HK40109052A - Processes for generating til products enriched for tumor antigen-specific t-cells - Google Patents

Description

Methods for producing TIL products rich in tumor antigen-specific T cells

This application is a divisional application of the invention patent application filed on January 8, 2019, with application number 201980017442.8 (international application number PCT/US2019/012729) entitled "Method for producing TIL products rich in tumor antigen-specific T cells".

Related applications

This application claims priority to U.S. Provisional Patent Application No. 62/614,887, filed January 8, 2018; U.S. Provisional Patent Application No. 62/664,034, filed April 27, 2018; U.S. Provisional Patent Application No. 62/669,319, filed May 9, 2018; U.S. Provisional Patent Application No. 62/697,921, filed July 13, 2018; U.S. Provisional Patent Application No. 62/734,868, filed September 21, 2018; and U.S. Provisional Patent Application No. 62/773,715, filed November 30, 2018, the entire contents of which are incorporated herein by reference.

sequence list

This application contains a sequence list, which has been electronically submitted in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy was created on January 7, 2019, and is named 116983-5034-WO_ST25.txt, with a size of 122KB.

Background Technology

Using adoptive metastatic tumor-infiltrating lymphocytes (TILs) to treat large, refractory cancers represents an effective approach for treating patients with poor prognoses. (Gattinoni et al., Nat. Rev. I mmunol., 2006, 6, 383-393). Successful immunotherapy requires large quantities of TILs, and commercialization necessitates robust and reliable methods. This presents a challenge due to technical, logistical, and regulatory issues surrounding cell expansion. Due to its speed and efficiency, IL-2-based TIL expansion followed by a rapid expansion process (REP) has become the preferred method for TIL expansion. Dudley et al., Science, 2002, 298, 850-54; Dudley et al., J. Clin. Oncol., 2005, 23, 2346-57; Dudley et al., J. Clin. Oncol., 2008, 26, 5233-39; Riddell et al., Science 1992, 257, 238-41; Dudley et al., J. I. mmunother., 2003, 26, 332-42. REP can amplify TILs by 1000-fold within 14 days, although it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as monocytes (MNCs)) as feeder cells, usually from multiple donors, as well as anti-CD3 antibody (OKT3) and high doses of IL-2. Dudley et al. J.I. mmunother. 2003, 26, 332-42. TILs undergoing the REP procedure yielded successful adoptive cell therapy in melanoma patients following host immunosuppression.

There is an urgent need for more potent or efficient methods for producing TILs and therapies based on such methods, suitable for commercial-scale production and regulatory approval for use in human patients across multiple clinical centers. This invention addresses this need by providing a transient genetic alteration method for reprogramming TILs to prepare a population of therapeutically potent TILs.

Summary of the Invention

This invention provides an improved and/or shortened method for amplifying TILs and generating therapeutic TIL populations.

This invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (i) obtaining a first TIL population from a tumor resected from a patient; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 and optionally OKT-3 to generate a second TIL population; (iii) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen-presenting cells (APCs) to generate a third TIL population; wherein the number of the third TIL population is at least 100 times greater than the number of the second TIL population; wherein the second expansion is performed for at least 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; and (iv) exposing the second TIL population and/or the third TIL population to transcription factors (TFs) and/or other molecules capable of transiently altering protein expression; wherein the TFs and/or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and/or the number of tumor antigen-specific T cells in the therapeutic TIL population.

In some embodiments, the method further includes exposing a second TIL population and/or a third TIL population to transcription factors (TFs) and/or other molecules capable of transiently altering protein expression; wherein the TFs and/or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and/or the number of tumor antigen-specific T cells in the therapeutic TIL population.

The present invention also provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) A first amplification is performed by culturing a first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 3 to 14 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (b) to step (c) occurs without opening the system;

(d) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (d) occurs without opening the system;

(e) Exposing the second and/or third TIL groups to transcription factors (TFs) and/or other molecules capable of transiently altering protein expression; wherein the TFs and/or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and/or the number of tumor antigen-specific T cells in the therapeutic TIL groups.

(f) Harvest the therapeutic TIL cluster obtained from step (d); wherein the transition from step (d) to step (e) occurs without opening the system; and

(g) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system.

In some embodiments, the method further includes: performing a second expansion before or after step (iv) by supplementing the cell culture medium of the third TIL population with additional IL-2, additional OKT-3 and additional APC; wherein the second expansion is performed for at least 14 days to obtain a larger therapeutic TIL population than that obtained in step (iii); wherein the larger therapeutic TIL population exhibits an alteration in the number of tumor antigen-specific T cells.

In some embodiments, the method further includes the step of: cryopreserving the infusion bag containing the TIL clusters harvested from step (f) using a cryopreservation method.

In some implementations, the cryopreservation method uses a 1:1 ratio of harvested TILs to the cryopreservation medium.

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, the PBMCs are added to the cell culture on any day from day 9 to day 14 of step (d). In some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

In some implementations, a membrane-based cell processing system is used to perform the harvest in step (e).

In some implementations, the LOVO cell processing system is used to perform the harvest in step (e).

In some implementations, the multiple fragments include about 4 to about 50 fragments, each fragment having a volume of about 27 ^mm³ .

In some embodiments, the multiple fragments include about 30 to about 60 fragments, with a total volume of about 1300 ^mm³ to about 1500 ^mm³ .

In some implementations, the multiple fragments include approximately 50 fragments with a total volume of approximately 1350 ^mm³ .

In some implementations, the multiple fragments include about 50 fragments 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 G containers and Xuri cell bags.

In some embodiments, the cell culture medium in step (d) further contains IL-15 and/or IL-21.

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

In some implementations, the IL-15 concentration is from about 500 IU/mL to about 100 IU/mL.

In some implementations, the IL-21 concentration is from about 20 IU/mL to about 0.5 IU/mL.

In some implementations, the infusion bag in step (f) is an infusion bag containing HypoThermosol.

In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO). In some embodiments, the cryopreservation medium contains 7% to 10% dimethyl sulfoxide (DMSO).

In some implementations, the first stage in step (c) and the second stage in step (e) are performed for 10 days, 11 days, or 12 days, respectively.

In some implementations, the first stage in step (c) and the second stage in step (e) each last for 11 days.

In some implementations, steps (a) through (f) take approximately 10 to approximately 22 days.

In some implementations, steps (a) through (f) take approximately 20 to approximately 22 days.

In some implementations, steps (a) through (f) take approximately 15 to approximately 20 days.

In some implementations, steps (a) through (f) take approximately 10 to approximately 20 days.

In some implementations, steps (a) through (f) take approximately 10 to 15 days.

In some implementations, steps (a) through (f) are performed for less than 22 days.

In some implementations, steps (a) through (f) are performed for less than 20 days.

In some implementations, steps (a) through (f) are performed for less than 15 days.

In some implementations, steps (a) through (f) are performed for less than 10 days.

In some implementations, steps (a) through (f) and cryopreservation are performed for less than 22 days.

In some implementations, the therapeutic TIL clusters harvested in step (e) contain sufficient TILs for a therapeutically effective dose of TILs.

In some implementations, the number of TILs sufficient for a therapeutically effective dose is about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ .

In some implementations, steps (b) through (e) are performed in a single container; wherein performing steps (b) through (e) in a single container increases the TIL yield per resected tumor compared to performing steps (b) through (e) in more than one container.

In some implementations, during the second phase of step (d), antigen-presenting cells are added to the TIL without opening the system.

In some implementations, when administered to a subject, the third TIL group in step (d) provides at least 5 times the interferon-γ production.

In some implementations, the risk of microbial contamination is reduced compared to open systems.

In some implementations, the TIL from step (f) or step (g) is injected into the patient.

In some implementations, the multiple fragments include approximately four fragments.

The present invention also provides a method for treating a subject suffering from cancer, the method comprising administering expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) First TIL clusters are obtained by processing tumor samples obtained from patients into multiple tumor fragments and tumors removed by the subject;

(b) Adding tumor fragments to the closed system;

(c) A first amplification is performed by culturing a first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 3 to 14 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (b) to step (c) occurs without opening the system;

(d) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (d) occurs without opening the system;

(e) Exposing second and/or third TIL groups to transcription factors (TFs) and/or other molecules capable of transiently altering protein expression; wherein TFs and/or other molecules capable of transiently altering protein expression provide an increase in tumor antigen expression and/or an increase in the number of tumor antigen-specific T cells in the therapeutic TIL group;

(f) Harvest the therapeutic TIL cluster obtained from step (d); wherein the transition from step (d) to step (e) occurs without opening the system; and

(g) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system;

(h) Optionally, the infusion bag containing the TIL clusters harvested from step (f) is cryopreserved using a cryopreservation method; and

(i) Administer the therapeutically effective dose of the third TIL group in the infusion bag of step (g) to the patient.

In some implementations, the therapeutic TIL cluster harvested in step (f) contains sufficient TILs for administering a therapeutically effective dose of TILs in step (h).

In some embodiments, the amount of TIL sufficient for administering a therapeutically effective dose in step (h) is about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ .

In some implementations, antigen-presenting cells (APCs) are PBMCs.

In some implementations, PBMCs are added to the cell culture on any day from day 9 to day 14 of step (d).

In some implementations, a non-myeloablative lymphodepletion regimen has been administered to the patient prior to the administration of a therapeutically effective dose of TIL cells in step (h).

In some implementations, the non-myeloablative lymphocyte depletion regimen includes the steps of administering cyclophosphamide at a dose of 60 mg/ ^m² /day for 2 days, followed by administering fludarabine at a dose of 25 mg/ ^m² /day for 5 days.

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

In some implementations, high-dose IL-2 regimens include administering 600,000 or 720,000 IU/kg via 15-minute intravenous infusion every 8 hours until tolerated.

In some implementations, the cancer is selected from: melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal epithelial cell carcinoma.

In some implementations, the cancer is selected from melanoma, HNSCC, cervical cancer, and NSCLC.

In some implementations, the cancer is melanoma.

In some implementations, the cancer is HNSCC.

In some implementations, the cancer is cervical cancer.

In some implementations, the cancer is NSCLC.

The present invention also provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(a) Processed tumor fragments from a patient’s excised tumor are added to a closed system to obtain the first TIL group;

(b) A first amplification is performed by culturing a first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 3 to 14 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (a) to step (b) occurs without opening the system;

(c) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (b) to step (c) occurs without opening the system;

(d) Harvest the therapeutic TIL cluster obtained from step (c); wherein the transition from step (c) to step (d) occurs without opening the system; and

(e) The TIL clusters harvested in step (d) are transferred to an infusion bag; wherein the transfer from step (d) to step (e) occurs without opening the system.

In some implementations, the therapeutic TIL clusters harvested in step (d) contain sufficient TILs for a therapeutically effective dose of TILs.

In some implementations, the number of TILs sufficient for a therapeutically effective dose is about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ .

In some embodiments, the method further includes the step of cryopreserving the infusion bag containing the harvested TIL clusters using a cryopreservation method.

In some implementations, the cryopreservation method uses a 1:1 ratio of harvested TILs to the cryopreservation medium.

In some implementations, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

In some implementations, the PBMC is an irradiated and allogeneic organism.

According to the method of claim 68, PBMCs are added to the cell culture on any one day from day 9 to day 14 of step (c).

In some implementations, the antigen-presenting cells are artificial antigen-presenting cells.

In some implementations, the LOVO cell processing system is used for harvesting in step (d).

In some implementations, the multiple fragments comprise about 4 to about 50 fragments, wherein each fragment has a volume of about 27 ^mm³ .

In some embodiments, the multiple fragments include about 30 to about 60 fragments, with a total volume of about 1300 ^mm³ to about 1500 ^mm³ .

In some implementations, the multiple fragments include approximately 50 fragments with a total volume of approximately 1350 ^mm³ .

In some implementations, the multiple fragments include about 50 fragments with a total mass of about 1 gram to about 1.5 grams.

In some implementations, the multiple fragments include approximately four fragments.

In some embodiments, the second cell culture medium is provided in a container selected from G containers and Xuri cell bags.

In some implementations, the infusion bag in step (e) is an infusion bag containing HypoThermosol.

In some implementations, the first stage in step (b) and the second stage in step (c) are performed for 10 days, 11 days, or 12 days, respectively.

In some implementations, the first stage in step (b) and the second stage in step (c) each last for 11 days.

In some implementations, steps (a) through (e) take approximately 10 to approximately 22 days.

In some implementations, steps (a) through (e) take approximately 10 to approximately 20 days.

In some implementations, steps (a) through (e) take approximately 10 to 15 days.

In some implementations, steps (a) through (e) are performed for less than 22 days.

In some implementations, steps (a) through (e) and cryopreservation are performed for less than 22 days.

In some implementations, steps (b) through (e) are performed in a single container; wherein performing steps (b) through (e) in a single container increases the TIL yield per resected tumor compared to performing steps (b) through (e) in more than one container.

In some implementations, antigen-presenting cells are added to the TIL during the second phase of step (c) without opening the system.

In some implementations, the risk of microbial contamination is reduced compared to open systems.

In some implementations, the TIL from step (e) is injected into the patient.

In some implementations, the closed container comprises a single bioreactor.

In some implementations, the sealed container includes G-REX-10.

In some implementations, the enclosed container includes G-REX-100.

In some embodiments, in step (d), antigen-presenting cells (APCs) are added to the cell culture of the second TIL population at an APC:TIL ratio of 25:1 to 100:1.

In some implementations, the cell culture has a ratio of 2.5 × ^10⁹ APC to 100 × ^10⁶ TIL.

In some embodiments, in step (c), antigen-presenting cells (APCs) are added to the cell culture of the second TIL population at an APC:TIL ratio of 25:1 to 100:1.

In some implementations, the cell culture has a ratio of 2.5 × ^10⁹ APC to 100 × ^10⁶ TIL.

The present invention also provides an expanded TIL population for treating subjects with cancer, wherein the expanded TIL population is a third TIL population obtained by a method comprising the following steps:

(a) First TIL clusters are obtained by processing tumor samples obtained from patients into multiple tumor fragments and tumors removed by the subject;

(b) Adding tumor fragments to the closed system;

(c) A first amplification is performed by culturing a first TIL population in a cell culture medium containing IL-2 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 3 to 14 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (b) to step (c) occurs without opening the system;

(d) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (d) occurs without opening the system;

(e) Exposing second and/or third TIL groups to transcription factors (TFs) and/or other molecules capable of transiently altering protein expression; wherein TFs and/or other molecules capable of transiently altering protein expression provide an increase in tumor antigen expression and/or an increase in the number of tumor antigen-specific T cells in the therapeutic TIL group;

(f) Harvest the therapeutic TIL cluster obtained from step (d); wherein the transition from step (d) to step (e) occurs without opening the system; and

(g) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

(h) Optionally, the infusion bag containing the TIL clusters harvested from step (f) may be cryopreserved using a cryopreservation method.

In some implementations, the TIL group is used to treat a subject with cancer according to the methods described above and herein, wherein the method further includes one or more of the features described above and herein.

This invention also provides a method for determining TIL activity. This disclosure provides a method for determining TIL activity by expanding tumor-infiltrating lymphocytes (TILs) into a larger TIL population, comprising:

(i) Obtain the previously expanded first TIL population;

(ii) A second TIL population is generated by first expansion through culturing the first TIL population in a cell culture medium containing IL-2; and

(iii) A second expansion was performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen-presenting cells (APC) to generate a third TIL population; wherein the number of the third TIL population was at least 100 times greater than that of the second TIL population; wherein the second expansion was performed for at least 14 days to obtain the third TIL population; wherein the activity of the third TIL population was further analyzed.

In some implementations, the method further includes:

(iv) A second amplification was performed by supplementing the cell culture medium of the third TIL population with additional IL-2, additional OKT-3 and additional APC; wherein the second amplification was performed for at least 14 days to obtain a larger TIL population than that obtained in step (iii); wherein the activity of the third TIL population was further analyzed.

In some implementations, the cells are cryopreserved prior to step (i).

In some implementations, the cells are thawed before step (i).

In some implementations, step (iv) 1 to 4 is repeated to obtain sufficient TIL for analysis.

In some implementations, steps (i) through (iii) or (iv) are performed for approximately 40 to approximately 50 days.

In some implementations, steps (i) through (iii) or (iv) are performed for approximately 42 to approximately 48 days.

In some implementations, steps (i) through (iii) or (iv) are performed for approximately 42 to approximately 45 days.

In some implementations, steps (i) through (iii) or (iv) take approximately 44 days.

In some implementations, cells from step (iii) or (iv) express similar levels of CD4, CD8, and TCRαβ as freshly harvested cells.

In some implementations, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

In some implementations, PBMCs are added to the cell culture on any day from day 9 to day 17 of step (iii).

In some implementations, the APC is an artificial APC (aAPC).

In some embodiments, the method further includes the step of transducing a first TIL population with an expression vector containing nucleic acid encoding a high-affinity T cell receptor.

In some implementations, the transduction step occurs before step (i).

In some embodiments, the method further includes the step of transducing a first TIL group with an expression vector containing nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to at least one intracellular domain of a T cell signaling molecule.

In some implementations, the transduction step occurs before step (i).

In some implementations, the activity of TIL is measured.

In some implementations, the activity of the TIL is determined after cryopreservation.

In some implementations, the activity of TILs is determined after cryopreservation and after step (iv).

The present invention also provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising exposing TILs to transcription factors (TFs) and/or other molecules capable of transiently altering protein expression to generate a therapeutic TIL population; wherein the TFs and/or other molecules capable of transiently altering protein expression provide an increase in tumor antigen expression and/or an increase in the number of tumor antigen-specific T cells in the therapeutic TIL population.

In some implementations, transient changes in protein expression lead to the induction of protein expression.

In some implementations, transient changes in protein expression lead to a decrease in protein expression.

In some implementations, more than one sd-RNA is used to reduce transient protein expression.

The present invention also provides a method for evaluating transcription factors (TFs) and/or other molecules capable of transiently altering protein expression; wherein the method comprises expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, exposing the TILs to transcription factors (TFs) and/or other molecules capable of transiently altering protein expression, thereby generating a therapeutic TIL population; wherein the TFs and/or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and/or the number of tumor antigen-specific T cells in the therapeutic TIL population.

In some embodiments, transient alterations in protein expression target genes selected from the group consisting of: PD-1, TGFBR2, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, and cAMP protein kinase A (PKA).

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 the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Contact the first TIL group with at least one sd-RNA; wherein the sd-RNA is added at the following concentrations: 0.1 μM sd-RNA/10,000 TIL/100 μL medium, 0.5 μM sd-RNA/10,000 TIL/100 μL medium, 0.75 μM sd-RNA/10,000 TIL/100 μL medium, 1 μM sd-RNA/10,000 TIL/100 μL medium, 1.25 μM sd-RNA/10,000 TIL The medium solutions are: 1.5 μM sd-RNA/10,000 TIL/100 μL medium, 2 μM sd-RNA/10,000 TIL/100 μL medium, 5 μM sd-RNA/10,000 TIL/100 μL medium, or 10 μM sd-RNA/10,000 TIL/100 μL medium; wherein, sd-RNA is used to inhibit the expression of the molecule, which is selected from PD-1, LAG-3, TIM-3, CISH, and CBLB and combinations thereof;

(e) Optionally, the first TIL group is subjected to a sterile electroporation step; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

(f) Adding OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (f) occurs without opening the system.

(g) Let the second TIL group stand for about 1 day;

(h) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (h) occurs without opening the system;

(i) Harvest the therapeutic TIL clusters obtained from step (h), providing the harvested TIL clusters; wherein the transition from step (h) to step (i) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

(j) The TIL cluster harvested in step (i) is transferred to an infusion bag; wherein the transfer from step (i) to step (j) occurs without opening the system; and

(k) Cryopreservation of harvested TIL groups using dimethyl sulfoxide-based cryopreservation medium.

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 the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Contact the first TIL group with at least one sd-RNA, wherein the sd-RNA is added at the following concentrations: 0.1 μM sd-RNA/10,000 TIL, 0.5 μM sd-RNA/10,000 TIL, 0.75 μM sd-RNA/10,000 TIL, 1 μM sd-RNA/10,000 TIL, 1.25 μM sd-RNA/10,000 TIL, 1.5 μM sd-RNA/10,000 TIL, 2 μM sd-RNA/10,000 TIL, 5 μM sd-RNA/10,000 TIL, or 10 μM sd-RNA/10,000 TIL; wherein the sd-RNA is used to inhibit the expression of a molecule selected from PD-1, LAG-3, TIM-3, CISH, and CBLB, and combinations thereof;

(e) Optionally, a sterile electroporation step is performed on the first TIL group; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

(f) Adding OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (f) occurs without opening the system.

(g) Let the second TIL group stand for about 1 day;

(h) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (g) to step (h) occurs without opening the system;

(i) Harvest the therapeutic TIL clusters obtained from step (h), providing the harvested TIL clusters; wherein the transition from step (h) to step (i) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

(j) The TIL cluster harvested in step (i) is transferred to an infusion bag; wherein the transfer from step (i) to step (j) occurs without opening the system; and

(k) Cryopreservation of harvested TIL groups using dimethyl sulfoxide-based cryopreservation medium.

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 the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

(e) Let the second TIL group stand for about 1 day;

(f) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody and antigen-presenting cells (APC) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (f) occurs without opening the system;

(g) During any of steps (d), (e), and/or (f), the second TIL group is contacted with at least one sd-RNA; wherein the sd-RNA is added at the following concentrations: 0.1 μM sd-RNA/10,000 TIL/100 μL medium, 0.5 μM sd-RNA/10,000 TIL/100 μL medium, 0.75 μM sd-RNA/10,000 TIL/100 μL medium, 1 μM sd-RNA/10,000 TIL/100 μL medium, 1.25 ... The medium contained d-RNA/10,000 TIL/100 μL, 1.5 μM sd-RNA/10,000 TIL/100 μL, 2 μM sd-RNA/10,000 TIL/100 μL, 5 μM sd-RNA/10,000 TIL/100 μL, or 10 μM sd-RNA/10,000 TIL/100 μL. The sd-RNA was used to inhibit the expression of a molecule selected from PD-1, LAG-3, TIM-3, CISH, and CBLB, and combinations thereof.

(h) Optionally, a sterile electroporation step is performed on the second TIL group; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

(i) Harvest the therapeutic TIL clusters obtained from step (g) or (h), and provide the harvested TIL clusters; wherein the transition from step (g) to step (i) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

(j) The TIL cluster harvested in step (i) is transferred to an infusion bag; wherein the transfer from step (i) to step (j) occurs without opening the system; and

(k) Cryopreservation of harvested TIL groups using dimethyl sulfoxide-based cryopreservation medium.

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 the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

(e) Let the second TIL group stand for about 1 day;

(f) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (e) to step (f) occurs without opening the system;

(g) During any of steps (d), (e), and/or (f), the second TIL group is contacted with at least one sd-RNA; wherein the sd-RNA is added at the following concentrations: 0.1 μM sd-RNA/10,000 TIL, 0.5 μM sd-RNA/10,000 TIL, 0.75 μM sd-RNA/10,000 TIL, 1 μM sd-RNA/10,000 TIL, 1.2 μM sd-RNA/10,000 TIL. 5 μM sd-RNA/10,000 TIL, 1.5 μM sd-RNA/10,000 TIL, 2 μM sd-RNA/10,000 TIL, 5 μM sd-RNA/10,000 TIL, or 10 μM sd-RNA/10,000 TIL; wherein the sd-RNA is used to inhibit the expression of the molecule, which is selected from PD-1, LAG-3, TIM-3, CISH, and CBLB and combinations thereof;

(h) Optionally, a sterile electroporation step is performed on the second TIL group; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

(i) Harvest the therapeutic TIL clusters obtained from step (g) or (h), providing the harvested TIL clusters; wherein the transition from step (e) to step (h) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

(j) The TIL cluster harvested in step (i) is transferred to an infusion bag; wherein the transfer from step (h) to step (i) occurs without opening the system; and

(k) Cryopreservation of harvested TIL groups using dimethyl sulfoxide-based cryopreservation medium.

In some implementations, during the first amplification period, sdRNA is added to the first cell population twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

In some implementations, during the first amplification period, sdRNA is added to the second cell population twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

In some implementations, two sdRNAs are added to suppress the expression of two molecules selected from PD-1, LAG-3, TIM-3, CISH, and CBLB.

In some implementations, two sdRNAs are added to suppress the expression of two molecules, wherein the two molecules are selected from:

i.PD-1 and LAG-3;

ii. PD-1 and TIM-3;

iii. PD-1 and CISH;

iv. PD-1 and CBLB;

v.LAG-3 and TIM-3;

vi. LAG-3 and CISH;

vii. LAG-3 and CBLB;

viii.TIM-3 and CISH;

ix.TIM-3 and CBLB; and

X.CISH and CBLB.

In some implementations, more than two sd-RNAs are added to suppress the expression of more than two molecules selected from PD-1, LAG-3, TIM-3, CISH, and CBLB.

In some embodiments, in TILs that have been in contact with at least one sd-RNA, the expression of at least one molecule is reduced by at least 80%, 85%, 90%, or 95%, said at least one molecule being selected from PD-1, LAG-3, TIM-3, CISH, and CBLB.

In some embodiments, in TILs in contact with at least one sd-RNA, the expression of at least one molecule is reduced by at least 80%, 85%, 90%, or 95% for at least 12 hours, at least 24 hours, or at least 48 hours, said at least one molecule being selected from PD-1, LAG-3, TIM-3, CISH, and CBLB.

Attached Figure Description

Figure 1: A diagram showing an implementation of process 2A (a 22-day process for producing TIL).

Figure 2 shows a comparison of the implementation methods of process 1C and process 2A for producing TIL.

Figure 3 shows the timeline of the 1C process.

Figure 4: This illustrates the implementation process of a TIL therapy (including administration and combination therapy steps) using process 2A to produce TILs with high cell counts.

Figure 5: This illustrates the implementation process of a TIL therapy (including administration and combination therapy steps) using process 2A to produce TILs when cell counts are low.

Figure 6: A detailed schematic diagram showing the implementation of the 2A process.

Figures 7A-7C illustrate the main steps of an implementation of process 2A (including the cryopreservation step).

Figure 8: Exemplary process 2A diagram, providing an overview of steps A through F.

Figure 9: Process flow diagram for the data collection plan of process 2A.

Figure 10: Exemplary implementation of the Rapid Expansion Protocol (REP). Upon tumor arrival, the tumor is lysed and placed in a G-Rex flask containing IL-2 for TIL amplification (pre-REP amplification) for 11 days. For triplet studies, IL-2/IL-15/IL-21 are added at the start of pre-REP. For the Rapid Expansion Protocol (REP), TILs are cultured with feeder cells and OKT3 for an additional 11 days for REP amplification.

Figure 11: Exemplary production process of cryopreserved TIL (approximately 22 days).

Figure 12: A diagram showing an implementation of process 2A (a 22-day process for producing TIL).

Figure 13: Comparison table of steps A to F of exemplary embodiments of process 1C and process 2A.

Figure 14: Detailed comparison of the implementation methods of process 1C and process 2A.

Figure 15: Description of an implementation of the cryopreserved TIL production process (22 days).

Figure 16: Table of process improvements from Gen 1 to Gen 2.

Figure 17: Scheme for the production process of Gen 2 cryopreserved LN-144.

Figure 18: A diagram showing an implementation of process 2A (a 22-day process for producing TIL).

Figure 19: A schematic diagram showing the aseptic welding (see process note 5.11 of Example 16) of the TIL suspension transfer pack to the bottom (single line) of the gravity blood filter.

Figure 20: A schematic diagram showing the process of aseptically welding the red culture medium removal tubing from the GRex100MCS (see process note 5.11 in Example 16) to the “supernatant” transfer pack.

Figure 21 shows a schematic diagram of welding 4S-4M60 (see process note 5.11 of Example 16) to CC2 CellConnect, replacing the single tip of CellConnect device (B) with the four spike ends of the 4S-4M60 manifold at (G).

Figure 22: A schematic diagram showing the soldering (see process note 5.11 of Example 16) of the repeater fluid transfer set to one of the male Luer interface terminals of the 4S-4M60.

Figure 23: A schematic diagram showing the aseptic welding of the long end of the gravity blood filter (see process note 5.11 of Example 16) to the LOVO source bag.

Figure 24: A schematic diagram showing the aseptic welding (see process note 5.11 of Example 16) of one of the two source lines of the filter to the “combined TIL suspension” collection bag.

Figure 25 shows a schematic diagram of aseptically soldering a 4S-4M60 (see process note 5.11 of Example 16) to a CC2CellConnect, replacing the single tip of the Cell Connect device (B) with the four tips of the 4S-4M60 manifold at (G).

Figure 26: shows a schematic diagram of aseptically welding the CS750 cryopreservation bag (see process note 5.11 of Example 16) to the harness prepared in step 8.14.8, replacing one of the four male Luer interface ends (E) with the bag.

Figure 27: A schematic diagram showing the welding of the CS-10 bag (see process note 5.11 of Example 16) to the nozzle of the 4S-4M60.

Figure 28: A schematic diagram showing the welding of the “Formulated TIL” bag (see process note 5.11 of Example 16) to the remaining tip (A) on the device prepared in step 8.14.10.

Figure 29: A schematic diagram of heat sealing at F (see process note 5.12 in Example 16), with the empty retentate bag and CS-10 bag removed.

Figure 30: A schematic diagram showing an implementation of the TIL process for transient gene editing.

Figure 31: A schematic diagram showing an implementation of the TIL process for transient gene editing.

Figure 32: A schematic diagram showing the incorporation of the RNA transfer step into the TIL process for the purpose of transient gene reprogramming.

Figure 33: Shows an overview of the proposed genetic engineering method for transiently altering gene expression in TILs.

Figure 34 shows an overview of chemokines and chemokine receptors, and transient changes in the gene expression of these chemokines and chemokine receptors can be used to improve TIL transport to tumor sites.

Figure 35: Shows a second profile of chemokines and chemokine receptors, whose transient changes in gene expression can be used to improve TIL transport to tumor sites.

Figure 36: A schematic structural representation of an exemplary self-delivered ribonucleic acid (sdRNA) implementation. See Ligtenberg et al., Mol. Therapy, 2018.

Figure 37: A schematic structural representation of an exemplary sd-RNA implementation. See U.S. Patent Publication No. 2016/0304873.

Figure 38 illustrates an exemplary scheme for mRNA synthesis using a DNA template obtained by PCR using specially designed primers. The forward primer contains a phage promoter suitable for in vitro transcription, and the reverse primer contains a polyT stretch. The PCR product is an expression cassette suitable for in vitro transcription. The polyadenylated nucleotide at the 3' end of the nascent mRNA prevents runoff RNA synthesis and the production of double-stranded RNA products. After transcription, the polyA tail can be further extended using poly(A) polymerase (see U.S. Patent No. 8,859,229).

Figure 39: A diagram showing Sd-rxRNA-mediated silencing of PDCD1, TIM3, CBLB, LAG3, and CISH.

Figure 40: Sd-rxRNA-mediated gene silencing in TILs; exemplary protocol. Exemplary tumors include melanoma (fresh or frozen; n=6), breast tumors (fresh or frozen; n=5), lung tumors (n=1), sarcomas (n=1), and/or ovarian cancer (n=1).

Figure 41: Reduced protein expression was detected in 4 out of 5 targets. PD1: n=9, TIM3: n=8, LAG3/CISH: n=2, Cbl-b: n=2. Preparations derived from pre-REP melanoma and fresh breast cancer TILs (prep), 2 uM sd-rxRNA. Calculated as KD% (100 - (100 * (target gene/NTC))).

Figure 42: Sd-rxRNA-induced decrease in KD over time and stimulation. n=3, preparation from pre-REP melanoma TIL, 2 uM sd-rxRNA.

Figure 43: PDCD1 sd-rxRNA slightly affected TIL activity. PD1,TIM3 n>6, formulations from pre-REP melanoma/fresh breast cancer TILs. LAG3,CISH n=2, pre-REP melanoma and breast cancer TILs, 2uM sd-rxRNA.

Figure 44: Sd-rxRNA-mediated KD of PD1 and TIM3 is associated with phenotypic alterations (indicating TIL activation). n=3, formulation from pre-REP melanoma TIL, 2 uM sd-rxRNA.

Figures 45A and 45B: PD1 and TIM3 knockout by sd-rxRNA does not affect the expression of other inhibition/exhaustion markers. A) and B) n=3, TIM3: n=2, formulation from pre-REP melanoma TIL, 2 uM sd-rxRNA.

Figure 46: PD1 and TIM3 KD did not significantly improve IFNγ secretion. n=3, preparations derived from pre-REP melanoma TILs, 2 uM sd-rxRNA.

Figures 47A-47F: CD107a mobilization is unaffected by any sd-rxRNA. A) n=6, formulation from pre-REP melanoma TILs, 2 uM sd-rxRNA. B) n=2, formulation from pre-REP melanoma and breast cancer TILs, 2 uM sd-rxRNA. C) n=3, frozen melanoma and fresh breast cancer TILs. D) n=3, frozen melanoma and fresh breast and lung cancer TILs. E) and F) n=3, fresh formulation from breast cancer tumors.

Figure 48: xCELLigence Real-Time Cell Analysis (RTCA).

Figures 49A and 49B: PD1 KD TILs induce greater killing efficiency. A) Representative graph of killing efficiency. B) Representative graph of n=3, melanoma TILs, 2 uM sd-rxRNA.

Figures 50A and 50B: Sd-rxRNA dose-response experiments. A) n=3, fresh formulation from breast cancer tumors. B) n=3, formulation from pre-REP melanoma TILs.

Figure 51: Sd-rxRNA-mediated CBLB knockout could not be detected. A) Graph. B) Flow cytometry analysis. n=2, formulations from pre-REP melanoma and fresh breast cancer TILs. CBLB mRNA levels were unchanged compared to NTC. Cbl-b protein levels were unchanged as determined by flow cytometry.

Figures 52A and 52B: Show the detection of sd-rxRNA-mediated gene silencing during TIL production at Iovance, assessing the TIL phenotype. sd-rxRNA-mediated PD-1 knockout was associated with phenotypic alterations (indicating TIL activation). PD-1, n>6, formulation from pre-REP melanoma/fresh breast cancer TILs, 2 uM sd-rxRNA. A) CD25, CCR7, CD27, CD28, CD56, CD95, 4-1BB, and OX40. B) CD25, CD56, CCR7, 4-1BB, and OX40. N=12, fresh and frozen TILs; breast, melanoma, ovary, and lung.

Figures 53A and 53B: Addition of PD1 sd-rxRNA significantly reduced cell growth but did not reduce TIL activity. A) Fold expansion. B) Cell viability. n=7, breast TIL, sarcoma TIL, and lung TIL.

Figures 54A and 54B: PD1 KD did not improve CD107a mobilization and IFNγ secretion in response to nonspecific stimulation. A) Percentage of CD8 cells expressing CD107a before and after stimulation. B) IFNγ secretion before and after stimulation. n=6, melanoma TIL.

Figure 55 shows the experimental design of Example 13.

Sequence List Description

SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

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

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 adefovir.

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 4-1BB.

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

SEQ ID NO: 11 is the heavy chain of utomilumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO: 12 is the light chain of utolumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO: 13 is the heavy chain variable region (V _H ) of utolumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO: 14 is the light chain variable region (V _L ) of utolumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO: 15 is the heavy chain CDR1 of utolumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO: 16 is the heavy chain CDR2 of utolumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

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

SEQ ID NO: 18 is the light chain CDR1 of utolumab (PF-05082566), a monoclonal antibody that acts as a 4-1BB agonist.

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

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

SEQ ID NO: 21 is the heavy chain of urelumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO: 22 is the light chain of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO: 23 is the heavy chain variable region (V _H ) of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO: 24 is the light chain variable region (V _L ) of urilumab (BMS-663513), a 4-1BB agonist monoclonal antibody.

SEQ ID NO: 25 is the heavy chain CDR1 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO: 26 is the heavy chain CDR2 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO: 27 is the heavy chain CDR3 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO: 28 is the light chain CDR1 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO: 29 is the light chain CDR2 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO: 30 is the light chain CDR3 of urilumab (BMS-663513), a monoclonal antibody that acts as a 4-1BB agonist.

SEQ ID NO: 31 is the Fc domain of the TNFRSF agonist fusion protein.

SEQ ID NO: 32 is a linker for the TNFRSF agonist fusion protein.

SEQ ID NO: 33 is a linker for the TNFRSF agonist fusion protein.

SEQ ID NO: 34 is a linker for the TNFRSF agonist fusion protein.

SEQ ID NO: 35 is a linker for the TNFRSF agonist fusion protein.

SEQ ID NO: 36 is a linker for the TNFRSF agonist fusion protein.

SEQ ID NO: 37 is the linker for the TNFRSF agonist fusion protein.

SEQ ID NO: 38 is a linker for the TNFRSF agonist fusion protein.

SEQ ID NO: 39 is a linker for the TNFRSF agonist fusion protein.

SEQ ID NO: 40 is a linker for the TNFRSF agonist fusion protein.

SEQ ID NO: 41 is a linker for the TNFRSF agonist fusion protein.

SEQ ID NO: 42 is the Fc domain of the TNFRSF agonist fusion protein.

SEQ ID NO: 43 is a linker for the TNFRSF agonist fusion protein.

SEQ ID NO: 44 is a linker for the TNFRSF agonist fusion protein.

SEQ ID NO: 45 is a linker for the TNFRSF agonist fusion protein.

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

SEQ ID NO: 47 is the soluble portion of the 4-1BBL polypeptide.

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

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

SEQ ID NO: 50 is the heavy chain variable region (V _H ) of the 4-1BB agonist antibody 4B4-1-1 version 2.

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

SEQ ID NO: 52 is the heavy chain variable region (V _H ) of the 4-1BB agonist antibody H39E3-2.

SEQ ID NO: 53 is the light chain variable region (V _L ) of the 4-1BB agonist antibody H39E3-2.

SEQ ID NO: 54 is the amino acid sequence of human OX40.

SEQ ID NO: 55 is the amino acid sequence of mouse OX40.

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

SEQ ID NO: 57 is the light chain of talixizumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO: 58 is the heavy chain variable region (V _H ) of talixizumab (MEDI-0562), an OX40 agonist monoclonal antibody.

SEQ ID NO: 59 is the light chain variable region (V _L ) of talixizumab (MEDI-0562), an OX40 agonist monoclonal antibody.

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

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

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

SEQ ID NO: 63 is the light chain CDR1 of talixizumab (MEDI-0562), an OX40 agonist monoclonal antibody.

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

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

SEQ ID NO: 66 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 67 is the light chain of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 68 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 69 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 70 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 71 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 72 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 73 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 74 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 75 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

SEQ ID NO: 76 is the heavy chain of 18D8, an OX40 agonist monoclonal antibody.

SEQ ID NO: 77 is the light chain of 18D8, an OX40 agonist monoclonal antibody.

SEQ ID NO: 78 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 79 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 80 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 81 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 82 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 83 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 84 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 85 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

SEQ ID NO: 86 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 87 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 88 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 89 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 90 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 91 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 92 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 93 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

SEQ ID NO: 94 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO: 95 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO: 96 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO: 97 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO: 98 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO: 99 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO: 100 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

SEQ ID NO: 101 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

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

SEQ ID NO: 103 is the soluble portion of the OX40L polypeptide.

SEQ ID NO: 104 is an alternative soluble fraction of the OX40L polypeptide.

SEQ ID NO: 105 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 008.

SEQ ID NO: 106 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 008.

SEQ ID NO: 107 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 011.

SEQ ID NO: 108 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody 011.

SEQ ID NO: 109 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 021.

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

SEQ ID NO: 111 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody 023.

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

SEQ ID NO: 113 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody.

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

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

SEQ ID NO: 116 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody.

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

SEQ ID NO: 118 is the heavy chain variable region ( _VH ) of a humanized OX40 agonist monoclonal antibody.

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

SEQ ID NO: 120 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO: 121 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO: 122 is the heavy chain variable region (V _H ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO: 123 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO: 124 is the light chain variable region ( _VL ) of a humanized OX40 agonist monoclonal antibody.

SEQ ID NO: 125 is the heavy chain variable region (V _H ) of the OX40 agonist monoclonal antibody.

SEQ ID NO: 126 is the light chain variable region (V _L ) of the OX40 agonist monoclonal antibody.

Detailed Implementation

I. Introduction

Adoptive cell therapy using TILs cultured in vitro via a rapid expansion protocol (REP) has yielded successful adoptive cell treatments in melanoma patients following host immunosuppression. Current infusion acceptance parameters depend on the TIL composition readings (e.g., CD28, CD8, or CD4 positive) and the fold increase and viability of the REP product.

Current REP protocols rarely consider the health status of the TILs to be injected into the patient. T cells undergo a profound metabolic transformation during their maturation from naive T cells to effector T cells (see Chang et al., Nat. Immunol. 2016, 17, 364, explicitly incorporated herein in its entirety, particularly the discussion and markers of anaerobic and aerobic metabolism). For example, naive T cells rely on mitochondrial respiration to produce ATP, while mature, healthy effector T cells such as TILs are highly glycolytic, relying on aerobic glycolysis to provide the bioenergetic substrates required for their proliferation, migration, activation, and antitumor efficacy.

Current TIL manufacturing processes are limited by length, cost, aseptic issues, and other factors described herein, severely restricting the commercialization potential of such methods; for these and other reasons, no commercially available methods are currently available. This invention provides a TIL manufacturing method and a therapy based on such a method, the TIL manufacturing method utilizing a transient protein expression alteration approach, which is suitable for commercial-scale production and regulatory approval for use in human patients at multiple clinical centers. This invention also provides a transient gene alteration method for reprogramming TILs to prepare a therapeutic TIL population with enhanced therapeutic efficacy.

II. Definition

Unless otherwise defined, 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 pertains. All patents and publications cited herein are incorporated herein by reference in their entirety.

The term "in vivo" refers to events that occur within the body of a subject.

The term "in vitro" refers to events that occur outside the body of a subject. In vitro assays include cell-based assays using live or dead cells, and may also include cell-free assays that do not use intact cells.

