 37 wherein in the step of initiating a first expansion, the cell culture medium comprises Antigen Presenting Cells (APCs), and the APCs are layered onto the first gas permeable surface region at an average thickness of about one cell layer to about three cell layers.

39. The method of claim 36 wherein in the step of initiating a first expansion, the APCs are layered onto the first gas permeable surface region at an average thickness of about 1.5 cell layers to about 2.5 cell layers.

40. The method of claim 38 wherein in the step of initiating a first expansion, the APCs are layered onto the first gas permeable surface region at an average thickness of about 2 cell layers.

41. The method of any one of claims 38 to 40 wherein in the step of rapid second expansion, the APCs are laminated to the first gas permeable surface region at a thickness of about 3 cell layers to about 5 cell layers.

42. The method of claim 41 wherein in the step of rapid second expansion, the APCs are laminated to the first gas permeable surface region at a thickness of about 3.5 cell layers to about 4.5 cell layers.

43. The method of claim 42 wherein in the step of rapid second expansion, the APCs are laminated to the first gas permeable surface region at a thickness of about 4 cell layers.

44. The method of any one of claims 2 to 29, wherein in the step of priming a first amplification, the priming first amplification is performed in a first container comprising a first gas-permeable surface region, and in the step of rapid second amplification, the rapid second amplification is performed in a second container comprising a second gas-permeable surface region.

45. The method of claim 44, wherein the second container is larger than the first container.

46. The method of claim 42 or 43 wherein in the step of initiating a first expansion, the cell culture medium comprises Antigen Presenting Cells (APCs) and the APCs are layered onto the first gas permeable surface region at an average thickness of about one cell layer to about three cell layers.

47. The method of claim 46 wherein in the step of initiating a first expansion, the APCs are laminated to the first gas permeable surface region at an average thickness of about 1.5 cell layers to about 2.5 cell layers.

48. The method of claim 48 wherein in the step of initiating a first expansion, the APCs are laminated to the first gas permeable surface region at an average thickness of about 2 cell layers.

49. The method of any one of claims 44 to 48 wherein in the step of rapid second expansion the APCs are laminated to the second gas permeable surface region at an average thickness of about 3 cell layers to about 5 cell layers.

50. The method of claim 49 wherein in the step of rapid second expansion, the APCs are laminated to the second gas permeable surface region at an average thickness of about 3.5 cell layers to about 4.5 cell layers.

51. The method of claim 49 wherein in the step of rapid second expansion, the APCs are laminated to the second gas permeable surface region at an average thickness of about 4 cell layers.

52. The method of any one of claims 2-43, wherein for each vessel in which said primed first amplification is performed on a first TIL population, said rapid second amplification is performed on said second TIL population produced from such first TIL population in the same vessel.

53. The method of claim 52 wherein each container includes a first gas-permeable surface region.

54. The method of claim 53 wherein in the step of initiating a first expansion, the cell culture medium comprises Antigen Presenting Cells (APCs) and the APCs are layered onto the first gas permeable surface region at an average thickness of about one cell layer to about three cell layers.

55. The method of claim 54 wherein in the step of initiating a first expansion, the APCs are laminated to the first gas permeable surface region at an average thickness of about 1.5 cell layers to about 2.5 cell layers.

56. The method of claim 55 wherein in the step of initiating a first expansion, the APCs are laminated to the first gas permeable surface region at an average thickness of about 2 cell layers.

57. The method of any one of claims 53 to 56 wherein in the step of rapid second expansion, the APCs are laminated to the first gas permeable surface region at an average thickness of about 3 cell layers to about 5 cell layers.

58. The method of claim 57 wherein in the step of rapid second expansion, the APCs are laminated to the first gas permeable surface region at an average thickness of about 3.5 cell layers to about 4.5 cell layers.

59. The method of claim 58 wherein in the step of rapid second expansion, the APCs are laminated to the first gas permeable surface region at an average thickness of about 4 cell layers.

60. The method of any one of claims 2 to 36, 44, 46 and 52, wherein for each container in which the priming first expansion is performed on a first population of TILs in the priming first expansion step, the first container comprises a first surface region, the cell culture medium comprises Antigen Presenting Cells (APCs), and the APCs are layered onto the first gas permeable surface region, and wherein the ratio of the average number of layers of APCs layered in the priming first expansion step to the average number of layers of APCs layered in the rapid second expansion step is selected from the range of about 1:1.1 to about 1: 10.