The term "ex vivo" refers to an event involving the processing or procedure of cells, tissues, and/or organs that have been removed from a subject's body. Appropriately, these cells, tissues, and/or organs may be returned to the subject during surgery or treatment.

The term "rapid amplification" refers to an increase in the number of antigen-specific TILs by at least approximately 3 times (or 4, 5, 6, 7, 8, or 9 times) within one week, more preferably by at least approximately 10 times (or 20, 30, 40, 50, 60, 70, 80, or 90 times) within one week, or most preferably by at least approximately 100 times within one week. Some rapid amplification protocols are outlined below.

In this article, “tumor-infiltrating lymphocytes” or “TILs” refers to a population of cells initially obtained as leukocytes that have left the subject’s bloodstream and migrated to the tumor. TILs include, but are not limited to, CD8 ⁺ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include primary TILs and secondary TILs. “Primary TILs” are those obtained from patient tissue samples as described herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any population of TIL cells that has expanded or proliferated as discussed herein, including, but not limited to, bulk TILs and expanded TILs (“REP TILs” or “post-REP TILs”). TIL cell populations may include genetically modified TILs.

In this article, a “cell population” (including TILs) refers to a group of cells sharing common characteristics. Typically, the population size is between 1 × ^10⁶ and 1 × ^10¹⁰ , with different TIL populations containing different numbers. For example, in the presence of IL-2, the initial growth of primary TILs produces a large TIL population of approximately 1 × ^10⁸ cells. Typically, REP expansion is performed to provide a population of 1.5 × ^10⁹ to 1.5 × ^10¹⁰ cells for infusion.

As used herein, “cryopreserved TIL” refers to primary, large-volume, or expanded TILs (REP TILs) that have been processed and stored at approximately -150°C to -60°C. General methods for cryopreservation are also described elsewhere herein, including in the examples. For clarity, “cryopreserved TIL” can be distinguished from frozen tissue samples that can be used as a source of primary TILs.

In this article, "thawed cryopreserved TILs" refers to a group of TILs that were previously cryopreserved and then processed to be restored to a temperature above room temperature (including but not limited to cell culture temperature or temperature at which TILs can be applied to patients).

Tumor-associated lymphoid tissue (TILs) can typically be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and influence treatment. TILs are generally classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into the patient.

The term "cryopreservation media" or "cryopreservation medium" refers to any culture medium that can be used for the cryopreservation of cells. Such media may include those containing 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to a commercially available cryopreservation medium from Stemcell Technologies or Biolife Solutions. CS10 medium may be referred to by the trade name "CS10". CS10 medium is a serum-free and animal-free culture medium containing DMSO.

The term "central memory T cells" refers to a subset of human T cells that are CD45R0+ and constitutively express CCR7 ( ^high CCR7) and CD62L ( ^high CD62). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Upon TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells are predominantly found in the CD4 compartment in the blood and are proportionally enriched in lymph nodes and tonsils in the human body.

The term "effective memory T cells" refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+, but lack constitutive expression of CCR7 ( ^low CCR7) and exhibit heterogeneity or low expression of CD62L ( ^low CD62L). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines, including interferon-γ, IL-4, and IL-5, upon antigen stimulation. Effector memory T cells are predominantly found in the CD8 compartment of the blood and are proportionally enriched in the lungs, liver, and intestines in humans. CD8 ⁺ effector memory T cells carry a large amount of perforin.

The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of this invention. Closed systems include, for example, but not limited to, closed G-containers. Once tumor fragments are added to the closed system, the system is not opened to the external environment until TIL is ready for administration to the patient.

The terms “fragmenting,” “fragment,” and “fragmented” used in this article to describe methods of destroying tumors include mechanical fragmentation methods, such as breaking, slicing, dividing, and pulverizing tumor tissue, as well as any other methods that destroy the physical structure of tumor tissue.

The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. Preferably, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMCs are antigen-presenting cells.

The term "anti-CD3 antibody" refers to an antibody or its variants (such as monoclonal antibodies), including human, humanized, chimeric, or mouse antibodies against the CD3 receptor in the T-cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as moromuzumab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

The term “OKT-3” (also referred to herein as “OKT3”) refers to a monoclonal antibody or its biosimilar or variant, including human, humanized, chimeric, or murine antibodies against the CD3 receptor in the T-cell antigen receptor of mature T cells, as well as commercially available forms such as OKT-3 (30 ng/mL, pure MACS GMP CD3, Miltenyi Biotech, Inc., San Diego, California, USA) and moromumab or its variants, conserved amino acid substitutions, glycoforms, or biosimilars. The amino acid sequences of the heavy and light chains of moromumab are given in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). Hybridomas capable of generating OKT-3 are deposited at the American Type Culture Collection (ATCC) with the assigned ATCC accession number CRL 8001. Hybridomas capable of producing OKT-3 are also deposited at the European Collection of Authenticated Cell Cultures (ECACC), with the catalog number 86022706.

Table 1: Amino acid sequence of moromuzumab

The term “IL-2” (also referred to herein as “IL2”) refers to the T cell growth factor known as interleukin-2, including all forms of IL-2, including human and mammalian forms, conserved amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J.I. mmunol. 2004, 172, 3983-88 and Malek, Annu. Rev. I. mmunol. 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 this invention is given in Table 2 (SEQ ID NO: 3). For example, the term IL-2 includes recombinant human IL-2, such as des-alanyl-1 (proleukin, commercially available from multiple suppliers, 22 million IU vials per individual use), and other commercially available recombinant IL-2 from CellGenix, Inc. (CELLGRO GMP) of Portsmouth, New Hampshire, USA, or ProSpec-Tany TechnoGene Ltd. (catalog number CYT-209-b) of East Brunswick, New Jersey, USA, and other commercial equivalents from other suppliers. Des-alanyl-1 is a non-glycosylated recombinant human form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of the des-alanyl-1 suitable for use in this invention is given in Table 2 (SEQ ID NO: 4). The term IL-2 also includes PEGylated forms of IL-2 as described herein, including the PEGylated IL-2 prodrug NKTR-214, commercially available from Nektar Therapeutics, Inc., South San Francisco, California, USA. NKTR-214 and PEGylated IL-2 suitable for use in this invention are described in U.S. Patent Application Publication No. US2014/0328791A1 and International Patent Application Publication No. WO2012/065086A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in this invention are described in U.S. Patents 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference. IL-2 formulations suitable for use in this invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

Table 2: Amino acid sequence of interleukins

The term "IL-4" (also referred to as "IL4" in this text) refers to a cytokine called interleukin-4, produced by Th2 T cells and eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. (Steinke and Borish, Respir. Res. 2001, 2, 66-70). Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, and induces B cells to convert to IgE and IgG1 expression. The recombinant human IL-4 suitable for use in this invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd. (catalog number CYT-211) in East Brunswick, New Jersey, USA, and ThermoFisher Scientific, Inc. (recombinant human IL-15 protein, catalog number Gibco CTP0043) in Waltham, MA, USA. The amino acid sequence of the recombinant human IL-4 suitable for use in this invention is given in Table 2 (SEQ ID NO: 5).

The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue-derived cytokine called interleukin-7, which is available from stromal cells, epithelial cells, and dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell growth. IL-7 binds to the IL-7 receptor, a heterodimer composed of IL-7 receptor α and a common gamma chain receptor, which plays an important role in the thymic development and peripheral survival of T cells in a series of signaling pathways. The recombinant human IL-7 suitable for use in this invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd. of East Brunswick, NJ (catalog number CYT-254) and ThermoFisher Scientific, Inc. of Waltham, MA (recombinant human IL-15 protein, catalog number GibcoPHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in this invention is given in Table 2 (SEQ ID NO: 6).

The term "IL-15" (also referred to herein as "IL15") refers to the T-cell growth factor called interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conserved amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a non-glycosylated polypeptide single chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd. (catalog number CYT-230-b) in East Brunswick, New Jersey, USA, and ThermoFisher Scientific, Inc. (recombinant human IL-15 protein, catalog number 34-8159-82) in Waltham, MA, USA. The amino acid sequence of the recombinant human IL-15 suitable for use in this invention is given in Table 2 (SEQ ID NO: 7).

The term "IL-21" (also referred to herein as "IL21") refers to a pleiotropic cytokine protein called interleukin-21 and includes all forms of IL-21, including human and mammalian forms, conserved amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 is primarily produced by natural killer T cells and activated human CD4 ⁺ T cells. Recombinant human IL-21 is a non-glycosylated polypeptide single chain containing 132 amino acids and a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd. (catalog number CYT-408-b) in East Brunswick, New Jersey, USA, and ThermoFisher Scientific, Inc. (recombinant human IL-21 protein, catalog number 14-8219-80) in Waltham, MA, USA. The amino acid sequence of the recombinant human IL-21 suitable for use in this invention is given in Table 2 (SEQ ID NO: 8).

When the instructions are “effective antitumor dose”, “effective tumor-suppressing dose” or “therapeutic dose”, the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis and patient (subject) condition. Generally speaking, a pharmaceutical composition comprising the tumor-infiltrating lymphocytes described herein (e.g., second-generation TILs or genetically modified cytotoxic lymphocytes) may be administered at doses of ^10⁴ to ^10¹¹ cells/kg body weight (e.g., ^10⁵ to ^10⁶ , ^10⁵ to ^10¹⁰ , ^10⁵ to ^10¹¹ , ^10⁶ to 10¹⁰, 10⁶ to ^{10¹¹ , 10⁷ to 10¹¹} , ^10⁷ to ^10¹⁰ , ^10⁸ to ^10¹¹ , ^10⁸ to ^10¹⁰ , ^10⁹ to ^10¹¹ , or ^10⁹ to ^10¹⁰ cells/kg body weight), including all integer values within these ranges. The tumor-infiltrating lymphocyte composition ⁽ in some cases, including genetically modified cytotoxic lymphocytes) may also be administered multiple times at these doses. Tumor-infiltrating lymphocytes (in some cases, including genetically induced ones) can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., “New” Eng. J. of Med. 319: 1676, 1988). By monitoring a patient’s disease signs and adjusting treatment accordingly, medical professionals can easily determine the optimal dosage and treatment regimen for a particular patient.

The term "hematologic malignancies" refers to cancers and tumors of mammals affecting hematopoietic and lymphatic tissues (including but not limited to blood, bone marrow, lymph nodes, and tissues of the lymphatic system). Hematologic malignancies are also known as "liquid tumors." Hematologic 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 malignancies" refers to hematologic malignancies affecting B cells.

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

The term "liquid tumor" refers to an abnormal cluster of cells that is essentially a fluid. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematologic malignancies. TILs obtained from liquid tumors may also be referred to as bone marrow infiltrating lymphocytes (MILs) in this article.

As used herein, the term “microenvironment” can refer to the entire solid tumor or hematologic malignancy microenvironment, or to a single cell subset within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of “cells, soluble factors, signaling molecules, extracellular matrix, and mechanistic clues that promote tumor transformation, support tumor growth and invasion, protect tumors from host immunity, promote resistance to treatment, and provide a niche for the proliferation of overt metastases,” as described by Swartz et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, the immune system rarely clears tumors due to immunosuppression of the microenvironment.

In one embodiment, the present invention includes a method of treating cancer with a group of triglycerides (TILs); wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TILs of the present invention. In some embodiments, a group of TILs may be provided; wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TILs of the present invention. In one embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/day for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/ ^m² /day for 5 days (days 27 to 23 prior to TIL infusion). In one embodiment, following the non-myeloablative chemotherapy and TIL infusion according to the present invention (on day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU/kg every 8 hours until physiologically tolerated.

Experimental findings have shown that lymphocyte depletion prior to adoptive metastasis of tumor-specific T lymphocytes plays a key role in enhancing therapeutic efficacy by eliminating competing components of the regulatory T cells and the immune system (“cytokine precipitation”). Therefore, some embodiments of the present invention employ a lymphocyte depletion step (sometimes referred to as “immunosuppressive modulation”) on the patient prior to the introduction of the rTIL of the present invention.

As used herein, the terms “co-administered,” “combined with,” “in combination with,” “simultaneously,” and “co-” encompass administering two or more active pharmaceutical ingredients (in a preferred embodiment of the invention, for example, at least one potassium channel agonist in combination with multiple TILs) to a subject such that the active pharmaceutical ingredients and/or their metabolites are simultaneously present in the subject. Co-administration includes: administering different compositions simultaneously, administering different compositions at different times, or administering a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration of different compositions and administration of compositions in which two pharmaceutical ingredients are present are preferred.

The term "effective amount" or "therapeutic effective amount" refers to an amount of compound or combination of compounds as described herein that is sufficient to achieve the intended application, including but not limited to the treatment of a disease. Therapeutic effective amounts can vary depending on the intended application (in vitro or in vivo), the subject being treated and the disease condition (e.g., the subject's weight, age, and sex), the severity of the disease condition, or the method of administration. This term also applies to doses that induce a specific response in target cells (e.g., a reduction in platelet adhesion and/or cell migration). The specific dose will depend on the specific compound chosen, the administration regimen to be followed, whether the compound is administered in combination with other compounds, the time of administration, the tissue of administration, and the physical delivery system carrying the compound.

The terms “treatment,” “treating,” “treat,” etc., refer to achieving the desired pharmacological and/or physiological effect. This effect may be preventative in relation to the complete or partial prevention of a disease or its symptoms, and/or therapeutic in relation to the partial or complete cure of a disease and/or adverse reactions caused by the disease. As used herein, “treatment” includes any treatment of a disease in mammals, particularly humans, including: (a) preventing the onset of the disease in subjects who may be susceptible to it but have not yet been diagnosed with it; (b) suppressing the disease, i.e., preventing its development or progression; and (c) alleviating the disease, i.e., causing disease remission and/or alleviating one or more disease symptoms. “Treatment” also means including the delivery of agents to provide a pharmacological effect, even in the absence of disease or symptom. For example, “treatment” includes the delivery of compositions that can elicit an immune response or confer immunity in the absence of disease, such as in the case of vaccines.

When referring to nucleic acid or protein components, the term "heterologous" means that the nucleic acid or protein contains two or more subsequences that are not naturally identical to each other. For example, nucleic acids are often produced through recombination and have two or more sequences from unrelated genes that are arranged to produce new functional nucleic acids, such as a promoter from one source and a coding region from another, or coding regions from different sources. Similarly, a heterologous protein means that the protein contains two or more subsequences that are not naturally identical to each other (e.g., a fusion protein).

In the case of two or more nucleic acids or peptides, the terms "sequence identity," "percentage identity," and "sequence percentage identity" (or their synonyms, such as "99% identical") refer to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotide or amino acid residues when compared and aligned (introducing vacancies if necessary) to obtain maximum correspondence, regardless of any conserved amino acid substitutions as part of sequence identity. The percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art for obtaining alignments of amino acid or nucleotide sequences. Suitable procedures for determining the percentage of sequence identity include, for example, the BLAST program suite, available from the National Center for Biotechnology Information (NCBI) BLAST website of the U.S. government. The BLASTN or BLASTP algorithms can be used for comparisons between two sequences. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign, available from DNASTAR, are other publicly available software programs that can be used for sequence alignment. Those skilled in the art can determine appropriate parameters for maximizing alignment using specific alignment software. In some implementations, the default parameters of the comparison software are used.

As used herein, the term "variant" includes, but is not limited to, antibodies or fusion proteins that contain an amino acid sequence different from that of a reference antibody by means of one or more substitutions, deletions, and/or additions at positions within or near the amino acid sequence of the reference antibody. A variant may contain one or more conserved substitutions in its amino acid sequence compared to that of the reference antibody. Conserved substitutions may involve, for example, substituting similar charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference antibody. The term "variant" also includes polyethylene glycol-modified antibodies or proteins.

In this document, “tumor-infiltrating lymphocytes” or “TILs” refers to a population of cells initially obtained as leukocytes that have left the subject’s bloodstream and migrated to the tumor. TILs include, but are not limited to, CD8 ⁺ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 ⁺ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include primary TILs and secondary TILs. “Primary TILs” are those obtained from patient tissue samples as described herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any population of TIL cells that have been expanded or proliferated as discussed herein, including, but not limited to, large numbers of TILs, expanded TILs (“REP TILs”), and “reREP TILs”. For example, reREP TILs may include second-amplified TILs or additional second-amplified TILs (e.g., those described in step D of Figure 8, including TILs referred to as reREP TILs).

Tumor-associated lymphoid tissue (TILs) can generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and influence treatment. TILs are typically classified by expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors after reintroduction into the patient. TILs can also be characterized by potency—for example, TILs can be considered effective if, for example, the release of interferon (IFN) is greater than about 50 pg/mL, greater than about 100 pg/mL, greater than about 150 pg/mL, or greater than about 200 pg/mL.

The term "deoxyribonucleic acid" encompasses both natural and synthetic unmodified and modified deoxyribonucleic acids. Modifications include changes to the sugar moiety, base moiety, and/or bonds between deoxyribonucleic acids in oligonucleotides.

The term "RNA" defines a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA, substantially pure RNA, synthetic RNA, recombinant RNA, and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or modification of more than one nucleotide). The nucleotides in the RNA molecules described herein may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

The term "modified nucleotide" refers to a nucleotide that has undergone one or more modifications to its nucleoside, nucleotide base, pentose ring, or phosphate group. For example, modified nucleotides do not include ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate, nor deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. Modifications include those naturally occurring due to modifications by enzymes that modify nucleotides (e.g., methyltransferases).

Modified nucleotides also include synthetic nucleotides or nucleotides that are not naturally occurring. Synthetic or non-natural modifications in nucleotides include modifications having a 2' modification (e.g., 2'-O-methyl, 2'-methoxyethoxy, 2'-fluorine, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'- _CH₂ -O-2'-bridge, 4'-( _CH₂ ) _₂ -O-2'-bridge, 2'-LNA, and 2'-O--(N-carbamate)) or those containing base analogues. With respect to 2'-modified nucleotides as described in this disclosure, "amino" refers to modified or unmodified 2'- _NH₂ or 2'-O-- _NH₂ . Such modifying groups are described, for example, in U.S. Patent Nos. 5,672,695 and 6,248,878; which are incorporated herein by reference.

The term "microRNA" or "miRNA" refers to a nucleic acid that forms a single-stranded RNA. When the miRNA is expressed in the same cell as a gene or target gene, the single-stranded RNA has the ability to alter the expression of the gene or target gene (reduce or suppress expression; regulate expression; directly or indirectly enhance expression). In one embodiment, miRNA refers to a nucleic acid that is substantially or completely identical to a target gene and forms a single-stranded miRNA. In some embodiments, miRNA may be in the form of pre-miRNA, wherein the pre-miRNA is a double-stranded RNA. The sequence of the miRNA may correspond to the full-length target gene or its subsequence. Typically, the length of miRNA is at least about 15 to 50 nucleotides (e.g., each sequence of a single-stranded miRNA is 15 to 50 nucleotides long, and the length of a double-stranded pre-miRNA is about 15 to 50 base pairs). In some embodiments, miRNA is 20 to 30 base nucleotides long. In some embodiments, miRNA is 20 to 25 nucleotides long. In some implementations, the miRNA is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.

The term “target gene” includes genes known or identified as regulating the expression of genes involved in immune resistance mechanisms. These genes may be one of several groups of genes, such as inhibitory receptors like CTLA4 and PD1; cytokine receptors that inactivate immune cells, such as TGF-β receptor, LAG3 and/or TIM3, and combinations thereof. In some embodiments, the target genes include one or more of PD-1, TGFBR2, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, NOTCH 1/2 intracellular domain (ICD), NOTCH ligand mDLL1, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, and/or cAMP protein kinase A (PKA).

The terms “small interfering RNA” or “siRNA” or “short interfering RNA” or “silent RNA” define a group of double-stranded RNA molecules, consisting of a sense RNA strand and an antisense RNA strand, each typically about 1022 nucleotides in length, optionally containing a 3' overhang of 1 to 3 nucleotides. siRNAs are active in the RNA interference (RNAi) pathway and interfere with the expression of specific target genes through complementary nucleotide sequences.

The term sd-RNA refers to a "self-delivering" RNAi reagent that forms an asymmetric double-stranded RNA-antisense oligonucleotide hybrid. The double-stranded RNA comprises a lead (sense) strand of approximately 19 to 25 nucleotides and a follower (antisense) strand of approximately 10 to 19 nucleotides, forming a double helix that results in a single-stranded phosphorothiolated tail of approximately 5 to 9 nucleotides. In some embodiments, the RNA sequence can be modified with stabilizing and hydrophobic modifications (e.g., sterols, such as cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan, and phenyl) that provide stability and efficient cellular uptake in the absence of any transfection reagents or formulations. In some embodiments, IFN-induced protein immunoreactivity assays have shown that sd-RNA produces reduced immunostimulatory activity compared to other RNAi reagents. See, for example, Byrne et al., December 2013, J. Ocular Pharmacology and Therapeutics, 29(10): 855-864, which is incorporated herein by reference. In some embodiments, the sd-RNA described herein is available from Advirna LLC (Worcester, MA, USA).

The terms "pharmaceutically acceptable carrier" or "pharmaceuticalally acceptable excipient" are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents, and inert components. The use of such pharmaceutically acceptable carriers or excipients for the active pharmaceutical ingredient is well known in the art. Unless any conventional pharmaceutically acceptable carrier or excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the present invention is contemplated. Other active pharmaceutical ingredients (e.g., other drugs) may also be incorporated into the described compositions and methods.

The terms “about” and “approximately” indicate a statistically significant range of values. Such a range may be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, more preferably within 10%, and even more preferably within 5%. The permissible variation included in the terms “about” or “approximately” depends on the specific system under study and will be readily understood by those skilled in the art. Furthermore, as used herein, the terms “about” and “approximately” mean that size, dimension, formulation, parameter, shape, and other properties and characteristics are not, and need not be, precise, but may be approximate and/or larger or smaller as needed, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art. Generally, size, dimension, formulation, parameter, shape, or other properties or characteristics are “about” or “approximate”, whether explicitly stated or not. It should be noted that embodiments with very different sizes, shapes, and dimensions may employ the aforementioned arrangements.

When used in the appended claims in both original and modified forms, the transitional terms “comprising,” “substantially consisting of,” and “consisting of” define the scope of the claims with respect to any additional, unlisted claim elements or steps (if any) excluded from the scope of the claims. The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unlisted elements, methods, steps, or materials. The term “consisting of” excludes any elements, steps, or materials other than those described in the claims, and in the case of materials, the term “consisting of” excludes common impurities associated with the specified material. The term “substantially consisting of” limits the scope of the claims to the specified elements, steps, or materials and those that do not substantially affect the essential and novel features 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,” “substantially consisting of,” and “consisting of.”

III. Methods for transiently altering protein expression in TILs

In some embodiments, the amplified TILs of the present invention are further manipulated to alter protein expression before, during, or after the amplification step (including during the closed aseptic production method as described herein). In some embodiments, the transiently altered protein expression is due to transient gene editing. In some embodiments, the amplified TILs of the present invention are treated with transcription factors (TFs) and/or other molecules capable of transiently altering protein expression in TILs. In some embodiments, TFs and/or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and/or the number of tumor antigen-specific T cells in the TIL population.

In some embodiments, the present invention includes gene editing via nucleotide insertion (e.g., via ribonucleic acid (RNA) insertion, including messenger RNA (mRNA) or small (or short) interfering RNA (siRNA) insertion) TIL groups to promote or inhibit the expression of one or more proteins, as well as combinations of simultaneously promoting one group of proteins and inhibiting another group of proteins.

In some embodiments, the amplified TILs of the present invention undergo transient changes in protein expression. In some embodiments, the transient change in protein expression occurs within a large population of TILs prior to the first amplification, including, for example, the TIL population obtained by step A as shown in FIG. 8. In some embodiments, the transient change in protein expression occurs during the first amplification, including, for example, the TIL population amplified in step B as shown in FIG. 8. In some embodiments, the transient change in protein expression occurs after the first amplification, including, for example, the transitional TIL population between the first and second amplifications, obtained by step B as shown in FIG. 8 and included in step C. In some embodiments, the transient change in protein expression occurs within a large population of TILs prior to the second amplification, including, for example, the TIL population obtained by step C as shown in FIG. 8 and prior to the amplification in step D. In some embodiments, the transient change in protein expression occurs during the second amplification, including, for example, the TIL population amplified in step D as shown in FIG. 8. In some embodiments, the transient change in protein expression occurs after the second amplification, including, for example, the TIL population obtained by the amplification by step D as shown in FIG. 8.

In one embodiment, the method for transiently altering protein expression in a TIL population includes an electroporation step. In one embodiment, the method for transiently altering protein expression in a TIL population is performed according to the methods shown in Figures 30 and 31. Electroporation methods are known in the art and described, for example, in Tsong, Biophys. J. 1991, 60, 297-306 and U.S. Patent Application Publication No. 2014/0227237A1, the disclosures of which are incorporated herein by reference. In one embodiment, the method for transiently altering protein expression in a TIL population includes a calcium phosphate transfection step. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and U.S. Patent No. 5,593,875, the disclosures of which are incorporated herein by reference. In one embodiment, a method for transiently altering protein expression in a TIL population includes a liposome transfection step. Liposome transfection methods, such as those using a 1:1 (w/w) lipid formulation of the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanolamine (DOPE) in filtered water, are known in the art and described in Rose et al., Biotechniques 1991, 10, 520-525 and Felgner et al., Proc. Natl. Acad. Sci. U.S., 1987, 84, 7413-7417 and U.S. Patent Nos. 5,279,833; 5,908,635; 6,056,938; 6,110,490; 6,534,484 and 7,687,070, the disclosures of which are incorporated herein by reference. In one embodiment, a method for transiently altering protein expression in a TIL population includes a transfection step using the methods described in U.S. Patent Nos. 5,766,902; 6,025,337; 6,410,517; 6,475,994 and 7,189,705, the disclosures of which are incorporated herein by reference.

In some embodiments, transient changes in protein expression lead to an increase in T memory stem cells (TSCMs). TSCMs are early progenitor cells of central memory T cells that have experienced antigens. TSCMs typically exhibit defining stem cell long-term survival, self-renewal, and pluripotency, often required to produce potent TIL products. TSCMs have shown enhanced antitumor activity compared to other T cell subsets in mouse models of adoptive cell transfer (Gattinoni et al., Nat Med 2009, 2011; Gattinoni, Nature Rev. Cancer, 2012; Cieri et al., Blood 2013). In some embodiments, transient changes in protein expression result in a TIL population comprising a high proportion of TSCMs. In some embodiments, the transient change in protein expression causes an increase in the percentage of TSCM of at least 5%, at least 10%, 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 change in protein expression causes an increase in TSCM of at least 1, 2, 3, 4, 5, or 10 times in the TIL population. In some embodiments, the transient change in protein expression results in a TSCM of at least 5%, at least 10%, 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% for the TIL population. In some embodiments, the transient change in protein expression results in a TSCM of at least 5%, at least 10%, 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% for the therapeutic TIL population.

In some embodiments, transient changes in protein expression cause rejuvenation of T cells that have experienced an antigen. In some embodiments, rejuvenation includes, for example, increased proliferation, increased T cell activation, and/or increased antigen recognition.

In some embodiments, transient changes in protein expression alter expression in most T cells to preserve the tumor-derived TCR repertoire. In some embodiments, transient changes in protein expression do not alter the tumor-derived TCR repertoire. In some embodiments, transient changes in protein expression maintain the tumor-derived TCR repertoire.

In some embodiments, transient changes in protein expression lead to alterations in the expression of specific genes. In some embodiments, the genes targeted by these transient changes in protein expression include, but are not limited to, PD-1 (also known 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 1/2 ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), 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 of protein expression targets genes selected from PD-1, TGFBR2, CCR4/5, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), 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 of protein expression targets PD-1. In some embodiments, the transient alteration of protein expression targets TGFBR2. In some embodiments, transient changes in protein expression target CCR4/5. In some embodiments, transient changes in protein expression target CBLB. In some embodiments, transient changes in protein expression target CISH. In some embodiments, transient changes in protein expression target CCR (chimeric co-stimulatory receptor). In some embodiments, transient changes in protein expression target IL-2. In some embodiments, transient changes in protein expression target IL-12. In some embodiments, transient changes in protein expression target IL-15. In some embodiments, transient changes in protein expression target IL-21. In some embodiments, transient changes in protein expression target NOTCH 1/2 ICD.

In some implementations, transient alterations in protein expression target the NOTCH signaling pathway, for example, via NOTCH1/2 ICD and/or via other NOTCH ligands, such as mDLL1 (see, for example, Kondo, T. et al., NOTCH-mediated conversion of activated T cells into stem cell memory-like T cells for adaptive immunotherapy, Nature Communications, Vol. 8, Article number: 15338 (2017), the entire contents of which are incorporated herein by reference).

In some embodiments, transient changes in protein expression target TIM3. In some embodiments, transient changes in protein expression target LAG3. In some embodiments, transient changes in protein expression target TIGIT. In some embodiments, transient changes in protein expression target TGFβ. In some embodiments, transient changes in protein expression target CCR1. In some embodiments, transient changes in protein expression target CCR2. In some embodiments, transient changes in protein expression target CCR4. In some embodiments, transient changes in protein expression target CCR5. In some embodiments, transient changes in protein expression target CXCR1. In some embodiments, transient changes in protein expression target CXCR2. In some embodiments, transient changes in protein expression target CSCR3. In some embodiments, transient changes in protein expression target CCL2 (MCP-1). In some embodiments, transient changes in protein expression target CCL3 (MIP-1α). In some embodiments, transient changes in protein expression target CCL4 (MIP1-β). In some embodiments, transient changes in protein expression target CCL5 (RANTES). In some embodiments, transient changes in protein expression target CXCL1. In some embodiments, transient changes in protein expression target CXCL8. In some embodiments, transient changes in protein expression target CCL22. In some embodiments, transient changes in protein expression target CCL17. In some embodiments, transient changes in protein expression target VHL. In some embodiments, transient changes in protein expression target CD44. In some embodiments, transient changes in protein expression target PIK3CD. In some embodiments, transient changes in protein expression target SOCS1. In some embodiments, transient changes in protein expression target cAMP protein kinase A (PKA).

In some embodiments, transient changes in protein expression result in an increase and/or overexpression of chemokine receptors. In some embodiments, the chemokine receptors overexpressed by transient protein expression include receptors with ligands, including but not limited to CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1, CXCL8, CCL22, and/or CCL17. In some embodiments, the chemokine receptors overexpressed by transient protein expression include receptors with ligands, including but not limited to IL-2, IL-7, IL-10, IL-15, and IL-21, and the NOTCH1/2 intracellular domain (ICD). In some implementations, transient alterations in protein expression target the NOTCH signaling pathway, for example, via NOTCH 1/2 ICD and/or via other NOTCH ligands, such as mDLL1 (see, for example, Kondo, T. et al., NOTCH-mediated conversion of activated T cells into stem cell memory-like T cells for adaptive immunotherapy, Nature Communications, Vol. 8, Article number: 15338 (2017), the entire contents of which are incorporated herein by reference).

In some embodiments, transient changes in protein expression result in decreased and/or reduced expression of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGFβR2, and/or TGFβ (including, for example, TGFβ pathway blockade). In some embodiments, transient changes in protein expression result in decreased and/or reduced CBLB (CBL-B) expression. In some embodiments, transient changes in protein expression result in decreased and/or reduced CISH expression.

In some embodiments, transient changes in protein expression lead to an increase and/or overexpression of chemokine receptors, thereby, for example, improving TIL transport or motility to tumor sites. In some embodiments, transient changes in protein expression lead to an increase and/or overexpression of CCRs (chimeric co-stimulatory receptors). In some embodiments, transient changes in protein expression lead to an increase and/or overexpression of chemokine receptors selected from CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, and/or CSCR3.

In some embodiments, transient changes in protein expression lead to an increase and/or overexpression of interleukins. In some embodiments, transient changes in protein expression lead to an increase and/or overexpression of interleukins selected from IL-2, IL-12, IL-15, and/or IL-21.

In some embodiments, transient alterations in protein expression target the NOTCH signaling pathway, for example, via NOTCH1/2 ICD and/or via other NOTCH ligands, such as mDLL1 (see, for example, Kondo, T. et al., NOTCH-mediated conversion of activated T cells into stem cell memory-like T cells for adaptive immunotherapy, Nature Communications, Vol. 8, Article No. 15338 (2017), the entire contents of which are incorporated herein by reference). In some embodiments, transient alterations in protein expression result in increased and/or overexpression of NOTCH 1/2 ICD. In some embodiments, transient alterations in protein expression result in increased and/or overexpression of NOTCH ligands (e.g., mDLL1). In some embodiments, transient alterations in protein expression result in increased and/or overexpression of VHL. In some embodiments, transient alterations in protein expression result in increased and/or overexpression of CD44. In some embodiments, transient changes in protein expression result in increased and/or overexpression of PIK3CD. In some embodiments, transient changes in protein expression result in increased and/or overexpression of SOCS1.

In some implementations, transient changes in protein expression lead to a decrease and/or reduction in cAMP protein kinase A (PKA) expression.

In some embodiments, the transient change in protein expression results in a decrease and/or reduction in the expression of one molecule selected from PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the transient change in protein expression results in a decrease and/or reduction in the expression of two molecules selected from PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the transient change in protein expression results in a decrease and/or reduction in the expression of PD-1 and one molecule selected from LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the transient change in protein expression results in a decrease and/or reduction in the expression of PD-1, LAG-3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, transient changes in protein expression result in a decrease and/or reduction in the expression of PD-1 and one of LAG3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, transient changes in protein expression result in a decrease and/or reduction in the expression of PD-1 and LAG3. In some embodiments, transient changes in protein expression result in a decrease and/or reduction in the expression of PD-1 and CISH. In some embodiments, transient changes in protein expression result in a decrease and/or reduction in the expression of PD-1 and CBLB. In some embodiments, transient changes in protein expression result in a decrease and/or reduction in the expression of LAG3 and CISH. In some embodiments, transient changes in protein expression result in a decrease and/or reduction in the expression of LAG3 and CBLB. In some embodiments, transient changes in protein expression result in a decrease and/or reduction in the expression of CISH and CBLB. In some embodiments, transient changes in protein expression result in a decrease and/or reduction in the expression of TIM3 and PD-1. In some embodiments, transient changes in protein expression result in a decrease and/or reduction in the expression of TIM3 and LAG3. In some embodiments, transient changes in protein expression result in a decrease and/or reduction in the expression of TIM3 and CISH. In some embodiments, transient changes in protein expression result in a decrease and/or reduction in the expression of TIM3 and CBLB.

In some implementations, adhesion molecules selected from CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof are inserted into a first TIL group, a second TIL group, or a harvested TIL group via gamma-retroviral or lentiviral methods (e.g., increased expression of adhesion molecules).

In some embodiments, transient changes in protein expression result in decreased and/or reduced expression of molecules selected from PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof, and increased and/or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof. In some embodiments, transient changes in protein expression result in decreased and/or reduced expression of molecules selected from PD-1, LAG3, TIM3, CISH, CBLB, and combinations thereof, and increased and/or enhanced expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

In some embodiments, expression is reduced by about 5%, about 10%, 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, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some implementations, expression is reduced by at least about 90%. In some implementations, expression is reduced by at least about 95%. In some implementations, expression is reduced by at least about 99%.

In some embodiments, expression is increased by about 5%, about 10%, 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, expression is increased by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%. In some embodiments, expression is increased by at least about 85%. In some implementations, expression is increased by at least about 90%. In some implementations, expression is increased by at least about 95%. In some implementations, expression is increased by at least about 99%.

In some embodiments, transient alterations in protein expression are induced by treating the TIL with transcription factors (TFs) and/or other molecules capable of transiently altering protein expression in the TIL. In some embodiments, transcription factors (TFs) and/or other molecules capable of transiently altering protein expression are delivered intracellularly using a microfluidic platform without the SQZ carrier. Such methods, demonstrating the ability to deliver proteins (including transcription factors) to a variety of primary human cells (including T cells), have been described (Sharei et al., PNAS 2013, and Sharei et al., PLOS ONE 2015 and Greisbeck et al., J. Immunology 2015, Vol. 195), including rapid methods that use microfluidic constriction to deform cells, thereby allowing TF or other molecules to enter the cells; see, for example, International Patent Application Publication Nos. WO2013/059343A1, WO2017/008063A1 or WO2017/123663A1 or U.S. Patent Application Publication Nos. US2014/0287509A1, US2018/0201889A1 or US2018/0245089A1, all of which are incorporated herein by reference in their entirety. The methods described in International Patent Application Publications WO2013/059343A1, WO2017/008063A1, or WO2017/123663A1, or U.S. Patent Application Publications US2014/0287509A1, US2018/0201889A1, or US2018/0245089A1, can be used in conjunction with this invention to expose TIL populations 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 provide an increase in tumor antigen expression and/or an increase in the number of tumor antigen-specific T cells in the TIL populations, thereby resulting in the reprogramming of the TIL populations, and the reprogrammed TIL populations exhibit improved therapeutic efficacy compared to unreprogrammed TIL populations. In some embodiments, as described herein, reprogramming results in an increase in subsets of effector T cells and/or central memory T cells relative to the initial TIL population or the prior (i.e., prior to reprogramming) TIL populations.

In some embodiments, transcription factors (TFs) 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 together with induced pluripotent stem cell cultures (iPSCs) (e.g., commercially available KNOCKOUT serum alternatives (Gibco/ThermoFisher)) to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is administered together with a mixture of iPSCs to induce additional TIL reprogramming. In some embodiments, the transcription factor (TF) is administered without an iPSC mixture. In some embodiments, reprogramming results in an increase in the percentage of TSCM. In some implementations, reprogramming results in an increase in the TSCM percentage of approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95%.

In some embodiments, methods for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population include:

(i) Obtaining the first TIL group from the tumor removed from the patient;

(ii) A second TIL population is generated by first expansion of the first TIL population by culturing the first TIL population in a cell culture medium containing IL-2 and optional OKT-3;

(iii) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the number of the third TIL population is at least 100 times greater than that of the second TIL population; wherein the second expansion is performed for at least 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; and

(iv) Expose the second and/or third TIL groups to transcription factors (TFs) and/or other molecules capable of transiently altering protein expression; wherein the TFs and/or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and/or the number of tumor antigen-specific T cells in the therapeutic TIL groups.

In one embodiment, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population includes:

(a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

(e) Performing a sterile electroporation step on the second TIL group; wherein the sterile electroporation step mediates the transfer of at least one short interfering RNA or a messenger RNA;

(f) Let the second TIL group stand for about 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody and antigen-presenting cells (APC) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (f) to step (g) occurs without opening the system;

(h) Harvest the therapeutic TIL clusters obtained from step (g), providing the harvested TIL clusters; wherein the transition from step (g) to step (h) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

(i) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

(j) Cryopreservation of harvested TIL populations using a dimethyl sulfoxide-based cryopreservation medium;

The aseptic electroporation step includes delivering short interfering RNA to inhibit the expression of molecules selected from PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof.

According to one embodiment, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population includes:

(a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

(e) Performing an SQZ microfluidic membrane disruption step on the second TIL group; wherein the SQZ microfluidic membrane disruption step mediates the transfer of at least one short interfering RNA or a messenger RNA;

(f) Let the second TIL group stand for about 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody and antigen-presenting cells (APC) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (f) to step (g) occurs without opening the system;

(h) Harvest the therapeutic TIL clusters obtained from step (g), providing the harvested TIL clusters; wherein the transition from step (g) to step (h) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

(i) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

(j) Cryopreservation of harvested TIL populations using a dimethyl sulfoxide-based cryopreservation medium;

The SQZ microfluidic membrane disruption step includes delivering short interfering RNA to inhibit the expression of molecules selected from PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof.

In some embodiments, the method for transiently altering protein expression as described above can be combined with a method for genetically modifying TIL groups, including a step of stably incorporating a gene for producing more than one protein. In one embodiment, the method for genetically modifying TIL groups includes a retroviral transduction step. In one embodiment, the method for genetically modifying TIL groups includes a lentiviral transduction step. Lentiviral transduction systems are known in the art and described, for example, in Levine et al., Proc. Nat’l Acad. Sci. 2006, 103, 17372-77; Zufferey et al., Nat. Biotechnol. 1997, 15, 871-75; Dull et al., J. Virology 1998, 72, 8463-71 and U.S. Patent No. 6,627,442, the contents of which are incorporated herein by reference. In one embodiment, the method for genetically modifying TIL groups includes a γ-retroviral transduction step. Gamma-retroviral transduction systems are known in the art and described, for example, by Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In one embodiment, a method for genetically modifying the TIL group includes a transposon-mediated gene transfer step. Transposon-mediated gene transfer systems are known in the art and include systems in which the transposase is provided in the form of a DNA expression vector or an expressible RNA or protein such that the transposase is not expressed long-term in transgenic cells, for example, a transposase provided in the form of mRNA (e.g., mRNA containing a cap and a poly-A tail). Suitable transposon-mediated gene transfer systems, including salmonid-type Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x, as well as engineered enzymes with enhanced enzyme activity, are described, for example, in Hackett et al., Mol. Therapy 2010, 18, 674-83 and U.S. Patent No. 6,489,458, the contents of which are incorporated herein by reference.

In one embodiment, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population includes:

(a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

(e) Performing a sterile electroporation step or an SQZ microfluidic membrane disruption step on the second TIL group; wherein the sterile electroporation step or the SQZ microfluidic membrane disruption step mediates the transfer of at least one short interfering RNA or a messenger RNA.