61. The method of claim 60, wherein the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapidly second amplification is selected from the range of about 1:1.2 to about 1: 8.

62. The method of claim 60, wherein the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapidly second amplification is selected from the range of about 1:1.3 to about 1: 7.

63. The method of claim 60, wherein the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapidly second amplification is selected from the range of about 1:1.4 to about 1: 6.

64. The method of claim 60, wherein the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapidly second amplification is selected from the range of about 1:1.5 to about 1: 5.

65. The method of claim 60, wherein the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapidly second amplification is selected from the range of about 1:1.6 to about 1: 4.

66. The method of claim 60, wherein the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapidly second amplification is selected from the range of about 1:1.7 to about 1: 3.5.

67. The method of claim 60, wherein the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapidly second amplification is selected from the range of about 1:1.8 to about 1:3.

68. The method of claim 60, wherein the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapidly second amplification is selected from the range of about 1:1.9 to about 1: 2.5.

69. The method of claim 60, wherein the ratio of the average number of stacked APCs in the step of initiating a first amplification to the average number of stacked APCs in the step of rapidly second amplification is about 1:2.

70. The method of any one of the preceding claims, wherein the cell culture medium is supplemented with additional IL-2 after 2 to 3 days in the step of rapid second expansion.

71. The method of any one of the preceding claims, further comprising cryopreserving the collected TIL population using a cryopreservation method in the step of collecting the therapeutic TIL population.

72. The method of claim 1 or 29, further comprising the step of cryopreserving the infusion bag.

73. The method of claim 71 or 72, wherein the cryopreservation process is performed using the collected TIL population and cryopreservation media in a ratio of 1: 1.

74. The method of any one of the preceding claims, wherein the antigen presenting cells are Peripheral Blood Mononuclear Cells (PBMCs).

75. The method of claim 74, wherein the PBMCs are irradiated and allogeneic.

76. The method of any one of the preceding claims, wherein in the step of priming a first expansion, the cell culture medium comprises Peripheral Blood Mononuclear Cells (PBMCs), and wherein the total number of PBMCs in the cell culture medium in the step of priming a first expansion is 2.5 x 10⁸。

77. The method according to any one of the preceding claims, wherein in the step of rapid second expansion the Antigen Presenting Cells (APCs) in the cell culture medium are Peripheral Blood Mononuclear Cells (PBMCs), and wherein the total number of PBMCs added to the cell culture medium in the step of rapid second expansion is 5 x 10 ⁸。

78. The method of any one of claims 1 to 70, wherein the antigen presenting cells are artificial antigen presenting cells.

79. The method of any one of the preceding claims, wherein the collecting in the step of collecting the therapeutic TIL population is performed using a membrane-based cell processing system.

80. The method of any one of the preceding claims, wherein the collecting in step (d) is performed using a LOVO cell processing system.

81. The method of any one of the preceding claims, wherein in the step of initiating a first amplification, the plurality of fragments comprises about 60 fragments per container, wherein the volume of each fragment is about 27mm³。

82. The method of any one of the preceding claims, wherein the plurality of fragments comprises about 30 to about 60 fragments, wherein the total volume is about 1300mm³To about 1500mm³。

83. The method of claim 82, wherein the plurality of fragments comprises about 50 fragments, wherein the total volume is about 1350mm³。

84. The method of any one of the preceding claims, wherein the plurality of fragments comprises about 50 fragments, wherein the total mass is from about 1 gram to about 1.5 grams.

85. The method of any one of the preceding claims, wherein the cell culture medium is provided in a container selected from the group consisting of a G container and a Xuri cell bag.

86. The method of any one of the preceding claims, wherein the cell culture medium is supplemented with additional IL-2 after 2 to 3 days in step (d).

87. The method of any one of the preceding claims, wherein the IL-2 concentration is from about 10,000IU/mL to about 5,000 IU/mL.