(f) Let the second TIL group stand for about 1 day;

(g) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody and antigen-presenting cells (APC) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (f) to step (g) occurs without opening the system;

(h) Harvest the therapeutic TIL clusters obtained from step (g), providing the harvested TIL clusters; wherein the transition from step (g) to step (h) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

(i) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

(j) Cryopreservation of harvested TIL populations using a dimethyl sulfoxide-based cryopreservation medium;

The electroporation step includes delivering short interfering RNA to inhibit the expression of a molecule selected from PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof; further, the adhesion molecule is inserted into a first TIL group, a second TIL group, or a harvested TIL group via a γ-retroviral or lentiviral method, the adhesion molecule being selected from CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

In some embodiments, the transient change in protein expression is induced by a decrease in expression due to self-delivering RNA interference (sd-RNA), which is a chemically synthesized asymmetric siRNA duplex with a high percentage of 2'-OH substitutions (typically fluorine or _-OCH3 ). The sd-RNA comprises a 20-nucleotide sense (guide) strand and a 13- to 15-base sense (lackey) strand conjugated to cholesterol at its 3' end using a tetraethyl ethylene glycol (TEG) linker. Methods using sd-RNA have been described in Khvorova and Watts, Nat. Biotechnol. 2017, 35, 238–248; Byrne et al., J. Ocul. Pharmacol. Ther. 2013, 29, 855-864; and Ligtenberg et al., Mol. Therapy, 2018, in print, the disclosures of which are incorporated herein by reference. In one embodiment, the delivery of sd-RNA to TILs is accomplished not by electroporation, SQZ, or other methods, but by the following steps: exposing TILs to sd-RNA at a concentration of 1 μM/10,000 TILs in a culture medium for 1 to 3 days. In some embodiments, the delivery of sd-RNA to TILs is accomplished by the following steps: exposing TILs to sd-RNA at a concentration of 10 μM/10,000 TILs in a culture medium for 1 to 3 days. In one embodiment, the delivery of sd-RNA to TILs is accomplished by the following steps: exposing TILs to sd-RNA at a concentration of 50 μM/10,000 TILs in a culture medium for 1 to 3 days. In one embodiment, the delivery of sd-RNA to TILs is accomplished by the following steps: exposing TILs to sd-RNA at concentrations ranging from 0.1 μM/10,000 TILs to 50 μM/10,000 TILs in a culture medium for 1 to 3 days. In one embodiment, the delivery of sd-RNA to TIL clusters is accomplished using the following steps: exposing TIL clusters to sd-RNA at concentrations of 0.1 μM/10,000 TILs to 50 μM/10,000 TILs in a culture medium for 1 to 3 days; wherein the exposure to sd-RNA is performed two, three, four, or five times by adding fresh sd-RNA to the culture medium. Other suitable methods are described, for example, in U.S. Patent Application Publications US2011/0039914A1, US2013/0131141A1, and US2013/0131142A1 and U.S. Patent No. 9,080,171, the disclosures of which are incorporated herein by reference.

In some embodiments, sd-RNA is inserted into the TIL group during production using the method shown in Figure 32. In some embodiments, the sd-RNA encodes RNA that interferes with NOTCH 1/2 ICD, NOTCH ligand mDLL1, PD-1, CTLA-4TIM-3, LAG-3, TIGIT, TGFβ, 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, for example, by flow cytometry and/or qPCR. In some embodiments, expression is reduced by approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or approximately 95%. In some embodiments, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some embodiments, expression is reduced by at least about 90%. In some embodiments, expression is reduced by at least about 95%. In some embodiments, expression is reduced by at least about 99%.

In one embodiment, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population includes:

(a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

(e) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, one or more self-delivering RNAs (sd-RNAs), optional OKT-3 antibody, optional OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (f) to step (g) occurs without opening the system;

(h) Harvest the therapeutic TIL clusters obtained from step (g), providing the harvested TIL clusters; wherein the transition from step (g) to step (h) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

(i) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

(j) Cryopreservation of harvested TIL populations using a dimethyl sulfoxide-based cryopreservation medium;

Among them, one or more sd-RNA transiently inhibits the expression of molecules selected from PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3) and combinations thereof.

In one embodiment, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population includes:

(a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

(e) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, one or more self-delivering RNAs (sd-RNAs), optional OKT-3 antibody, optional OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (f) to step (g) occurs without opening the system;

(h) Harvest the therapeutic TIL clusters obtained from step (g), providing the harvested TIL clusters; wherein the transition from step (g) to step (h) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

(i) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

(j) Cryopreservation of harvested TIL populations using a dimethyl sulfoxide-based cryopreservation medium;

The expression of one or more sd-RNA transient inhibitory molecules is selected from PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof; and the adhesion molecules are inserted into the first TIL group, the second TIL group, or the harvested TIL group by a γ-retroviral or lentiviral method, and the adhesion molecules are selected from CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

A. sdRNA method

Chemically modified siRNA-based self-delivered RNAi technology can be used in conjunction with the methods of this invention to successfully deliver sd-RNA to the TIL described herein. Backbone modifications to the asymmetric siRNA structure and the binding of hydrophobic ligands (see, for example, Ligtenberg et al., Mol. Therapy, 2018 and US20160304873, and Figures 36 and 37 herein) enable sd-RNA to penetrate cultured mammalian cells by simply adding it to the culture medium without the need for other formulations and methods, leveraging the nuclease stability of sd-RNA. This stability can support constant levels of RNAi-mediated reduction in target gene activity simply by maintaining the active concentration of sd-RNA in the culture medium. Unbound by theory, the backbone stabilization of sd-RNA can prolong the reduction in gene expression effects, potentially lasting for months in non-dividing cells.

In one embodiment, the sd-RNA targeting the gene disclosed herein has the structure shown in Figure 36 or Figure 37.

In some embodiments, TIL transfection efficiency exceeding 95% and target expression of various specific sd-RNAs are reduced. In some embodiments, sd-RNAs containing several unmodified ribose residues are replaced with fully modified sequences to increase the titer and/or lifetime of RNAi action. 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 longer. In some embodiments, the expression reduction effect decreases more than 10 days after TIL sd-RNA treatment. In some embodiments, the reduction in expression maintaining target expression exceeds 70%. In some embodiments, the reduction in expression maintaining TIL target expression exceeds 70%. In some embodiments, the reduction in expression in the PD-1/PD-L1 pathway allows TILs to exhibit more effective in vivo action, which in some embodiments is due to the avoidance of PD-1/PD-L1 pathway inhibition. In some embodiments, the reduction in PD-1 expression via sd-RNA leads to increased TIL proliferation.

1. Selection and characteristics of sdRNA

a. sd-RNA oligonucleotide structure

Small interfering RNA (siRNA), sometimes called short interfering RNA or silent RNA, is a double-stranded RNA molecule that is typically 19 to 25 base pairs in length. siRNA is used for RNA interference (RNAi), in which it interferes with the expression of specific genes that have complementary nucleotide sequences.

Double-stranded DNA (dsRNA) is generally used to define any molecule containing a pair of complementary RNA strands, typically a sense (lagging) and an antisense (leading) strand, and may include single-stranded overhang regions. In contrast to siRNA, the term dsRNA typically refers to a precursor molecule containing the siRNA molecular sequence, which is released from a larger dsRNA molecule by the action of a cleavage enzyme system, including Dicer.

sd-RNA (self-delivered RNA) is a novel class of covalently modified RNAi compounds that do not require a delivery vector for cell entry and exhibit improved pharmacology compared to conventional siRNA. "Self-delivered RNA" or "sd-RNA" is a hydrophobically modified RNA interference-antisense hybrid that has been shown to be highly efficient in vitro in primary cells and in vivo upon topical administration. Robust uptake and/or silencing without toxicity have been demonstrated. sd-RNA is typically an asymmetrically modified nucleic acid molecule with a minimal double-stranded region. sd-RNA molecules generally contain both single-stranded and double-stranded regions and can incorporate various chemical modifications within these regions. Furthermore, as described herein, sd-RNA molecules can be linked to hydrophobic conjugates, such as conventional and higher sterol-type molecules. sd-RNA (and/or RNA that can be used in a manner similar to sd-RNA) and related methods for preparing such sd-RNA have been extensively described in, for example, U.S. Patent Publication No. US2016/0304873, International Patent Application Publication No. WO2010/033246, International Patent Application Publication No. WO2017/070151, International Patent Application Publication No. WO2009/102427, International Patent Application Publication No. WO201/1119887, International Patent Application Publication No. WO2010/033247, International Patent Application Publication No. WO2009045457, International Patent Application Publication No. WO2011/119852, International Patent Application Publication No. WO2011/119871, and U.S. Patent Publication No. US201 1/0263680, International Patent Application Publication No. WO2010/033248, International Patent Application Publication No. WO2010/078536, International Patent Application Publication No. WO2010/090762, US Patent Publication No. US20110039914, International Publication No. WO2011/109698, International Patent Application Publication No. WO2010/090762, US Patent No. US8,815,818, International Patent Application Publication No. WO2016/094845, International Patent Application Publication No. WO2017/193053, US Patent Publication No. US2006/0276635, International Patent Application Publication No. WO2001/009312, US Patent Publication No. US2017/004302 4. US Patent Publication No. US2017/0312367, US Patent Publication No. US2016/0319278, US Patent Publication No. US2017/0369882, US Patent No. US8,501,706, US Patent No. US2004/0224405, US Patent No. US8,252,755, US Patent No. US2007/0031844, US Patent No. US2007/0039072, US Patent Publication No. US2007/0207974, US Patent Publication No. US2007/0213520, US Patent Publication No. US2007/0213521, US Patent Publication No. US2007/0219362, US Patent Publication No. US2007/0238 U.S. Patent Publication No. 868, US2014/0148362, US2016/0193242, US2016/01946461, US2016/0201058, US2016/0201065, US2017/0349904, US2018/0119144, US7,834,170, US8,090,542 and US2012/0052487, the entire contents of which are incorporated herein by reference for all purposes; sd-RNA is also commercially available from Advirna LLC (Worcester, MA, USA). To optimize the structure, chemical properties, target location, and sequence preference of sd-RNA, proprietary algorithms have been developed and used for sd-RNA titer prediction (see, for example, US20160304873). Based on these analyses, a functional sd-RNA sequence is typically defined as one whose expression decreases by more than 70% at a concentration of 1 μM with a probability exceeding 40%.

b. sd-RNA oligonucleotide structure

In some embodiments, more than one sd-RNA for use in this invention can be generated from a linear double-stranded DNA template. In some embodiments, the linear double-stranded DNA template used to generate more than one sd-RNA is a template as described in U.S. Patent No. 8,859,229 and hereinafter.

In some embodiments, the linear double-stranded DNA template obtained by polymerase chain reaction (PCR) and suitable for in vitro transcription of mRNA comprises, from 5' to 3': an RNA polymerase promoter on the coding strand of the double-stranded DNA, a 5' untranslated region (less than 3,000 nucleotides in length, capable of efficiently translating mRNA into a detectable polypeptide after transfection into eukaryotic cells), and an open reading frame encoding the polypeptide; wherein the polypeptide is heterologous to the cells to be transfected; wherein the polypeptide is selected from: ligands or receptors of immune cells, polypeptides that stimulate or inhibit the function of the immune system, polypeptides that inhibit the function of oncogenic polypeptides, a 3' untranslated region capable of efficiently translating mRNA into a detectable polypeptide after transfection into eukaryotic cells, and a poly(A) segment of 50 to 5,000 nucleotides on the coding strand of the double-stranded DNA; wherein the promoter and the open reading frame are heterologous; wherein the DNA template is not contained within a DNA vector and terminates at the 3' end of the poly(A) segment. In some embodiments, the RNA polymerase promoter contains a common binding sequence of the RNA polymerase selected from T7, T3, or SP6 RNA polymerases. In some embodiments, the open reading frame encodes a fusion polypeptide. In some embodiments, the open reading frame encodes a polypeptide selected from the group consisting of: PD-1, TGFBR2, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, cAMP protein kinase A (PKA), and combinations thereof. In some embodiments, the open reading frame encodes a polypeptide selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof. In some embodiments, the linear double-stranded DNA template further includes an internal ribosome entry site. In some embodiments, the poly(A) segment is 300-400 nucleotides in length.

In some embodiments, the linear double-stranded DNA template of claim 1 is comprising, from 5' to 3', an RNA polymerase promoter on the coding strand of the double-stranded DNA, a 5' untranslated region (less than 3,000 nucleotides in length, capable of efficiently translating mRNA into a detectable polypeptide after transfection into eukaryotic cells), and an open reading frame encoding the polypeptide; wherein the polypeptide is heterologous to the cells to be transfected; wherein the polypeptide is selected from: ligands or receptors of immune cells, polypeptides that stimulate or inhibit the function of the immune system, polypeptides that inhibit the function of oncogenic polypeptides, a 3' untranslated region capable of efficiently translating mRNA into a detectable polypeptide after transfection into eukaryotic cells, and a poly(A) segment of 50 to 5,000 nucleotides on the coding strand of the double-stranded DNA; wherein the promoter and the open reading frame are heterologous; wherein the DNA template is not contained within a DNA vector and terminates at the 3' end of the poly(A) segment. In some embodiments, the 3' untranslated region is at least 100 nucleotides in length.

In some embodiments, the present invention provides a method for generating the above-described linear double-stranded DNA template; wherein the method includes: generating a forward primer and a reverse primer; wherein the forward primer comprises: a plurality of nucleotides substantially complementary to the non-coding strand of the target double-stranded DNA, and a plurality of nucleotides serving as an RNA polymerase binding site; wherein the reverse primer comprises: a plurality of nucleotides substantially complementary to the coding strand of the target double-stranded DNA, and a plurality of deoxythymidine nucleotides; and, performing a polymerase chain reaction amplification on the target DNA using the forward primer and the reverse primer to form a linear double-stranded DNA template. In some embodiments, the present invention provides a method for generating the above-described linear double-stranded DNA template, wherein the method includes: generating a forward primer and a reverse primer; wherein the forward primer comprises a plurality of nucleotides substantially complementary to a nucleotide region directly upstream of the target double-stranded DNA; wherein the reverse primer comprises a plurality of nucleotides substantially complementary to a nucleotide region directly downstream of the target double-stranded DNA; and, performing a polymerase chain reaction amplification on the target DNA using the forward primer and the reverse primer to form a linear double-stranded DNA template. In some embodiments, the primers comprise nucleotide sequences substantially complementary to the stretches of nucleotides in the 5' and 3' untranslated regions of the target double-stranded DNA. In some embodiments, the primers comprise nucleotide sequences substantially complementary to the stretches of nucleotides within the open reading frame of the target double-stranded DNA. In some embodiments, the primers comprise nucleotide sequences substantially complementary to the stretches of nucleotides within the open reading frame of the target double-stranded DNA; wherein the primers also comprise nucleotide stretches containing both the 5' and 3' untranslated regions; wherein the nucleotide stretch in the forward primer containing the 5' untranslated region is between a nucleotide containing the RNA polymerase promoter and a nucleotide substantially complementary to the non-coding strand of the target double-stranded DNA; wherein the nucleotide stretch in the reverse primer containing the 3' untranslated region is between a plurality of deoxythymidine nucleotides and a nucleotide substantially complementary to the coding strand of the target double-stranded DNA. In some embodiments, the forward primer and the open reading frame comprise a shared Kozak sequence.

In some embodiments, the present invention provides a method for generating one or more RNAs for transfecting cells, the method comprising in vitro transcription from a linear double-stranded DNA template. In some embodiments, the method further comprises using a poly(A) polymerase to extend the poly(A) tail of the RNA with one or more adenine nucleotides or their analogues. In some embodiments, the method further comprises adding a nucleotide during transcription, the nucleotide acting as a 5' cap on the transcribed RNA. In some embodiments, RNA targets peptides selected from the group consisting of: PD-1, TGFBR2, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, cAMP protein kinase A (PKA), and combinations thereof. In some embodiments, RNA targets peptides selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof.

In some embodiments, the invention utilizes more than one isolated RNA, said isolated RNA being generated from a linear double-stranded DNA template and containing more than one open reading frame. In some embodiments, the invention provides a method for expressing more than one RNA in cells, the method comprising contacting the cells with more than one RNA generated from a linear double-stranded DNA template. In some embodiments, RNA is present in the cells in unequal molar amounts to provide different levels of RNA expression. In some embodiments, one or more RNAs target peptides selected from the group consisting of: PD-1, TGFBR2, CBLB (CBL-B), CISH, CCRs (chimeric co-stimulatory receptors), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TGFβ, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, cAMP protein kinase A (PKA), and combinations thereof. In some embodiments, one or more RNA targets a polypeptide selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF(BR3), and combinations thereof.

(i) Non-translation area

Chemical structures that promote stability and/or translation efficiency may also be used. RNA preferably has a 5'UTR and a 3'UTR. The examples below show that including a 44-base-pair 5'UTR in a PCR template results in higher translation efficiency of transcribed CFP RNA compared to a PCR template containing only a 6-base-pair 5'UTR. The examples also show that adding a 113-base-pair 3'UTR results in higher translation efficiency of transcribed GFP RNA compared to a PCR template containing only an 11-base-pair 3'UTR. Typically, the 3'UTR is longer than 100 nucleotides, therefore a 3'UTR longer than 100 nucleotides is preferred. In one embodiment, the 3'UTR sequence is 100 to 5000 nucleotides. The length of the 5'UTR is less important than the length of the 3'UTR, and the 5'UTR can be shorter. In one embodiment, the 5'UTR is 0 to 3000 nucleotides long. The lengths of the 5'UTR and 3'UTR sequences to be added to the coding region can be varied using different methods, including, but not limited to, PCR primers designed for annealing to different UTR regions. Using this method, those skilled in the art can modify the desired 5'UTR and 3'UTR lengths to achieve optimal translation efficiency after transfection of the transcribed RNA.

The 5'UTR and 3'UTR can be naturally occurring endogenous 5'UTR and 3'UTR of the target gene. Alternatively, non-endogenous UTR sequences of the target gene can be added by incorporating the UTR sequence into the forward and reverse primers or through any other modification of the template. The use of non-endogenous UTR sequences of the target gene can be used to alter RNA stability and/or translation efficiency. For example, it is known that AU-rich elements in the 3'UTR sequence can reduce mRNA stability. Therefore, based on the characteristics of UTRs well known in the art, 3'UTRs can be selected or designed to increase the stability of transcribed RNA.

In one embodiment, the 5'UTR may contain the Kozak sequence of an endogenous gene. Alternatively, when a non-endogenous 5'UTR of the target gene is added via PCR as described above, the shared Kozak sequence can be redesigned by adding a 5'UTR sequence. Kozak sequences can improve the translation efficiency of some RNA transcripts, but it appears that not all RNAs require a Kozak sequence for efficient translation. The requirement for a Kozak sequence in many mRNAs is known in the art. In other embodiments, the 5'UTR may be derived from an RNA virus whose RNA genome is stable in the cell. In other embodiments, various nucleotide analogs may be used in the 3'UTR or 5'UTR to prevent exonuclease degradation of the mRNA.

(ii) RNA polymerase promoter

To synthesize RNA from a DNA template without gene cloning, a transcription promoter should be ligated to the DNA template upstream of the sequence to be transcribed. A phage RNA polymerase promoter sequence can be ligated to the St UTR using various genetic engineering methods (e.g., DNA ligation), or the phage RNA polymerase promoter sequence can be added to a forward primer (5') of a sequence substantially complementary to the target DNA. When the sequence acting as the RNA polymerase promoter is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In a preferred embodiment, the promoter is the T7 polymerase promoter as described above. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. The common nucleotide sequences of the T7, T3, and SP6 promoters are known in the art.

(iii) Poly(A) tail and 5' cap

In a preferred embodiment, the mRNA has caps at both the 5' end and the 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability in the cell. On a circular DNA template (e.g., plasmid DNA), RNA polymerase produces a long concatameric product unsuitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR end produces a normal-sized mRNA that, even if polyadenylated post-transcriptionally, is ineffective in eukaryotic transfection.

On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc. Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)). This can lead to runoff transcript bending, followed by template exchange with the second DNA strand or transcription of the RNA itself (Triana-Alonso et al., J. Biol. Chem., 270:6298-307 (1995); Dunn and Studier, J. Mol. Biol., 166:477-535 (1983); Arnaud-Barbe et al., Nuc. Acids Res., 26:3550-54 (1998); Macdonald et al., 1993), which then leads to reverse aberrant transcription and accumulation of double-stranded RNA, thereby suppressing gene expression. DNA linearization alone is insufficient for proper transcription (Triana-Alonso et al., J. Biol. Chem., 270:6298-307 (1995); Dunn and Studier, J. Mol. Biol., 166:477-535 (1983); Arnaud-Barbe et al., 1998 Nuc. Acids Res., 26:3550-54 (1998); Macdonald et al., J. Mol. Biol., 232:1030-47 (1993); Nakano et al., Biotechn ol. Bioeng., 64:194-99 (1999)), linearized plasmid DNA downstream of a poly(A/T) segment of 64 to 100 nucleotides produces a good template (Saeboe-Larssen et al., J. Immunol. Meth., 259:191-203 (2002); Boczkowski et al., Cancer Res., 60:1028-34 (2000); Elango et al., Biochem Riophys Res Commun., 330:958-966 2005). The endogenous termination signal of T7 RNA polymerase encodes RNA that folds into a stem-loop structure after uridine residues are traced (Dunn and J. Mol. Biol., 166:477-535 (1983); Arnaud-Barbe et al., 1998 Nuc. Acids Res., 26:3550-54 (1998)). Even without hairpins, traces of synthetic uridine can attenuate transcription (Kiyama and Oishi, Nuc. Acids Res., 24:4577-4583 (1996)). It is hypothesized that plasmid DNA linearization downstream of the poly(A/T) segment may form a “dynamic” terminator to prevent potentially aberrant transcription: 3' extension and reverse transcription of the RNA transcript at the poly(A/T) segment would produce a continuously growing terminal-like signal—an extended poly(U) segment and a poly(A/U) hairpin. Therefore, reverse PCR primers were designed, which have a 3' anchoring sequence downstream of the GFP gene and a 5' segment of a 100-base poly(T) (Figure 38).

The conventional method for integrating a polyA/T segment into a DNA template is molecular cloning. However, the polyA/T sequence integrated into plasmid DNA leads to plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes the cloning process not only time-consuming and laborious but also often unreliable. This is why there is a strong need for a method that can construct DNA templates with a polyA/T 3' segment without cloning.

The poly(A)/T region of the transcribed DNA template can be generated during PCR using a reverse primer containing a poly(T) tail (e.g., a 100T tail, the size of which can be 50T to 5000T)), or generated after PCR by any other method, including but not limited to DNA ligation or in vitro recombination. The poly(A) tail can also provide stability to the RNA and reduce RNA degradation. Generally, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is 100 to 5000 adenosine. The examples below show that a 100-base-pair poly(A) segment is sufficient for efficient translation of RNA transcripts.

Following in vitro transcription, the poly(A) tail of the RNA can be further extended using a poly(A) polymerase (e.g., E. coli polyA polymerase (E-PAP)). The examples below demonstrate that increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides improves RNA translation efficiency by approximately two-fold. Furthermore, attaching different chemical groups to the 3' end can increase mRNA stability. This attachment can include modified/artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be added to the poly(A) tail using a poly(A) polymerase. ATP analogs can further enhance RNA stability. Suitable ATP analogs include, but are not limited to, cordiocipin and 8-azaadenosine.

The 5' cap can also provide stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein includes a 5' cap. For example, the 5' cap can be an ^m7G (5')ppp(5')G, ^m7G (5')ppp(5')A, G(5')ppp(5')G, or G(5')ppp(5')A' cap analogue, all of which are commercially available. The 5' cap can also be an anti-reverse-cap-analog (ARCA) (see Stepinski et al., RNA, 7:1468-95 (2001)) or any other suitable analogue. A 5' cap is provided using techniques known in the art and described herein (Cougot et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski et al., RNA, 7:1468-95 (2001); Elango et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNA generated by the methods disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and promotes the initiation of translation. It may contain any solute suitable for cell electroporation, including factors that promote cell permeability and viability, such as carbohydrates, peptides, lipids, proteins, antioxidants, and surfactants.

In some embodiments, the sd-RNA sequence used in this invention shows a 70% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 75% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 80% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 85% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 90% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 95% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 99% reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.5 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.25 μM/10,000 TIL to about 10 μM/10,000 TIL or about 0.25 μM/10,000 TIL to about 4 μM/10,000 TIL. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.25 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.5 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.75 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.0 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.25 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.5 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.75 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.0 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.25 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.5 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.75 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.0 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.25 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.5 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.75 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 4.0 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 5.0 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM/10,000 TIL. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM/10,000 TIL. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM/10,000 TIL. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM/10,000 TIL. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM/10,000 TIL. The sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.25 μM/10,000 TIL/100 μL of culture medium. The sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.5 μM/10,000 TIL/100 μL of culture medium. The sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.75 μM/10,000 TIL/100 μL of culture medium. When delivered at a concentration of approximately 1.0 μM/10,000 TIL/100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 1.25 μM/10,000 TIL/100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 1.5 μM/10,000 TIL/100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 1.75 μM/10,000 TIL/100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 2.0 μM/10,000 TIL/100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 2.25 μM/10,000 TIL/100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 2.5 μM/10,000 TIL/100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 2.75 μM/10,000 TIL/100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. When delivered at a concentration of approximately 3.0 μM/10,000 TIL/100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. In some embodiments, when delivered at a concentration of approximately 3.25 μM/10,000 TIL/100 μL of culture medium, the sd-RNA sequence used in this invention showed decreased target gene expression. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.5 μM/10,000 TIL/100 μL of culture medium. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.75 μM/10,000 TIL/100 μL of culture medium. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 4.0 μM/10,000 TIL/100 μL of culture medium. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 5.0 μM/10,000 TIL/100 μL of culture medium. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM/10,000 TIL/100 μL of culture medium. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM/10,000 TIL/100 μL of culture medium. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM/10,000 TIL/100 μL of culture medium. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM/10,000 TIL/100 μL of culture medium. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM/10,000 TIL/100 μL of culture medium.

c. sd-RNA modification

In some embodiments, the oligonucleotide reagent comprises more than one modification to increase the stability and/or efficacy of the therapeutic agent and to enable efficient delivery of the oligonucleotide to the cells or tissues to be treated. Such modifications may include 2'-O-methyl modifications, 2'-O-fluoro modifications, dithiophosphate modifications, 2'F-modified nucleotides, 2'-O-methyl-modified nucleotides, and/or 2'-deoxynucleotides. In some embodiments, the oligonucleotide is modified to include more than one hydrophobic modification, including, for example, sterols, cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan, and/or phenyl. In another specific embodiment, the chemically modified nucleotide is a combination of thiophosphate, 2'-O-methyl, 2'-deoxy, hydrophobic modifications, and thiophosphate. In some embodiments, the sugar may be modified, including but 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'-halogen (including 2'-fluorine), T-methoxyethoxy, 2'-allyloxy ( _-OCH₂CH = _CH₂ ), 2'-propynyl, 2'-propyl, ethynyl, vinyl, propenyl, and cyano. In one embodiment, the sugar moiety may be a hexose and incorporated into an oligonucleotide as described (Augustyns, K. et al., Nucl. Acids. Res. 18: 4711 (1992)).

In some embodiments, the double-stranded oligonucleotides of the present invention are double-stranded throughout their entire length, i.e., without any protruding single-stranded sequences at either end of the molecule, i.e., blunt-ended. In some embodiments, the individual nucleic acid molecules may have different lengths. In other words, the double-stranded oligonucleotides of the present invention are not double-stranded throughout their entire length. For example, when using two separate nucleic acid molecules, one of the molecules (e.g., the first molecule containing the antisense sequence) may be longer than the second molecule with which it hybridizes (resulting in a portion of the molecule being single-stranded). In some embodiments, when using a single nucleic acid molecule, a portion of that molecule may remain single-stranded at either end.

In some embodiments, the double-stranded oligonucleotides of the present invention contain mismatches and/or loops or protrusions, but at least about 70% of the length of the oligonucleotide is double-stranded. In some embodiments, the double-stranded oligonucleotides of the present invention are double-stranded at least about 80% of their length. In another embodiment, the double-stranded oligonucleotides of the present invention are double-stranded at least about 90%-95% of their length. In some embodiments, the double-stranded oligonucleotides of the present invention are double-stranded at least about 96%-98% of their length. In some embodiments, the double-stranded oligonucleotides of the present invention contain 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 protected substantially from nucleases, for example, by modifying the 3' or 5' bond (e.g., U.S. Patent Nos. 5,849,902 and WO98/13526). For example, oligonucleotides can be made resistant by including a "blocking group." As used herein, the term "blocking group" refers to a substituent (e.g., a substituent other than an OH group) that can be attached to an oligonucleotide or nucleomonomer as a protecting group or coupling group for synthesis (e.g., FITC, propyl ( _CH₂ - _CH₂ - _CH₃ ), ethylene glycol (-O- _CH₂ - ^CH₂ _- O-), phosphate ( _PO₃²⁺ ), phosphonate, or phosphoramide). The "blocking group" may also include a "terminal blocking group" or "exonuclease blocking group" that protects the 5' and 3' ends of the oligonucleotide, including modified nucleotide and non-nucleotide exonuclease-resistant structures.

In some implementations, at least a portion of the continuous polynucleotides within the sd-RNA are linked by substitutional bonds (e.g., phosphate thioester bonds).

In some embodiments, the chemical modification can lead to enhanced cellular uptake by 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, and 500. In some embodiments, at least one of the C or U residues includes a hydrophobic modification. In some embodiments, multiple 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, sd-RNA or sd-rxRNA exhibits enhanced endosome release of the sd-rxRNA molecule by incorporating a protonable amine. In some embodiments, a protonable amine is incorporated into the sense strand (in a portion of the molecule discarded after RISC loading). In some embodiments, the sd-RNA compounds of the present invention comprise an asymmetric compound comprising a double-stranded region (required for a valid RISC entry of 10-15 bases) and a single-stranded region of 4-12 nucleotides; a double-stranded region having 13 nucleotides. In some embodiments, a single-stranded region of 6 nucleotides is employed. In some embodiments, the single-stranded region of the sd-RNA comprises 2-12 phosphate-thioester nucleotide inter-bonds (referred to as phosphate-thioester modification). In some embodiments, 6-8 phosphate-thioester nucleotide inter-bonds are employed. In some embodiments, the sd-RNA compounds of the present invention also include a unique chemical modification pattern that provides stability and compatibility with RISC entries.

For example, the guide chain can also be modified by any chemical modification that enhances stability without interfering with the RISC entry. In some embodiments, the chemical modification pattern in the guide chain includes: most of the C and U nucleotides being 2'F modified and the 5' end being phosphorylated.

In some embodiments, at least 30% of the nucleotides in the sd-RNA 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% of the nucleotides in the sd-RNA or sd-rxRNA are modified. % , 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 are modified. In some embodiments, 100% of the nucleotides in the sd-RNA or sd-rxRNA are modified.

In some embodiments, the sd-RNA molecule has a minimal double-stranded region. In some embodiments, the double-stranded region of the molecule is 8-15 nucleotides long. In some embodiments, the double-stranded region of the molecule is 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides long. In some embodiments, the double-stranded region is 13 nucleotides long. The leader and lagging strands may have 100% complementarity, or there may be more than one mismatch between the leader and lagging strands. In some embodiments, at one end of the double-stranded molecule, the molecule is blunt-ended or has a single nucleotide overhang. In some embodiments, the single-stranded region of the molecule is 4-12 nucleotides long. In some embodiments, the single-stranded region may be 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides long. In some embodiments, the single-stranded region may also be less than 4 nucleotides or greater than 12 nucleotides long. In some embodiments, the single-stranded region is 6 or 7 nucleotides long.

In some embodiments, the sd-RNA molecules exhibit increased stability. In certain cases, the chemically modified sd-RNA or sd-rxRNA molecules have a half-life in culture medium longer than 1, 2, 3, 4, 5, 6, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours or more, including any intermediate values. In some embodiments, the sd-rxRNA has a half-life in culture medium longer than 12 hours.

In some embodiments, sd-RNA is optimized to improve potency and/or reduce toxicity. In some embodiments, the nucleotide length of the guide and/or lagging strands and/or the amount of phosphate thioester modifications in the guide and/or lagging strands can affect the potency of the RNA molecule in some ways, while replacing 2'-fluoro(2'F) modifications with 2'-O-methyl (2'OMe) modifications can affect the toxicity of the molecule in some ways. In some embodiments, a reduction in the 2'F content in the molecule is expected to reduce the toxicity of the molecule. In some embodiments, the amount of phosphate thioester modifications in the RNA molecule can affect the molecule's uptake into cells, for example, the efficiency of passive uptake into cells. In some embodiments, sd-RNA does not have 2'F modifications but has equal efficacy in cellular uptake and tissue penetration.

In some embodiments, the guide chain is approximately 18 to 19 nucleotides in length and has approximately 2 to 14 phosphate ester modifications. For example, the guide chain may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 phosphate-modified nucleotides. The guide chain may contain more than one modification that imparts increased stability without interfering with RISC entries. Phosphate-modified nucleotides, such as thiophosphate-modified nucleotides, may be located at the 3' end, the 5' end, or throughout the entire guide chain. In some embodiments, the 3' terminal 10 nucleotides of the guide chain contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 thiophosphate-modified nucleotides. The guide chain may also contain 2'F and/or 2'OMe modifications, which may be located throughout the molecule. In some embodiments, the first nucleotide of the guide chain (the nucleotide at the 5' position of the guide chain) is 2'OMe modified and/or phosphorylated. The C and U nucleotides within the guide chain may be 2'F modified. For example, the C and U nucleotides at positions 2-10 (or corresponding positions in guide strands of different lengths) of a 19nt leader strand may be 2'F modified. The C and U nucleotides within the leader strand may also be 2'OMe modified. For example, the C and U nucleotides at positions 11-18 (or corresponding positions in guide strands of different lengths) of a 19nt leader strand may be 2'OMe modified. In some embodiments, the nucleotide at the 3' end of the leader strand is unmodified. In some embodiments, most of the C and U nucleotides within the leader strand are 2'F modified, and the 5' end of the leader strand is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 are 2'OMe modified, and the 5' end of the leader strand is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 are 2'OMe modified, the 5' end of the leader strand is phosphorylated, and the C or U at positions 2-10 are 2'F modified.

d.sdRNA delivery

Self-delivered RNAi technology provides a method for directly transfecting cells 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 characteristics of compositions and methods, offering significant functional advantages compared to conventional siRNA-based technologies. Therefore, the sd-RNA method has been employed in some embodiments relating to methods for reducing target gene expression in TILs of the present invention. The sd-RNAi method can directly deliver chemically synthesized compounds to a variety of primary cells and tissues in vivo and in vitro. The sd-RNAs described in some embodiments of the present invention are available from Advirna LLC (Worcester, MA, USA).

The general structure of the sd-RNA molecule is shown in Figure 36. The sd-RNA is formed as a hydrophobically modified siRNA-antisense oligonucleotide hybrid structure, as disclosed in, for example, Byrne et al., December 2013, J. Ocular Pharmacology and Therapeutics, 29(10): 855-864, the contents of which are incorporated herein by reference.

In some implementations, sterile electroporation can be used to deliver sd-RNA oligonucleotides to the TIL described herein.

In some embodiments, the oligonucleotide may be bound to a transmembrane delivery system for delivery to cells. In some embodiments, the transmembrane delivery system comprises lipids, viral vectors, etc. In some embodiments, the oligonucleotide agent is a self-delivering RNAi agent that requires no delivery agent.

In implementation, various methods can be used to introduce oligonucleotides (e.g., RNA or sd-RNA as described herein) into target cells. These methods include, for example, commercially available methods, including but not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, MIT), Neon ^™ Transfection System (available from ThermoFisher Scientific, Waltham, MIT) and/or Gene Pulser II (BioRad, Denver, Colorado), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipid transfection, polymer encapsulation, peptide-mediated transfection, or bioprojectile particle delivery systems, such as “gene guns” (see, for example, Nishikawa et al., Hum Gene). Ther., 12(8): 861-70 (2001), the entire contents of which are incorporated herein by reference. See also U.S. Patent No. 8,859,229, U.S. Patent Application No. 2016/0230188 and the Amaxa II manual (available at http://icob.sinica.edu.tw/pubweb/bio-chem/Core％20Facilities/Data/R401-core/Nucleofector_Manual_II_Apr06.pdf).

In some embodiments, electroporation can be performed using Amaxa NUCLEOFECTOR.TM.-II as recommended by the manufacturer. In some embodiments, TILs can be transfected using NUCLEOFECTOR.TM.-II solution V and a set of recommended electroporation protocols. In some embodiments, TILs can be transfected using solutions V, T, and R, as well as different electroporation protocols. In some embodiments, TILs can be transfected using T cell NUCLEOFECTOR.TM.-II solution and different electroporation protocols. Alternative methods of nucleic acid delivery can also be used for transfection of the oligonucleotides described herein: cationic liposome-mediated transfection using LIPOFECTIN or LIPOFECTAMIN (Invitrogen). Electroporation can also be performed using an ECM 830 (BTX) (Harvard Instruments, Boston, MA), Gene Pulser II (BioRad, Denver, Colorado), Multiporator (Eppendorf, Hamburg, Germany), and/or the Neon ^™ Transfection System (available from ThermoFisher Scientific, Waltham, MIT). In some embodiments, pmaxGFP plasmid DNA (Amaxa Biosystems) can be used as a DNA control. In some embodiments, transfection efficiency (ET) can be determined by fluorescence-activated cell sorting (FACS) at approximately 3, 6, 9, 12, 15, and/or 18 hours post-transfection. In some experiments, transfectants can be further analyzed every 12 to 24 hours until GFP is undetectable in the GFP control. In some embodiments, cell viability can be determined by trypan blue exclusion.

Oligonucleotides and oligonucleotide compositions are contacted (e.g., contacted with, also referred to herein as being administered or delivered to) and absorbed by the TIL as described herein, including passive uptake via the TIL. sd-RNA may be added to the TIL as described herein during the first amplification (e.g., step B), after the first amplification (e.g., during step C), before or during the second amplification (e.g., before or during step D, after step D and before harvest in step E, during or after harvest in step F, before or during final formulation and/or transfer to the infusion bag in step F, and before any optional cryopreservation step in step F). Furthermore, sd-RNA may be added after thawing in any cryopreservation step in step F. In one embodiment, one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing TIL and other reagents at concentrations of 100 nM to 20 mM, 200 nM to 10 mM, 500 nM to 1 mM, 1 μM to 100 μM, and 1 μM to 100 μM. In one embodiment, one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing TIL and other reagents in amounts selected from the group consisting of: 0.1 μM sd-RNA/10,000 TIL/100 μL medium, 0.5 μM sd-RNA/10,000 TIL/100 μL medium, 0.75 μM sd-RNA/10,000 TIL/100 μL medium, etc. 1 μM sd-RNA/10,000 TIL/100 μL medium, 1.25 μM sd-RNA/10,000 TIL/100 μL medium, 1.5 μM sd-RNA/10,000 TIL/100 μL medium, 2 μM sd-RNA/10,000 TIL/100 μL medium, 5 μM sd-RNA/10,000 TIL/100 μL medium, or 10 μM sd-RNA/10,000 TIL/100 μL medium. In one embodiment, during the pre-REP or REP phase, one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to the TIL culture twice daily, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days. In one embodiment, one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing TIL and other reagents in amounts selected from the group consisting of: 0.1 μM sd-RNA/10,000 TIL/100 μL medium, 0.5 μM sd-RNA/10,000 TIL/100 μL medium, 0.75 μM sd-RNA/10,000 TIL/100 μL medium, 1 The following media are available in medium: μM sd-RNA/10,000TIL/100μL, 1.25μM sd-RNA/10,000TIL/100μL, 1.5μM sd-RNA/10,000TIL/100μL, 2μM sd-RNA/10,000TIL/100μL, 5μM sd-RNA/10,000TIL/100μL, or 10μM sd-RNA/10,000TIL/100μL. In one implementation, during the first amplification, second amplification, and/or additional amplification phases, one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to the TIL culture twice daily, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

The oligonucleotide compositions comprising sd-RNA of the present invention can be contacted with TILs during amplification as described herein, for example by dissolving a high concentration of sd-RNA in cell culture medium and allowing sufficient time for passive uptake. In some embodiments, the high concentration includes 0.1 μM sd-RNA/10,000 TILs, 0.5 μM sd-RNA/10,000 TILs, 0.75 μM sd-RNA/10,000 TILs, 1 μM sd-RNA/10,000 TILs, 1.25 μM sd-RNA/10,000 TILs, 1.5 μM sd-RNA/10,000 TILs, 2 μM sd-RNA/10,000 TILs, 5 μM sd-RNA/10,000 TILs, or 10 μM sd-RNA/10,000 TILs. In some embodiments, high concentrations include 2 μM sd-RNA/10,000 TIL, 5 μM sd-RNA/10,000 TIL, or 10 μM sd-RNA/10,000 TIL. In some embodiments, high concentrations include 5 μM sd-RNA/10,000 TIL or up to 10 μM sd-RNA/10,000 TIL.

In some embodiments, methods known in the art (see, for example, WO90/14074; WO91/16024; WO91/17424; U.S. Patent No. 4,897,355; Bergan et al., Nucleic Acids Research 1993, 21:3567) can be used to enhance the delivery of oligonucleotides into cells by suitable, art-recognized methods (including calcium phosphate, DMSO, glycerol or dextran, electroporation or by transfection, such as using cationic, anionic or neutral lipid compositions or liposomes).

e.sd-RNA combination

In some embodiments, more than one sd-RNA is used to reduce the expression of a target gene. In some embodiments, one or more sd-RNAs targeting PD-1, TIM-3, CBLB, LAG3, and/or CISH are used together. In some embodiments, PD-1 sd-RNA is used in combination with one or more of TIM-3, CBLB, LAG3, and/or CISH to reduce the expression of more than one gene target. In some embodiments, LAG3 sd-RNA is used in combination with a CISH-targeting sd-RNA to reduce the expression of two target genes. In some embodiments, the sd-RNAs targeting one or more of PD-1, TIM-3, CBLB, LAG3, and/or CISH described herein are commercially available from Advirna LLC (Worcester, MA, USA). In some embodiments, the sd-RNAs targeting one or more of PD-1, TIM-3, CBLB, LAG3, and/or CISH have the structures shown in Figure 36 or Figure 37.