88. The method of any one of the preceding claims, wherein the IL-2 concentration is about 6,000 IU/mL.

89. The method of claim 1 or 29, wherein in the step of transferring the collected therapeutic TIL population to an infusion bag, the infusion bag is a hypo thermosol-containing infusion bag.

90. The method of any one of claims 71-73, wherein the cryopreservation media comprises dimethyl sulfoxide (DMSO).

91. The method of claim 90, wherein the cryopreservation media comprises 7% to 10% DMSO.

92. The method of any one of the preceding claims, wherein the first time period in the step of priming first amplification and the second time period in the step of rapid second amplification are each performed separately over a time period of 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

93. The method of any one of claims 1-92, wherein the first time period in the step of priming for first amplification is performed over a period of 5 days, 6 days, or 7 days.

94. The method of any one of claims 1-92, wherein the second time period in the step of rapid second amplification is performed over a period of 7 days, 8 days, or 9 days.

95. The method of any one of claims 1-92, wherein the first time period in the step of priming a first amplification and the second time period in the step of rapid second amplification are each performed separately over a period of 7 days.

96. The method of any one of claims 1-92, wherein the step of priming a first expansion is performed over a period of about 14 days to about 16 days by collecting the therapeutic TIL population.

97. The method of any one of claims 1-92, wherein the step of priming a first expansion is performed over a period of about 15 days to about 16 days by collecting the therapeutic TIL population.

98. The method of any one of claims 1-92, wherein the step of priming a first expansion is performed over a period of about 14 days by collecting the therapeutic TIL population.

99. The method of any one of claims 1-92, wherein the step of priming a first expansion is performed over a period of about 15 days by collecting the therapeutic TIL population.

100. The method of any one of claims 1-92, wherein the step of priming a first expansion is performed over a period of about 16 days by collecting the therapeutic TIL population.

101. The method of any one of claims 1-92, further comprising the step of cryopreserving the collected therapeutic TIL population using a cryopreservation process, wherein the step of initiating a first expansion is performed in 16 days or less by collecting the therapeutic TIL population and cryopreserving.

102. The method of any one of claims 1-101, wherein the therapeutic TIL population collected in the step of collecting the therapeutic TIL population comprises sufficient TIL for a therapeutically effective dose of TIL.

103. The method of claim 102, wherein the amount of TIL sufficient for a therapeutically effective dose is about 2.3 x 10¹⁰To about 13.7X 10¹⁰。

104. The method of any one of claims 1-103, wherein the third TIL population in the step of rapid second amplification provides increased efficacy, increased interferon gamma production, and/or increased polyclonality.

105. The method of any one of claims 1-103, wherein the third TIL population in the step of rapidly second amplifying provides at least one to five or more times interferon gamma production as compared to TILs prepared by a process longer than 16 days.

106. The method of any one of claims 1-103, wherein effector T cells and/or central memory T cells obtained from the third TIL population in the step of rapidly second expanding exhibit increased expression of CD8 and CD28 relative to effector T cells and/or central memory T cells obtained from the second TIL population in the step of initiating first expanding.

107. The method of any one of claims 1-106, wherein the therapeutic TIL population from the step of collecting the therapeutic TIL population is infused into a patient.

108. The method of claim 1 or 2 or 5 or 6, further comprising the step of cryopreserving the infusion bag comprising the collected TIL population in step (f) using a cryopreservation process.

109. The method of claim 1 or 2 or 5 or 6, wherein the cryopreservation process is performed using the collected TIL population and cryopreservation media in a ratio of 1: 1.

110. The method of claim 1 or 2 or 5 or 6, wherein the antigen presenting cells are Peripheral Blood Mononuclear Cells (PBMCs).

111. The method of claim 110, wherein the PBMCs are irradiated and allogeneic.

112. The method of claim 1 or 2 or 6 or 7, wherein the antigen presenting cell is an artificial antigen presenting cell.

113. The method of claim 1 or 2 or 6 or 7, wherein the collecting in step (e) is performed using a membrane-based cell processing system.

114. The method of claim 1 or 2 or 6 or 7, wherein the collecting in step (e) is performed using a LOVO cell processing system.