In some embodiments, the sdRNA targets genes selected from PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the sdRNA targets genes selected from PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, one sdRNA targets PD-1, and another sdRNA targets genes selected from LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the sdRNA targets genes selected from PD-1, LAG-3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, the sdRNA targets genes selected from PD-1 and LAG3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, one sdRNA targets PD-1, and another targets LAG3. In some embodiments, one sdRNA targets PD-1, and another targets CISH. In some embodiments, one sdRNA targets PD-1, and another targets CBLB. In some embodiments, one sdRNA targets LAG3, and another targets CISH. In some embodiments, one sdRNA targets LAG3, and another targets CBLB. In some embodiments, one sdRNA targets CISH, and another targets CBLB. In some embodiments, one sdRNA targets TIM3, and another targets PD-1. In some embodiments, one sdRNA targets TIM3, and another targets LAG3. In some embodiments, one sdRNA targets TIM3, and another targets CISH. In some implementations, one sd-RNA targets TIM3, and another sd-RNA targets CBLB.

f. Overexpression of co-stimulatory receptors or adhesion molecules

According to other embodiments, altering the protein expression of TILs during the TIL amplification method may also allow for enhanced expression of more than one immune checkpoint gene in at least a subset of therapeutic TIL populations. For example, altering protein expression may result in enhanced expression of stimulatory receptors, meaning that the stimulatory receptor is overexpressed compared to its unmodified form. Non-limiting examples of immune checkpoint genes that can exhibit enhanced expression by transiently altering protein expression in the TILs of the present invention include certain chemokine receptors and interleukins, such as CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH 1/2 intracellular domain (ICD), and/or the NOTCH ligand mDLL1.

(i) CCR and CCL

For adoptive T-cell immunotherapy to be effective, T cells need to be appropriately transported to the tumor via chemokines. The matching between chemokines secreted by tumor cells, surrounding chemokines, and chemokine receptors expressed by T cells is crucial for the successful delivery of T cells to the tumor bed.

According to certain embodiments, the method of altering protein expression of the present invention can be used to increase the expression of certain chemokine receptors in TILs, such as one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, and/or CX3CR1. Following adoptive transfer, overexpression of CCRs may contribute to promoting effector function and TIL proliferation. In some embodiments, the method of altering protein expression of the present invention can be used to increase the expression of CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1, CXCL8, CCL22, and/or CCL17 in TILs.

According to specific embodiments, the compositions and methods of the present invention enhance the expression of one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, and/or CX3CR1 in TILs. For example, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population can be performed according to any embodiment of the methods described herein (e.g., process 2A or the method shown in Figures 20 and 21), wherein the method includes gene editing at least a portion of the TILs by enhancing the expression of one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, and/or CX3CR1. As described in more detail below, the gene editing process may include the use of a programmable nuclease that mediates double-strand or single-strand breaks in a chemokine receptor gene. For example, CRISPR, TALE, or zinc finger methods can be used to enhance the expression of certain chemokine receptors in TILs.

In one embodiment, CCR4 and/or CCR5 adhesion molecules are inserted into the TIL group using the γ-retroviral or lentiviral methods described herein. In one embodiment, CXCR2 adhesion molecules are inserted into the TIL group using the γ-retroviral or lentiviral methods described by Forget et al. (Frontiers Immunology 2017, 8, 908 or Peng et al., Clin. Cancer Res. 2010, 16, 5458, the disclosure of which is incorporated herein by reference).

In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(i) Obtaining the first TIL group from the tumor removed from the patient;

(ii) A second TIL population is generated by first expansion of the first TIL population by culturing the first TIL population in a cell culture medium containing IL-2 and optional OKT-3;

(iii) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the number of the third TIL population is at least 100 times greater than that of the second TIL population; wherein the second expansion is performed for at least 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; and

(iv) Expose the second and/or third TIL groups to transcription factors (TFs) and/or other molecules capable of transiently altering protein expression; wherein the TFs and/or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and/or the number of tumor antigen-specific T cells in the therapeutic TIL groups; wherein the alteration in expression is an increase in the expression of one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 (RANTES), CXCL1, CXCL8 and/or CCL22.

(ii) Interleukins and others

According to another embodiment, the gene editing method of the present invention can be used to increase the expression of certain interleukins (e.g., one or more of IL-2, IL-4, IL-7, IL-10, IL-15, and IL-21) and the NOTCH 1/2 intracellular domain (ICD). Some interleukins have been shown to enhance T cell effector function and regulate tumor control.

According to specific embodiments, the compositions and methods of the present invention enhance the expression of one or more of IL-2, IL-4, IL-7, IL-10, IL-15, and IL-21, as well as the NOTCH 1/2 intracellular domain (ICD), in tumor-infiltrating lymphocytes (TILs). In some embodiments, the present invention provides a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(i) Obtaining the first TIL group from the tumor removed from the patient;

(ii) A second TIL population is generated by first expansion of the first TIL population by culturing the first TIL population in a cell culture medium containing IL-2 and optional OKT-3;

(iii) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the number of the third TIL population is at least 100 times greater than that of the second TIL population; wherein the second expansion is performed for at least 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; and

(iv) Expose the second and/or third TIL groups to transcription factors (TFs) and/or other molecules capable of transiently altering protein expression; wherein the TFs and/or other molecules capable of transiently altering protein expression provide alterations in tumor antigen expression and/or the number of tumor antigen-specific T cells in the therapeutic TIL groups; wherein the alteration in expression is an increase in the expression of one or more of IL-2, IL-4, IL-7, IL-10, IL-15 and IL-21 and the NOTCH 1/2 intracellular domain (ICD).

IV. TIL Production Process

Figure 1 illustrates an exemplary TIL procedure (referred to as procedure 2A) that includes some of these features, and Figure 2 illustrates some advantages of this embodiment of the invention relative to procedure 1C, as shown in Figures 13 and 14. Figure 3 shows procedure 1C for comparison. Figures 4 (higher cell count) and 5 (lower cell count) show two alternative timelines for TIL treatment based on procedure 2A. Figures 6 and 8 show implementations of procedure 2A. Figures 13 and 14 also provide an exemplary 2A procedure compared to exemplary procedure 1C.

As described herein, the present invention may include steps related to the restimulation of cryopreserved TILs to increase their metabolic activity and thus relative health prior to transplantation into a patient, and methods for detecting said metabolic health. As generally summarized herein, TILs are typically obtained from patient samples and manipulated to amplify their quantity prior to transplantation into a patient. In some embodiments, optionally, TILs may be genetically manipulated as described below.

In some implementations, TILs can be cryopreserved. Once thawed, they can also be restimulated to increase their metabolism before being infused into the patient.

In some embodiments, as detailed below and as illustrated in the examples and figures, the first amplification (including a process called pre-REP and shown as step A in Figure 8) is shortened to 3 to 14 days, and the second amplification (including a process called REP and shown as step B in Figure 8) is shortened to 7 to 14 days. In some embodiments, as described in the examples and as shown in Figures 4, 5, 6, and 7, the first amplification (e.g., the amplification described in step B in Figure 8) is shortened to 11 days, and the second amplification (e.g., the amplification described in step D in Figure 8) is shortened to 11 days. In some embodiments, as detailed below and as illustrated in the examples and figures, the sum of the first and second amplifications (e.g., the amplifications described in steps B and D in Figure 8) is shortened to 22 days.

The "steps" denoted as A, B, C, etc. below refer to Figure 8 and certain embodiments described herein. The order of the steps in the following description and Figure 8 is exemplary, and any combination or order of steps, as well as additional steps, repetitions of steps, and/or omissions of steps, are contemplated in this application and the methods disclosed herein.

Step A: Obtaining a tumor sample from the patient.

Typically, TILs are initially obtained from patient tumor samples (“primary TILs”), then expanded into a larger population for further manipulation as described herein, optionally cryopreserved, restimulated as described herein, and optionally assessed for phenotypic and metabolic parameters as indicators of TIL health.

Patient tumor samples can be obtained using methods known in the art, typically through surgical resection, needle aspiration biopsy, or other means of obtaining a sample containing a mixture of tumor and TIL cells. Generally, tumor samples can be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be liquid tumors, such as those obtained from hematologic malignancies. Solid tumors can be any type of cancer, including but not limited to breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, gastric cancer, and skin cancer (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, useful TILs are obtained from malignant melanoma tumors because they are reported to have particularly high levels of TILs.

The term "solid tumor" refers to an abnormal mass of tissue that does not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. The term "solid tumor carcinoma" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor carcinomas include, but are not limited to, sarcomas, malignant epithelial tumors (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 (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. The histological structure of a solid tumor includes interdependent tissue compartments, including the parenchyma (cancer cells) and supporting stromal cells (the microenvironment in which cancer cells are dispersed and can provide support).

The term "hematologic malignancies" refers to cancers and tumors of the hematopoietic and lymphatic tissues of mammals, including but not limited to tissues of the blood, bone marrow, lymph nodes, and lymphatic system. Hematologic malignancies are also known as "liquid tumors." Hematologic 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 malignancies" refers to hematologic malignancies affecting B cells.

Once obtained, tumor samples are typically broken into small fragments of 1 ^mm³ to approximately 8 ^mm³ using sharp dissection, with approximately 2 ^mm³ to 3 ^mm³ being particularly useful. TILs are cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in an enzyme medium (e.g., Roswell Park Memorial Institute 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be produced by placing the tumor in the enzyme medium, mechanically dissociating it for approximately 1 minute, then incubating it at 5% _CO₂ and 37°C for 30 minutes, and repeating this mechanical dissociation and incubation process under the same conditions until only small tissue fragments remain. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, these cells can be removed using density gradient separation with FICOLL branched hydrophilic polysaccharides. 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 implementation of the methods described herein for amplifying TILs or treating cancer.

Typically, the harvested cell suspension is referred to as a "primary cell population" or a "freshly harvested" cell population.

In some embodiments, fragmentation includes physical fragmentation, including, for example, dissection and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is dissection. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may be initially cultured from enzymatically digested tumor digests and tumor fragments obtained from the patient. In one embodiment, TILs may be initially cultured from enzymatically digested tumor digests and tumor fragments obtained from the patient.

In some embodiments, when the tumor is a solid tumor, the tumor is physically fragmented after obtaining the tumor sample, for example, in step A (as shown in Figure 8). In some embodiments, fragmentation occurs before cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after obtaining the tumor without any cryopreservation. In some embodiments, the tumor is fragmented, and 10, 20, 30, 40, or more fragments or pieces are placed in each container for the first amplification. In some embodiments, the tumor is fragmented, and 30 or 40 fragments or pieces are placed in each container for the first amplification. In some embodiments, the tumor is fragmented, and 40 fragments or pieces are placed in each container for the first amplification. In some embodiments, the multiple fragments comprise about 4 to about 50 fragments, wherein each fragment has a volume of about 27 ^mm³ . In some embodiments, the multiple fragments comprise about 30 to about 60 fragments, with a total volume of about 1300 ^mm³ to about 1500 ^mm³ . In some embodiments, the multiple fragments comprise about 50 fragments, with a total volume of about 1350 ^mm³ . In some embodiments, the plurality of fragments comprises about 50 fragments 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 tumor fragment is obtained from tumor debris. In some embodiments, the tumor debris is obtained through sharp dissection. In some embodiments, the tumor debris is about 1 ^mm³ to 10 ^mm³ . In some embodiments, the tumor debris is about 1 ^mm³ to 8 ^mm³ . In some embodiments, the tumor debris is about 1 ^mm³ . In some embodiments, the tumor debris is about 2 ^mm³ . In some embodiments, the tumor debris is about 3 ^mm³ . In some embodiments, the tumor debris is about 4 ^mm³ . In some embodiments, the tumor debris is about 5 ^mm³ . In some embodiments, the tumor debris is about 6 ^mm³ . In some embodiments, the tumor debris is about 7 ^mm³ . In some embodiments, the tumor debris is about 8 ^mm³ . In some embodiments, the tumor debris is about 9 ^mm³ . In some embodiments, the tumor debris is about 10 ^mm³ . In some embodiments, the tumor is 1 mm to 4 mm × 1 mm to 4 mm × 1 mm to 4 mm. In some embodiments, the tumor is 1 mm × 1 mm × 1 mm. In some embodiments, the tumor is 2mm × 2mm × 2mm. In some embodiments, the tumor is 3mm × 3mm × 3mm. In some embodiments, the tumor is 4mm × 4mm × 4mm.

In some embodiments, the tumor is removed to minimize the amount of hemorrhagic tissue, necrotic tissue, and/or adipose tissue in each small area. In some embodiments, the tumor is removed to minimize the amount of hemorrhagic tissue in each small area. In some embodiments, the tumor is removed to minimize the amount of necrotic tissue in each small area. In some embodiments, the tumor is removed to minimize the amount of adipose tissue in each small area.

In some embodiments, tumor fragmentation is performed to preserve the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without a scalpel-like sawing motion. In some embodiments, TIL is obtained from tumor digests. In some embodiments, tumor digests are generated by incubation in an enzyme medium (e.g., but not limited to RPMI 1640, 2 mm GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase) followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Aubum, California). After placing the tumor in the enzyme medium, it can be mechanically dissociated for approximately 1 minute. The solution is then incubated at 37°C and 5% _CO2 for 30 minutes, followed by mechanical fragmentation again for approximately 1 minute. After incubation again at 37°C and 5% _CO2 for 30 minutes, the tumor can be mechanically fragmented a third time for approximately 1 minute. In some embodiments, after the third mechanical disruption, if large tissue fragments are present, the sample is subjected to one or two additional mechanical dissociations, with or without an additional 30-minute incubation at 37°C and 5% _CO2 . 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, Ficoll density gradient separation can be used to remove these cells.

In some implementations, the cell suspension harvested prior to the first amplification step is referred to as the “primary cell population” or “freshly harvested” cell population.

In some implementations, cells may optionally be frozen after the sample is harvested and cryopreserved before proceeding to the amplification described in step B, which will be described in further detail below and illustrated by example in Figure 8.

B. Step B: First amplification

In some embodiments, the method of the present invention provides the acquisition of young TILs, which, compared to older TILs (i.e., TILs that have undergone more rounds of replication prior to administration to the subject/patient), are capable of increasing the replication cycle after administration to the subject/patient, thus providing additional therapeutic benefits. Characteristics of young TILs have been described in the literature, for example, Donia et al., Scandinavian Journal of Immunology, 75:157-167 (2012); Dudley et al., Clin Cancer Res, 16:6122-6131 (2010); Huang et al., J Immunother, 28(3):258-267 (2005); Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013); Besser et al., J Immunother, 28(3):258-267 (2005); Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013); Besser et al., J Immunother, 28(3):258-267 (2005); Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013); Besser et al., J Immunother, 28(3):258-267 (2005). Other, 32:415-423 (2009); Bunds et al., J Immunother, 32:415-423 (2009); Robbins et al., J Immunol, 2004; 173:7125-7130; Shen et al., J Immunother, 30:123-129 (2007); Zhou et al., J Immunother, 28:53-62 (2005) and Tran et al., J Immunother, 31:742-751 (2008), the entire contents of which are incorporated herein by reference.

Multiple antigen receptors for T and B lymphocytes are generated through somatic recombination of a limited but large number of gene fragments. These gene fragments—V (variable segment), D (diverse segment), J (connector segment), and C (constant segment)—determine the binding specificity of immunoglobulins and T cell receptors (TCRs) and their downstream applications. This invention provides a method for generating TILs that exhibit and increase the diversity of the T-cell repertoire. In some embodiments, TILs obtained by this method exhibit an increase in T-cell repertoire diversity. In some embodiments, TILs obtained by the method of this invention exhibit an increase in T-cell repertoire diversity compared to freshly harvested TILs and/or TILs prepared using methods other than those provided herein (including, for example, methods other than those shown in Figure 8). In some embodiments, TILs obtained by this method exhibit an increase in T-cell repertoire diversity compared to freshly harvested TILs and/or TILs prepared using a method referred to as Process 1C as shown in Figure 13. In some embodiments, TILs obtained from the first amplification exhibit an increase in T-cell repertoire diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T-cell receptor diversity. In some embodiments, immunoglobulin diversity is immunoglobulin heavy chain diversity. In some embodiments, immunoglobulin diversity is immunoglobulin light chain diversity. In some embodiments, diversity is T cell receptor diversity. In some embodiments, diversity is the diversity of T cell receptors selected from one of α, β, γ, and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and/or β is increased. In some embodiments, the expression of T cell receptor (TCR) α is increased. In some embodiments, the expression of T cell receptor (TCR) β is increased. In some embodiments, the expression of TCRab (i.e., TCRα/β) is increased.

After dissecting or digesting tumor fragments (e.g., as described in step A of Figure 8), the resulting cells are cultured in serum containing IL-2 under conditions more favorable to TIL growth compared to tumor and other cells. In some embodiments, the tumor digest is incubated in 2 mL wells in a medium containing inactivated human AB serum and 6000 IU/mL IL-2. This primary cell population is cultured for a period of time, typically 3 to 14 days, producing a large population of TILs, typically about 1 × ^10⁸ TIL cells. In some embodiments, this primary cell population is cultured for 7 to 14 days, producing a large population of TILs, typically about 1 × ^10⁸ TIL cells. In some embodiments, this primary cell population is cultured for 10 to 14 days, producing a large population of TILs, typically about 1 × ^10⁸ TIL cells. In some embodiments, this primary cell population is cultured for about 11 days, producing a large population of TILs, typically about 1 × ^10⁸ TIL cells.

In a preferred embodiment, TIL amplification can be performed as follows: using an initial large-scale TIL amplification step as described below and herein (e.g., those described in step B of Figure 8, which may include a process called pre-REP), followed by a second amplification step as described below and herein (step D, including a process called the Rapid Amplification Protocol (REP) step), optionally followed by cryopreservation, and then a second step D as described below and herein (including a process called the Restimulation REP step). Optionally, the TILs obtained from this process can be characterized for phenotypic features and metabolic parameters as described herein.

In embodiments where TIL culture is initiated using a 24-well plate, for example, a Costar 24-well cell culture plate with a flat bottom (Corning, New York), 1 × ^10⁶ tumor digested cells or one tumor fragment can be seeded in each well in 2 mL of complete medium (CM) containing IL-2 (6000 IU/mL; Chiron Corp., Emeryville, CA). In some embodiments, the tumor fragment is approximately 1 ^mm³ to 10 ^mm³ .

In some embodiments, the initial amplification medium is referred to as "CM," where CM is an abbreviation for culture medium. In some embodiments, the CM in step B consists of RPMI 1640 containing GlutaMAX, supplemented with 10% human AB serum, 25 mmHEPES, and 10 mg/mL gentamicin. In embodiments where culture is initiated using 40 mL permeable flasks with a 10 ^cm² permeable silica bottom (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), each flask contains 10 × ^10⁶ to 40 × 10⁶ live tumor cells or 5 to ³⁰ tumor fragments in 10 to 40 mL of CM containing IL-2. G-Rex10 and 24-well plates are incubated in a humidified incubator at 37°C and 5% _CO₂ . After 5 days of incubation, half of the culture medium is removed and replaced with fresh CM and IL-2. After day 5, half of the culture medium is replaced every 2 to 3 days.

Following the preparation of tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2 under conditions more favorable to TIL growth compared to tumor cells and other cells. In some embodiments, the tumor fragments are incubated in 2 mL wells of medium containing inactivated human AB serum (or, in some cases, as described herein, in the presence of an aAPC cell population) and 6000 IU/mL IL-2. This primary cell population is cultured for a period of time, typically 10 to 14 days, to produce a large population of TILs, typically approximately 1 × ^10⁸ TIL cells. In some embodiments, the growth medium during the initial expansion contains IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, a 1 mg vial of IL-2 stock solution has a specific activity of ²⁰ × ^10⁶ IU/mg to 30 ^{× 10⁶} IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 25 × ^10⁶ IU/mg. In some embodiments, the 1 mg vial of IL-2 stock solution has a specific activity of 30 × ^10⁶ IU/mg. In some embodiments, the final concentration of the IL-2 stock solution is from 4 × ^10⁶ IU/mg to 8 × ^10⁶ IU/mg IL-2. In some embodiments, the final concentration of the IL-2 stock solution is from 5 × ^10⁶ IU/mg to 7 × ^10⁶ IU/mg IL-2. In some embodiments, the final concentration of the IL-2 stock solution is 6 × ^10⁶ IU/mg IL-2. In some embodiments, the IL-2 stock solution is prepared as described in Example 4. In some embodiments, the first amplification medium contains about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7,000 IU/mL IL-2, about 6,000 IU/mL IL-2, or about 5,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 9,000 IU/mL to about 5,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 8,000 IU/mL to about 6,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 7,000 IU/mL to about 6,000 IU/mL IL-2. In some embodiments, the first amplification medium contains about 6,000 IU/mL IL-2. In one embodiment, the cell culture medium also contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU/mL IL-2. In one embodiment, the cell culture medium further contains IL-2. In a preferred embodiment, the cell culture medium contains about 3000 IU/mL IL-2. In one embodiment, the cell culture medium contains about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL IL-2. In one embodiment, the cell culture medium contains 1000 to 2000 IU/mL, 2000 to 3000 IU/mL, 3000 to 4000 IU/mL, 4000 to 5000 IU/mL, 5000 to 6000 IU/mL, 6000 to 7000 IU/mL, 7000 to 8000 IU/mL, or about 8000 IU/mL IL-2.

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

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

In one embodiment, the cell culture medium contains OKT-3 antibody. In a preferred embodiment, the cell culture medium contains about 30 ng/mL of OKT3 antibody. In one embodiment, the cell culture medium contains about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL of OKT3 antibody. In one embodiment, the cell culture medium contains OKT3 antibody at concentrations of 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, or 50 ng/mL to 100 ng/mL. In some embodiments, the cell culture medium does not contain OKT-3 antibody.

In some embodiments, the cell culture medium contains more than one TNFRSF agonist. In some embodiments, the TNFRSF agonist includes a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist selected from urogenumab, utorumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve 20 μg/mL to 40 μg/mL in the cell culture medium.

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

In some embodiments, the initial amplification medium is referred to as "CM (abbreviation for culture medium)". In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 and GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin. In some embodiments, culture is initiated in permeable flasks (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) with a 40 mL capacity and a 10 ^cm² permeable silica bottom, each flask containing 10 mL to 40 mL of CM supplemented with IL-2, the CM containing 10 × ^10⁶ to 40 × ^10⁶ live tumor cells or 5 to 30 tumor fragments. Both the G-Rex10 and 24-well plates are incubated in a humidified incubator at 37°C and 5% _CO₂ . After 5 days of culture, half of the culture medium is removed and replaced with fresh CM and IL-2. After day 5, half of the culture medium is replaced every 2 to 3 days. In some embodiments, CM is CM1 as described in the examples, see Example 5. In some embodiments, the first amplification occurs in the initial cell culture medium or the first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium contains IL-2.

In some implementations, as described in the embodiments and figures, the first amplification process (including, for example, those described in step B of FIG8, which may include those sometimes referred to as pre-REP) is shortened to 3 to 14 days. In some implementations, as described in the embodiments and figures 4 and 5, and including, for example, the amplification described in step B of FIG8, the first amplification process (including, for example, those described in step B of FIG8, which may include those sometimes referred to as pre-REP) is shortened to 7 to 14 days. In some implementations, as described in the embodiments and figures 4 and 5, the first amplification in step B is shortened to 10 to 14 days. In some implementations, in the first embodiment, as described in the embodiments and figures 4 and 5, and including, for example, the amplification described in step B of FIG8, the first amplification is shortened to 11 days.

In some embodiments, the first TIL amplification can be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the first TIL amplification can be performed for 1 to 14 days. In some embodiments, the first TIL amplification can be performed for 2 to 14 days. In some embodiments, the first TIL amplification can be performed for 3 to 14 days. In some embodiments, the first TIL amplification can be performed for 4 to 14 days. In some embodiments, the first TIL amplification can be performed for 5 to 14 days. In some embodiments, the first TIL amplification can be performed for 6 to 14 days. In some embodiments, the first TIL amplification can be performed for 7 to 14 days. In some embodiments, the first TIL amplification can be performed for 8 to 14 days. In some embodiments, the first TIL amplification can be performed for 9 to 14 days. In some embodiments, the first TIL amplification can be performed for 10 to 14 days. In some embodiments, the first TIL amplification can be performed for 11 to 14 days. In some embodiments, the first TIL amplification may take 12 to 14 days. In some embodiments, the first TIL amplification may take 13 to 14 days. In some embodiments, the first TIL amplification may take 14 days. In some embodiments, the first TIL amplification may take 1 to 11 days. In some embodiments, the first TIL amplification may take 2 to 11 days. In some embodiments, the first TIL amplification may take 3 to 11 days. In some embodiments, the first TIL amplification may take 4 to 11 days. In some embodiments, the first TIL amplification may take 5 to 11 days. In some embodiments, the first TIL amplification may take 6 to 11 days. In some embodiments, the first TIL amplification may take 7 to 11 days. In some embodiments, the first TIL amplification may take 8 to 11 days. In some embodiments, the first TIL amplification may take 9 to 11 days. In some embodiments, the first TIL amplification may take 10 to 11 days. In some embodiments, the first TIL amplification may take 11 days.

In some embodiments, during the first amplification, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination. In some embodiments, during the first amplification, including, for example, during step B according to FIG8 and as described herein, IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof, may be included. In some embodiments, during the first amplification, a combination of IL-2, IL-15, and IL-21 is used as a combination. In some embodiments, during step B according to FIG8 and as described herein, IL-2, IL-15, and IL-21, and any combination thereof, may be included.

In some embodiments, as described in the examples and shown in Figures 4 and 5, the first amplification process (including a process referred to as pre-REP; for example, step B according to Figure 8) is shortened to 3 to 14 days. In some embodiments, as described in the examples and shown in Figures 4 and 5, the first amplification in step B is shortened to 7 to 14 days. In some embodiments, as described in the examples and shown in Figures 4, 5, 6, and 7, the first amplification in step B is shortened to 10 to 14 days. In some embodiments, as described in the examples and shown in Figures 4, 5, 6, and 7, the first amplification is shortened to 11 days.

In some embodiments, the first amplification (e.g., according to step B of FIG8) is performed in a closed-system bioreactor. In some embodiments, as described herein, a closed system is used for TIL amplification. In some embodiments, a single bioreactor is used. In some embodiments, for example, the single bioreactor used is a G-REX-10 or a G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some embodiments, during the first amplification (e.g., according to step B of FIG8), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing amounts of TIL and other reagents selected from the group consisting of: 0.1 μM sd-RNA/10,000 TIL/100 μL medium, 0.5 μM sd-RNA/10,000 TIL/100 μL medium, 0.75 μM sd-RNA/10,000 TIL/100 μL medium, etc. 0 TIL/100 μL medium, 1 μM sd-RNA/10,000 TIL/100 μL medium, 1.25 μM sd-RNA/10,000 TIL/100 μL medium, 1.5 μM sd-RNA/10,000 TIL/100 μL medium, 2 μM sd-RNA/10,000 TIL/100 μL medium, 5 μM sd-RNA/10,000 TIL/100 μL medium, or 10 μM sd-RNA/10,000 TIL/100 μL medium. In some implementations, during the first amplification period (e.g., according to step B of Figure 8), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to the TIL culture twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days. In one embodiment, during the first amplification (e.g., according to step B of FIG8), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing amounts of TIL and other reagents selected from the group consisting of: 0.1 μM sd-RNA/10,000 TIL, 0.5 μM sd-RNA/10,000 TIL, 0.75 μM sd-RNA/10,000 TIL, 1 μM sd-RNA/10,000 TIL, 1.25 μM sd-RNA/10,000 TIL, 1.5 μM sd-RNA/10,000 TIL, 2 μM sd-RNA/10,000 TIL, 5 μM sd-RNA/10,000 TIL, or 10 μM sd-RNA/10,000 TIL. In one implementation, during the first amplification period (e.g., according to step B of FIG8), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to the TIL culture twice daily, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

C. Step C: Transition from the first amplification to the second amplification

In some cases, the schemes discussed below can be used to immediately cryopreserve the large number of TIL clusters obtained from the first amplification, including, for example, the TIL clusters obtained from step B as shown in Figure 8. Optionally, the TIL clusters harvested from the first amplification (referred to as the second TIL cluster) can be subjected to a second amplification (which may include an amplification sometimes referred to as REP), and then cryopreserved as described below. Similarly, in cases where genetically modified TILs will be used for treatment, the first TIL cluster (sometimes referred to as the large number of TIL clusters) or the second TIL cluster (which in some embodiments may include a cluster referred to as the REP TIL cluster) can be genetically modified before amplification or after the first amplification and before the second amplification for use in appropriate treatment.

In some embodiments, the TIL obtained from the first amplification (e.g., step B as shown in FIG. 7) is stored until phenotypic selection is performed. In some embodiments, the TIL obtained from the first amplification (e.g., step B as shown in FIG. 7) is not stored but is directly subjected to a second amplification. In some embodiments, the TIL obtained from the first amplification is not cryopreserved after the first amplification and before the second amplification. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 3 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 4 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 4 to 10 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs approximately 7 to 14 days after fragmentation. In some implementations, the transition from the first amplification to the second amplification occurs approximately 14 days after fragmentation begins.

In some embodiments, the transition from the first amplification to the second amplification occurs approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after fragmentation. In some embodiments, the transition occurs from 1 to 14 days after fragmentation. In some embodiments, the first TIL amplification may take 2 to 14 days. In some embodiments, the transition occurs from 3 to 14 days after fragmentation. In some embodiments, the transition occurs from 4 to 14 days after fragmentation. In some embodiments, the transition occurs from 5 to 14 days after fragmentation. In some embodiments, the transition occurs from 6 to 14 days after fragmentation. In some embodiments, the transition occurs from 7 to 14 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 8 to 14 days after fragmentation. In some embodiments, the transition occurs 9 to 14 days after fragmentation. In some embodiments, the transition occurs 10 to 14 days after fragmentation. In some embodiments, the transition occurs 11 to 14 days after fragmentation. In some embodiments, the transition occurs 12 to 14 days after fragmentation. In some embodiments, the transition occurs 13 to 14 days after fragmentation. In some embodiments, the transition occurs 14 days after fragmentation. In some embodiments, the transition occurs 1 to 11 days after fragmentation. In some embodiments, the transition occurs 2 to 11 days after fragmentation. In some embodiments, the transition from the first amplification to the second amplification occurs 3 to 11 days after fragmentation. In some embodiments, the transition occurs 8 to 11 days after fragmentation. In some embodiments, the transition occurs 9 to 11 days after fragmentation. In some embodiments, the transition occurs 10 to 11 days after fragmentation. In some embodiments, the transition occurs 11 days after fragmentation.

In some implementations, TILs are not stored after the first amplification and before the second amplification, and the TILs are directly amplified in the second amplification (e.g., in some implementations, as shown in Figure 8, no storage is performed during the transition from step B to step D). In some implementations, as described herein, the transition occurs in a closed system. In some implementations, TILs from the first amplification (i.e., the second TIL group) do not undergo a transition period and directly enter the second amplification.

In some embodiments, the transition from the first amplification to the second amplification is performed in a closed-system bioreactor (e.g., according to step C of Figure 8). In some embodiments, as described herein, a closed system is used for TIL amplification. In some embodiments, a single bioreactor is used. In some embodiments, for example, the single bioreactor used is a G-REX-10 or a G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some embodiments, during the transition from the first amplification to the second amplification (e.g., according to step C of Figure 8), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing amounts of TIL and other reagents selected from the group consisting of: 0.1 μM sd-RNA/10,000 TIL/100 μL medium, 0.5 μM sd-RNA/10,000 TIL/100 μL medium, 0.75 μM sd-RNA/100 μL medium, etc. 10,000 TIL/100 μL medium, 1 μM sd-RNA/10,000 TIL/100 μL medium, 1.25 μM sd-RNA/10,000 TIL/100 μL medium, 1.5 μM sd-RNA/10,000 TIL/100 μL medium, 2 μM sd-RNA/10,000 TIL/100 μL medium, 5 μM sd-RNA/10,000 TIL/100 μL medium, or 10 μM sd-RNA/10,000 TIL/100 μL medium. In some embodiments, during the transition from the first amplification to the second amplification (e.g., according to step C of FIG8), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to the TIL culture twice daily, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days. In one embodiment, during the transition from the first amplification to the second amplification (e.g., according to step C of FIG8), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing amounts of TIL and other reagents selected from the group consisting of: 0.1 μM sd-RNA/10,000 TIL, 0.5 μM sd-RNA/10, 0.000 TIL, 0.75 μM sd-RNA/10,000 TIL, 1 μM sd-RNA/10,000 TIL, 1.25 μM sd-RNA/10,000 TIL, 1.5 μM sd-RNA/10,000 TIL, 2 μM sd-RNA/10,000 TIL, 5 μM sd-RNA/10,000 TIL, or 10 μM sd-RNA/10,000 TIL. In some embodiments, during the transition from the first amplification to the second amplification (e.g., according to step C of Figure 8), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to the TIL culture twice daily, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

1. Cytokines

As is known in the art, the amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2).

Alternatively, combinations of cytokines can be used for rapid expansion and/or secondary expansion of TILs; wherein combinations of two or more of IL-2, IL-15, and IL-21 are as outlined in International Publications WO2015/189356 and WO2015/189357, the entire contents of which are expressly incorporated herein by reference. 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, the latter being found particularly useful in many embodiments. The use of combinations of cytokines is particularly beneficial for the production of lymphocytes (especially T cells as described herein).

Step D: Second amplification

In some implementations, after harvesting and initial mass treatment, such as after steps A and B, the number of TIL cells increases; this transition is referred to as step C, as shown in Figure 8. This further expansion is referred to herein as a second expansion, which may include an expansion process commonly known in the art as the Rapid Expansion Process (REP; and the process shown in step D of Figure 8). The second expansion is typically performed in an aerated container using a culture medium containing multiple components, including feeder cells, a cytokine source, and an anti-CD3 antibody.

In some embodiments, the second amplification or second TIL amplification (which may include amplification sometimes referred to as REP; and the process shown in step D of FIG8) can be performed using any TIL flask or container known to those skilled in the art. In some embodiments, the second TIL amplification may be performed for 10, 11, 12, 13, or 14 days. In some embodiments, the second TIL amplification may be performed for about 7 to about 14 days. In some embodiments, the second TIL amplification may be performed for about 8 to about 14 days. In some embodiments, the second TIL amplification may be performed for about 9 to about 14 days. In some embodiments, the second TIL amplification may be performed for about 10 to about 14 days. In some embodiments, the second TIL amplification may be performed for about 11 to about 14 days. In some embodiments, the second TIL amplification may be performed for about 12 to about 14 days. In some embodiments, the second TIL amplification may be performed for about 13 to about 14 days. In some embodiments, the second TIL amplification may be performed for about 14 days.

In one embodiment, the second amplification can be performed in a breathable container using the methods of this disclosure (including, for example, an amplification referred to as REP; and the process shown in step D of FIG7). For example, TILs can be rapidly amplified using nonspecific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Nonspecific T cell receptor stimulation may include, for example, an anti-CD3 antibody, such as about 30 ng/mL of OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). During the second expansion, TILs can be rapidly expanded in vitro by further stimulation with a cancer antigen (including its antigenic motif, such as an epitope) that can optionally be expressed by a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, for example 0.3 μM MART-L26-35 (27L) or gp 100:209-217 (210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase carcinoma antigen, MAGE-A3, SSX-2, and VEGFR2, or their antigenic motifs. TILs can also be rapidly expanded by restimulating them with the same cancer antigen pulsed to an HLA-A2-expressing antigen. Alternatively, TILs can be further restimulated with, for example, irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, restimulation occurs as part of the second expansion. In some implementations, a second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

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

In one embodiment, the cell culture medium contains OKT3 antibody. In some embodiments, the cell culture medium contains about 30 ng/mL of OKT3 antibody. In one embodiment, the cell culture medium contains about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL of OKT3 antibody. In one embodiment, the cell culture medium contains OKT3 antibody at concentrations of 0.1 ng/mL to 1 ng/mL, 1 ng/mL to 5 ng/mL, 5 ng/mL to 10 ng/mL, 10 ng/mL to 20 ng/mL, 20 ng/mL to 30 ng/mL, 30 ng/mL to 40 ng/mL, 40 ng/mL to 50 ng/mL, or 50 ng/mL to 100 ng/mL. In some embodiments, the cell culture medium does not contain OKT-3 antibody.

In some embodiments, the initial amplification cell culture medium contains one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist includes a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist selected from: urogenumab, utorumab, EU-101, fusion proteins, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve 0.1 μg/mL to 100 μg/mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve 20 μg/mL to 40 μg/mL in the cell culture medium.

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

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is used as a combination during the second amplification. In some embodiments, the second amplification period (including, for example, step D of FIG8, and as described herein) may include IL-2, IL-7, IL-15, and/or IL-21, and any combination thereof. In some embodiments, a combination of IL-2, IL-15, and IL-21 is used as a combination during the second amplification. In some embodiments, step D of FIG8, and as described herein, may include IL-2, IL-15, and IL-21, and any combination thereof.

In some embodiments, the second expansion can be performed in a supplemental cell culture medium containing IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemental cell culture medium. In some embodiments, the supplemental cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium contains IL-2, OKT-3, and antigen-presenting cells (APCs; also known as antigen-presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen-presenting cells).

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

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

In some embodiments, the antigen-presenting feeder cells (APCs) are PBMCs. In one embodiment, during rapid expansion and/or secondary expansion, the ratio of TILs to PBMCs and/or antigen-presenting cells is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In one embodiment, during rapid expansion and/or secondary expansion, the ratio of TILs to PBMCs is from 1:50 to 1:300. In one embodiment, during rapid expansion and/or secondary expansion, the ratio of TILs to PBMCs is from 1:100 to 1:200.

In one embodiment, the REP and/or second amplification are performed in a flask; wherein a large amount of TIL is mixed with 100 or 200 times excess of inactivated feeder cells, 30 mg/mL of OKT3 anti-CD3 antibody, and 3000 IU/mL of IL-2 in 150 mL of culture medium. The culture medium is replaced (typically by aspirating fresh medium to replace 2/3 of the medium) until the cells are transferred to a replacement growth chamber. As discussed more fully below, the replacement growth chamber comprises a G-REX flask and a ventilated container.

In some implementations, as described in the examples and figures, the second amplification (which may include a process referred to as the REP process) is shortened to 7 to 14 days. In some implementations, the second amplification is shortened to 11 days.

In one embodiment, the REP and/or second amplification can be performed using T-175 flasks and aeration bags as described above (Tran et al., J. I. ^M _... In some implementations, on day 7, cells from two T-175 flasks can be combined into a 3L bag, and 300mL of AIM V containing 5% human AB serum and 3000 IU/mL IL-2 can be added to 300mL of TIL suspension. Cell counts in each bag are counted daily or every two days, and fresh culture medium is added to maintain the cell count at 0.5 × ^10⁶ to 2.0 × ^10⁶ cells/mL.

In one embodiment, the second amplification (which may include amplification referred to as REP, as well as those mentioned in step D of Figure 7) can be performed in a 500 mL gas-permeable flask with a 100 ^cm² gas-permeable silica bottom (G-Rex 100, available from WilsonWolf Manufacturing Corporation, New Brighton, MN, USA). 5 × ^10⁶ or 10 × ^10⁶ TILs can be cultured with PBMCs in 400 mL of 50/50 medium supplemented with 5% human AB serum, 3000 IU/mL IL-2, and 30 ng/mL anti-CD3 (OKT3). The G-REX 100 flask can be incubated at 37°C in 5% _CO₂ . On day 5, 250 mL of the supernatant can be removed and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL precipitate can be resuspended in 150 mL of fresh culture medium containing 5% human AB serum and 3000 IU/mL IL-2, and then added back to the original G-REX 100 flask. When TILs are continuously amplified in the G-REX 100 flasks, on day 7, the TILs from each G-REX 100 flask can be resuspended in the 300 mL of culture medium present in each flask, and the cell suspension can be aliquoted into three 100 mL aliquots suitable for inoculating three G-REX 100 flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 can be added to each flask. The G-REX 100 flasks can be incubated at 37°C and 5% _CO2 for 4 days. After that, 150 mL of AIM-V containing 3000 IU/mL IL-2 can be added to each G-REX 100 flask. Cells can be harvested on day 14 of culture.

In one embodiment, the second amplification (including an amplification termed REP) is performed in a flask; wherein a large amount of TIL is mixed with 100 or 200 times excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody, and 3000 IU/mL IL-2 in 150 mL of culture medium. In some embodiments, the culture medium is replaced until the cells are transferred to a replacement growth chamber. In some embodiments, two-thirds of the culture medium is replaced by respiration with fresh culture medium. In some embodiments, as discussed more fully below, the replacement growth chamber comprises a G-REX flask and a vented container.

In one embodiment, a second amplification (including an amplification termed REP) is performed, and the process further includes a step of selecting tumor-responsive TILs. Any selection method known in the art can be used. For example, the method described in U.S. Patent Application Publication No. 2016/0010058A1 (the disclosure of which is incorporated herein by reference) can be used to select tumor-responsive TILs.

Optionally, standard detection methods known in the art can be used to determine cell viability after the second amplification (including amplification known as REP amplification). For example, a trypan blue exclusion assay can be performed on a large number of TIL samples, which selectively labels dead cells and allows for viability assessment. In some embodiments, TIL samples can be counted and viability detected using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to, for example, the Cellometer K2 ImageCytometer Automatic Cell Counter protocol described in Example 15.

In some embodiments, the second amplification of TIL (including an amplification referred to as REP) can be performed using T-175 flasks and aeration bags as described above (Tran KQ, Zhou J, Durflinger KH et al., 2008, J. Immunother., 31:742-751 and Dudley ME, Wunderlich JR, Shelton TE et al., 2003, J. Immunother., 26:332-342) or aeration-permeable G-REX flasks. In some embodiments, the second amplification is performed using flasks. In some embodiments, the second amplification is performed using aeration-permeable G-REX flasks. In some embodiments, the second amplification is performed in T-175 flasks, with approximately 1 × ^10⁶ TIL suspended in approximately 150 mL of culture medium added to each T-175 flask. TILs were co-cultured with irradiated (50 Gy) allogeneic PBMCs (as "feeder" cells) at a 1:100 ratio in a 1:1 mixture of CM and AIM-V media (50/50 medium), supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. T-175 flasks were incubated at 37°C in 5% _CO2 . In some embodiments, on day 5, half of the medium was replaced with 50/50 medium containing 3000 IU/mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks were combined into a 3L bag, and 300 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 was added to 300 mL of TIL suspension. The number of cells in each bag can be counted daily or every two days, and fresh culture medium can be added to maintain the cell count at approximately 0.5 × ^10⁶ to approximately 2.0 × ^10⁶ cells/mL.