115. The method of claim 1 or 2 or 6 or 7, wherein in step (c), the plurality of fragments comprises about 60 fragments per first gas-permeable surface area, wherein the volume of each fragment is about 27mm³。

116. The method of claim 1 or 2 or 6 or 7, wherein the plurality of fragments comprises about 30 to about 60 fragments, wherein the total volume is about 1300mm³To about 1500mm³。

117. The method of claim 116, wherein the plurality of fragments comprises about 50 fragments, wherein the total volume is about 1350mm ³。

118. The method of claim 1 or 2 or 6 or 7, wherein the plurality of fragments comprises about 50 fragments, wherein the total mass is about 1 gram to about 1.5 grams.

119. The method of claim 1 or 2 or 6 or 7, wherein the cell culture medium is provided in a container selected from the group consisting of a G container and a Xuri cell bag.

120. The method of any one of the preceding claims, wherein the IL-2 concentration is from about 10,000IU/mL to about 5,000 IU/mL.

121. The method of any one of the preceding claims, wherein the IL-2 concentration is about 6,000 IU/mL.

122. The method of claim 1 or 2 or 6 or 7, wherein the infusion bag in step (d) is a HypoThermosol-containing infusion bag.

123. The method of claim 122, wherein the cryopreservation media comprises dimethyl sulfoxide (DMSO).

124. The method of claim 123, wherein the cryopreservation media comprises 7% to 10% DMSO.

125. The method of claim 1 or 2 or 6 or 7, wherein the first time period in step (c) and the second time period in step (c) are each performed separately over a period of 5 days, 6 days, or 7 days.

126. The method of claim 1 or 2 or 6 or 7, wherein the first period of time in step (c) is performed over a period of 5 days, 6 days or 7 days.

127. The method of claim 1, wherein the second period of time in step (d) is performed over a period of 7 days, 8 days, or 9 days.

128. The method of claim 1 or 2 or 6 or 7, wherein the first time period in step (c) and the second time period in step (c) are each performed separately over a 7 day period.

129. The method of claim 1 or 2 or 6 or 7, wherein steps (a) through (f) are performed over a period of about 14 days to about 16 days.

130. The method of claim 1 or 2 or 6 or 7, wherein steps (a) through (f) are performed over a period of about 15 days to about 16 days.

131. The method of claim 1 or 2 or 6 or 7, wherein steps (a) through (f) are performed over a period of about 14 days.

132. The method of claim 1 or 2 or 6 or 7, wherein steps (a) to (f) are performed over a period of about 15 days.

133. The method of claim 1 or 2 or 6 or 7, wherein steps (a) through (f) are performed over a period of about 16 days.

134. The method of claim 133, wherein steps (a) through (f) and cryopreservation are performed in 16 days or less.

135. The method of any one of claims 1-134, wherein the therapeutic TIL population collected in step (f) comprises sufficient TIL for a therapeutically effective dose of TIL.

136. The method of claim 135, wherein the amount of TIL sufficient for a therapeutically effective dose is about 2.3 x 10¹⁰To about 13.7X 10¹⁰。

137. The method of any one of claims 1-136, the container in step (c) being larger than the container in step (b).

138. The method of any one of claims 1-137, wherein the third TIL population in step (d) provides increased efficacy, increased interferon-gamma production, and/or increased polyclonality.

139. The method of any one of claims 1-138, wherein the third TIL population in step (d) provides at least one to five or more times interferon gamma production as compared to TILs prepared by a process longer than 16 days.

140. The method of any one of claims 1 to 139, wherein the effector T cells and/or central memory T cells obtained from the third TIL population in step (d) exhibit increased expression of CD8 and CD28 relative to the effector T cells and/or central memory T cells obtained from the second cell population in step (c).

141. The method of any one of claims 1-140, wherein the TIL from step (f) is infused into a patient.

142. A method for treating a subject having cancer, the method comprising administering expanded Tumor Infiltrating Lymphocytes (TILs), the method comprising:

(e) Collecting the therapeutic TIL population obtained from step (c);

143. The method of claim 142, wherein the amount of TIL sufficient to administer a therapeutically effective dose in step (g) is about 2.3 x 10¹⁰To about 13.7X 10¹⁰。

144. The method of claim 142 or 143, wherein the PD-1 positive TIL is PD-1 high TIL.

145. The method of any one of claims 142-144, wherein the selecting of step (b) comprises the steps of: (i) exposing the first TIL population to an excess of monoclonal anti-PD-1 IgG4 antibody that binds to PD-1 through the N-terminal loop outside the IgV domain of PD-1; (ii) adding an excess of anti-IgG 4 antibody conjugated to a fluorophore; and (iii) performing flow-based cell sorting based on the fluorophore to obtain a population of PD-1 enriched TILs.

146. The method of claim 145, wherein the monoclonal anti-PD-1 IgG4 antibody is nivolumab or a variant, fragment, or conjugate thereof.

147. The method of claim 146, wherein said anti-IgG 4 antibody is clone anti-human IgG4, clone HP 6023.

148. The method of claim 147, wherein the Antigen Presenting Cells (APCs) are PBMCs.

149. The method of any one of claims 145-148, wherein the patient has been administered a non-myeloablative lymphocyte depletion regimen prior to administering a therapeutically effective dose of TIL cells in step (g).

150. The method of claim 151, wherein the non-myeloablative lymphocyte depletion regimen comprises the steps of: at 60 mg/m²Cyclophosphamide was administered for two days at a dose of 25 mg/m²The dose of fludarabine was administered for five days.

151. The method of any one of claims 145-150, further comprising the step of treating the patient with a high-dose IL-2 regimen beginning the day after administering the TIL cells to the patient in step (g).

152. The method of claim 151, wherein the high-dose IL-2 regimen comprises administering 600,000 or 720,000IU/kg every eight hours in 15 minute bolus intravenous infusion until tolerated.

153. The method of any one of claims 145-152, wherein the third TIL population in step (c) provides increased efficacy, increased interferon gamma production, and/or increased polyclonality.

154. The method of any one of claims 145-153, wherein the third TIL population in step (d) provides at least one to five or more times interferon gamma production as compared to TILs prepared by a process longer than 16 days.

155. The method of any one of claims 145 to 154, wherein the effector T cells and/or central memory T cells obtained from the third TIL population in step (d) exhibit increased expression of CD8 and CD28 relative to the effector T cells and/or central memory T cells obtained from the second population of cells in step (c).

156. The method of any one of the preceding claims, wherein the cancer is selected from the group consisting of: melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer (including Head and Neck Squamous Cell Carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, renal cancer, and renal cell carcinoma.

157. The method of any one of the preceding claims, wherein the cancer is selected from the group consisting of: melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

158. The method of any one of the preceding claims, wherein the cancer is melanoma.

159. The method of any one of the preceding claims, wherein the cancer is HNSCC.

160. The method of any one of the preceding claims, wherein the cancer is cervical cancer.

161. The method of any one of the preceding claims, wherein the cancer is NSCLC.

162. The method of any one of the preceding claims, wherein the cancer is glioblastoma (comprising GBM).

163. The method of any one of the preceding claims, wherein the cancer is a gastrointestinal cancer.

164. The method of any one of the preceding claims, wherein the cancer is a highly mutated cancer.

165. The method of any one of the preceding claims, wherein the cancer is a pediatric hypermutation cancer.

166. The method of any preceding claim, wherein the container is GREX-10.

167. The method of any of the preceding claims, wherein the containment vessel comprises GREX-100.

168. The method of any of the preceding claims, wherein the containment vessel comprises GREX-500.

169. The method of any one of the preceding claims, wherein the subject has been previously treated with an anti-PD-1 antibody.

170. The method of any one of the preceding claims, wherein the subject has not been previously treated with an anti-PD-1 antibody.

171. The method of any one of the preceding claims, wherein in step (b), the PD-1 positive TIL is selected from the first TIL population by performing the following steps: performing the step of contacting the first TIL population with an anti-PD-1 antibody to form a first complex of the anti-PD-1 antibody and the TIL cells in the first TIL population; and then performing the step of separating the first complex to obtain the population of PD-1 enriched TILs.

172. The method of claim 165, wherein the anti-PD-1 antibody comprises an Fc region, wherein after the step of forming the first complex and before the step of isolating the first complex, the method further comprises the step of contacting the first complex with an anti-Fc antibody to form a second complex of the anti-Fc antibody and the first complex, the anti-Fc antibody binding to the Fc region of the anti-PD-1 antibody, and wherein the step of isolating the first complex is performed by isolating the second complex.