In some embodiments, the second amplification (including an amplification referred to as REP) is performed in a 500 mL flask (G-REX 100, Wilson Wolf) with a 100 ^cm² permeable silica bottom (Figure 1). Approximately 5 × ^10⁶ or 10 × ^10⁶ TILs are incubated with irradiated allogeneic PBMCs at a 1:100 ratio in 400 mL of 50/50 medium supplemented with 3000 IU/mL IL-2 and 30 ng/mL anti-CD3. The G-REX 100 flask is incubated at 37°C in 5% _CO₂ . In some embodiments, on day 5, 250 mL of the supernatant is collected and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL precipitate can then be resuspended in 150 mL of fresh 50/50 medium containing 3000 IU/mL IL-2 and added back to the original G-REX 100 flask. In the embodiment of continuous TIL amplification in G-REX 100 flasks, on day 7, the TIL from each G-REX 100 flask is resuspended in 300 mL of medium present in each flask, and the cell suspension is divided into three 100 mL aliquots for inoculating three G-REX 100 flasks. Then, 150 mL of AIM-V containing 5% human AB serum and 3000 IU/mL IL-2 is added to each flask. The G-REX 100 flasks are incubated at 37°C and 5% _CO2 for 4 days, after which 150 mL of AIM-V containing 3000 IU/mL IL-2 is added to each G-REX 100 flask. Cells are harvested on day 14 of culture.

Multiple antigen receptors on T and B lymphocytes are generated through somatic recombination of a limited but large number of gene fragments. These gene fragments—V (variable region), D (multivariable region), J (connector region), and C (constant region)—determine the binding specificity and downstream applications of immunoglobulins and T cell receptors (TCRs). This invention provides a method for generating TILs that demonstrate and increase the diversity of the T cell repertoire. In some embodiments, TILs obtained by this method demonstrate an increase in T cell repertoire diversity. In some embodiments, TILs obtained through a second amplification demonstrate an increase in T cell repertoire diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, immunoglobulin diversity is immunoglobulin heavy chain diversity. In some embodiments, immunoglobulin diversity is immunoglobulin light chain diversity. In some embodiments, diversity is T cell receptor diversity. In some embodiments, diversity is the diversity of T cell receptors selected from one of α, β, γ, and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and/or β is increased. In some embodiments, the expression of T cell receptor (TCR) α is increased. In some implementations, the expression of T-cell receptor (TCR)β is increased. In some implementations, the expression of TCRab (i.e., TCRα/β) is increased.

In some implementations, the second amplification medium (e.g., sometimes referred to as CM2 or second cell culture medium) contains IL-2, OKT-3, and antigen-presenting feeder cells (APCs), as discussed in more detail below.

In some embodiments, the second amplification (e.g., according to step D of FIG8) is performed in a closed-system bioreactor. In some embodiments, as described herein, a closed system is used for TIL amplification. In some embodiments, a single bioreactor is used. In some embodiments, for example, the single bioreactor used is a G-REX-10 or a G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some embodiments, during the second amplification (e.g., according to step D of FIG8), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing amounts of TIL and other reagents selected from the group consisting of: 0.1 μM sd-RNA/10,000 TIL/100 μL medium, 0.5 μM sd-RNA/10,000 TIL/100 μL medium, 0.75 μM sd-RNA/10,000 TIL/100 μL medium, etc. 0 TIL/100 μL medium, 1 μM sd-RNA/10,000 TIL/100 μL medium, 1.25 μM sd-RNA/10,000 TIL/100 μL medium, 1.5 μM sd-RNA/10,000 TIL/100 μL medium, 2 μM sd-RNA/10,000 TIL/100 μL medium, 5 μM sd-RNA/10,000 TIL/100 μL medium, or 10 μM sd-RNA/10,000 TIL/100 μL medium. In some implementations, during the second amplification (e.g., according to step D of FIG8), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to the TIL culture twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days. In one embodiment, during the second amplification (e.g., according to step D of FIG8), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium containing amounts of TIL and other reagents selected from the group consisting of: 0.1 μM sd-RNA/10,000 TIL, 0.5 μM sd-RNA/10,000 TIL, 0.75 μM sd-RNA/10,000 TIL, 1 μM sd-RNA/10,000 TIL, 1.25 μM sd-RNA/10,000 TIL, 1.5 μM sd-RNA/10,000 TIL, 2 μM sd-RNA/10,000 TIL, 5 μM sd-RNA/10,000 TIL, or 10 μM sd-RNA/10,000 TIL. In some implementations, during the second amplification (e.g., according to step D of FIG8), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to the TIL culture twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

1. Feeder cells and antigen-presenting cells

In one implementation, during REP TIL amplification and/or during the second amplification, the second amplification step described herein (e.g., including those described in step D of Figure 8 and those referred to as REP) requires an excess of feeder cells. In many implementations, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation.

Typically, allogeneic PBMCs are inactivated by irradiation or heat treatment and used in the REP step, as described in the embodiments, which provides an exemplary scheme for evaluating the replication incompetence of irradiated allogeneic PBMCs.

In some implementations, if the total number of viable cells on day 14 is less than the initial number of viable cells introduced into culture on day 0 of the REP and/or day 0 of the second expansion (i.e., the start date of the second expansion), the PBMCs are considered to be incompletely replicating and are accepted for use in the TIL expansion step described herein.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells introduced into culture on day 0 of REP and/or day 0 of the second amplification (i.e., the start date of the second amplification), then PBMCs are considered to have impaired replication function and are accepted for the TIL amplification step described herein. In some embodiments, PBMCs are cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2.

In some embodiments, if the total number of viable cells cultured on days 7 and 14 in the presence of OKT3 and IL-2 does not increase compared to the initial number of viable cells introduced into culture on day 0 of the REP and/or day 0 of the second amplification (i.e., the start date of the second amplification), then PBMCs are considered to have impaired replication function and are accepted for the TIL amplification step described herein. In some embodiments, PBMCs are cultured in the presence of 5 ng/mL to 60 ng/mL OKT3 antibody and 1000 IU/mL to 6000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 10 ng/mL to 50 ng/mL OKT3 antibody and 2000 IU/mL to 5000 IU/mL IL-2. In some embodiments, PBMCs are cultured in the presence of 20 ng/mL to 40 ng/mL OKT3 antibody and 2000 IU/mL to 4000 IU/mL IL-2. In some implementations, PBMCs are cultured in the presence of 25 ng/mL to 35 ng/mL OKT3 antibody and 2500 IU/mL to 3500 IU/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 one embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500. In one embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is from 1:50 to 1:300. In one embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is from 1:100 to 1:200.

In one embodiment, the second expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁹ feeder cells to approximately 100 × ^10⁶ TILs. In another embodiment, the second expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁹ feeder cells to approximately 50 × ^10⁶ TILs. In yet another embodiment, the second expansion procedure described herein requires a ratio of approximately 2.5 × ^10⁹ feeder cells to approximately 25 × ^10⁶ TILs.

In one embodiment, the second amplification step described herein requires an excess of feeder cells during the second amplification. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In one embodiment, artificial antigen-presenting (aAPC) cells are used instead of PBMCs.

Typically, allogeneic PBMCs are inactivated by irradiation or heat treatment and used in the TIL amplification steps described herein, including, for example, the exemplary steps shown in Figures 4, 5, 6 and 7.

In one implementation, artificial antigen-presenting cells are used in the second expansion, either in place of PBMCs or in combination with PBMCs.

2. Cytokines

As is known in the art, the amplification methods described herein typically use culture media containing high doses of cytokines (particularly IL-2).

Alternatively, combinations of cytokines can be used for rapid expansion and/or secondary expansion of TILs; wherein combinations of two or more of IL-2, IL-15, and IL-21 are as outlined in International Publications WO2015/189356 and WO2015/189357, the entire contents of which are expressly incorporated herein by reference. 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, the latter being found particularly useful in many embodiments. The use of combinations of cytokines is particularly beneficial for the production of lymphocytes (especially T cells as described herein).

Step E: Harvesting TIL

Cells can be harvested after the second amplification step. In some embodiments, for example, TILs are harvested after one, two, three, four, or more amplification steps as shown in Figure 8. In some embodiments, for example, TILs are harvested after two amplification steps as shown in Figure 8.

TILs can be harvested in any suitable and aseptic manner, including, for example, by centrifugation. Methods for harvesting TILs are well known in the art, and any such known methods can be used in conjunction with the method of the present invention. In some embodiments, an automated system is used to harvest TILs.

Cell collectors and/or cell processing systems are commercially available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell-based collector can be used in the methods of this invention. In some embodiments, the cell collector and/or cell processing system is a membrane-based cell collector. In some embodiments, cells are harvested via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump a cell-containing solution through a membrane or filter (such as a rotating membrane or rotary filter) in a sterile and/or closed system environment, allowing for continuous flow and cell processing to remove supernatant or cell culture medium without precipitate. In some embodiments, the cell collector 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, the harvest (e.g., according to step E of FIG8) is carried out in a closed-system bioreactor. In some embodiments, as described herein, a closed system is used for TIL amplification. In some embodiments, a single bioreactor is used. In some embodiments, for example, the single bioreactor used is a G-REX-10 or a G-REX-100. In some embodiments, the closed-system bioreactor is a single bioreactor.

In some embodiments, step E of FIG8 is performed according to the process described in Example 16. In some embodiments, to maintain the sterility and closure of the system, the closed system is introduced under sterile conditions using a syringe. In some embodiments, a closed system as described in Example 16 is employed.

In some embodiments, TIL is harvested according to the method described in Example 16. In some embodiments, TIL from day 1 to day 11 is harvested using the method described in Section 8.5 (referred to as: Day 11 TIL Harvest in Example 16). In some embodiments, TIL from day 12 to day 22 is harvested using the method described in Section 8.12 (referred to as: Day 22 TIL Harvest in Example 16).

Step F: Final formulation/transfer to infusion bag

Following steps A through E as exemplarily provided in Figure 7 and as outlined above and in detail herein, the cells are transferred to a container for administration to a patient. In some embodiments, once a sufficient number of therapeutically adequate TILs have been obtained using the amplification method described above, they are transferred to a container for administration to a patient.

In one embodiment, TILs amplified by APCs of this disclosure are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified by PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

1. Drug composition, dosage and administration regimen

In one embodiment, TILs amplified using the methods of this disclosure are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs amplified using PBMCs of this disclosure can be administered via any suitable route known in the art. In some embodiments, T cells are administered via a single intra-arterial or intravenous infusion, preferably lasting about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TILs can be administered. In some embodiments, particularly when the cancer is melanoma, about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ TIL clusters are administered, with an average of about 7.8 × ^10¹⁰ TIL clusters. In one embodiment, about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TIL clusters are administered. In some embodiments, about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TIL clusters are administered. In some embodiments, about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TIL clusters are administered. In some embodiments, about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TIL clusters are administered. In some embodiments, about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TIL clusters are administered. In some embodiments, about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TIL clusters are administered. In some embodiments, the therapeutically effective dose is about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ . In some embodiments, particularly when the cancer is melanoma, the therapeutically effective dose is about 7.8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 1.2 × ^10¹⁰ to about 4.3 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 3 × ^10¹⁰ to about 12 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 4 × ^10¹⁰ to about 10 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 5 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 6 × ^10¹⁰ to about 8 × ^10¹⁰ TILs. In some embodiments, the therapeutically effective dose is about 7 × ^10¹⁰ to about 8 × ^10¹⁰ TILs.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention 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} , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ , and 9×10 ¹³ . In one embodiment, the amount of TIL provided in the pharmaceutical composition of the present invention ranges from 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5× ^10⁷ , 5× ^10⁷ to 1× ^10⁸ , 1× ^10⁸ to 5× ^10⁸ , 5× ^10⁸ to 1× ^10⁹ , 1× ^10⁹ to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1× ^10¹² to 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the concentration of TIL provided in the pharmaceutical composition of the present invention is less than, for example, 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%, or 0.07% of the pharmaceutical composition. 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 concentration of TIL provided in the pharmaceutical composition of the present 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% of the pharmaceutical composition. 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%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 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 concentration range of TIL provided in the pharmaceutical composition of the present invention is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, and from about 0.07% to about 24% of the pharmaceutical composition. %, 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/w, w/v or v/v.

In some embodiments, the concentration range of TIL provided in the pharmaceutical composition of the present invention is 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.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is 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, etc. g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.0 05g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g or 0.0001g.

In some embodiments, the amount of TIL provided in the pharmaceutical composition of the present invention is 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, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0.0 8g, 0.085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.45g , 0.5g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g , 2.5g, 3g, 3.5g, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g or 10g.

The TIL provided in the pharmaceutical compositions of the present invention is effective over a wide dosage range. The precise dosage depends on the route of administration, the form of the compound being administered, the sex and age of the patient being treated, the patient's weight, and the preferences and experience of the attending physician. Clinically determined TIL dosages may also be used if appropriate. The amount of pharmaceutical composition administered using the methods described herein (e.g., the dosage of TIL) will depend on the person or mammal being treated, the condition or severity of the condition, the rate of administration, the formulation of the active pharmaceutical ingredient, and the prescribing physician's discretion.

In some implementations, TIL can be administered as a single dose. This administration can be by injection, such as intravenous injection. In some implementations, TIL can be administered in multiple doses. The dose can be once, twice, three times, four times, five times, six times, or more than six times per year. The dose can be once a month, once every two weeks, once a week, or once every two days. TIL administration can continue as long as necessary.

In some implementations, the effective dose of TIL is approximately 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 ^{. 9} , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 2×10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 ¹³ , 7×10 ¹³ , 8×10 ¹³ , and 9×10 ¹³ . In some implementations, the effective dose of TIL ranges from 1× ^10⁶ to 5× ^10⁶ , 5× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 5×10⁷, 5×10⁷ to 1 ^{×10⁸ ,} 1× ^10⁸ to 5×10⁸, 5× ^10⁸ to 1× ^10⁹ , 1 ^{× 10⁹} to 5× ^10⁹ , 5× ^10⁹ to 1× ^10¹⁰ , 1× ^10¹⁰ to 5× ^10¹⁰ , 5× ^10¹⁰ to 1× ^10¹¹ , 5× ^10¹¹ to 1× ^10¹² , 1 ^{×10¹² to} 5× ^10¹² , and 5× ^10¹² to 1× ^10¹³ .

In some embodiments, the effective dose range of TIL is about 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about 2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg/kg to about 2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg to about 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kg to about 0.8 mg/kg, about 0.7 mg/kg, etc. From about 0.75 mg/kg, from about 0.7 mg/kg to about 2.15 mg/kg, from about 0.85 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 1.85 mg/kg, from about 1.15 mg/kg to about 1.7 mg/kg, from about 1.3 mg/kg to about 1.6 mg/kg, from about 1.35 mg/kg to about 1.5 mg/kg, from about 2.15 mg/kg to about 3.6 mg/kg, from about 2.3 mg/kg to about 3.4 mg/kg, from about 2.4 mg/kg to about 3.3 mg/kg, from about 2.6 mg/kg to about 3.15 mg/kg, from about 2.7 mg/kg to about 3 mg/kg, from about 2.8 mg/kg to about 3 mg/kg, or from about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, the effective dose range of TIL is about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg. g, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 mg to about 207 mg.

An effective amount of TIL can be administered in single or multiple doses via any acceptable route of administration of a similar agent, including intranasal and transdermal routes, via intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, local, via transplantation, or via inhalation.

G. Optional cell culture medium components

1. Anti-CD3 antibody

In some embodiments, the culture medium used in the amplification methods described herein (including those referred to as REP, see, for example, Figure A) also contains anti-CD3 antibodies. The binding of anti-CD3 antibodies to IL-2 induces T cell activation and cell division in the TIL population. This effect can be observed with both full-length antibodies and Fab and F(ab′)2 fragments, with the former generally preferred. See, for example, Tsoukas et al., J. Immunol. 1985, 135, 1719, the entire contents of which are incorporated herein by reference.

As those skilled in the art will understand, there are many suitable anti-human CD3 antibodies that can be used in this invention, including polyclonal and monoclonal antibodies against human CD3 from various mammals, including but not limited to antibodies against murine, human, primate, rat, and canine animals. In a specific embodiment, OKT3 anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Aubum, CA) is used.

2,4-1BB (CD137) agonist

In one embodiment, the TNFRSF agonist is a 4-1BB (CD137) agonist. The 4-1BB agonist can be any 4-1BB binding molecule known in the art. The 4-1BB binding molecule can be a monoclonal antibody or fusion protein capable of binding human or mammalian 4-1BB. The 4-1BB agonist or 4-1BB binding molecule can comprise any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG3, IgG4, IgA1, and IgA2), or any subclass of immunoglobulin heavy chain. The 4-1BB agonist or 4-1BB binding molecule can 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 antibodies, humanized antibodies, or chimeric antibodies, as well as antibody fragments (e.g., Fab fragments, F(ab') fragments, fragments generated from Fab expression libraries, epitope-binding fragments of any of the above), and engineered forms of antibodies (e.g., scFv molecules). In one embodiment, the 4-1BB agonist is an antigen-binding protein that is a fully human antibody. In one embodiment, the 4-1BB agonist is an antigen-binding protein that is a humanized antibody. In some embodiments, the 4-1BB agonist used in the methods and compositions of this disclosure includes: anti-4-1BB antibodies, human anti-4-1BB antibodies, mouse anti-4-1BB antibodies, mammalian anti-4-1BB antibodies, monoclonal anti-4-1BB antibodies, polyclonal anti-4-1BB antibodies, chimeric anti-4-1BB antibodies, anti-4-1BB fibronectin, anti-4-1BB domain antibodies, single-chain anti-4-1BB fragments, heavy-chain anti-4-1BB fragments, light-chain anti-4-1BB fragments, anti-4-1BB fusion proteins, and fragments, derivatives, conjugates, variants, or bioanalytes thereof. Agonisttic anti-4-1BB antibodies are known to induce strong immune responses. (Lee et al., PLOS One 2013, 8, e69677). In a preferred embodiment, the 4-1BB agonist is an agonisttic anti-4-1BB humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line). In one embodiment, the 4-1BB agonist is EU-101 (Eutilex Co. Ltd.), utolumab or urogenumab or a fragment, derivative, conjugate, variant or biosimilar thereof. In a preferred embodiment, the 4-1BB agonist is utolumab or urogenumab or a fragment, derivative, conjugate, variant or biosimilar thereof.

In a preferred embodiment, the 4-1BB agonist or 4-1BB binding molecule may also be a fusion protein. In a preferred embodiment, compared to agonistic monoclonal antibodies (which typically have two ligand-binding domains), multimeric 4-1BB agonists (e.g., trimer or hexamer 4-1BB agonists having three or six ligand-binding domains) can induce superior receptor (4-1BBL) aggregation and the formation of internal cell signaling complexes. Fusion proteins comprising three TNFRSF binding domains and IgG1-Fc, and optionally further linked to two or more of these fusion proteins, in trimeric (trivalent) or hexamer (or hexavalent) or larger forms, are described, for example, in Gieffers et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

It is known that agonistic 4-1BB antibodies and fusion proteins induce strong immune responses. In a preferred embodiment, the 4-1BB agonist is a monoclonal antibody or fusion protein that specifically binds to the 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 eliminates antibody-dependent cytotoxicity (ADCC) (e.g., NK cell cytotoxicity). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates antibody-dependent phagocytosis (ADCP). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates complement-dependent cytotoxicity (CDC). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that eliminates Fc region functionality.

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 one embodiment, the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO: 9). In one embodiment, the 4-1BB agonist is a binding molecule that binds to mouse 4-1BB (SEQ ID NO: 10). Table 3 summarizes the amino acid sequences of the 4-1BB antigen bound to the 4-1BB agonist or binding molecule.

Table 3: Amino acid sequence of 4-1BB antigen

In some embodiments, the compositions, processes, and methods include a 4-1BB agonist that binds to human or mouse 4-1BB with a _KD of less than 100 pM, a _KD of less than 90 pM, a _KD of less than 80 pM, _{a KD of} less than 70 pM, a _KD of less than 60 pM, a _KD of less than 50 pM, a KD of less than 40 pM, or _{a KD of} less than 30 pM.

In some embodiments, the compositions, processes, and methods include 4-1BB agonists binding to human or mouse 4-1BB with a _kassoc of about 7.5 × ^10⁵ 1/M·s or more, _kassoc of about 7.5 × ^10⁵ 1/M·s or more, _kassoc of about 8 × ^10⁵ 1/M·s or more, _kassoc of about 8.5 × ^10⁵ 1/M·s or more, kassoc of about 9 × ^10⁵ 1/M·s or more, or _kassoc of about 1 × ^{10⁶ 1} /M· _s or more _. 4-1BB agonists that bind to human or mouse 4-1BB at a rate of 1/M·s or higher.

In some embodiments, the compositions, processes, and methods include 4-1BB agonists binding to human or mouse 4-1BB with _{a kdissoc} of less than about 2 × ^10⁻⁵ lb/s, _kdissoc of less than 2.1 × ^10⁻⁵ lb/s, _kdissoc of less than 2.2 × 10⁻⁵ lb/s, _kdissoc of less than 2.3 × ^10⁻⁵ lb/s, _kdissoc of less than 2.4 × ^10⁻⁵ lb/s, _kdissoc of less than 2.5 × ^10⁻⁵ lb/s, and _kdissoc of less than 2.6 × ^10⁻⁵ lb/ ^s. 4-1BB agonists that bind to human or mouse 4-1BB at a rate of less than 1/s ^{, 4-1BB agonists that bind to human or mouse 4-1BB at a rate of less than 2.7 × 10⁻⁵} ₁ ^/ s, _4-1BB agonists that bind to human or mouse 4-1BB at a rate of less than 2.8 × ^10⁻⁵ 1/s, 4-1BB agonists that _bind to human or mouse 4-1BB at a rate of less than 2.9 × ^{10⁻⁵ 1/s, or 4-1BB agonists that bind to human or mouse 4-1BB at a rate of less than 3 × 10⁻⁵} ₁ /s.

In some embodiments, the compositions, processes, and methods include _IC50 of less than 10 nM of a 4-1BB agonist binding to human or mouse 4-1BB, _IC50 of less than 9 nM of a 4-1BB agonist binding to human or mouse 4-1BB, _IC50 of less than 8 nM of a 4-1BB agonist binding to human or mouse 4-1BB, _IC50 of less than 7 nM of a 4-1BB agonist binding to human or mouse 4-1BB, _IC50 of less than 6 nM of a 4-1BB agonist binding to human or mouse 4-1BB, _IC50 of less than 5 nM of a 4-1BB agonist binding to human or mouse 4-1BB, _IC50 of less than 4 nM of a 4-1BB agonist binding to human or mouse 4-1BB, _IC50 of less than 3 nM of a 4-1BB agonist binding to human or mouse 4-1BB, IC50, etc. ₅₀ is a 4-1BB agonist of human or mouse 4-1BB at a concentration of less than 2 nM or _IC50 is a 4-1BB agonist of human or mouse 4-1BB at a concentration of less than 1 nM.

In a preferred embodiment, the 4-1BB agonist is utolumab (also known as PF-05082566 or MOR-7480) or a fragment, derivative, variant, or biosimilar thereof. Utolumab is commercially available from Pfizer, Inc. Utolumab is an immunoglobulin G2-λ, 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 utolumab is shown in Table 4. Utolumab contains: glycosylation sites at Asn59 and Asn292; intrachain disulfide bonds in the heavy chain at positions 22-96 ( _VH - _VL ), 143-199 ( _CH1 - _CL ), 256-316 ( _CH2 ), and 362-420 ( _CH3 ); and intrachain disulfide bonds at positions 22'-87' ( _VH - _VL ) and 136'-195' ( _CH1 -CL _). Intrachain disulfide bonds of the light chain of IgG2A isoforms at positions 218-218, 219-219, 222-222 and 225-225, positions 218-130, 219-219, 222-222 and 225-225, positions 219-130(2), 222-222 and 225-225, and positions 219-130(2), 222-222 and 225-225 of IgG2B isoforms; and interchain heavy chain-light chain disulfide bonds at positions 130-213′(2), 218-213′ and 130-213′ of IgG2A/B isoforms and positions 218-213′(2) of IgG2B isoforms. The preparation and properties of utorumab, its variants, and fragments are described in U.S. Patent Nos. 8,821,867, 8,337,850, and 9,468,678; and International Patent Application Publication No. WO2012/032433A1, the disclosures of which are incorporated herein by reference. The preclinical characteristics of utorumab are described in Fisher et al., Cancer Immunolog. & Immunother. 2012, 61, 1721-33. Current clinical trials of utorumab in various hematologic and solid tumor indications include those identified by US National Institutes of Health clinicaltrials.gov: NCT02444793, NCT01307267, NCT02315066, and NCT02554812.

In one embodiment, the 4-1BB agonist comprises the heavy chain given in SEQ ID NO: 11 and the light chain given in SEQ ID NO: 12. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, or their antigen-binding fragments, Fab fragments, single-chain variable fragments (scFv), variants thereof, or conjugates thereof, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain having at least 99% identity with the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain having at least 98% identity with the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain having at least 97% identity with the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain having at least 96% identity with the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In another embodiment, the 4-1BB agonist comprises a heavy chain and a light chain having at least 95% identity with the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

In one embodiment, the 4-1BB agonist comprises a heavy chain CDR and a light chain CDR or variable region (VR) of utolumab. In one embodiment, the 4-1BB agonist heavy chain variable region ( _VR ) comprises the sequence shown in SEQ ID NO: 13, and the 4-1BB agonist light chain variable region ( _VR ) comprises the sequence shown in SEQ ID NO: 14, and their conserved amino acid substitutions. In one embodiment, the 4-1BB agonist comprises _VR and _VR regions having at least 99% identity with the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In one embodiment, the 4-1BB agonist comprises _VR and _VR regions having at least 98% identity with the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In one embodiment, the 4-1BB agonist comprises _VR and _VR regions having at least 97% identity with the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In one embodiment, the 4-1BB agonist comprises _VH and _VL regions having at least 96% identity with the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In one embodiment, the 4-1BB agonist comprises _VH and _VL regions having at least 95% identity with the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In one embodiment, the 4-1BB agonist comprises an scFv antibody having _VH and _VL regions having at least 99% identity with the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

In one embodiment, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 and their conserved amino acid substitutions, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 and their conserved amino acid substitutions, respectively.

In one embodiment, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by a drug regulatory agency with reference to utoluma. In one embodiment, the biosimilar monoclonal antibody comprises a 4-1BB antibody containing an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) with respect to an amino acid sequence of a reference drug product or reference biological product, and containing one or more post-translational modifications compared to the reference drug product or reference biological product, wherein the reference drug product or reference biological product is utoluma. 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 biosimilar is an authorized or submitted-for-authorization 4-1BB agonist antibody, wherein the 4-1BB agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference drug product or reference biological product is utoluma. The 4-1BB agonist antibody may be authorized by a drug regulatory agency (e.g., the US FDA and/or the EU EMA). In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is utolumab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is utolumab.

Table 4: Amino acid sequences of 4-1BB agonist antibodies associated with utolumab

In a preferred embodiment, the 4-1BB agonist is the monoclonal antibody urogenumab (also known as BMS-663513 and 20H4.9.h4a) or a fragment, derivative, variant, or biosimilar thereof. Urigumab is commercially available from Bristol-Myers Squibb, Inc. and Creative Biolabs, Inc. Urigumab is an immunoglobulin G4-κ, 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 urogenumab is listed in Table 5. Urelimab contains: N-glycosylation sites at positions 298 (and 298'); intrachain disulfide bonds in the heavy chain at positions 22–95 ( _VH – _VL ), 148–204 ( _CH1 – _CL ), 262–322 ( _CH2 ), and 368–426 ( _CH3 ) (as well as at positions 22”–95”, 148”–204”, 262”–322”, and 368”–426”); and intrachain disulfide bonds in the heavy chain at positions 23”–88” ( _VH – _VL ) and 136”–196” ( _CH1 –CL _). (and intrachain disulfide bridges at positions 23"'-88"' and 136"'-196"'; interchain heavy chain-heavy chain disulfide bonds at positions 227-227" and 230-230"; and interchain heavy chain-light chain disulfide bonds at positions 135-216" and 135"-216". The preparation and properties of urogenumab and its variants and fragments are described in U.S. Patent Nos. 7,288,638 and 8,962,804, the disclosures of which are incorporated herein by reference. The preclinical and clinical characteristics of urogenumab are described in Segal et al., Clin. Cancer Res. 2016, available at http://dx.doi.org/10.1158/1078-0432.CCR-16-1272. Current clinical trials of urogenumab in various hematologic and solid tumor indications include those at the U.S. National Institutes of Health. The identifiers from clinicaltrials.gov are NCT01775631, NCT02110082, NCT02253992, and NCT01471210.

In one embodiment, the 4-1BB agonist comprises the heavy chain given in SEQ ID NO: 21 and the light chain given in SEQ ID NO: 22. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, or their antigen-binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain having at least 99% identity with the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain having at least 98% identity with the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain having at least 97% identity with the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In one embodiment, the 4-1BB agonist comprises a heavy chain and a light chain having at least 96% identity with the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In another embodiment, the 4-1BB agonist comprises a heavy chain and a light chain having at least 95% identity with the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively.

In one embodiment, the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of urilumab. In one embodiment, the 4-1BB agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO: 23, and the 4-1BB agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO: 24, and its conserved amino acid substitutions. In one embodiment, the 4-1BB agonist comprises _VH and VL regions having at least 99% identity with the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. In one embodiment, the 4-1BB agonist comprises VH and _VL regions having at least 98% identity with the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. In one embodiment, the 4-1BB agonist comprises _VH and _{VL regions} having at least 97% identity with the sequences shown in SEQ ID NO: 23 and SEQ ID _NO : 24, respectively. In one embodiment, the 4-1BB agonist comprises _VH and _VL regions having at least 96% identity with the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. In one embodiment, the 4-1BB agonist comprises _VH and _VL regions having at least 95% identity with the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively. In one embodiment, the 4-1BB agonist comprises an scFv antibody having _VH and _VL regions having at least 99% identity with the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

In one embodiment, the 4-1BB agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively, and their conserved amino acid substitutions, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively, and their conserved amino acid substitutions.

In one embodiment, the 4-1BB agonist is a biosimilar monoclonal antibody of a 4-1BB agonist approved by a regulatory agency with reference to uregluzumab. In one embodiment, the biosimilar monoclonal antibody comprises a 4-1BB antibody containing an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) with a reference pharmaceutical product or reference biological product and containing one or more post-translational modifications compared to the reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is uregluzumab. 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 biosimilar is an authorized or submitted-for-authorization 4-1BB agonist antibody formulation that differs from a reference pharmaceutical product or reference biological product formulation, wherein the reference pharmaceutical product or reference biological product is uregluzumab. The 4-1BB agonist antibody may be authorized by a regulatory agency (e.g., the US FDA and/or the EU EMA). In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is uroreglumab.

Table 5: Amino acid sequences of 4-1BB agonist antibodies associated with urilumab

In one embodiment, the 4-1BB agonist is selected from: 1D8, 3Elor, 4B4 (BioLegend 309809), H4-1BB-M127 (BD Pharmingen 552532), BBK2 (Thermo Fisher MS621PABX), 145501 (Leinco Technologies B591), and the anti-4-1BB agonist produced by a cell line deposited at ATCC No. HB-11248 and disclosed in US Patent No. 6,974,863. Antibodies disclosed in US Patent Application Publication No. US2005/0095244, 5F4 (BioLegend 31 1503), C65-485 (BD Pharmingen 559446), US Patent No. 7,288,638 (e.g., 20H4.9-IgG1 (BMS-663031)), US Patent No. 6,887,673 (e.g., 4E9 or BMS-554271), and US Patent No. 7,214,493 Antibodies disclosed in U.S. Patent No. 6,303,121, U.S. Patent No. 6,569,997, U.S. Patent No. 6,905,685 (e.g., 4E9 or BMS-554271), U.S. Patent No. 6,362,325 (e.g., 1D8 or BMS-469492; 3H3 or BMS-469497; or 3E1), U.S. Patent No. 6,974,863 (e.g., 53A2), and U.S. Patent No. 6,210,669. (e.g., 1D8, 3B8, or 3E1), antibodies described in U.S. Patent 5,928,893, antibodies disclosed in U.S. Patent 6,303,121, antibodies disclosed in U.S. Patent 6,569,997, antibodies disclosed in International Patent Application Publication Nos. WO2012/177788, WO2015/119923, and WO2010/042433, and fragments, derivatives, conjugates, variants, or bioanalytes thereof, wherein the respective disclosures in the aforementioned patents or patent application publications are incorporated herein by reference in their entirety.

In one embodiment, the 4-1BB agonist is a 4-1BB agonist fusion protein described in International Patent Application Publications WO2008/025516A1, WO2009/007120A1, WO2010/003766A1, WO2010/010051A1, and WO2010/078966A1; U.S. Patent Application Publications US2011/0027218A1, US2015/0126709A1, US2011/0111494A1, US2015/0110734A1, and US2015/0126710A1; and U.S. Patents 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference.

In one embodiment, the 4-1BB agonist is a 4-1BB agonist fusion protein as described 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 bioanalyte thereof:

In structures IA and IB, the cylinder refers to a single polypeptide-binding domain. Structures IA and IB contain three linearly linked TNFRSF-binding domains derived from, for example, antibodies binding to 4-1BBL or 4-1BB. These domains fold to form a trivalent protein, which is then linked to a second trivalent protein via IgG1-Fc (including _CH3 and _CH2 domains). The two trivalent proteins are then linked together via disulfide bonds (small, elongated ellipses), thereby stabilizing the structure and providing an agonist capable of binding the intracellular signaling domains and signaling proteins of six receptors to form a signaling complex. The TNFRSF-binding domain (represented as a cylinder) can be an scFv domain, which includes, for example, _VH and _VL chains linked by a linker. This linker may contain hydrophilic residues and Gly and Ser sequences for flexibility, and Glu and Lys for solubility. Any scFv domain can be used in the design, such as those described in de Marco, Microbial Cell Factories, 2011, 10, 44; Ahmad et al., Clin. & Dev. Immunol. 2012, 980250; Monnier et al., Antibodies, 2013, 2, 193-208; and those elsewhere incorporated herein by reference. The structure of this form of fusion protein is 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.

Table 6 provides the amino acid sequences of the other polypeptide domains of structures I-A. The Fc domain preferably comprises a complete constant domain (amino acids 17-230 of SEQ ID NO: 31), a complete hinge domain (amino acids 1-16 of SEQ ID NO: 31), or a portion of a hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 31). Preferred adapters for linking C-terminal Fc antibodies may be selected from the embodiments given in SEQ ID NO: 32 to SEQ ID NO: 41, including adapters suitable for fusing other polypeptides.

Table 6: Amino acid sequences of TNFRSF fusion proteins (including 4-1BB fusion proteins) designed with C-terminal Fc-antibody fragments (structural I-A)

Table 7 shows the amino acid sequences of the other polypeptide domains of structures I-B. If the Fc antibody fragment is fused to the N-terminus of the TNRFSF fusion protein (as in structures I-B), the sequence of the Fc module is preferably the sequence shown in SED ID NO: 42, and the adapter sequence is preferably selected from those embodiments shown in SED ID NO: 43 to SEQ ID NO: 45.

Table 7: Amino acid sequences of TNFRSF fusion proteins (including 4-1BB fusion proteins) designed with N-terminal Fc antibody fragments (structures I-B)

In one embodiment, the 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: variable heavy and variable light chains of urinumab, variable heavy and variable light chains of urinumab, variable heavy and variable light chains of urinumab, variable heavy and variable light chains selected from the variable heavy and variable light chains described in Table 8, any combination of the aforementioned variable heavy and variable chains, and fragments, derivatives, conjugates, variants and bioanalysts thereof.

In one embodiment, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a 4-1BBL sequence. In one embodiment, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a sequence according to SEQ ID NO: 46. In one embodiment, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a soluble 4-1BBL sequence. In one embodiment, the 4-1BB agonist fusion protein according to structure I-A or I-B comprises one or more 4-1BB binding domains containing a sequence according to SEQ ID NO: 47.

In one embodiment, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains, which are scFv domains comprising FH and VL regions, the _VH and _VL regions having at least 95% identity with the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively, wherein the _VH and _VL domains are connected by a linker. In another embodiment, the 4-1BB agonist fusion protein according to structure IA or IB comprises one or more 4-1BB binding domains, which are scFv domains comprising _VH and _VL regions, the _VH and _VL regions having at least 95% identity with the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively, wherein the _VH and _VL domains are connected by a linker. In one embodiment, the 4-1BB agonist fusion protein according to structure IA or IB includes one or more 4-1BB binding domains, which are scFv domains containing _VH and _VL regions that have at least 95% identity with _{the VH and VL} sequences given in Table 8 _, respectively, wherein the _VH and _VL domains are connected by a linker.

Table 8: Other peptide domains used as 4-1BB binding domains in fusion proteins or as scFv 4-1BB agonist antibodies

In one embodiment, 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, and further comprising other domains located at the N-terminus and/or C-terminus, wherein such other domains are Fab or Fc fragment domains. In one embodiment, 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 soluble 4-1BB domain lacks a stalk region (which facilitates trimerization and provides a certain distance from 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 to 8 amino acids.

In one 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 soluble TNF superfamily cytokine domain lacks a stem region, the first and second peptide linkers independently have a length of 3 to 8 amino acids, and each TNF superfamily cytokine domain is a 4-1BB binding domain.

In one embodiment, the 4-1BB agonist is a 4-1BB agonist scFv antibody that contains any of the aforementioned _VH domains linked to any of the aforementioned _VL domains.

In one embodiment, the 4-1BB agonist is the 4-1BB agonist antibody of catalog number 79097-2 from BPS Bioscience, commercially available from BPS Bioscience (San Diego, CA, USA). In another embodiment, the 4-1BB agonist is the 4-1BB agonist antibody of catalog number MOM-18179 from Creative Biolabs, commercially available from Creative Biolabs (Shirley, NY, USA).

3. OX40 (CD134) agonist

In one embodiment, 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 human or mammalian OX40. The OX40 agonist or OX40-binding molecule can comprise any isotype (e.g., IgG1, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass of immunoglobulin heavy chain. The OX40 agonist or OX40-binding molecule can 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 antibodies, humanized antibodies, or chimeric antibodies, as well as antibody fragments (e.g., Fab fragments, F(ab') fragments, fragments generated from Fab expression libraries, epitope-binding fragments of any of the above), and engineered forms of antibodies (e.g., scFv molecules). In one embodiment, the OX40 agonist is an antigen-binding protein that is a fully human antibody. In one embodiment, the OX40 agonist is an antigen-binding protein that is a humanized antibody. In some embodiments, the OX40 agonist used in the methods and compositions of this disclosure includes: anti-OX40 antibodies, human anti-OX40 antibodies, mouse anti-OX40 antibodies, mammalian anti-OX40 antibodies, monoclonal anti-OX40 antibodies, polyclonal anti-OX40 antibodies, chimeric anti-OX40 antibodies, anti-OX40 fibronectin, anti-OX40 domain antibodies, single-chain anti-OX40 fragments, heavy-chain anti-OX40 fragments, light-chain anti-OX40 fragments, anti-OX40 fusion proteins, and fragments, derivatives, conjugates, variants, or bioanalytes thereof. In a preferred embodiment, the OX40 agonist is an agonistic anti-OX40 humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line).

In a preferred embodiment, 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, by Sadun et al., J. Immunother. J. Immunol. 2009, 182, 1481-89. In a preferred embodiment, multimeric OX40 agonists (e.g., trimers or hexamers of OX40 agonists having three or six ligand-binding domains) induce superior receptor (OX40L) clustering and formation of internal cell signaling complexes, compared to agonistic monoclonal antibodies (which typically have two ligand-binding domains). Trimers (trivalent) or hexamers (or hexamers) or larger fusion proteins comprising three TNFRSF binding domains and IgG1-Fc and optionally further linked with two or more of these fusion proteins are described, for example, by Gieffers et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.

It is known that agonistic OX40 antibodies and fusion proteins induce strong immune responses. (Curti et al., Cancer Res. 2013, 73, 7189-98). In a preferred embodiment, the OX40 agonist is a monoclonal antibody or fusion protein that specifically binds to the OX40 antigen in a manner sufficient to reduce toxicity. In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates antibody-dependent cytotoxicity (ADCC) (e.g., NK cell cytotoxicity). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates antibody-dependent phagocytosis (ADCP). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates complement-dependent cytotoxicity (CDC). In some embodiments, the OX40 agonist is an agonistic OX40 monoclonal antibody or fusion protein that eliminates Fc region functionality.

In some embodiments, the OX40 agonist is characterized by binding to human OX40 (SEQ ID NO: 54) with high affinity and agonistic activity. In one embodiment, the OX40 agonist is a binding molecule that binds to human OX40 (SEQ ID NO: 54). In one embodiment, the OX40 agonist is a binding molecule that binds to mouse OX40 (SEQ ID NO: 55). Table 9 summarizes the amino acid sequences of the OX40 antigen bound to the OX40 agonist or binding molecule.

Table 9: Amino acid sequence of OX40 antigen

In some embodiments, the compositions, processes, and methods include an OX40 agonist that binds to human or mouse OX40 with a _KD of less than 100 pM, a _KD of less than 90 pM, a KD of less than 80 pM, a _KD of less than 70 pM, a _KD of less than 60 pM, _{a KD} of less than 50 pM, a _KD of less than 40 pM, or a _KD of less than 30 pM.

In some embodiments, the compositions, processes, and methods include OX40 agonists that bind to human or mouse OX40 with a _kassoc of about 7.5 × ^10⁵ 1/M·s or more, _kassoc of about 8 × ^10⁵ 1/M·s or more, _kassoc of about 8.5 × ^10⁵ 1/M·s or more, kassoc of about 9 × ^{10⁵ 1} /M·s or more, _kassoc of about 9.5 × 10⁵ 1/M·s _{or more, or kassoc of} about 1 × ^10⁶ 1/M·s or more.

In some embodiments, the compositions, processes, and methods include OX40 agonists that bind to human or mouse OX40 at _{a kdissoc ratio} of less than about 2 × ^10⁻⁵ 1/s, _kdissoc at a kdissoc ratio of less than about 2.1 × ^10⁻⁵ 1/s, _kdissoc at a kdissoc ratio of less than about 2.2 × ^10⁻⁵ 1/s, _kdissoc at a kdissoc ratio of less than about 2.3 × ^10⁻⁵ 1/s, _kdissoc at a kdissoc ratio of less than about 2.4 × ^10⁻⁵ 1/s, _kdissoc at a kdissoc ratio of less than about 2.5 × ^10⁻⁵ 1/s, and _kdissoc at a kdissoc ratio of less than 2.6 × ^10⁻⁵ 1/s. OX40 agonists that bind to human or mouse OX40 at a rate of less than 1/s, OX40 _agonists that bind to human or mouse OX40 at ^{a rate of less than 2.7 × 10⁻⁵ 1} /s, OX40 _agonists that bind to human or mouse OX40 at a rate of less than 2.8 × ^10⁻⁵ 1/s, OX40 agonists that _bind to human or mouse OX40 at a rate of less than 2.9 × ^10⁻⁵ 1/s, or OX40 agonists that _bind to human or mouse OX40 at a rate of less than 3 × 10⁻⁵ 1/s.

In some embodiments, the compositions, processes, and methods include an _IC50 of less than 10 nM of a human or mouse OX40 agonist, an _IC50 of less than 9 nM of a human or mouse OX40 agonist, an _IC50 of less than 8 nM of a human or mouse OX40 agonist, an _IC50 of less than 7 nM of a human or mouse OX40 agonist, an _IC50 of less than 6 nM of a human or mouse OX40 agonist, an _IC50 of less than 5 nM of a human or mouse OX40 agonist, an _IC50 of less than 4 nM of a human or mouse OX40 agonist, an _IC50 of less than 3 nM of a human or mouse OX40 agonist, an _IC50 of less than 2 nM of a human or mouse OX40 agonist, or an IC50 agonist. ₅₀ is an OX40 agonist that binds to human or mouse OX40 at a concentration of less than 1 nM.

In some embodiments, the OX40 agonist is taliximab (also known as MEDI0562 or MEDI-0562). Taliximab is commercially available from MedImmune, a subsidiary of AstraZeneca, Inc. Taliximab is an immunoglobulin G1-kappa, anti-[Homo sapiens TNFRSF4 (tumor necrosis factor receptor (TNFR) superfamily member 4, OX40, CD134)], humanized, and chimeric monoclonal antibody. The amino acid sequence of taliximab is listed in Table 10. Talixizumab comprises: a complex biantennary CHO-type polysaccharide with fucosylation at N-glycosylation sites at positions 301 and 301"; intrachain disulfide bonds in the heavy chain at positions 22-95 ( _VH - _VL ), 148-204 ( _CH1 - _CL ), 265-325 ( _CH2 ), and 371-429 ( _CH3 ) (as well as at positions 22"-95", 148"-204", 265"-325", and 371"-429); and intrachain disulfide bonds at positions 23"-88" ( _VH - _VL ) and 134"-194" ( _CH1 -CL _). (and positions 23"'-88"' and 134"'-194"') intra-light chain disulfide bonds; inter-chain heavy chain-heavy chain disulfide bonds at positions 230-230' and 233-233"'; and inter-chain heavy chain-light chain disulfide bonds at positions 224-214' and 224"'. Current clinical trials of taliximab in various solid tumor indications include US National Institutes of Health clinicaltrials.gov (identification codes NCT02318394 and NCT02705482).

In one embodiment, the OX40 agonist comprises a heavy chain as given in SEQ ID NO: 56 and a light chain as given in SEQ ID NO: 57. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO: 56 and SEQ ID NO: 57, or their antigen-binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates, respectively. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain having at least 99% identity with the sequences shown in SEQ ID NO: 56 and SEQ ID NO: 57, respectively. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain having at least 98% identity with the sequences shown in SEQ ID NO: 56 and SEQ ID NO: 57, respectively. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain having at least 97% identity with the sequences shown in SEQ ID NO: 56 and SEQ ID NO: 57, respectively. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain having at least 96% identity with the sequences shown in SEQ ID NO: 56 and SEQ ID NO: 57, respectively. In another embodiment, the OX40 agonist comprises a heavy chain and a light chain having at least 95% identity with the sequences shown in SEQ ID NO: 56 and SEQ ID NO: 57, respectively.

In one embodiment, the OX40 agonist comprises the heavy chain CDR and light chain CDR or variable region (VR) of talixizumab. In one embodiment, the OX40 agonist heavy chain variable region ( _VR ) comprises the sequence shown in SEQ ID NO: 58 and its conserved amino acid substitutions, and the OX40 agonist light chain variable region ( _VR ) comprises the sequence shown in SEQ ID NO: 59 and its conserved amino acid substitutions. In one embodiment, the OX40 agonist comprises _VR and _VR regions having at least 99% identity with the sequences shown in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. In one embodiment, the OX40 agonist comprises _VR and _VR regions having at least 98% identity with the sequences shown in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. In one embodiment, the OX40 agonist comprises _VR and _VR regions having at least 97% identity with the sequences shown in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. In one embodiment, the OX40 agonist comprises _VH and _VL regions having at least 96% identity with the sequences shown in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. In one embodiment, the OX40 agonist comprises _VH and _VL regions having at least 95% identity with the sequences shown in SEQ ID NO: 58 and SEQ ID NO: 59, respectively. In one embodiment, the OX40 agonist comprises an scFv antibody having _VH and _VL regions having at least 99% identity with the sequences shown in SEQ ID NO: 58 and SEQ ID NO: 59, respectively.

In one embodiment, the OX40 agonist comprises: heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62 and their conserved amino acid substitutions, respectively; and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65 and their conserved amino acid substitutions, respectively.

In one embodiment, the OX40 agonist is a biosimilar monoclonal antibody of an OXA40 agonist approved by a regulatory agency with reference to taliximab. In one embodiment, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) with an amino acid sequence having one or more post-translational modifications compared to the reference drug product or reference biologic product, wherein the reference drug product or reference biologic product is taliximab. 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 biosimilar is an authorized or submitted-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biologic product, wherein the reference drug product or reference biologic product is taliximab. The OX40 agonist antibody may be authorized by a regulatory agency (e.g., the US FDA and/or the EU EMA). In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is taliximab. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is taliximab.

Table 10: Amino acid sequences of OX40 agonist antibodies associated with talixizumab

In some embodiments, the OX40 agonist is 11D4, a fully human antibody commercially available from Pfizer, Inc. The preparation and properties of 11D4 are described in U.S. Patent Nos. 7,960,515, 8,236,930, and 9,028,824, the disclosures of which are incorporated herein by reference. Table 11 lists the amino acid sequence of 11D4.

In one embodiment, the OX40 agonist comprises a heavy chain as given in SEQ ID NO: 66 and a light chain as given in SEQ ID NO: 67. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO: 66 and SEQ ID NO: 67, or their antigen-binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates, respectively. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain having at least 99% sequence identity with the sequences shown in SEQ ID NO: 66 and SEQ ID NO: 67, respectively. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain having at least 98% sequence identity with the sequences shown in SEQ ID NO: 66 and SEQ ID NO: 67, respectively. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain having at least 97% sequence identity with the sequences shown in SEQ ID NO: 66 and SEQ ID NO: 67, respectively. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain having at least 96% sequence identity with the sequences shown in SEQ ID NO: 66 and SEQ ID NO: 67, respectively. In another embodiment, the OX40 agonist comprises a heavy chain and a light chain having at least 95% sequence identity with the sequences shown in SEQ ID NO: 66 and SEQ ID NO: 67, respectively.

In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of 11D4. In one embodiment, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO: 68, and the OX40 agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO: 69, and its conserved amino acid substitutions. In one embodiment, the OX40 agonist comprises _VH and VL regions having at least 99% identity with the sequences shown in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. In one embodiment, the OX40 agonist comprises VH and _VL regions having at least 98% identity with the sequences shown in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. In one embodiment, the OX40 agonist comprises _VH and _VL regions having at least 97% identity with the sequences shown in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. In one embodiment, the OX40 agonist comprises _VH and _VL regions having at least 96% identity with the sequences shown in _SEQ ID NO: 68 and _SEQ ID NO: 69, respectively. In one embodiment, the OX40 agonist comprises _VH and _VL regions having at least 95% identity with the sequences shown in SEQ ID NO: 68 and SEQ ID NO: 69, respectively.

In one embodiment, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72 and their conserved amino acid substitutions, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75 and their conserved amino acid substitutions, respectively.

In one embodiment, the OX40 agonist is a biosimilar monoclonal antibody approved by a drug regulatory agency with reference to 11D4. In one embodiment, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) with an amino acid sequence that is 11D4 and contains one or more post-translational modifications compared to the reference drug product or reference biological product. In some embodiments, the post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or submitted-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 11D4. The OX40 agonist antibody may be authorized by a drug regulatory agency (e.g., the US FDA and/or the EU EMA). In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is 11D4. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is 11D4.

Table 11: Amino acid sequences of OX40 agonist antibodies associated with 11D4

In some embodiments, the OX40 agonist is 18D8, a fully human antibody commercially available from Pfizer, Inc. The preparation and properties of 18D8 are described in U.S. Patent Nos. 7,960,515, 8,236,930, and 9,028,824, the disclosures of which are incorporated herein by reference. Table 12 lists the amino acid sequence of 18D8.

In one embodiment, the OX40 agonist comprises a heavy chain as given in SEQ ID NO: 76 and a light chain as given in SEQ ID NO: 77. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO: 76 and SEQ ID NO: 77, or their antigen-binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates, respectively. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain having at least 99% identity with the sequences shown in SEQ ID NO: 76 and SEQ ID NO: 77, respectively. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain having at least 98% identity with the sequences shown in SEQ ID NO: 76 and SEQ ID NO: 77, respectively. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain having at least 97% identity with the sequences shown in SEQ ID NO: 76 and SEQ ID NO: 77, respectively. In one embodiment, the OX40 agonist comprises a heavy chain and a light chain having at least 96% identity with the sequences shown in SEQ ID NO: 76 and SEQ ID NO: 77, respectively. In another embodiment, the OX40 agonist comprises a heavy chain and a light chain having at least 95% identity with the sequences shown in SEQ ID NO: 76 and SEQ ID NO: 77, respectively.

In one embodiment, the OX40 agonist comprises a heavy chain and a light chain CDR or variable region (VR) of 18D8. In one embodiment, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO: 78, and the OX40 agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO: 79, and its conserved amino acid substitutions. In one embodiment, the OX40 agonist comprises _VH and _VL regions having at least 99% identity with the sequences shown in SEQ ID NO: 78 and SEQ ID NO: 79, respectively. In one embodiment, the OX40 agonist comprises VH and _VL regions having at least 98% identity with the sequences shown in SEQ ID NO: 78 and SEQ ID NO: 79, respectively. In one embodiment, the OX40 agonist comprises _VH and VL regions having at least 97% identity with the sequences shown in SEQ ID NO: 78 and SEQ ID NO: 79, respectively. In one embodiment, the OX40 agonist comprises _VH and _VL regions having at least 96% identity with the sequences shown in SEQ ID NO: 78 and SEQ ID NO: 79 _{, respectively} . In one embodiment, the OX40 agonist comprises _VH and _VL regions having at least 95% identity with the sequences shown in SEQ ID NO: 78 and SEQ ID NO: 79, respectively.

In one embodiment, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82 and their conserved amino acid substitutions, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85 and their conserved amino acid substitutions, respectively.

In one embodiment, the OX40 agonist is a biosimilar monoclonal antibody approved by a drug regulatory agency with reference to 18D8. In one embodiment, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) with an amino acid sequence that is 18D8 and contains one or more post-translational modifications compared to the reference drug product or reference biological product. In some embodiments, the post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or submitted-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference drug product or reference biological product, wherein the reference drug product or reference biological product is 18D8. The OX40 agonist antibody may be authorized by a drug regulatory agency (e.g., the US FDA and/or the EU EMA). In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is 18D8. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is 18D8.

Table 12: Amino acid sequences of OX40 agonist antibodies associated with 18D8

In some embodiments, the OX40 agonist is Hu119-122, a commercially available humanized antibody 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. WO2012/027328, the disclosures of which are incorporated herein by reference. Table 13 lists the amino acid sequence of Hu119-122.

In one embodiment, the OX40 agonist comprises the heavy chain CDR and light chain CDR or variable region (VR) of Hu119-122. In one embodiment, the heavy chain variable region ( _VH ) of the OX40 agonist comprises the sequence shown in SEQ ID NO: 86, and the light chain variable region ( _VL ) of the OX40 agonist comprises the sequence shown in SEQ ID NO: 87 and its conserved amino acid substitutions. In one embodiment, the OX40 agonist comprises _VH and _{VL regions having at least 99% identity with the sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. In one embodiment, the OX40 agonist comprises VH and VL regions having} at least 98% identity with the sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. In one embodiment, the OX40 agonist comprises _VH and _VL regions having at least 97% identity with the sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. In one embodiment, the OX40 agonist comprises _VH and _VL regions having at least 96% identity with the sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. In another embodiment, the OX40 agonist comprises _VH and _VL regions having at least 95% identity with the sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87, respectively.

In one embodiment, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90 and their conserved amino acid substitutions, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 91, SEQ ID NO: 92, and SEQ ID NO: 93 and their conserved amino acid substitutions, respectively.

In one embodiment, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a regulatory agency with reference to Hu119-122. In one embodiment, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) with an amino acid sequence having one or more post-translational modifications compared to the reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu119-122. 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 biosimilar is an authorized or submitted-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from that of the reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu119-122. OX40 agonist antibodies are available for authorization by regulatory agencies such as the US FDA and/or the EU EMA. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu119-122. In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu119-122.

Table 13: Amino acid sequences of OX40 agonist antibodies associated with Hu119-122

In some embodiments, the OX40 agonist is Hu106-222, a commercially available humanized antibody from GlaxoSmithKline plc. 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. WO2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu106-222 is listed in Table 14.

In one embodiment, the OX40 agonist comprises the heavy chain CDR and light chain CDR or variable region (VR) of Hu106-222. In one embodiment, the OX40 agonist heavy chain variable region ( _VH ) comprises the sequence shown in SEQ ID NO: 94, and the OX40 agonist light chain variable region ( _VL ) comprises the sequence shown in SEQ ID NO: 95, and its conserved amino acid substitutions. In one embodiment, the OX40 agonist comprises _VH and _VL regions having at least 99% identity with the sequences shown in SEQ ID NO: 94 and SEQ ID NO: 95, respectively. In one embodiment, the OX40 agonist comprises VH and _VL regions having at least 98% identity with the sequences shown in SEQ ID NO: 94 and SEQ ID NO: 95 _{, respectively. In one embodiment, the OX40 agonist comprises VH and VL} regions having at least 97% identity with the sequences shown in SEQ ID NO: 94 and SEQ ID NO: 95, respectively. In one embodiment, the OX40 agonist comprises _VH and _VL regions having at least 96% identity with the sequences shown in SEQ ID NO: 94 and SEQ ID NO: 95, respectively. In another embodiment, the OX40 agonist comprises _VH and _VL regions having at least 95% identity with the sequences shown in SEQ ID NO: 94 and SEQ ID NO: 95, respectively.

In one embodiment, the OX40 agonist comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98 and their conserved amino acid substitutions, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101 and their conserved amino acid substitutions, respectively.

In one embodiment, the OX40 agonist is a biosimilar monoclonal antibody of an OX40 agonist approved by a regulatory agency with reference to Hu106-222. In one embodiment, the biosimilar monoclonal antibody comprises an OX40 antibody having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) with an amino acid sequence that is identical to the reference pharmaceutical product or reference biological product and contains one or more post-translational modifications compared to the reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu106-222. 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 biosimilar is an authorized or submitted-for-authorization OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a form different from the reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu106-222. The OX40 agonist antibody may be authorized by a regulatory agency (e.g., the US FDA and/or the EU EMA). In some embodiments, the biosimilar is provided as a composition further comprising one or more excipients, wherein the excipients are the same as or different from those contained in a reference pharmaceutical product or reference biological product, wherein the reference pharmaceutical product or reference biological product is Hu106-222.

Table 14: Amino acid sequences of OX40 agonist antibodies associated with Hu106-222

In some embodiments, the OX40 agonist antibody is MEDI6469 (also known as 9B12). MEDI6469 is a mouse monoclonal antibody. Weinberg et al., J. Immunother. 2006, 29, 575-585. In some embodiments, the OX40 agonist is an antibody produced from a 9B12 hybridoma deposited at Biovest Inc. (Malvern, MA, USA), as described in Weinberg et al., J. Immunother. 2006, 29, 575-585, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the antibody comprises the CDR sequence of MEDI6469. In some embodiments, the antibody comprises the heavy chain variable region sequence and/or the light chain variable region sequence of MEDI6469.

In one embodiment, the OX40 agonist is L106 BD (Pharmingen product #340420). In some embodiments, the OX40 agonist comprises the CDR of antibody L106 (BD Pharmingen product #340420). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody L106 (BD Pharmingen product #340420). In one embodiment, the OX40 agonist is ACT35 (Santa Cruz Biotechnology, catalog number 20073). In some embodiments, the OX40 agonist comprises the CDR of antibody ACT35 (Santa Cruz Biotechnology, 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, catalog number 20073). In one embodiment, the OX40 agonist is a mouse monoclonal antibody anti-mCD134/mOX40 (clone OX86), commercially available from InVivoMAb, BioXcell Inc, West Lebanon, NH.

In one embodiment, the OX40 agonist is selected from international patent application publications WO95/12673, WO95/21925, WO2006/121810, WO2012/027328, WO2013/028231, WO2013/038191 and WO2014/148895; European patent application EP0672141; and US patent application publications US2010/136030 and US2014/377284. The OX40 agonists described in US2015/190506 and US2015/132288 (including clone 20E5 and 12H3); and US 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 one embodiment, the OX40 agonist is an OX40 agonistic fusion protein as described in structures I-A (C-terminal Fc antibody fragment fusion protein) or I-B (N-terminal Fc antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or bioanalyte thereof. The properties of structures I-A and I-B are as described above and 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. Table 6 provides the amino acid sequences of the polypeptide domains of structures I-A. The Fc domain preferably comprises a complete constant domain (amino acids 17-230 of SEQ ID NO: 31), a complete hinge domain (amino acids 1-16 of SEQ ID NO: 31), or a portion of a hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 31). Preferred adapters for linking C-terminal Fc antibodies may be selected from the embodiments given in SEQ ID NO: 32 to SEQ ID NO: 41, including adapters suitable for fusing other peptides. Similarly, the amino acid sequences of the peptide domains of structures I-B are given in Table 7. If the Fc antibody fragment is fused to the N-terminus of a TNRFSF fusion protein (as in structures I-B), the sequence of the Fc module is preferably the sequence shown in SEQ ID NO: 42, and the adapter sequence is preferably selected from those embodiments shown in SEQ ID NO: 43 to SEQ ID NO: 45.

In one embodiment, the 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: variable heavy and variable light chains of talixizumab, variable heavy and variable light chains of 11D4, variable heavy and variable light chains of 18D8, variable heavy and variable light chains of Hu119-122, variable heavy and variable light chains of Hu106-222, variable heavy and variable light chains selected from the variable heavy and variable light chains described in Table 15, any combination of the aforementioned variable heavy and variable light chains, and fragments, derivatives, conjugates, variants and bioanalysts thereof.

In one embodiment, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the OX40L sequence. In one embodiment, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the sequence SEQ ID NO: 102. In one embodiment, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the soluble OX40L sequence. In one embodiment, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the sequence SEQ ID NO: 103. In one embodiment, the OX40 agonist fusion protein according to structure I-A or I-B comprises one or more OX40-binding domains containing the sequence SEQ ID NO: 104.

In one embodiment, the OX40 agonist fusion protein according to structure 1A or 1B comprises one or more OX40-binding domains, which are scFv domains comprising _VH and _VL regions, wherein the _VH and _VL regions have at least 95% identity with the sequences shown in SEQ ID NO: 58 and SEQ ID NO: 59, respectively, wherein the _VH and _VL domains are connected by a linker. In another embodiment, the OX40 agonist fusion protein according to structure 1A or 1B comprises one or more OX40-binding domains, which are scFv domains comprising _FH and _VL regions, wherein the _VH and _VL regions have at least 95% identity with the sequences shown in SEQ ID NO: 68 and SEQ ID NO: 69, respectively, wherein the _VH and _VL domains are connected by a linker. In one embodiment, the OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40-binding domains, which are scFv domains comprising _VH and _VL regions, the _VH and _VL regions having at least 95% identity with the sequences shown in SEQ ID NO: 78 and SEQ ID NO: 79, respectively, wherein the _VH and _VL domains are connected by a linker. In another embodiment, the OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40-binding domains, which are scFv domains comprising _FH and _VL regions, the _VH and _VL regions having at least 95% identity with the sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87, respectively, wherein the _VH and _VL domains are connected by a linker. In one embodiment, the OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40-binding domains, which are scFv domains comprising _VH and _VL regions, the _VH and _VL regions having at least 95% identity with the sequences shown in SEQ ID NO: 94 and SEQ ID NO: 95, respectively, wherein the _VH and _VL domains are connected by a linker. In another embodiment, the OX40 agonist fusion protein according to structure IA or IB comprises one or more OX40-binding domains, which are scFv domains comprising _FH and _VL regions, the _VH and _VL regions having at least 95% identity with the _VH and _VL sequences shown in Table 15, wherein the _VH and _VL domains are connected by a linker.

Table 15: Other peptide domains used as OX40 binding domains in fusion proteins (e.g., structures I-A and I-B) or as scFv OX40 agonist antibodies

In one embodiment, the OX40 agonist is an OX40-activating 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 other domains at the N-terminus and/or C-terminus, wherein said other domains are Fab or Fc fragment domains. In one embodiment, the OX40 agonist is an OX40-activating 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, and further comprising other domains at the N-terminus and/or C-terminus, wherein such other domains are Fab or Fc fragment domains, wherein each soluble OX40-binding domain lacks a stem region (which facilitates trimerization and provides a certain 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 to 8 amino acids.

In one embodiment, the OX40 agonist is an OX40-inducing 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 soluble TNF superfamily cytokine domain lacks a stem region, and the first and second peptide linkers independently have a length of 3 to 8 amino acids, wherein the TNF superfamily cytokine domain is an OX40-binding domain.

In some embodiments, the OX40 agonist is MEDI6383. MEDI6383 is an OX40 agonistic fusion protein that can be prepared as described in U.S. Patent No. 6,312,700, the disclosure of which is incorporated herein by reference.

In one embodiment, the OX40 agonist is an OX40-agonistic scFv antibody that includes any of the aforementioned _VH domains linked to any of the aforementioned _VL domains.

In one embodiment, the OX40 agonist is Creative Biolabs OX40 agonist monoclonal antibody MOM-18455, commercially available from Creative Biolabs, Inc., Shirley, NY, USA.

In one embodiment, the OX40 agonist is the OX40 agonist antibody clone Ber-ACT35, which is commercially available from BioLegend, Inc. (San Diego, CA, USA).

H. Optional cell viability analysis

Optionally, cell viability assays can be performed after the first amplification in step B using standard assays known in the art. Alternatively, cell viability assays can be performed after the first amplification (sometimes referred to as initial large-scale amplification) using standard assays known in the art. For example, a trypan blue exclusion assay can be performed on a large number of TIL samples, which selectively labels dead cells and allows for viability assessment. Other assays for detecting viability may include, but are not limited to, the Alamar blue assay and MTT assay.

1. Cell counting, viability, and flow cytometry

In some implementations, cell count and/or viability are measured. The expression of markers (e.g., but not limited to CD3, CD4, CD8, and CD56) and any other markers disclosed or described herein can be measured by flow cytometry using antibodies, such as, but not limited to, those commercially available from BD Bio-Sciences (BD Biosciences, San Jose, CA) using a FACSCanto ^™ flow cytometer (BD Biosciences). Cells can be manually counted using a disposable c-chip hematology counter (VWR, Batavia, IL), and viability can be assessed using any method known in the art, including but not limited to trypan blue staining.

In some cases, large numbers of TIL clusters can be cryopreserved immediately using the methods described below. Alternatively, large numbers of TIL clusters can be re-prepared (REP) and then cryopreserved. Similarly, in cases where genetically modified TILs are used for treatment, large or re-prepared TIL clusters can be genetically modified for appropriate treatment.

2. Cell Culture

In one embodiment, a method for amplifying TILs may include using about 5,000 mL to about 25,000 mL of cell culture medium, about 5,000 mL to about 10,000 mL of cell culture medium, or about 5,800 mL to about 8,700 mL of cell culture medium. In one embodiment, no more than one type of cell culture medium is used to amplify the number of TILs. Any suitable cell culture medium can be used, such as AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad CA). In this respect, the method of the present invention advantageously reduces the amount of culture medium and the number of culture medium types required to amplify the number of TILs. In one embodiment, amplifying the number of TILs may include adding fresh cell culture medium (also known as feeder cells) to the cells at a frequency of no more than once every three or four days. Amplifying cell numbers using a breathable container simplifies the steps required for amplifying cell numbers by reducing the feeder frequency required for cell amplification.

In one embodiment, the cell culture medium in the first and/or second vented containers is unfiltered. Using unfiltered cell culture medium simplifies the steps required to expand cell numbers. In one embodiment, the cell culture medium in the first and/or second vented containers is free of β-mercaptoethanol (BME).

In one embodiment, the duration of the method is from about 14 days to about 42 days (e.g., about 28 days), and the method includes: obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first ventilated container containing cell culture medium therein; obtaining TILs from the tumor tissue sample; and amplifying the number of TILs using aAPC in a second ventilated container containing cell culture medium therein.

In one embodiment, TILs are amplified in a breathable container. Using methods, compositions, and apparatus known in the art, including those described in U.S. Patent Application Publication No. US2005/0106717A1 (the disclosure of which is incorporated herein by reference), a breathable container is used to amplify TILs with PBMCs. In one embodiment, TILs are amplified in a breathable bag. In one embodiment, TILs are amplified using a cell expansion system that amplifies TILs in a breathable bag, such as the Xuri Cell Expansion System W25 (GE Healthcare). In one embodiment, TILs are amplified using a cell expansion system that amplifies TILs in a breathable bag, such as the WAVE bioreactor system, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In one embodiment, the cell expansion system includes a breathable cell bag selected from the group consisting of: about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L.

In one embodiment, TILs can be amplified in G-REX flasks (commercially available from Wilson Wolf Manufacturing). This type of implementation allows the cell population to expand from approximately 5 × ^10⁵ cells/ ^cm² to 10 × ^10⁶ to 30 × ^10⁶ cells/ ^cm² . In one embodiment, this amplification is performed without adding fresh cell culture medium (also known as feeder cells). In one embodiment, feeding is not required as long as the culture medium remains at a height of approximately 10 cm in the G-REX flask. In one embodiment, feeding is not performed, but more than one cytokine is added. In one embodiment, the cytokines can be added as a bolus injection without mixing the cytokines with the culture medium. Such containers, devices, and methods are known in the art and have been used to amplify TILs, including those described in U.S. Patent Application Publication No. US2014/0377739A1, International Patent Application Publication No. WO2014/210036A1, U.S. Patent Application Publication No. US2013/0115617A1, International Publication No. WO2013/188427A1, U.S. Patent Application Publication No. US2011/0136228A1, U.S. Patent No. 8,809,050, and International Patent Application Publication No. WO2011/072. The disclosures of those described in U.S. Patent Application Publication No. 088A2, U.S. Patent Application Publication No. US2016/0208216A1, U.S. Patent Application Publication No. US2012/0244133A1, International Patent Application Publication No. WO2012/129201A1, U.S. Patent Application Publication No. US2013/0102075A1, U.S. Patent No. 8,956,860, International Patent Application Publication No. WO2013/173835A1, and U.S. Patent Application Publication No. US2015/0175966A1, the contents of which are incorporated herein by reference. Similar methods are also described in Jin et al., JI mmunotherapy 2012, 35, 283-292, the disclosures of which are incorporated herein by reference.

I. Optional TIL genetic engineering

In some implementations, TILs may optionally be genetically engineered to include other functions, including but not limited to: high-affinity T-cell receptors (TCRs), such as TCRs that target tumor-associated antigens (e.g., MAGE, HER2, or NY-ESO-1); or chimeric antigen receptors (CARs) that bind to tumor-associated cell surface molecules (e.g., mesothelin) or lineage-restricted cell surface molecules (e.g., CD19).

J. Optional cryopreservation of TILs

As described above and illustrated in steps A through E of Figure 8, cryopreservation can occur at many points throughout the TIL population expansion process. In some embodiments, the expanded TIL population can be cryopreserved after a second expansion (e.g., according to step D of Figure 8). Typically, TIL populations can be cryopreserved by placing them in a freezing solution (e.g., 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO)). Cells in the solution are placed in cryovials and stored at -80°C for 24 hours, optionally transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986. In some embodiments, TILs are cryopreserved in 5% DMSO. In some embodiments, TILs are cryopreserved in cell culture medium supplemented with 5% DMSO. In some embodiments, TILs are cryopreserved according to the methods provided in Examples 8 and 9.

When appropriate, remove the cells from the freezer and thaw them in a 37°C water bath until approximately 4/5 of the solution is thawed. The cells are typically resuspended in complete culture medium, optionally washed once or multiple times. In some embodiments, as known in the art, the thawed TILs can be counted and their viability assessed.

K. Phenotypic characterization methods for amplified TILs

Granzyme B production: Granzyme B is another measure of the ability of TILs to kill target cells. Following the manufacturer's instructions, the level of granzyme B in the supernatant of the restimulated culture medium was assessed using the Human Granzyme B DuoSet ELISA Kit (R&D Systems, Minneapolis, MN) with antibodies against CD3, CD28, and CD137/4-1BB, as described above. In some embodiments, a second amplification of the TIL or a second additional amplification of the TIL (e.g., those described in step D of Figure 8, including TILs referred to as reREP TILs) resulted in increased granzyme B production.

In some embodiments, telomere length can be used as a measure of cell viability and/or cell function. In some embodiments, the telomere length of TILs produced by the present invention is unexpectedly similar to that of TILs prepared using methods other than those described herein (including, for example, those other than those described in Figure 8). Telomere length measurement: Various methods have been used to measure the length of telomeres in genomic DNA and cell preparations. Telomere restriction fragment (TRF) analysis is the gold standard for measuring telomere length (de Lange et al., 1990). However, a major limitation of TRF is that it requires a large amount of DNA (1.5 μg). The present invention utilizes two widely used techniques for measuring telomere length: fluorescence in situ hybridization (FISH; Agilent Technologies, Santa Clara, CA) and quantitative PCR.

In some embodiments, TIL health is measured by IFN-γ secretion. In some embodiments, IFN-γ secretion indicates active TILs. In some embodiments, an IFN-γ titer assay is employed. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in TIL culture media stimulated with antibodies against CD3, CD28, and CD137/4-1BB. The IFN-γ level in the culture media of these stimulated TILs can be determined by measuring IFN-γ release.

In some embodiments, the cytotoxic potential of TIL in lysing target cells is assessed using a co-culture assay of TIL with the bioluminescent retargeting lysis assay (potency assay) (which measures the cytotoxicity of TIL in a highly sensitive dose-dependent manner).

In some embodiments, this method provides an assay for assessing TIL viability using the methods described above. In some embodiments, TILs are amplified as described above (including, for example, in Figure 8). In some embodiments, TILs are cryopreserved before viability assessment. In some embodiments, viability assessment includes thawing TILs before performing a first amplification, a second amplification, and a further second amplification. In some embodiments, this method provides assays for assessing cell proliferation, cytotoxicity, cell death, and/or other terms related to the viability of TIL populations. Viability can be measured by any of the TIL metabolic assays described above and any known methods for assessing cell viability known in the art. In some embodiments, this method provides analytical methods for assessing cell proliferation, cytotoxicity, cell death, and/or other terms related to the viability of TILs amplified using the methods described herein, including the method shown in Figure 8.

This invention also provides a method for determining TIL viability. In some embodiments, the TIL has the same viability compared to freshly harvested TIL and/or TIL prepared using methods other than those provided herein (including, for example, methods other than those described in Figure 8). In some embodiments, the TIL has increased viability compared to freshly harvested TIL and/or TIL prepared using methods other than those provided herein (including, for example, methods other than those described in Figure 8). This invention provides a method for determining TIL viability by expanding tumor-infiltrating lymphocytes (TILs) into a larger TIL population, the method comprising:

(i) Obtain the previously expanded first TIL population;

(ii) A first expansion of the first TIL population was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optional OKT-3, resulting in a second TIL population; and

(iii) A second expansion was performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen-presenting cells (APC) to generate a third TIL population; wherein the number of the third TIL population was at least 100 times greater than that of the second TIL population; wherein the second expansion was performed for at least 14 days to obtain the third TIL population; wherein the activity of the third TIL population was further analyzed.

In some implementations, the method further includes:

(iv) A second amplification was performed by supplementing the cell culture medium of the third TIL population with additional IL-2, additional OKT-3 and additional APC; wherein the second amplification was performed for at least 14 days to obtain a larger TIL population than that obtained in step (iii); wherein the activity of the third TIL population was further determined.

In some implementations, the cells are cryopreserved prior to step (i).

In some implementations, the cells are thawed before step (i).

In some implementations, step (iv) 1 to 4 is repeated to obtain sufficient TIL for analysis.

In some implementations, steps (i) through (iii) or (iv) are performed for approximately 40 to approximately 50 days.

In some implementations, steps (i) through (iii) or (iv) are performed for approximately 42 to approximately 48 days.

In some implementations, steps (i) through (iii) or (iv) take approximately 42 to approximately 45 days.

In some implementations, steps (i) through (iii) or (iv) take approximately 44 days.

In some implementations, cells from step (iii) or step (iv) express similar levels of CD4, CD8, and TCRαβ as freshly harvested cells.

In some implementations, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

In some implementations, PBMCs are added to the cell culture on any day from day 9 to day 17 of step (iii).

In some implementations, the APC is an artificial APC (aAPC).

In some embodiments, the method further includes the step of transducing a first TIL population with an expression vector containing nucleic acid encoding a high-affinity T cell receptor.

In some implementations, the transduction step occurs before step (i).

In some embodiments, the method further includes the step of transducing a first TIL group with an expression vector containing a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to at least one intracellular domain of a T cell signaling molecule.

In some implementations, the transduction step occurs before step (i).

In some implementations, the activity of TIL is measured.

In some implementations, the activity of the TIL is determined after cryopreservation.

In some implementations, the activity of TILs is determined after cryopreservation and after step (iv).

Multiple antigen receptors for T and B lymphocytes are generated through somatic recombination of a limited but large number of gene fragments. These gene fragments—V (variable segment), D (diverse segment), J (connector segment), and C (constant segment)—determine the binding specificity of immunoglobulins and T cell receptors (TCRs) and their downstream applications. This invention provides a method for generating TILs that exhibit and increase the diversity (sometimes referred to as polyclonalness) of the T cell repertoire. In some embodiments, the increase in T cell repertoire diversity is compared to freshly harvested TILs and/or TILs prepared using methods other than those provided herein (including, for example, methods other than those shown in Figure 8). In some embodiments, TILs obtained by the methods of this invention exhibit an increase in T cell repertoire diversity. In some embodiments, TILs obtained from the first amplification exhibit an increase in T cell repertoire diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, immunoglobulin diversity is immunoglobulin heavy chain diversity. In some embodiments, immunoglobulin diversity is immunoglobulin light chain diversity. In some embodiments, diversity is T cell receptor diversity. In some embodiments, diversity refers to the diversity of T cell receptors selected from one of the α, β, γ, and δ receptors. In some embodiments, the expression of T cell receptor (TCR) α and/or β is increased. In some embodiments, the expression of T cell receptor (TCR) α is increased. In some embodiments, the expression of T cell receptor (TCR) β is increased. In some embodiments, the expression of TCRab (i.e., TCRα/β) is increased.

According to this disclosure, methods for determining TIL activity and/or further administration to a subject are described. In some embodiments, methods for determining tumor-infiltrating lymphocytes (TILs) include:

(i) Obtain the first TIL group;

(ii) A first expansion of the first TIL population was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optional OKT-3, resulting in a second TIL population; and

(iii) A second expansion was carried out by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen-presenting cells (APC) to generate a third TIL population; wherein the number of the third TIL population was at least 50 times that of the second TIL population.

(iv) Harvesting, washing and cryopreservation of Group 3 TILs;

(v) Preserve cryopreserved TILs at freezing temperatures;

(vi) Unfreeze the third TIL group and provide the unfrozen third TIL group;

(vii) A portion of the thawed third TIL population was further amplified by supplementing the cell culture medium of the third population with IL-2, OKT-3 and APC, in an additional amplification phase lasting at least 3 days (sometimes called the reREP phase); wherein, a third amplification was performed to obtain the fourth TIL population; wherein, the number of TILs in the fourth TIL population was compared with the number of TILs in the third TIL population to obtain the ratio;

(viii) Based on the ratio in step (vii), determine whether the thawed TIL clusters are suitable for administration to the patient;

(ix) When it is determined in step (viii) that the ratio of the number of TILs in the fourth TIL group to the number of TILs in the third TIL group is greater than 5:1, a therapeutically effective dose of the thawed third TIL group is administered to the patient.

In some implementations, an additional amplification phase (sometimes called the reREP phase) is performed until the ratio of the number of TILs in the fourth TIL group to the number of TILs in the third TIL group is greater than 50:1.

In some implementations, the number of TILs sufficient for a therapeutically effective dose is about 2.3 × ^10¹⁰ to about 13.7 × ^10¹⁰ .

In some embodiments, steps (i) to (vii) take approximately 40 to approximately 50 days. In some embodiments, steps (i) to (vii) take approximately 42 to approximately 48 days. In some embodiments, steps (i) to (vii) take approximately 42 to approximately 45 days. In some embodiments, steps (i) to (vii) take approximately 44 days.

In some embodiments, cells from step (iii) or (vii) express similar levels of CD4, CD8, and TCRαβ as freshly harvested cells. In some embodiments, the cells are TILs.

In some embodiments, the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, PBMCs are added to the cell culture on any day from day 9 to day 17 of step (iii).

In some implementations, the APC is an artificial APC (aAPC).

In some implementations, the step of transducing the first TIL group with an expression vector containing nucleic acid encoding a high-affinity T cell receptor is included.

In some implementations, the transduction step occurs before step (i).

In some embodiments, the step of transducing a first TIL group with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single-chain variable fragment antibody fused to at least one intracellular domain of a T cell signaling molecule.

In some implementations, the transduction step occurs before step (i).

In some implementations, the activity of TIL is determined after step (vii).

This disclosure also provides other methods for determining TIL. In some embodiments, this disclosure provides a method for determining TIL, the method comprising:

(i) Obtain a portion of the first cryopreserved TIL group;

(ii) Thawing this portion of the first TIL group that has been cryopreserved;

(iii) A second TIL population is generated by first amplification of this portion of the first TIL population in a cell culture medium containing IL-2, OKT-3 and antigen-presenting cells (APCs), followed by an additional amplification phase (sometimes called the reREP phase) lasting at least 3 days; wherein this portion of the first TIL population is compared with the second TIL population to obtain a ratio of TIL numbers; wherein the ratio of the number of TILs in the second TIL population to the number of TILs in this portion of the first TIL population is greater than 5:1;

(iv) Based on the ratio in step (iii), determine whether the first TIL group is suitable for therapeutic administration to the patient;

(v) When it is determined in step (iv) that the ratio of the number of TILs in the second TIL group to the number of TILs in the first TIL group is greater than 5:1, the first TIL group is determined to be suitable for therapeutic application.

In some implementations, the ratio of the number of TILs in the second TIL group to the number of TILs in that portion of the first TIL group is greater than 50:1.

In some embodiments, the method further includes amplifying the entire first TIL group cryopreserved in step (i) according to the method described in any of the embodiments provided herein.

In some embodiments, the method further includes administering the cryopreserved entire first TIL cluster from step (i) to the patient.

L. Closed systems used in TIL production

This invention provides the application of a closed system in TIL (Total Liquid Incubation) culture. Such a closed system can prevent and/or reduce microbial contamination, allow the use of fewer flasks, and reduce costs. In some embodiments, the closed system uses two containers.

Such closed systems are well known in the art and can be found, for example, at http://www.fda.gov/cber/guidelines.htm and https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779.htm.

In some embodiments, the closure system includes a Luer lock and a heat-sealing system as described in Example 16. In some embodiments, the closure system is introduced under aseptic conditions via a syringe to maintain the sterility and closure of the system. In some embodiments, the closure system as described in Example 16 is employed. In some embodiments, the TIL is formulated into the final product formulation container according to the method described in Section 8.14, “Final Formulation and Filling,” of Example 16.

As stated on the FDA website, closed systems using aseptic methods are known and well-described. See also https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779.htm, as described above and provided in the relevant section below.

introduction

The Sterile Connection Device (STCD) creates a sterile weld between two compatible tubes. This procedure allows for the aseptic connection of various containers and tube diameters. This guidance describes recommended practices and procedures for using these devices. This guidance does not cover data or information that manufacturers of sterile connection devices must submit to the FDA for approval or permission to market. It is also important to note that, under the Federal Food, Drug and Cosmetic Act, using an approved or authorized sterile connection device for a purpose not authorized on the label may result in the device being considered adulterated and counterfeit.