173. The method of any one of the preceding claims, wherein the anti-PD-1 antibody used for selection in step (b) is selected from the group consisting of: EH12.2H7, PD1.3.1, M1H4, nivolumab (BMS-936558, Bristol-Myers Squibb);) Pembrolizumab (lambrolizumab), MK03475 or MK-3475 Merck (Merck);) H12.1, PD1.3.1, NAT 105, humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR-042(Tesaro, Inc.), Pidilizumab (Pidilizumab) (anti-PD-1 mAb CT-011, mediwesson medical company (Medivation)), anti-PD-1 monoclonal antibody BGB-a317 (BeiGene)), and/or anti-PD-1 antibody SHR-1210 (ShangHai henderui), human monoclonal antibody REGN2810 (regenerlon)), human monoclonal antibody MDX-1106 (nikamura), humanized anti-PD-1 antibody 4 PDR001 (novanis) and RMP1-14 (catalog number xp) -0146 rat (biox).

174. The method of any one of the preceding claims, wherein the anti-PD-1 antibody used for selection in step (b) is EH12.2H7.

175. The method of any one of the preceding claims, wherein the anti-PD-1 antibody used for selection in step (b) binds to an epitope other than nivolumab or pembrolizumab.

176. The method of any one of the preceding claims, wherein the anti-PD-1 antibody used for selection in step (b) binds the same epitope as EH12.2H7 or nivolumab.

177. The method of any one of the preceding claims, wherein the anti-PD-1 antibody used for selection in step (b) is nivolumab.

178. The method of any one of claims 1-177, wherein the subject has been previously treated with a first anti-PD 1 antibody, wherein in step (b), the PD-1 positive TIL is selected by contacting the first TIL population with a second anti-PD-1 antibody, and wherein the second anti-PD-1 antibody does not block binding to the first TIL population by the first anti-PD-1 antibody that is insoluble in the first TIL population.

179. The method of claims 1-177, wherein the subject has been previously treated with a first anti-PD 1 antibody, wherein in step (b), the PD-1 positive TIL is selected by contacting the first TIL population with a second anti-PD-1 antibody, and wherein the second anti-PD-1 antibody blocks binding to the first TIL population by the first anti-PD-1 antibody that is insoluble in the first TIL population.

180. The method of any one of claims 1 to 177, wherein the subject has been previously treated with a first anti-PD 1 antibody, wherein in step (b), the PD-1 positive TIL is selected by performing the steps of: performing the step of contacting the first TIL population with a second anti-PD-1 antibody to form a first complex of the second anti-PD-1 antibody and the first TIL population, wherein the second anti-PD-1 antibody does not block binding to the first TIL population by the first anti-PD-1 antibody that is insoluble in the first TIL population; and then performing the step of separating the first complex to obtain the population of PD-1 enriched TILs.

181. The method of claims 1-177, wherein the first anti-PD-1 antibody and the second anti-PD-1 antibody comprise Fc regions, wherein after the step of forming the first complex and before the step of isolating the first complex, the method further comprises the step of contacting the first complex with an anti-Fc antibody that binds to the Fc regions of the first anti-PD-1 antibody and the second anti-PD-1 antibody to form a second complex of the anti-Fc antibody and the first complex, and wherein the step of isolating the first complex is performed by isolating the second complex.

182. The method of any one of claims 1 to 177, wherein the subject has been previously treated with a first anti-PD 1 antibody, wherein in step (b), the PD-1 positive TIL is selected by performing the steps of: performing a step of contacting the first TIL population with a second anti-PD-1 antibody to form a first complex of the second anti-PD-1 antibody and the first TIL population, wherein the second anti-PD-1 antibody is blocked from binding to the PD-1 positive TIL by the first anti-PD-1 antibody that is insoluble in the first TIL population, wherein the first anti-PD-1 antibody and the second anti-PD-1 antibody comprise an Fc region, wherein after the step of forming the first complex and before the step of obtaining the PD-1 enriched TIL population, the method further comprises the steps of: contacting the first complex with an anti-Fc antibody to form a second complex of the anti-Fc antibody and the first complex, the anti-Fc antibody binding to the Fc region of the second anti-PD-1 antibody; and contacting the first anti-PD-1 antibody that is insoluble in the first TIL population with the anti-Fc antibody to form a third complex of the anti-Fc antibody and the first anti-PD-1 antibody that is insoluble in the first TIL population, and performing the step of separating the second complex and the third complex to obtain the PD-1 enriched TIL population.