In some embodiments, the closed system uses a single container from the time tumor fragments are obtained until the TILs are 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 to an infusion bag without opening the first closed G container. In some embodiments, when two containers are used, the infusion bag is an infusion bag containing HypoThermosol. The closed system or closed TIL cell culture system is characterized in that once the tumor sample and/or tumor fragments are added, the system is tightly sealed to the outside environment, forming a closed environment free from intrusion by bacteria, fungi, and/or any other microbial contaminants.

In some embodiments, microbial contamination is reduced by about 5% to about 100%. In some embodiments, microbial contamination is reduced by about 5% to about 95%. In some embodiments, microbial contamination is reduced by about 5% to about 90%. In some embodiments, microbial contamination is reduced by about 10% to about 90%. In some embodiments, microbial contamination is reduced by about 15% to about 85%. In some embodiments, microbial contamination is reduced by 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 the growth of TILs in the absence of microbial contamination and/or with a significant reduction in microbial contamination.

Furthermore, the pH, partial pressure of carbon dioxide, and partial pressure of oxygen in the TIL cell culture environment all change with cell culture. Therefore, even after circulating a culture medium suitable for cell culture, it is still necessary to continuously maintain the closed environment as the optimal environment for TIL proliferation. For this purpose, it is desirable to monitor the physical factors of pH, partial pressure of carbon dioxide, and partial pressure of oxygen in the culture medium within the closed environment using sensors. The signals from these sensors are used to control a gas exchanger installed at the inlet of the culture environment, adjusting the gas partial pressure in the closed environment in real time according to changes in the culture medium to optimize the cell culture environment. In some embodiments, the present invention provides a closed cell culture system that incorporates a gas exchanger equipped with a monitoring device at the inlet of the closed environment. This monitoring device measures the pH, partial pressure of carbon dioxide, and partial pressure of oxygen in the closed environment and optimizes the cell culture environment by automatically adjusting the gas concentration based on signals from the monitoring device.

In some implementations, the pressure within the closed environment is controlled continuously or intermittently. That is, for example, the pressure within the closed environment can be varied using a pressure maintaining device to ensure that the space is under positive pressure suitable for TIL growth, or under negative pressure to promote fluid exudation, thereby promoting cell proliferation. Furthermore, by intermittently applying negative pressure, the circulating fluid within the closed environment can be uniformly and effectively replaced through temporary contraction of the closed environment's volume.

In some implementations, the optimal culture components for TIL proliferation may be replaced or added, and factors such as IL-2 and/or OKT3 and combinations thereof may be added.

C. Cell Culture

In one embodiment, the method for amplifying TILs (including those described above and illustrated in Figure 8) may include using about 5,000 mL to about 25,000 mL of cell culture medium, about 5,000 mL to about 10,000 mL of cell culture medium, or about 5,800 mL to about 8,700 mL of cell culture medium. In some embodiments, such as those described in Example 21, the culture medium is serum-free. In some embodiments, the culture medium in the first amplification is serum-free. In some embodiments, the culture medium in the second amplification is serum-free. In some embodiments, both the first and second amplifications are serum-free. In one embodiment, the number of TILs amplified uses no more than one type of cell culture medium. Any suitable cell culture medium may be used, such as AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad CA). In this respect, the method of the present invention advantageously reduces the amount of culture medium and the number of types of culture medium required to amplify the number of TILs. In one implementation, increasing the number of TILs may include feeding the cells at a frequency of no more than once every three days or once every four days. By reducing the feeding frequency required for cell expansion, the procedure for increasing cell numbers in a ventilated container can be simplified.

In one embodiment, the cell culture medium in the first and/or second vented containers is unfiltered. Using unfiltered cell culture medium simplifies the procedures required to expand cell numbers. In one embodiment, the cell culture medium in the first and/or second vented containers is β-mercaptoethanol (BME)-free.

In one embodiment, the method lasts for about 7 to 14 days (e.g., about 11 days), and includes: obtaining a tumor tissue sample from a mammal; culturing the tumor tissue sample in a first ventilated container containing cell culture medium; obtaining TILs from the tumor tissue sample; and amplifying the number of TILs in a second ventilated container containing cell culture medium. In some embodiments, the pre-REP is about 7 to 14 days, for example, about 11 days. In some embodiments, the REP is about 7 to 14 days, for example, about 11 days.

In one embodiment, TILs are amplified in a breathable container. Breathable containers have been used to amplify TILs using PBMCs with methods, compositions, and apparatus known in the art, including those described in U.S. Patent Application Publication No. 2005/0106717A1, the disclosure of which is incorporated herein by reference. In one embodiment, TILs are amplified in a breathable bag. In one embodiment, a cell amplification system for amplifying TILs, such as the Xuri Cell Amplification System W25 (GE Healthcare), is used in the breathable bag to amplify TILs. In one embodiment, a cell amplification system for amplifying TILs, such as the WAVE Bioreactor System (also known as the Xuri Cell Amplification System W5) (GE Healthcare), is used in the breathable bag to amplify TILs. In one embodiment, the cell expansion system includes a breathable cell bag with a volume selected from: about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L.

In one embodiment, TILs can be expanded in G-Rex flasks (commercially available from Wilson Wolf Manufacturing). This type of implementation allows the cell population to expand from approximately 5 × ^10⁵ cells/ ^cm² to 10 × ^10⁶ cells/ ^cm² to 30 × ^10⁶ cells/ ^cm² . In one embodiment, no feed is required. In one embodiment, feed is not required as long as the culture medium remains at a height of approximately 10 cm in the G-Rex flask. In one embodiment, feed is not required; instead, more than one cytokine is added. In one embodiment, cytokines can be added as a bolus injection without mixing the cytokines with the culture medium. Such containers, devices, and methods are known in the art and have been used to amplify TILs, including U.S. Patent Application Publication No. US2014/0377739A1, International Publication No. WO2014/210036A1, U.S. Patent Application Publication No. US2013/0115617A1, International Publication No. WO2013/188427A1, U.S. Patent Application Publication No. US2011/0136228A1, U.S. Patent No. US8,809,050B2, International Publication No. WO2011/072088A2, U.S. Patent Application Publication No. US2016/0208216A1, U.S. Patent Application Publication No. US2012/0244133A1, International Publication No. WO2012/129201A1, and U.S. Patent Application Publication No. 2013. The disclosures of those described in 2013/0102075A1, U.S. Patent No. 8,956,860B2, International Publication No. WO2013/173835A1, and U.S. Patent Application Publication No. US2015/0175966A1 are incorporated herein by reference. Such processes are also described in Jin et al., J. Immunotherapy, 2012, 35: 283-292.

D. Optional TIL genetic engineering

In some implementations, TILs may optionally be genetically engineered to include other functions, including but not limited to: high-affinity T-cell receptors (TCRs), such as TCRs that target tumor-associated antigens (e.g., MAGE, HER2, or NY-ESO-1); or chimeric antigen receptors (CARs) that bind to tumor-associated cell surface molecules (e.g., mesothelin) or lineage-restricted cell surface molecules (e.g., CD19).

E. Optional cryopreservation of TIL

Optionally, large numbers of TIL populations or expanded TIL populations can be cryopreserved. In some embodiments, therapeutic TIL populations are cryopreserved. In some embodiments, TILs harvested after a second expansion are cryopreserved. In some embodiments, TILs are cryopreserved in exemplary step F of Figure 8. In some embodiments, TILs are cryopreserved in an infusion bag. In some embodiments, TILs are cryopreserved before being placed in an infusion bag. In some embodiments, TILs are cryopreserved without being placed in an infusion bag. In some embodiments, cryopreservation is performed using a cryopreservation medium. In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO). This is typically done by placing the TIL populations in a freezing solution (e.g., 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO)). The cells in the solution are placed in cryovials and stored at -80°C for 24 hours, and optionally transferred to a gaseous nitrogen cryopreservation system. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986.

When appropriate, remove the cells from the freezer and thaw them in a 37°C water bath until approximately 4/5 of the solution is thawed. The cells are typically resuspended in complete culture medium and optionally washed once or several times. In some embodiments, as known in the art, the thawed TILs can be counted and their viability assessed.

In one preferred embodiment, the TIL population is cryopreserved using CS10 cryopreservation medium (CryoStor 10, BioLifeSolutions). In another preferred embodiment, the TIL population is cryopreserved using a cryopreservation medium containing dimethyl sulfoxide (DMSO). In yet another preferred embodiment, the TIL population is cryopreserved using a 1:1 (volume:volume) ratio of CS10 to cell culture medium. In yet another preferred embodiment, the TIL population is cryopreserved using an approximately 10:1 (volume:volume) ratio of CS10 to cell culture medium, wherein the cell culture medium further contains additional IL-2.

As discussed in steps A through E above, cryopreservation can occur at many points throughout the TIL population amplification process.

As described above, and illustrated by examples in steps A through E provided in Figure 8, cryopreservation can occur at many time points throughout the TIL population expansion process. In some embodiments, expanded TIL populations after a second expansion can be cryopreserved (e.g., as provided according to step D in Figure A). Cryopreservation is typically accomplished by placing the TIL populations in a freezing solution (e.g., 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO)). The cells in the solution are placed in cryovials and stored at -80°C for 24 hours, and optionally transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986. In some embodiments, TILs are cryopreserved in 5% DMSO. In some embodiments, TILs are cryopreserved in cell culture medium supplemented with 5% DMSO. In some embodiments, TILs are cryopreserved according to the method provided in Example 16.

In some embodiments, large populations of TILs following the first amplification according to step B or expanded TILs following one or more second amplifications according to step D can be cryopreserved. Typically, TILs can be cryopreserved by placing them in a freezing solution (e.g., 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO)). Cells in solution are placed in cryovials and stored at -80°C for 24 hours, or optionally transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al., Acta Oncologica 2013, 52, 978-986.

When appropriate, remove the cells from the freezer and thaw them in a 37°C water bath until approximately 4/5 of the solution is thawed. The cells are typically resuspended in complete culture medium, and may be washed once or multiple times. In some embodiments, as known in the art, the thawed TILs can be counted and their viability assessed.

In some cases, the TIL population from step B can be cryopreserved immediately using the protocol described below. Alternatively, steps C and D can be performed on a large TIL population, followed by cryopreservation after step D. Similarly, in cases where genetically modified TILs are used for treatment, the TIL population from steps B or D can be genetically modified for appropriate treatment.

V. Methods of treating patients

The treatment method begins with the initial collection and culture of TILs. These methods have been described in the art, for example by Jin et al. (J. Immunotherapy, 2012, 35(3): 283-292), the entire contents of which are incorporated herein by reference. The following section (including examples) describes the implementation of the treatment method.

Amplified TILs produced according to the methods described herein (including, for example, those described in steps A through F above or those described in steps A through F above (e.g., also as shown in Figure 8)) have specific uses in treating cancer patients (e.g., described in Goff et al., J. Clinical Oncology 2016, 34(20): 2389-239 and supplements; the contents of which are incorporated herein by reference). In some embodiments, as previously described, TILs are grown from excised metastatic melanoma deposits (see Dudley et al., J Immunother., 2003, 26: 332-342; the entire contents of which are incorporated herein by reference). Fresh tumors can be dissected under sterile conditions. Representative samples can be collected for formal pathological analysis. Single fragments of 2 ^mm³ to ³ mm³ can be used. In some embodiments, 5, 10, 15, 20, 25, or 30 samples are obtained per patient. In some embodiments, 20, 25, or 30 samples are obtained per patient. In some embodiments, 20, 22, 24, 26, or 28 samples are obtained per patient. In some embodiments, 24 samples are obtained per patient. Samples can be placed in the wells of a 24-well plate, maintained in growth medium with a high dose of IL-2 (6,000 IU/mL), and tumor destruction and/or TIL proliferation can be monitored. Any tumors with remaining viable cells after treatment can be enzymatically digested into a single-cell suspension and cryopreserved as described herein.

In some embodiments, successfully grown TILs may be sampled for phenotypic analysis (CD3, CD4, CD8, and CD56) and, where appropriate, for detection against autologous tumors. TILs are considered reactive if overnight co-culture produces interferon-γ (IFN-γ) levels >200 pg/mL and twice the background level. (Goff et al., J Immunother., 2010, 33: 840-847; the entire contents of which are incorporated herein by reference). In some embodiments, cultures exhibiting signs of autoreactivity or a sufficient growth pattern may be selected for a second amplification (e.g., the second amplification provided according to step D of Figure 8), including a second amplification sometimes referred to as rapid amplification (REP). In some embodiments, TILs amplified with high autoreactivity (e.g., high proliferation during the second amplification) are selected for an additional second amplification. In some embodiments, TILs with high autoreactivity (e.g., high proliferation during the second amplification provided in step D of Figure 8) are selected for an additional second amplification according to step D of Figure 8.

In some implementations, the patient does not undergo ACT (adoptive cell transfer) directly; for example, in some implementations, the cells are not used immediately after tumor harvest and/or initial expansion. In such implementations, the TIL can be cryopreserved and thawed 2 days before administration to the patient. In other implementations, the TIL can be cryopreserved and thawed 1 day before administration to the patient. In some implementations, the TIL can be cryopreserved and thawed immediately before administration to the patient.

Surface markers of cell phenotype, including CD3, CD4, CD8, CCR7, and CD45RA (BD BioSciences), in cryopreserved samples of infusion bag TILs can be analyzed by flow cytometry (e.g., FlowJo) and by any of the methods described herein. Serum cytokines are measured using standard enzyme-linked immunosorbent assay (ELISA). An increase in serum IFN-γ is defined as ≥100 pg/mL and greater than 43 baseline levels.

In some embodiments, TILs produced by the methods provided herein (e.g., those illustrated in Figure 8) offer a surprising improvement in the clinical efficacy of TILs. In some embodiments, TILs produced by the methods provided herein (e.g., those shown in Figure 8) exhibit increased clinical efficacy compared to TILs produced by methods other than those described herein (e.g., methods other than those shown in Figure 8). In some embodiments, methods other than those described herein include methods referred to as Process 1C and/or first-generation (first-generation) methods. In some embodiments, the increased efficacy is measured by DCR, ORR, and/or other clinical responses. In some embodiments, TILs produced by the methods provided herein (e.g., the TIL shown in Figure 8) exhibit similar response times and safety compared to TILs produced by methods other than those described herein (including, for example, methods other than those shown in Figure 8, such as the Gen 1 process).

In some embodiments, IFN-gamma (IFN-γ) indicates therapeutic efficacy and/or increased clinical efficacy. In some embodiments, IFN-γ in the blood of a subject treated with TIL indicates an active TIL. In some embodiments, an IFN-γ production titer assay 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, serum, or TIL of a subject treated with TIL prepared by the methods of the present invention, including, for example, those shown in Figure 8. In some embodiments, an increase in IFN-γ indicates therapeutic efficacy in patients treated with TIL produced by the methods of the present invention. In some embodiments, IFN-γ is increased by 1, 2, 3, 4, or 5 times or more compared to untreated patients and/or patients treated with TIL prepared using methods other than those provided herein (including, for example, those other than those shown in Figure 8). In some embodiments, IFN-γ is increased by 1-fold compared to untreated patients and/or patients treated with TILs prepared using methods other than those provided herein (including, for example, those other than those described in FIG8). In some embodiments, IFN-γ is increased by 2-fold compared to untreated patients and/or patients treated with TILs prepared using methods other than those provided herein (including, for example, those other than those described in FIG8). In some embodiments, IFN-γ is increased by 3-fold compared to untreated patients and/or patients treated with TILs prepared using methods other than those provided herein (including, for example, those other than those described in FIG8). In some embodiments, IFN-γ is increased by 4-fold compared to untreated patients and/or patients treated with TILs prepared using methods other than those provided herein (including, for example, those other than those described in FIG8). In some embodiments, IFN-γ is increased by 5-fold compared to untreated patients and/or patients treated with TILs prepared using methods other than those provided herein (including, for example, those other than those described in FIG8). In some embodiments, IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, IFN-γ is measured in ex vivo TILs prepared by methods of the present invention (including, for example, those described in Figure 8) from subjects treated with TILs. In some embodiments, IFN-γ is measured in the blood of subjects treated with TILs prepared by methods of the present invention (including, for example, those described in Figure 8). In some embodiments, IFN-γ is measured in the serum of subjects treated with TILs prepared by methods of the present invention (including, for example, those described in Figure 8).

In some embodiments, TILs prepared by the methods of the present invention (including, for example, those described in FIG8) exhibit increased polyclonalness compared to TILs prepared by other methods (including those not shown in FIG8, such as the method referred to as Process 1C). In some embodiments, significantly improved polyclonalness and/or increased polyclonalness indicate increased therapeutic efficacy and/or clinical efficacy. In some embodiments, polyclonalness refers to T cell repertoire diversity. In some embodiments, increased polyclonalness may indicate therapeutic efficacy with respect to TILs produced by the methods of the present invention. In some embodiments, TILs prepared using methods other than those provided herein (including, for example, those other than those described in FIG8) exhibit 1-fold, 2-fold, 10-fold, 100-fold, 500-fold, or 1000-fold increased polyclonalness compared to TILs prepared using methods other than those provided herein (including, for example, those other than those described in FIG8). In some embodiments, TILs prepared using methods other than those provided herein (including, for example, those other than those described in FIG8) exhibit 1-fold increased polyclonalness compared to TILs prepared using methods other than those provided herein (including, for example, those other than those described in FIG8). In some embodiments, TILs prepared using methods other than those provided herein (including, for example, those other than those described in FIG8) exhibit 2-fold increased polyclonalness compared to TILs prepared using methods other than those provided herein (including, for example, those other than those described in FIG8). In some embodiments, TILs prepared using methods other than those provided herein (including, for example, those other than those described in FIG8) exhibit a 10-fold increase in multiclonalizability compared to those prepared using methods other than those provided herein (including, for example, those other than those described in FIG8). In some embodiments, TILs prepared using methods other than those provided herein (including, for example, those other than those described in FIG8) exhibit a 100-fold increase in multiclonalizability compared to those prepared using methods other than those provided herein (including, for example, those other than those described in FIG8). In some embodiments, TILs prepared using methods other than those provided herein (including, for example, those other than those described in FIG8) exhibit a 1000-fold increase in multiclonalizability compared to those prepared using methods other than those provided herein (including, for example, those other than those described in FIG8).

As is known in the art and as described herein, measures of efficacy may include disease control rate (DCR) and overall response rate (ORR).

1. Methods for treating cancer and other diseases

The compositions and methods described herein can be used to treat diseases. In one embodiment, they are used to treat hyperproliferative diseases. They can also be used to treat other diseases described herein and in the following paragraphs.

In some embodiments, the hyperproliferative disease is cancer. In some embodiments, the hyperproliferative disease is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from: melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal epithelial renal cell carcinoma. In some embodiments, the hyperproliferative disease is a hematologic malignancy. In some embodiments, the solid tumor cancer is selected from chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, follicular lymphoma, and mantle cell lymphoma.

In one embodiment, the present invention includes a method of treating cancer with TILs, wherein, according to the present disclosure, the patient is pretreated with non-myeloablative chemotherapy prior to TIL infusion. In one embodiment, the non-myeloablative chemotherapy consists of cyclophosphamide at 60 mg/kg/day for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine at 25 mg/ ^m² /day for 5 days (days 27 to 23 prior to TIL infusion). In one embodiment, following the non-myeloablative chemotherapy and TIL infusion (on day 0) according to the present disclosure, the patient receives an intravenous infusion of IL-2 at 720,000 IU/kg every 8 hours until physiologically tolerated.

Various models known in the art can be used to test the efficacy of the compounds and combinations thereof described herein in treating, preventing, and/or controlling the diseases or conditions described, which provide guidance for the treatment of human diseases. For example, models for determining efficacy in treating ovarian cancer are described, for instance, in Mullany et al., Endocrinology 2012, 153, 1585-92; and Fong et al., J. Ovarian Res. 2009, 2, 12. Models for determining efficacy in treating pancreatic cancer are described, for instance, in Herreros-Villanueva et al., World J. Gastroenterol 2012, 18, 1286-1294. Models for determining efficacy in treating breast cancer are described, for instance, in Fantozzi, Breast Cancer Res. 2006, 8, 212. Models for determining efficacy in treating melanoma are described, for instance, in Damsky et al., Pigment Cell & Melanoma Res. 2010, 23, 853–859. Models for determining the efficacy of lung cancer treatments are described, for example, by Meuwissen et al., Genes & Development, 2005, 19, 643-664. Models for determining the efficacy of lung cancer treatments are also described, for example, by Kim, Clin. Exp. Otorhinolaryngol. 2009, 2, 55-60; and Sano, Head Neck Oncol. 2009, 1, 32.

In some embodiments, IFN-gamma (IFN-γ) indicates therapeutic efficacy in the treatment of hyperproliferative diseases. In some embodiments, IFN-γ in the blood of subjects treated with TILs indicates active TILs. In some embodiments, a titer 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 subjects treated with TILs prepared by the methods of the present invention, including, for example, those described in Figure 8. In some embodiments, TILs obtained by the methods of the present invention provide an increase in IFN-γ in the blood of subjects treated with TILs prepared using a method called Process 1C, as shown in Figure 13. In some embodiments, the increase in IFN-γ indicates therapeutic efficacy in patients treated with TILs produced by the methods of the present invention. In some embodiments, IFN-γ is increased by 1, 2, 3, 4, or 5 times or more compared to untreated patients and/or patients treated with TILs prepared using methods other than those described herein (including methods other than those shown in FIG8). In some embodiments, IFN-γ is increased by 1 time compared to untreated patients and/or patients treated with TILs prepared using methods other than those described herein (including methods other than those shown in FIG8). In some embodiments, IFN-γ is increased by 2 times compared to untreated patients and/or patients treated with TILs prepared using methods other than those described herein (including methods other than those shown in FIG8). In some embodiments, IFN-γ is increased by 3 times compared to untreated patients and/or patients treated with TILs prepared using methods other than those described herein (including methods other than those shown in FIG8). In some embodiments, IFN-γ is increased fourfold compared to untreated patients and/or patients treated with TILs prepared using methods other than those described herein (including methods other than those shown in Figure 8). In some embodiments, IFN-γ is increased fivefold compared to untreated patients and/or patients treated with TILs prepared using methods other than those described herein (including methods other than those shown in Figure 8). 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 ex vivo TILs from patients treated with TILs produced by the method of the present invention. In some embodiments, IFN-γ is measured in the blood of patients treated with TILs produced by the method of the present invention. In some embodiments, IFN-γ is measured in the serum of patients treated with TILs produced by the method of the present invention.

In some embodiments, TILs prepared by the methods of the present invention (including, for example, those described in FIG8) exhibit increased polyclonalness compared to TILs produced by other methods (not those shown in FIG8, such as the method referred to as Process 1C). In some embodiments, significantly improved polyclonalness and/or increased polyclonalness indicate increased therapeutic efficacy and/or clinical efficacy in cancer treatment. In some embodiments, polyclonalness refers to T cell repertoire diversity. In some embodiments, increased polyclonalness can indicate therapeutic efficacy with respect to the application of TILs produced by the methods of the present invention. In some embodiments, polyclonalness is increased by 1, 2, 10, 100, 500, or 1000 times compared to TILs prepared using methods other than those described herein (including, for example, those shown in FIG8). In some embodiments, polyclonalness is increased by 1 time compared to untreated patients and/or patients treated with TILs prepared using methods other than those described herein (including, for example, those shown in FIG8). In some embodiments, polyclonality is increased by 2-fold compared to untreated patients and/or patients treated with TILs prepared using methods other than those described herein (including, for example, those shown in Figure 8). In some embodiments, polyclonality is increased by 10-fold compared to untreated patients and/or patients treated with TILs prepared using methods other than those described herein (including, for example, those shown in Figure 8). In some embodiments, polyclonality is increased by 100-fold compared to untreated patients and/or patients treated with TILs prepared using methods other than those described herein (including, for example, those shown in Figure 8). In some embodiments, polyclonality is increased by 500-fold compared to untreated patients and/or patients treated with TILs prepared using methods other than those described herein (including, for example, those shown in Figure 8). In some embodiments, polyclonality is increased by 1000-fold compared to untreated patients and/or patients treated with TILs prepared using methods other than those described herein (including, for example, those shown in Figure 8).

2. Method of combined application

In some embodiments, the TILs generated as described herein, including, for example, those derived from the methods described in steps A through F of FIG8, can be administered in combination with one or more immune checkpoint modulators, such as antibodies described below. For example, antibodies targeting PD-1 and co-administered with the TILs of the present invention include, but are not limited to, nivolumab (BMS-936558, Bristol-Myers Squibb), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck), humanized anti-PD-1 antibody JS001 (Shanghai Junshi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), and Pid... The antibodies may include ilizumab (anti-PD-1 mAb CT-011, Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene), and/or anti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and/or humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is derived from clone: RMP1-14 (rat IgG) - BioXcell catalog number BP0146. Other suitable antibodies applicable to the co-administration method of the TIL produced according to steps A through F described herein are the anti-PD-1 antibody disclosed in U.S. Patent No. 8,008,449, which is incorporated herein by reference. In some embodiments, the antibody or its antigen-binding moiety 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, thereby stimulating an anti-tumor immune response, is suitable for co-administration of the TILs generated according to steps A through F described herein. For example, antibodies targeting PD-L1 and currently in clinical trials include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genentech). Other suitable antibodies targeting PD-L1 are disclosed in U.S. Patent No. 7,943,743, which is incorporated herein by reference. Those skilled in the art will understand 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 co-administration of the TILs generated according to steps A through F described herein. In some embodiments, when a patient has a type of cancer that is difficult to treat with anti-PD-1 antibodies alone, the subject receiving the TIL combination generated according to steps A through F is co-administered with an anti-PD-1 antibody. In some implementations, when a patient has refactored melanoma, a combination of TIL and anti-PD-1 is administered. In some implementations, when a patient has non-small cell lung cancer (NSCLC), a combination of TIL and anti-PD-1 is administered.

3. Optional pretreatment for patients with lymphocyte depletion

In one embodiment, the invention includes a method of treating cancer with a group of triglycerides (TILs), wherein, according to the present disclosure, a patient is pretreated with non-myeloablative chemotherapy prior to TIL infusion. In one embodiment, the invention includes cancer in a patient with a group of TILs who has been pretreated with non-myeloablative chemotherapy. In one embodiment, the TILs are administered by infusion. In one embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/day for 2 days (on days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/ ^m² /day for 5 days (on days 27 to 23 prior to TIL infusion). In one embodiment, following the non-myeloablative chemotherapy and TIL infusion according to the present disclosure (day 0), the patient receives an intravenous infusion of IL-2 (aldeleukin, commercially available as PROLEUKIN) at 720,000 IU/kg every 8 hours until physiologically tolerable. In some implementations, TILs are used in combination with IL-2 to treat cancer, wherein IL-2 is administered after the TILs.

Experimental results indicate that lymphocyte depletion prior to adoptive transfer of tumor-specific T lymphocytes plays a crucial role in enhancing therapeutic efficacy by eliminating competitive elements of the regulatory T cells and the immune system (“cytokine precipitation”). Therefore, some embodiments of the present invention employ a lymphocyte depletion step (sometimes referred to as “immunosuppressive modulation”) on the patient prior to the introduction of the TIL of the present invention.

Lymphocyte depletion is typically achieved by administering fludarabine or cyclophosphamide (the active form of which is called equiphosphamide) and combinations thereof. This approach is described in Gassner et al., Cancer I mmunol I mmunother. 2011, 60, 75–85; Muranski et al., Nat. Clin. Pract. Oncol., 2006, 3, 668–681; Dudley et al.; J. Clin. Oncol. 2008, 26, 5233–5239; and Dudley et al., J. Clin. Oncol. 2005, 23, 2346–2357, all of which are incorporated herein by reference in their entirety.

In some embodiments, fludarabine is administered at a concentration of 0.5 μg/mL to 10 μg/mL. In some embodiments, fludarabine is administered at a concentration of 1 μg/mL. In some embodiments, fludarabine treatment is administered for 1, 2, 3, 4, 5, 6, or 7 days or longer. In some embodiments, fludarabine is administered at doses of 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 2 to 7 days. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 4 to 5 days. In some embodiments, fludarabine treatment is administered at 25 mg/kg/day for 4 to 5 days.

In some embodiments, horsephosphamide (the active form of cyclophosphamide) is obtained at a concentration of 0.5 μg/mL to 10 μg/mL by administration of cyclophosphamide. In some embodiments, horsephosphamide (the active form of cyclophosphamide) is obtained at a concentration of 1 μg/mL by administration of cyclophosphamide. In some embodiments, cyclophosphamide treatment is administered for 1, 2, 3, 4, 5, 6, or 7 days or longer. In some embodiments, cyclophosphamide is administered at doses of 100 mg/ ^m² /day, 150 mg/ ^m² /day, 175 mg/ ^m² /day, 200 mg/ ^m² /day, 225 mg/ ^m² /day, 250 mg/ ^m² /day, 275 mg/ ^m² /day, or 300 mg/ ^m² /day. In some embodiments, cyclophosphamide is administered intravenously (i.e., IV). In some embodiments, cyclophosphamide treatment is administered at 35 mg/kg/day for 2 to 7 days. In some embodiments, cyclophosphamide treatment is administered intravenously at 250 mg/ ^m² /day for 4 to 5 days. In some embodiments, cyclophosphamide treatment is administered intravenously at 250 mg/ ^m² /day for 4 days.

In some embodiments, lymphocyte depletion is achieved by administering fludarabine and cyclophosphamide together to the patient. In some embodiments, fludarabine is administered intravenously at 25 mg/ ^m² /day and cyclophosphamide is administered intravenously at 250 mg/ ^m² /day for 4 days.

In one implementation, lymphocyte depletion was achieved by administering cyclophosphamide at a dose of 60 mg/ ^m² /day for 2 days, followed by administering fludarabine at a dose of 25 mg/ ^m² /day for 5 days.

4. IL-2 scheme

In one implementation, the IL-2 regimen includes a high-dose IL-2 regimen comprising adeleukin or a biosimilar or variant thereof, administered intravenously starting the day after the treatment-effective dose of the therapeutic TIL group, wherein a dose of 0.037 mg/kg or 0.044 mg/kg (patient weight) of adeleukin or a biosimilar or variant thereof is administered every 8 hours via intravenous bolus over 15 minutes until tolerated, for a maximum of 14 doses. After a 9-day rest period, this schedule may be repeated for another 14 doses, for a total of up to 28 doses.

In one implementation, the IL-2 regimen comprises a tapering IL-2 regimen. Tapering IL-2 regimens are described in O'Day et al., J. Clin. Oncol. 1999, 17, 2752-61 and Eton et al., Cancer 2000, 88, 1703-9, the disclosures of which are incorporated herein by reference. In one implementation, the tapering IL-2 regimen comprises an intravenous administration of 18 × ^10⁶ IU/ ^m² over 6 hours, followed by an intravenous administration of 18 × ^10⁶ IU/ ^m² over 12 hours, then an intravenous administration of 18 × ^10⁶ IU/ ^m² over 24 hours, and then an intravenous injection of 4.5 × ^10⁶ IU/ ^m² over 72 hours. This treatment cycle can be repeated every 28 days for a maximum of four cycles. In one implementation, the decreasing IL-2 regimen contains 18,000,000 IU/ ^m² on day 1, 9,000,000 IU/ ^m² on day 2, and 4,500,000 IU/ ^m² on days 3 and 4.

In one implementation, the IL-2 regimen includes administering polyethylene glycol-modified IL-2 at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

5. Adoptive cell transfer

Adoptive cell transfer (ACT) is a highly effective form of immunotherapy involving the transfer of immune cells with anti-tumor activity into a cancer patient. ACT is a therapeutic approach involving the in vitro identification of lymphocytes with anti-tumor activity, the large-scale in vitro expansion of these cells, and their infusion into a cancerous host. Lymphocytes used for adoptive transfer can be derived from the stroma of a resected tumor (tumor-infiltrating lymphocytes or TILs). TILs for ACT can be prepared as described herein. In some embodiments, TILs are prepared, for example, according to the method described in Figure 8. They can also be derived from or derived from blood if they are genetically engineered to express anti-tumor T-cell receptors (TCRs) or chimeric antigen receptors (CARs), enriched with mixed lymphocyte tumor cell cultures (MLTCs), or using autologous antigen-presenting cells and tumor-derived peptide clones. ACT in which lymphocytes are derived from the cancerous host to be infused is called autologous ACT. U.S. Publication No. 2011/0052530 relates to methods for performing adoptive cell therapy to promote cancer regression, primarily for treating patients with metastatic melanoma, and is incorporated herein by reference in whole. In some embodiments, TIL can be administered as described herein. In some embodiments, TIL can be administered as a single dose. Such administration can be by injection, such as intravenous injection. In some embodiments, TIL and/or cytotoxic lymphocytes can be administered in multiple doses. Administration can be once, twice, three times, four times, five times, six times, or more than six times per year. Administration can be once a month, once every two weeks, once a week, or every other day. TIL and/or cytotoxic lymphocytes can be continued as needed.

6. Exemplary Treatment Implementation Methods

In some embodiments, this disclosure provides a method for treating cancer with a population of tumor-infiltrating lymphocytes (TILs), comprising the steps of: (a) obtaining a first TIL population by resection of a tumor from a patient; (b) initially expanding the first TIL population in a first cell culture medium to obtain a second TIL population; wherein the number of the second TIL population is at least 5 times greater than the number of the first TIL population; wherein the first cell culture medium contains IL-2; (c) rapidly expanding the second TIL population in a second cell culture medium using a population of myeloid artificial antigen-presenting cells (myeloid aAPCs) to obtain a third TIL population; wherein, 7 days after the start of rapid expansion, the number of the third TIL population is at least 50 times greater than the number of the second TIL population; wherein the second cell culture medium contains IL-2 and OKT-3; and (d) administering a therapeutically effective portion of the third TIL population to a cancer patient. In some embodiments, the present invention discloses a tumor-infiltrating lymphocyte (TIL) population for treating cancer, wherein the TIL population is obtained by a method comprising the following steps: (b) initially expanding a first TIL population obtained from a resected tumor of a patient in a first cell culture medium to obtain a second TIL population; wherein the number of the second TIL population is at least 5 times greater than the number of the first TIL population; wherein the first cell culture medium contains IL-2; (c) rapidly expanding the second TIL population in a second cell culture medium using a population of myeloid artificial antigen-presenting cells (myeloid aAPCs) to obtain a third TIL population; wherein, 7 days after the start of rapid expansion, the number of the third TIL population is at least 50 times greater than the number of the second TIL population; wherein the second cell culture medium contains IL-2 and OKT-3; (d) administering the therapeutically effective portion of the third TIL population to a cancer patient. In some embodiments, the method includes the first step: (a) obtaining the first TIL population from a resected tumor of a patient. In some embodiments, IL-2 is present at an initial concentration of approximately 3000 IU/mL in the second cell culture medium, and OKT-3 antibody is present at an initial concentration of approximately 30 ng/mL. In some embodiments, the first amplification is performed for no more than 14 days. In some embodiments, a ventilated container is used for the first amplification. In some embodiments, a ventilated container is used for the second amplification. In some embodiments, during rapid amplification, the ratio of the second TIL group to the aAPC group is 1:80 to 1:400. In some embodiments, during rapid amplification, the ratio of the second TIL group to the aAPC group is approximately 1:300. In some embodiments, the cancer being treated is selected from melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal epithelial renal cell carcinoma. In some embodiments, the cancer being treated is selected from melanoma, ovarian cancer, and cervical cancer. In some embodiments, the cancer being treated is melanoma. In some embodiments, the cancer being treated is ovarian cancer. In some embodiments, the cancer being treated is cervical cancer. In some embodiments, the method of treating cancer further includes treating the patient with a non-myeloablative lymphocyte depletion regimen before administering the third TIL group. In some embodiments, the non-myeloablative lymphocyte depletion regimen includes the following steps: administering cyclophosphamide at a dose of 60 mg/ ^m² /day for two days, followed by administering fludarabine at a dose of 25 mg/ ^m² /day for five days. In some embodiments, the high-dose IL-2 regimen includes administering 600,000 or 720,000 IU/kg of aldehyde interleukin or its bioanalytes or variants via intravenous bolus injection every 8 hours over 15 minutes until tolerated. In some embodiments, the TIL for treatment has been contacted with one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB), and the TIL for treatment may be added to the cell culture medium during the first and/or second amplification (e.g., according to steps B, C, and/or D of FIG8); wherein the TIL and other reagents may be added in amounts selected from the group consisting of: 0.1 μM sd-RNA/10,000 TIL/100 μL culture medium, 0.5 μM sd-RNA/10,000 TIL/100 μL culture medium, 0.75 μM sd-RNA/10,000 TIL/100 μL culture medium, 1 μM sd-RNA/10,000 TIL/100 μL culture medium, 1.25 μM sd-RNA/10,000 TIL/100 μL culture medium, 1.5 μM The culture medium can be prepared using sd-RNA/10,000 TIL/100 μL, 2 μM sd-RNA/10,000 TIL/100 μL, 5 μM sd-RNA/10,000 TIL/100 μL, or 10 μM sd-RNA/10,000 TIL/100 μL. In some embodiments, the TILs used for treatment have been contacted with one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB); during the first and/or second amplification (e.g., according to steps B, C, and/or D of Figure 8), one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) can be added to the TIL culture twice daily, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days. In one embodiment, the TIL used for treatment has been contacted with one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB); wherein, during the first and/or second amplification (e.g., according to steps B, C, and/or D of FIG8), TIL and other reagents may be added in amounts selected from the group consisting of: 0.1 μM sd-RNA/10,000 TIL, 0.5 μM sd-RNA/10,000 TIL, 0.75 μM sd-RNA/10,000 TIL, 1 μM sd-RNA/10,000 TIL, 1.25 μM sd-RNA/10,000 TIL, 1.5 μM sd-RNA/10,000 TIL, 2 μM sd-RNA/10,000 TIL, 5 μM sd-RNA/10,000 TIL, or 10 μM sd-RNA/10,000 TIL. sd-RNA/10,000 TILs. In one embodiment, the TILs for treatment have been contacted with one or more sd-RNAs targeting the genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB); wherein, during the first and/or second amplification (e.g., according to steps B, C, and/or D of Figure 8), the TILs and other reagents may be added to the TIL culture twice daily, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

Example

The implementation methods included herein are now described with reference to the following embodiments. These embodiments are provided for illustrative purposes only, and the disclosure included herein should in no way be construed as limiting to these embodiments, but rather as including any and all variations that become apparent from the teachings provided herein.

Example 1: Measurement of a Closed System

As described in this article, a protocol and assay for generating TILs from patient tumors in a closed system were developed.

This embodiment describes a novel, simplified procedure for generating a clinically relevant number of TILs from excised tumor tissue in a G-REX device and cryopreserving the final cell product. Examples 2 through 8 describe other aspects of this procedure.

program

Advance preparation: On day 0 (up to 36 hours in advance), prepare TIL separation and washing buffer (TIWB) by adding 50 μg/mL gentamicin to 500 mL of Hank’s Balanced Salt Solution. Transfer 2.5 mL of 10 mg/mL gentamicin stock solution to HBSS. Transfer 0.5 mL of 50 mg/mL stock solution to HBSS.

Prepare CM1 medium using GlutaMax ^™ according to LAB-005's "PreREP and REP Medium Preparation" for CM2 operations. Store at 4°C for up to 24 hours. Warm at 37°C for at least 1 hour before use.

Remove the IL-2 aliquots from the -20°C freezer and place them in a freezer between 2°C and 8°C. Remove the tumor specimens and store them at 4°C until ready for processing.

Unused tumors were transported either in HypoThermasol or as cryopreserved fragments in CryoStor CS10 (both HypoThermasol and CryoStor CS10 are commercially available from BioLife Solutions, Inc.).

Tumor treatment of TIL

Aseptically transfer the following materials to the BSC as needed and label them according to Table 16 below.

Table 16: Materials used for tumor separation

Transfer 5 mL of gentamicin to an HBSS bottle. Label it TIWB. Mix by vortexing. Pipette 50 mL of TIWB into each culture dish. Using long forceps, remove the tumor from the specimen bottle and transfer it to wash dish 1. Incubate the tumor in the wash dish for 3 minutes at room temperature. Transfer the tumor to a wash dish and incubate it in the culture dish for 3 minutes at room temperature. Repeat the washing process in a new wash dish.

Measure and record the length of the tumor. Initially dissect the tumor mass into 10 intermediate blocks, preserving the tumor structure in each block. Process one intermediate tumor block at a time, carefully cutting the tumor into 3mm × 3mm × 3mm fragments. Repeat the same steps for the remaining intermediate tumor blocks.

If fewer than 4 tumor fragments are available, use other available fragments to reach the target of 40 fragments. When fewer than 40 fragments are available, place 10 to 40 fragments into a single G-Rex 100M flask.

Inoculation with G-Rex 100M flask

Aseptically transfer the following materials to the BSC as needed and label them according to Table 4 below.

Table 17: Other materials used for inoculation flasks

project Minimum quantity Process Labels G-Rex 100M Flask As needed batch number Warm CM1 As needed batch number IL-2 aliquots As needed batch number

Add 1 mL of IL-2 stock solution (6 × ^10⁶ IU/mL) to each liter of CM1.

As shown in Table 5, add 1000 mL of preheated CM1 containing 6,000 IU/mL IL-2 to each desired G-REX 100M bioreactor. Transfer an appropriate number of tumor fragments to each G-Rex 100M flask using a pipette, distributing the fragments according to Table 5. When more than one tumor fragment transferred to a G-Rex 100M flask floats, obtain another usable tumor fragment and transfer it to a G-Rex 100M flask. Record the total number of fragments added to each flask. Place each G-REX 100M bioreactor in a 37°C, 5% _CO2 incubator.

When >41 fragments are obtained, 1000 mL of preheated complete CM1 is placed in a second G-REX 100M bioreactor.

Table 18: Number of G-REX bioreactors required

Prepare in advance: Day 11 (prepare up to 24 hours in advance)

Prepare 6 L of CM2 using GlutaMax. Use the reference laboratory procedure for “PreREP and REP Culture Medium Preparation” for CM2 operation. Heat at 37°C for 1 hour before use. Thaw IL-2 aliquots: Remove IL-2 aliquots from the freezer and place at 4°C.