183. A therapeutic Tumor Infiltrating Lymphocyte (TIL) population prepared from PD-1 positive cells selected from a tumor tissue of a patient, wherein said therapeutic TIL population provides increased efficacy and/or increased interferon gamma production.

184. The therapeutic TIL population of claim 183, which provides increased interferon gamma production.

185. The therapeutic TIL population of claim 183 or claim 184, which provides increased efficacy.

186. The therapeutic TIL population of any one of claims 183-185, wherein the therapeutic TIL population is capable of producing at least one-fold more interferon gamma production as compared to a TIL prepared by a process longer than 16 days.

187. The therapeutic TIL population of any one of claims 183-186, wherein the therapeutic TIL population is capable of producing at least one-fold more interferon gamma production as compared to a TIL prepared by a process longer than 16 to 22 days.

188. The method of any one of the preceding claims, wherein selecting a PD-1 positive TIL from the first TIL population to obtain a population of TILs enriched in PD-1 comprises selecting a population of TILs from the first TIL population that is at least 11.27% to 74.4% of the PD-1 positive TIL.

189. The method according to any of the preceding claims, wherein the selection of steps comprises the steps of:

(ii) adding an excess of anti-IgG 4 antibody conjugated to a fluorophore;

190. The method of any one of the preceding claims, wherein the intensities of the fluorophores in both the first population and the PBMC population are used to establish FACS gates to establish low, intermediate and high levels of intensity corresponding to PD-1 negative TIL, PD-1 intermediate TIL and PD-1 positive TIL, respectively.

191. The method of any one of the preceding claims, wherein the FACS gates are established after step (a).

192. The method of any one of claims 1-4, wherein the PD-1 positive TIL is PD-1 high TIL.

193. The method of any one of claims 1-5, wherein at least 80% of the PD-1 enriched TIL population is PD-1 positive TIL.

194. A method for expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(e) Collecting the therapeutic TIL population obtained from step (d); and

(f) transferring the collected TIL population from step (e) to an infusion bag.

195. The method of claim 194, wherein the selecting of step (b) comprises the steps of:

(ii) adding an excess of anti-IgG 4 antibody conjugated to a fluorophore;

196. The method of any one of claims 194 to 195, wherein the intensities of the fluorophores in both the first population and the PBMC population are used to establish FACS gates to establish low, intermediate, and high levels of intensity corresponding to PD-1 negative TIL, PD-1 intermediate TIL, and PD-1 positive TIL, respectively.

197. The method of any one of claims 194 to 196, wherein the FACS gates are established after step (a).

198. The method of any one of claims 194 to 197, wherein the PD-1 positive TIL is PD-1 high TIL.

199. The method of any one of claims 194 to 198, wherein at least 80% of the population of PD-1-enriched TILs are PD-1 positive TILs.

200. The method of any one of claims 194-199, wherein the third TIL population comprises at least about 1 x 10 in the container⁸And (4) TIL.

201. The method of any one of claims 194-200, wherein the third TIL population comprises at least about 1 x 10 in the container⁹And (4) TIL.

202. The method of any one of claims 194 to 201, wherein the amount of PD-1-enriched TIL in the priming-first amplification is about 1 x 10⁴To about 1X 10⁶。

203. The method of any one of claims 194 to 202, wherein the amount of PD-1-enriched TIL in the priming-first amplification is about 5 x 10⁴To about 1X 10⁶。

204. The method of any one of claims 194 to 203, wherein the amount of PD-1-enriched TIL in the priming-first amplification is about 2 x 10⁵To about 1X 10⁶。

205. The method of any one of claims 194 to 204, further comprising the step of performing cryopreservation on the first TIL population from a tumor resected from the subject prior to performing step (a).