Harvesting TIL (Day 11)

Remove the G-REX-100M flask from the incubator and place it in BSC2. Do not disturb the cells at the bottom of the flask. Use a GatherRex or peristaltic pump to aspirate approximately 900 mL of cell culture supernatant from the flask. Resuspend the TILs by slowly rotating the flask. Observe that all cells have been released from the membrane. Transfer the remaining cell suspension to an appropriately sized blood transfer kit (300-1000 mL). Be careful not to transfer any debris into the blood transfer kit. Insert a 4-inch plasma transfer kit into the transfer kit. Mix the cell suspension and use a 3 mL syringe to extract 1 mL of TIL suspension for cell counting. Place the transfer kit in the incubator until usable.

Culture medium preparation

Incubate the culture medium at 37°C for >1 hour. Add 3 mL of 6 × ^10⁶ IU/mL rhIL-2 stock solution to 6 L CM₂ to obtain a final concentration of 3,000 IU/mL rhIL-2 (“Complete CM₂”). Aseptically weld a 4-inch plasma transfer kit with a mother Luer connector to a 1 L transfer pack. Transfer 500 mL of Complete CM₂ into the 1 L transfer pack. Using a 1.0 mL syringe with a needle, aspirate 150 μL of 1 mg/mL anti-CD3 (clone OKT3) and transfer it to 500 mL of “Complete CM₂”. Store at 37°C until used.

Flask preparation

Transfer 4.5L of “Complete CM2” to a G-REX-500M flask and place the flask in a 37°C incubator until ready.

Thawing irradiated feeder cells

Use 5.0 × ^10⁹ allogeneic irradiated feeder cells from two or more donors. Remove the feeder cells from the LN2 freezer. Thaw the feeder cells in a 37°C incubator or bead bath. When almost completely thawed but still very cold, remove the feeder cells from the warm bath. Add each feeder cell bag directly to an open G-Rex 500M, ensuring a sufficient number of irradiated cells (5.0 × ^10⁹ cells, +/- 20%). Transfer 500 mL of “Complete CM2” + OKT3 to a BSC. Draw the entire contents of the feeder cell bag into a syringe, record the volume, and dispense 5.0 × ^10⁹ allogeneic irradiated feeder cells into the transfer bag.

When the target cell number is reached at +/- 10% (5.0 × ^10⁹ ) and viability is >70%, continue. When the target cell number is reached at less than 90% (5.0 × ^10⁹ ) and viability is >70%, thaw another bag and repeat the above steps. When the target cell number is reached at more than 110%, calculate the appropriate volume required for the desired cell dose and continue.

TILs and feeder cells were co-cultured in G-REX 500M flasks.

Remove the G-REX 500M flask containing the prepared culture medium from the incubator. Connect the feeder cell transfer kit to the G-REX-500M, allowing the contents of the kit to drain into the 500M. Calculate the volume of TIL suspension to be added to achieve a total viable cell count of 200 × ^10⁶ .

(TVC/mL)/200× ^10⁶ = mL

When the total number of viable cells (TILs) is between 5 × ^10⁶ and 200 × ^10⁶ , add all TILs (total volume) to the G-REX-500M. When the TIL count is greater than 200 × ^10⁶ viable cells, add the calculated volume required to add 200 × ^10⁶ TILs to a single G-REX-500M. Rotate the remaining TILs and freeze them in CS10 vials at a concentration of up to ^10⁸ /mL using at least two cryovials, labeling them with TIL identification and the freezing date.

Place the G-REX-500M in an incubator at 37℃ and 5% _CO2 for 5 days.

Preparation in advance: Days 16-18

Heat one 10L bag of AIM V (for initiating cultures of less than 50 × ^10⁶ TIL) at 37°C for at least 1 hour, or heat two bags (for initiating cultures of greater than 50 × ^10⁶ TIL) until ready for use.

TIL cell counting: Days 16-18

Remove the G-REX-500M flask from the incubator, being careful not to disturb the cell culture at the bottom. Remove 4L of cell culture medium from the G-REX-500M flask and place it in a sterile container. Swirl the G-REX-500M until all TILs are resuspended from the membrane. Transfer the cell suspension to a 2L transfer pack. Retain the 500M flask for later use. Calculate the total number of flasks required for subculture using the following formula. Round up.

Total number of live cells / 1.0 x ^10⁹ = Number of flasks

Preparation of CM4

Prepare one 10L bag of AIM-V for every two required 500M flasks. Heat additional culture medium as needed. Prepare CM4 by adding 100mL of GlutaMAX to each required 10L AIM-V. Supplement the CM4 medium with rhIL-2 to a final concentration of 3,000 IU/mL rhIL-2. Aliquot the cell cultures. Supplement each G-REX-500M to 5L. Distribute the TIL volume evenly among the calculated amounts of G-REX-500M. Incubate the flasks at 37°C, 5% _CO2 until harvest on day 22 of the REP.

Preparation in advance: Days 22-24

Prepare 2 L of 1% HSA wash buffer by adding 40 mL of 25% HSA to each of two 1 L PlasmaLyte A 7.4 bags. Combine into one LOVO auxiliary bag. Replenish with 200 mL of CS10 at 600 IU/mL IL-2. Pre-chill four 750 mL aluminum freezer containers at 4 °C.

TIL harvest: Days 22-24

Remove the G-REX-500M flasks from the 37°C incubator, being careful not to disturb the cell culture at the bottom. Aspirate and discard 4.5 L of cell culture supernatant from each flask. Rotate the G-REX-500M flask to completely resuspend the TILs. Harvest the TILs into a bioprocessing bag. Thoroughly mix the bag and use a 3 mL syringe to collect 2 × 2 mL samples for cell counting. Weigh the bag and calculate the difference between the initial and final weight. Determine the cell suspension volume using the following formula.

Net weight of cell suspension (mL) / 1.03 = Volume (mL)

Filter TILs and prepare LOVO source bags. After transferring all cells to the LOVO source bags, close all clamps and seal the tubes of the LOVO source bags. Remove the filter and weigh the cells. Calculate the volume.

Prepare TIL 1:1 in cold CS10 containing 600 IU/mL rhIL-2.

Calculate the required number of cryopreservation bags.

(Volume of cell product × 2) / 100 = Number of bags required (round down)

Calculate the volume allocated to each bag.

(Volume of cell product × 2) / Number of bags required = Volume to be added in each bag

Aseptically transfer the following materials from Table 6 to the BSC.

Table 19: Materials used for TIL cryopreservation

project Minimum quantity Process Labels Cell products 1 batch number Aluminum Refrigerated Cabinet (750ml) 1 n/a Cold CS10 + 600 IU/mL IL-2 As needed batch number Cell Connect CC1 device 1 n/a 750mL cryopreservation bag Calculated Label: Equal sample 1 - Maximum quantity 100mL syringe Number of frozen bags +1 n/a Three-way plug valve 1 n/a cryopreservation tubes 5 TIL Frozen Products Included Bottles

TIL preparations

Connect the LOVO final product, CS10 bag Luer lock, and the appropriate number of cryopreservation bags. The required CS10 volume is equal to the volume of the LOVO final product bags. Mix the LOVO final product bags by inverting them.

Transfer 100 mL of the prepared product into each cryovial. Remove all air bubbles from the cryovial and seal it. Transfer the sealed bags to 4°C and place them in a pre-cooled aluminum freezer container.

TILs are cryopreserved using a controlled-rate freezer (CRF).

Follow the standard procedures for the controlled-rate freezer. After using CRF, store the cryopreservation bags in liquid nitrogen ( _LN2 ).

Example 2: Lymphocyte depletion

Cell counting can be performed on day 7 and before lymphocyte depletion. The final cell product comprises up to approximately 150 × ^10⁹ viable cells formulated with at least 50% HypoThermosol ^™ (volume/volume) in Plasma-Lyte A ^™ and up to 0.5% HSA (human-compatible infusion), which contains 300 IU/mL IL₂. The final product can be administered in one of two infusion volumes:

1) When the total amount of TILs harvested is ≤75× ^10⁹ , use 250mL (in a 300mL infusion bag).

or

2) When the total amount of TILs harvested is <150× ^10⁹ , use 500mL (in a 600mL infusion bag).

Due to the variability in T-cell expansion rates among patients during the REP step, the total number of cells that the final TIL infusion product may produce for each patient cannot be predicted. Based on the minimum number of cells required to deplete a patient's lymphocytes using a cyclophosphamide + fludarabine chemotherapy regimen, lower limits for cell counts on days 3, 4, 5, 6, and 7 of a 3- to 14-day REP were set. Once lymphocyte depletion was initiated based on this obtained minimum cell count, patients were treated with the number of available TILs produced on days 3 to 14 of the REP (and in many cases, day 7). The upper limit of the infusion range (150 × ^10⁹ viable cells) was based on known, publicly available upper limits of safe infusion (up to which clinical responses have been achieved). Radvanyi et al., Clin Cancer Res 2012, 18, 6758-6770.

Example 3: Process 2A – Day 0

This embodiment describes the detailed scheme for day 0 of the 2A process described in embodiments 3 to 6.

preparation.

Confirm that the tumor washing medium, CM1, and IL-2 are within their expiration dates. Place CM1 (cell culture medium 1) into the incubator.

method.

Prepare TIL medium CM1 containing 6000 IU/mL IL-2: 1 L CM1 and 1 mL IL-2 (6,000,000 IU/mL). While adding G-REX and preheating in a 37°C incubator, add 25 mL CM1 + IL2 to a 50 mL Erlenmeyer flask for fragmentation.

Pump 975 mL of preheated CM1 (containing 6,000 IU/mL IL-2) into each G-REX 100MCS bioreactor. Place the G-REX 100MCS in an incubator until needed.

tissue anatomy

Record the start time of tumor treatment. Pipette 3 to 5 mL of tumor washing medium into each well of a six-well plate for excess tumor masses. Pipette 50 mL of tumor washing medium into washing dishes 1 to 3 and a storage dish. Place two 150 mm dissecting dishes into the biosafety cabinet. Place three sterile 50 mL conical tubes into the BSC. Add 5 to 20 mL of tumor washing medium to each conical tube. During tumor washing and dissection, immerse forceps and scalpels in the tumor washing medium as needed.

Remove the tumor from the specimen bottle and transfer it to Wash 1 dish. Incubate the tumor in Wash 1 dish for ≥3 minutes. Transfer the tumor to Wash 2 dish. Incubate the tumor in Wash 2 dish for ≥3 minutes. Transfer the tumor to Wash 3 dish. Incubate the tumor in Wash 3 dish for ≥3 minutes. Transfer the tumor to a dissection dish, measure and record the tumor length.

Initially dissect the tumor mass in the dissecting dish into intermediate blocks, taking care to preserve the tumor structure of each intermediate block. Transfer any intermediate tumor masses that are not actively dissected to a tissue storage dish, ensuring that the tissue remains moist throughout the dissection.

Process one intermediate tumor block at a time, carefully cutting the tumor into approximately 3mm × 3mm × 3mm fragments in a dissecting dish. Continue dissecting fragments from the intermediate tumor block until all tissue from the intermediate block has been evaluated. Select good fragments and use a pipette to transfer up to four good fragments into a ring of wash culture medium drops in a "Tumor Fragments" culture dish. Use a pipette, scalpel, or forceps to transfer as much undesirable tissue and waste as possible into an "Undesirable Tissue" culture dish. Place all remaining tissue into one well of a six-well plate. (Yellow fatty tissue or necrotic tissue indicates undesirable tissue.) Continue processing the remaining intermediate tumor blocks, one block at a time, until the entire tumor has been processed.

Transfer up to 50 optimal tumor fragments to a 50 mL conical flask (labeled: Tumor fragments containing CM1). Remove any floating material from the 50 mL conical flask. Record the number of fragments and floating material. Rotate the conical flask containing the tumor fragments to pour the contents of the 50 mL conical flask into a G-Rex 100 MCS flask. If one or more tumor fragments transferred to the G-Rex 100 M flask float, obtain another tumor fragment from a good tissue dish and transfer it to the G-Rex 100 M flask.

Record the incubator number and the total number of fragments added to each flask. Place the G-REX 100M bioreactor in a 37°C, 5% _CO2 incubator.

Example 4: Process 2A - Day 11

This embodiment describes the detailed scheme for day 11 of process 2A described in embodiments 3 to 6.

Prepared in advance.

The day before processing:

CM2 can be prepared the day before the treatment. Store at 4°C.

Processing date.

Prepare the feeder cell harness. Prepare 5 mL of cryopreservation medium according to CTF-FORM-318 and store at 4°C until needed.

Prepare the G-Rex 500MCS flask. Using a 10mL syringe, aseptically transfer 0.5mL of IL-2 (stock solution 6× ^10⁶ IU/mL) (per liter of CM² (cell culture medium)) into the bioprocessing bag via an unused, sterile female Luer connector. Ensure all IL-2 is mixed with the culture medium. Pump 4.5L of CM² culture medium into the G-Rex 500MCS. Place the G-Rex 500MCS in an incubator.

Preparation of irradiated feeder cells

Record the dry weight of the 1L transfer pack (TP). Pump 500mL CM2 into the TP by weight. Thaw the feeder cells in a 37°C (+/-1°C) water bath. Thoroughly mix the final feeder cell preparation. Using a 5mL syringe with a needle-free port, flush the port with some cell solution to ensure accurate sampling, remove 1mL of cells, and place it into a labeled test tube for counting. Perform single-cell counting on the feeder cell sample, record the data, and append the raw count data to the batch record. If the cell count is <5× ^10⁹ , thaw more cells, count them, and add them to the feeder cell collection. Reweigh the feeder cell bag and calculate the volume. Calculate the volume of cells to be removed.

Add feeder cells to G-REX

Mix the cells thoroughly, and collect the calculated volume to reach 5.0 × ^10⁹ cells. Discard any unwanted cells. Using a 1 mL syringe and an 18G needle, draw 0.150 mL of OKT3, remove the needle, and transfer it to the feeder cells (TP) through the mother Luer interface. Aseptically weld the feeder cell bag to the red tubing of the G-Rex 500MCS. Loosen the tubing to allow the feeder cells to flow into the flask under gravity. Return the G-Rex 500MCS to the incubator and record the time.

Preparation of TIL: Record the start time of TIL harvest.

Carefully remove the G-Rex 100MCS from the incubator. Using GatheRex, transfer approximately 900 mL of culture supernatant to a 1 L transfer pack. Rotate the flask until all cells have detached from the membrane. Check the membrane to ensure all cells have separated. Tilt the flask away from the collection tube to allow tumor debris to settle along the edge. Slowly tilt the flask toward the collection tube, leaving debris on the opposite side of the flask. Using GatheRex, transfer the remaining cell suspension to a 300 mL transfer pack, avoiding tumor debris. Recheck that all cells have been removed from the membrane. If necessary, backwash by loosening the clamps on the GatheRex, allowing some culture medium to flow into the G-Rex 100MCS flask under gravity. Tap the flask firmly to release the cells and pump into 300 mL of TP. After collection, close the red tubing and heat seal.

Record the mass (including dry weight) of 300 mL TP containing cell suspension, and calculate the volume of the cell suspension. Mix the cells thoroughly. Aseptically connect a 5 mL syringe, aspirate 1 mL, and place in a cryovial. Repeat with a second syringe. These are used for cell counting and viability testing. Place in an incubator and record the incubation time. Perform single-cell counting for each sample and record the results. Adjust the total viable TIL density to ≤2 × ^10⁸ viable cells if necessary. Calculate the volume to be removed or record any unnecessary adjustments.

Transfer excess cells into appropriately sized conical tubes and place them in an incubator with the caps loose for later cryopreservation.

Remove the G-Rex 500MCS from the incubator and pump the cells into a flask. Place the G-Rex 500MCS back into the incubator and record the time spent in the G-Rex incubator.

Cryopreservation of excess (cells)

Calculate the amount of frozen culture medium added to the cells:

Table 20: Target cell concentration is 1× ^10⁸ /mL

A. Total cells (from step 15) mL B. Target cell concentration <![CDATA[1×10⁸ cells/mL]]> Volume of frozen culture medium to be added (A/B) mL

Rotate the TILs at 400×g for 5 minutes with full braking and full acceleration at 20°C. Aseptically aspirate the supernatant. Resuspend the cells in the remaining liquid, and slowly add the prepared cryo-culture medium while resuspending. Aliquot the samples and incubate at -80°C.

Example 5: Process 2A - Day 16

This embodiment describes the detailed scheme for day 16 of process 2A described in embodiments 3 to 6.

Harvest and count TILs.

Heat one 10L CM4 bag (for initiating cultures of less than 50 × ^10⁶ TILs) in a 37°C incubator for at least 30 minutes or until ready for use. Remove the G-Rex 500MCS flask from the incubator and transfer approximately 4L of culture supernatant to a 10L Labtainer using GatheRex. Harvest according to the appropriate GatheRex harvesting instructions.

After removing the supernatant, rotate the flask until all cells have detached from the membrane. Tilt the flask, ensuring the tubing is at the edge. Using GatheRex, transfer the remaining cell suspension to a 2L TP container, keeping the edge tilted, until all cells are collected. Examine the adherent cells on the membrane. Tap the flask firmly to release the cells. Add the cells to the 2L TP container. Heat-seal the 2L transfer package. Record the mass of the transfer package containing the cell suspension and calculate the volume of the cell suspension. Determine the volume of the cell suspension, including dry weight.

Gently mix the cells and aspirate 11 mL into equal portions as shown in Table 21.

Table 21: Detection Parameters

Calculate the new volume based on the cell suspension volume and the QC removal volume (11 mL), and record the volume in the 2L transfer pack.

Inoculate and perform sterility testing. Store the mycoplasma sample in a test rack for mycoplasma testing at 4°C. Let stand until TIL inoculation.

Cell count:

Perform single-cell counts, record the data, and append the raw count data to the batch record. Record the dilution. Record the Cellometer counting procedure. Enter the validated dilution into the Cellometer. Calculate the total number of flasks required for subculture.

Add IL-2 to CM

Place a 10L Aim V bag containing Glutamax. Inject 5mL of IL-2 into the syringe (final concentration 3000 IU/mL) and add the IL-2 into the bag. Repeat the same procedure for the remaining Aim V bags.

Prepare a G-REX500MCS flask

Determine the amount of CM4 to be added to the flask. Record the volume of cells added and the volume of CM4 5000 mL-A added to each flask. Place the flasks at 37°C and 5% _CO2 .

TIL inoculation flask

Place the cell product bag on an analytical balance and record the time of TIL addition to the G-REX flask. Mix the cells thoroughly. Repeat the cell transfer for all flasks. Place the flasks at 37°C and 5% _CO2 and record the time of TIL addition to the G-REX flask. Order sedimentation plate tests, as well as aerobic and anaerobic sterility tests, from the microbiology laboratory.

Flow cytometry or cryopreservation of excess cells:

Calculate the required volume of cryopreservation medium: target cell concentration is 1 × 10⁸ ^cells /mL; record the total number of cells removed. Target cell concentration is 1 × ^10⁸ cells/mL. Calculate the total volume of cryopreservation medium to be added.

Prepare cryopreservation medium and store at 40°C until needed. Rotate the TIL at 400×g for 5 minutes at 20°C with full braking and acceleration. Aspirate the supernatant. Gently tap the bottom of the test tube to resuspend the cells in the remaining liquid, and slowly add the prepared cryopreservation medium while gently tapping the tube. Aliquot into appropriately sized, labeled cryovials. Store at -80°C. Transfer to permanent storage within 72 hours, recording the date and time of placement at -80°C.

Example 6: Process 2A - Day 22

This embodiment describes the detailed scheme for day 22 of process 2A described in embodiments 3 to 6.

Prepare in advance

Place three 1L PlasmaLyte A bags into a BSC. Prepare for merging; label the bag as PlasmaLyte A containing 1% HSA. Load 120mL of 25% HSA for transfer. Transfer the HSA to the 3L PlasmaLyte bag. Mix thoroughly. Remove 5mL of PlasmaLyte containing 1% HSA from the needle-free port of the 3L bag. Label the bag with LOVO wash buffer and the date.

IL-2 preparation

Add PlasmaLyte/1% HSA from a 5 mL syringe into a labeled 50 mL sterile conical tube. Add 0.05 mL of IL-2 stock solution to the PlasmaLyte tube and label it: IL-2 6 × ^10⁴ . Store at 2°C to 8°C.

Cell preparation

Remove the G-REX 500M flask from 37°C. Using the GatheRex pump, reduce the volume of the first flask. Rotate the G-REX 500M flask until the TILs are completely resuspended, avoiding splashing or foaming. Ensure all cells have detached from the membrane. Tilt the G-REX flask so that the cell suspension is combined on the side where the collection pipette is located. Start the GatherRex to collect the cell suspension, ensuring all cells have been removed from the flask. If cells remain in the flask, add 100 mL of supernatant back into the flask, vortex, and collect into a cell suspension bag. Repeat for the remaining flasks. Heat seal and label: LOVO Source Bag. Record the dry weight.

Allow TILs to pass through a filter from the cell suspension bag into the LOVO source bag. After transferring all cells to the LOVO source bag, close all clamps, heat-seal above the label, and remove. Thoroughly mix the bag, and using two 3mL syringes, extract two independent 2mL samples from the syringe sample ports for cell counting and viability testing. Weigh the bag and determine the difference between the initial and final weight. Record the data and its position in the incubator, including dry weight.

Cell count.

Perform single-cell counting on each sample, record the data, and append the raw count data to the batch record. Record the Cellometer counting procedure. Enter the validated dilution into the Cellometer. Determine the total number of nucleated cells. Determine the number of TNCs to be removed for LOVO treatment: 1.5 × ^10¹¹ cells. Dispose of the removed cells in appropriately sized containers.

LOVO gains

The 10L Labtainer with Baxter amplification in the "Prepared" section is a replacement filtrate bag soldered to the LOVO kit. Track the LOVO display. To begin this procedure, select the "TIL G-Rex Harvest" protocol from the drop-down menu and follow the instructions.

When the final product volume (retention volume) screen is displayed, use the total nucleated cell count (TNC) values from Table 15 to determine the target final product volume for the following table (Table 16). Enter the final product volume (mL) corresponding to this cell range during the LOVO program setup process.

Table 22: Determination of Target Volume of Final Product

Cellular range Target volume (mL) of the final product (retained sample) 0 < (live + dead) cell total ≤ 7.1E10 150 7.1E10 < (live + dead) cell total ≤ 1.1E11 200 1.1E11 < (live + dead) cell total ≤ 1.5E11 250

Table 23: Target Volume of Product

To determine the target volume from Table 16, touch the Final Product Volume (mL) input field. The numeric keypad will appear. Enter the desired final product volume (in mL).

Record the volumes of the filtrate and solution 1 (read as PlasmaLyte) shown.

Pre-coated IP bags. Mixed source bags. During the LOVO procedure, the system will automatically pause to allow the operator to work with other bags. Different screens will be displayed during different pauses. Follow the instructions on each screen.

Source (bag) rinsing paused

After emptying the source bag, LOVO adds wash buffer to the source bag to rinse it. After adding the prepared volume of wash buffer to the source bag, LOVO automatically pauses and displays the "Source (Bag) Rinse" pause screen.

LOVO processes the rinsing fluid from the source bag and then continues the automated process.

Mixed IP bag pause

To allow the cells to pass through the vortexor again, the IP bag is diluted with wash buffer. After the wash buffer is added to the IP bag, LOVO automatically pauses and displays the "Mix IP Bags" pause screen.

When the pause screen displays "Mix IP Bag," the operator inverts the IP bag several times to thoroughly mix the cell suspension. Add LOVO treatment solution from the IP bag according to the instructions.

Pause "Massage IP Corners"

In the final wash cycle of the LOVO program, cells are pumped from the IP bag, passed through a spinner, and then transferred to a retention (final product) bag. When the IP bag is empty, 10 mL of wash buffer is added to the bottom of the IP bag to rinse it. After adding the rinse buffer, LOVO automatically pauses and displays the "Rub IP Corners" pause screen.

When the "Rub IP Corner" pause screen appears, the operator rubs the corner of the bag to suspend any remaining cells. Resume LOVO to pump the flushing fluid out of the IP bag.

When the LOVO program ends, the "Retrieve Product" screen will be displayed.

Record the results data, in the format shown in Table 17.

Table 24: Summary Table of LOVO Results

Close the LOVO shutdown program

Record the final volume of the prepared product. Calculate the amount of IL-2 required in the final product table.

Determine the number of cryopreservation bags and the retention volume.

Mark the target volume, and retain the number of cryopreservation bags and the retained sample volume of the product as shown in the table below.

Target volume/bag calculation: (Final preparation volume – volume adjusted due to failure to achieve 100% recovery = 10 mL)/#bag.

Cells were prepared using CS10 (CryoStor 10, BioLife Solutions) and IL-2 in a 1:1 (volume:volume) ratio.

Cells were prepared using IL-2 and the connected device. The cells and device were placed in a transport bag and incubated at 2°C to 8°C for ≤15 minutes.

Add CS10

Aspirate the amount of cold CS10 specified in the "Final Product Volume" table. Slowly and gently add the CS10 (1:1, volume:volume) to the cells.

Add the prepared cell products to the cryopreservation bag.

Replace the syringe with the appropriate-sized syringe and add the cell volume to each cryopreservation bag. Mix the cell product. Open the clips leading to the cell product bags and aspirate the appropriate volume.

Record the final product volume

Using a needleless port and an appropriately sized syringe, aspirate the previously determined retention volume. Place the retention into a 50 mL conical tube labeled "Retention". Using a syringe connected to the tubing, remove all air from the bag and aspirate the cells to approximately 1 inch above the bag into the tubing. Incubate at 2°C to 8°C. Mix the cells in the cell product bag and, using a new syringe on the stopcock valve, repeat steps 3 through 8 for the remaining CS750 bags to obtain the cell retention. Once the product enters the CRF, the retained product should be set aside for further processing.

Controlled-rate refrigeration (CRF) program (see also Example 16)

Keep the refrigerator at 4°C until you are ready to add the sample. Add the sample to the CRF.

Wait until the CRF returns to 4°C. Once the temperature is reached, proceed with the freezing process according to the CRF procedure. Perform the following visual inspections on the cryopreservation bags (Note: do not check for over- or under-temperature): container integrity, port integrity, seal integrity, presence of cell clumps, and presence of particles.

Place the cryopreservation bags into the pre-treated boxes and transfer them to the CRF. Arrange the boxes evenly on the CRF racks. Use a ribbon thermocouple for the central box or place a dummy bag in the center.

Close the CRF door. Once the chamber temperature reaches 4℃ +/- 1.5℃, record the time and chamber temperature at which the product is transferred to the CRF.

Processing of quality control samples

Aseptically transfer the following materials as needed, labeling them according to QC and Retention Table 25. One tube for quality control (cell counting, endotoxin, mycoplasma, Gram staining, restimulation, and flow cytometry) is for point-of-care testing. Place the remaining duplicate tubes in a rate-controlled freezer.

Table 25: Testing and Storage Instructions

Cell Count

Perform single-cell counts on each sample, record the data, and append the raw count data to the batch record. Record the Cellometer counting procedure. Enter the validated dilution into the Cellometer.

Cryopreservation of cells after preparation: Place the vial in a CRF. After freezing, transfer it to the storage location and record the date and time of placement in the CFR. Record the date and time of transfer to LN ₂ .

Microbiological testing: Perform aerobic and anaerobic sterility tests.

After cryopreservation of cell product bags

After the operation is complete, stop the refrigeration unit. Remove the cryopreservation bag from the container. Transfer the container to the gas phase LN ₂ .

Example 7: Using a mixture of IL-2, IL-15 and IL-21 cytokines

This embodiment describes the use of IL-2, IL-15, and IL-21 cytokines as additional T cell growth factors in conjunction with the TIL methods of Examples 1 to 10.

Using the methods of Examples 1 to 10, in one experimental arm, TILs were generated from colorectal cancer, melanoma, cervical cancer, triple-negative breast cancer, lung cancer, and renal cell carcinoma tumors in the presence of IL-2; and in the other arm, at the start of culture, a combination of IL-2, IL-15, and IL-21 was used instead of IL-2. At the completion of pre-REP, the amplification, phenotype, function (CD107a+ and IFNγ), and TCR Vβ repertoire of the cultures were evaluated. IL-15 and IL-21 are described elsewhere in this article and in Gruijl et al., IL-21 promotes the expansion of CD27+CD28+ tumor-infiltrating lymphocytes with high cytotoxic potential and low collateral expansion of regulatory T cells, Santegoets, S.J., J Transl Med., 2013, 11:37 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626797/).

The results showed that, compared to IL-2-only conditions, enhanced TIL amplification (>20%) was observed in CD4+ and CD8+ cells treated with IL-2, IL-15, and IL-21 in various histological studies. Compared to cultures treated with IL-2 alone, TILs obtained from cultures treated with IL-2, IL-15, and IL-21 exhibited a predominantly CD8+ population biased towards the TCR Vβ repertoire. IFNγ and CD107a levels were increased in TILs treated with IL-2, IL-15, and IL-21 compared to TILs treated with IL-2 alone.

Example 8: A Phase II, Multicenter, Three-Cohort Study of Melanoma

This phase II, multicenter, three-cohort study aimed to evaluate the safety and efficacy of TIL treatment produced according to method 1C (as described herein) in patients with metastatic melanoma. Cohorts 1 and 2 each enrolled 30 patients; cohort 3 was a retreatment cohort, with up to 10 patients receiving a second TIL infusion. The first two cohorts were evaluating two different production processes: one embodiment of process 1C and process 2A (described in Examples 1 through 10, respectively). Patients in cohort 1 received fresh, non-cryopreserved TILs, while patients in cohort 2 received products produced via the processes described in Examples 1 through 10 (resulting in cryopreserved products). The study design is shown in Figure 26. This phase II, multicenter, three-cohort study was designed to evaluate the safety and efficacy of autologous TIL treatment in a subset of patients with metastatic melanoma. Key inclusion criteria included: measurable metastatic melanoma and ≥1 resectable lesion for TIL production; at least one prior systemic therapy; age ≥18 years; and an ECOG performance status of 0–1. The treatment cohort included patients with unfrozen TIL products (prepared using method 1C), frozen TIL products (prepared using the implementation method of procedure 2A), and patients who did not respond to TIL products or who progressed after an initial response. The primary endpoint was safety, and the secondary endpoints were efficacy, defined as objective response rate (ORR), complete response rate (CRR), progression-free survival (PFS), duration of response (DOR), and overall survival (OS).

Example 9: A qualified single batch of peripheral mononuclear cells irradiated with gamma rays

This embodiment describes a novel simplified procedure for qualifying a single batch of γ-irradiated peripheral mononuclear cells (PBMCs, also known as MNCs) for use as allogeneic feeder cells in the exemplary method described herein.

Each batch of irradiated MNC feeder cells was prepared from a single donor. The ability of each batch or donor to amplify TILs in the REP was individually screened in the presence of purified anti-CD3 (clone OKT3) antibody and interleukin-2 (IL-2). Furthermore, each batch of feeder cells was tested without the addition of TILs to verify that the received gamma ray dose was sufficient to impair their replication function.

background

TIL regeneration requires growth-arrested MNC feeder cells exposed to gamma radiation. Membrane receptors on the feeder MNCs bind to anti-CD3 (clonal OKT3) antibodies and cross-link with TILs in the regeneration flask, thereby stimulating TIL expansion. Feeder cell batches are prepared using leukocyte apheresis from whole blood of various donors. Under GMP conditions, the leukocyte apheresis products are centrifuged, washed, irradiated, and cryopreserved on a Ficoll-Hypaque.

Importantly, patients receiving TIL treatment should not be infused with live feeder cells, as this could lead to graft-versus-host disease (GVHD). Therefore, by irradiating the cells with gamma rays to halt feeder cell growth, double-strand DNA breaks are caused, resulting in loss of cell viability of MNC cells upon reculture.

Evaluation criteria and experimental setup

Feeding batches were evaluated using two criteria: 1) their ability to amplify TILs >100-fold in co-culture, and 2) their impaired replication function.

Feeder cell batches were tested in mini-REP form using two primary pre-REP TIL lines grown in upright T25 tissue culture flasks. Feeder cell batches were tested against two different TIL lines because each TIL population exhibited proliferative capacity upon activation in response to the REP. As a control, large quantities of irradiated MNC feeder cells (which have previously been shown to meet the above criteria) were run concurrently with the test batches.

To ensure that all batches tested in a single experiment undergo the same testing, an adequate stock of the same pre-REP-TIL line can be used to test all conditions and all feeder cell batches.

For each batch of feeder cells tested, there were a total of six T25 flasks: Pre-REP TIL line #1 (2 flasks); Pre-REP TIL line #2 (2 flasks); and feeder cell control (2 flasks). The feeder cells' ability to expand TILs was assessed in the flasks containing TIL lines #1 and #2. The feeder cell control flasks were used to assess the replication incompleteness of the feeder cell batches.

Experimental protocol

Day 2/3, Thaw TIL series

Prepare CM2 medium. Heat CM2 in a 37°C water bath. Prepare 40 mL of CM2 supplemented with 3000 IU/mL IL-2. Keep warm until use. Place 20 mL of preheated CM2 (without IL-2) into two 50 mL conical tubes labeled with the intended TIL line. Remove two designated pre-REP TIL lines from LN ₂ storage and transfer the vials to the tissue culture room. Thaw the vials in a sealed zip-lock storage bag in a 37°C water bath until a small amount of ice remains.

Using a sterile pipette, immediately transfer the contents of the vial to 20 mL of CM2 in a prepared, labeled 50 mL conical tube. Wash the cells with 40 mL of CM2 without IL-2. Centrifuge at 400 x CF for 5 minutes. Aspirate the supernatant and resuspend the cells in 5 mL of warm CM2 supplemented with 3000 IU/mL IL-2.

Take two equal aliquots (20 μL) for cell counting using an automated cell counter. Record the counts. During counting, place the 50 mL conical tube containing TIL cells into a humidified incubator at 37°C and 5% _CO2 , and loosen the cap to allow gas exchange. Determine the cell concentration and dilute the TIL to 1 × ^10⁶ cells/mL in CM2 supplemented with 3000 IU/mL IL-2.

In a humidified 37°C incubator, culture any number of wells in a 24-well tissue culture plate at 2 mL/well as needed until day 0 of the mini-REP. Culture different TIL lines in separate 24-well tissue culture plates to avoid confusion and potential cross-contamination.

Day 0, Mini-REP begins

Prepare sufficient CM2 medium for the number of feeder cell batches to be tested (e.g., prepare 800 mL of CM2 medium for testing 4 feeder cell batches at a time). Divide a portion of the CM2 prepared above and supplement it with 3000 IU/mL IL-2 for cell culture (e.g., prepare 500 mL of CM2 medium supplemented with 3000 IU/mL IL-2 for testing 4 feeder cell batches at a time).

To prevent cross-contamination, each TIL culture was used separately. The 24-well plate containing the TIL culture was removed from the incubator and transferred to the BSC.

Using a sterile pipette or a 100-1000 μL pipette and tip, take about 1 mL of culture medium from each well of the TIL to be used and place it into an unused well of a 24-well tissue culture plate.

Using fresh, sterile pipettes or 100-1000 μL pipettes and tips, mix the remaining culture medium and TIL in the wells to resuspend the cells. Then transfer the cell suspension to a 50 mL conical tube labeled TIL and record the volume.

Wash the wells with the reserved culture medium and transfer the volume to the same 50 mL conical tube. Rotate the cells at 400 x CF to collect the cell pellet. Aspirate the culture supernatant and resuspend the cell pellet in 2–5 mL of CM2 medium containing 3000 IU/mL IL-2. The volume used depends on the number of wells harvested and the size of the pellet—the volume should be sufficient to ensure a concentration >1.3 × ^10⁶ cells/mL.

Thoroughly mix the cell suspension using a serum pipette and record the volume. Use an automated cell counter to dispense 200 μL of the solution for cell counting. During counting, place a 50 mL conical tube containing TIL cells into a 37°C, 5% _CO2 humidified incubator and loosen the cap to allow gas exchange. Record the cell count.

Remove the 50 mL conical tube containing TIL cells from the incubator and resuspend it in warm CM2 supplemented with 3000 IU/mL IL-2 at a concentration of 1.3 × ^10⁶ cells/mL. Place the 50 mL conical tube back into the incubator and loosen the cap.

Repeat the above steps for the second TIL system.

Before seeding TIL into the T25 flasks used for experiments, TIL will be diluted 1:10 to a final concentration of 1.3 × ^10⁵ cells/mL, as described below.

Preparation of MACS GMP CD3 pure (OKT3) working solution

Remove the OKT3 stock solution (1 mg/mL) from the 4°C freezer and place it in BSC. The final concentration used in the mini-REP medium is 30 ng/mL OKT3.

In each T25 flask of this experiment, 600 ng OKT3 is required per 20 mL; this is equivalent to 60 μL of 10 μg/mL solution per 20 mL, or 360 μL for all 6 flasks (testing each batch of feeder cells).

For each batch of feeder cells tested, prepare 400 μL of a 1:100 dilution of 1 mg/mL OKT3 at a working concentration of 10 μg/mL (e.g., for 4 batches of feeder cells tested at one time, prepare 1600 μL of a 1:100 dilution of 1 mg/mL OKT3: 16 μL of 1 mg/mL OKT3 + 1.584 mL of CM2 medium (with 3000 IU/mL IL-2)).

Prepare a T25 flask

Before preparing feeder cells, label each flask and fill it with CM2 medium. Place the flasks in a humidified 5% _CO2 incubator at 37°C to keep the medium warm while awaiting the addition of the remaining components. After preparing the feeder cells, add the components to the CM2 in each flask.

Table 26: Solutions

Preparation of feeder cells

This protocol requires a minimum of 78 × ^10⁶ feeder cells per batch. Each 1 mL vial frozen via SDBB contains 100 × ^10⁶ viable cells at freezing. Assuming a 50% recovery rate after thawing from LN ₂ storage, it is recommended to thaw at least two 1 mL feeder vials per batch, so that each REP is estimated to contain 100 × ^10⁶ viable cells. Alternatively, if supplied in 1.8 mL vials, one vial will provide sufficient feeder cells.

Before thawing the feeder cells, preheat approximately 50 mL of IL-2-free CM2 for each batch of feeder cells to be tested. Remove the designated feeder cell batch vial from LN ₂ storage, place it in a zip-lock storage bag, and keep it on ice. Thaw the vial in the sealed zip-lock bag by immersion in a 37°C water bath. Remove the vial from the zip-lock bag, spray or wipe it with 70% ethanol, and then transfer the vial to the BSC.

Immediately transfer the contents of the feeder cell vial to 30 mL of warm CM2 in a 50 mL conical tube using a transfer pipette. Wash the vial with a small amount of CM2 to remove any residual cells. Centrifuge at 400x CF for 5 minutes. Aspirate the supernatant and resuspend the cells in 4 mL of warm CM2 with 3000 IU/mL IL-2. Take 200 μL for cell counting using an automated cell counter. Record the cell count.

Cells were resuspended at 1.3 × ^10⁷ cells/mL in warm CM2 with 3000 IU/mL IL-2. TIL cells were diluted from 1.3 × ^10⁶ cells/mL to 1.3 × ^10⁵ cells/mL.

Set up co-cultivation

Dilute TIL cells from 1.3 × ^10⁶ cells/mL to 1.3 × ^10⁵ cells/mL. Add 4.5 mL of CM2 medium to a 15 mL conical tube. Remove the TIL cells from the incubator and resuspend them using a 10 mL serum pipette. Take 0.5 mL of cells from the 1.3 × ^10⁶ cells/mL TIL suspension and add it to 4.5 mL of medium in a 15 mL conical tube. Return the TIL stock vial to the incubator. Mix thoroughly. Repeat this process for a second TIL line.

Transfer the culture flasks containing preheated culture medium from the incubator to the BSC for a single batch of feeder cells. Mix the feeder cells by a few up-and-down pipette tips using a 1 mL pipette tip, then transfer 1 mL (1.3 × ^10⁷ cells) into each of the feeder flasks. Add 60 μL of OKT3 working stock solution (10 μg/mL) to each flask. Return the two control flasks to the incubator.

Transfer 1 mL (1.3 × ^10⁵ ) of each TIL group batch to the corresponding labeled T25 flask. Return the flask to the incubator and incubate upright. Do not disturb until the fifth day.

Repeat the process for all tested batches of feeder cells.

Day 5, culture medium changed

Prepare CM2 using 3000 IU/mL IL-2. 10 mL is needed for each flask. Using a 10 mL pipette, transfer 10 mL of warm CM2 containing 3000 IU/mL IL-2 into each flask. Return the flasks to the incubator and incubate upright until day 7. Repeat the procedure for all batches of feeder cells to be tested.

Day 7, Harvest

Remove the flasks from the incubator and transfer them to the BSC, being careful not to disturb the cell layer at the bottom of the flask. Without disturbing the cells growing at the bottom of the flask, remove 10 mL of culture medium from each test flask and 15 mL of culture medium from each control flask.

Using a 10 mL serum pipette, resuspend the cells in the remaining culture medium and mix thoroughly to break up any cell clumps. After thoroughly mixing the cell suspension by pipetting, take 200 μL for cell counting. Count TILs using appropriate standard operating procedures and an automated cell counting device. Record the counts on day 7.

Repeat the process for all batches of feeder cells tested.

Based on the TT table below, assess the replication insufficiency of the feeder cell control flasks and evaluate the fold expansion of the flasks containing TIL from day 0.

On day 7, the feeder cell control flasks were kept on until day 14.

After counting the feeder cells in the control flasks on day 7, add 15 mL of fresh CM2 medium containing 3000 IU/mL IL-2 to each control flask. Return the control flasks to the incubator and incubate upright until day 14.

On day 14, the non-proliferation of the feeder cell control flasks was prolonged.

Remove the flasks from the incubator and transfer them to BSC, being careful not to disturb the cell layer at the bottom of the flask. Without disturbing the cells growing at the bottom of the flask, remove approximately 17 mL of culture medium from each control flask. Using a 5 mL serum pipette, resuspend the cells in the remaining culture medium and mix thoroughly to break up any cell clumps. Record the volume of each flask.

After thoroughly mixing the cell suspension by pipetting, take 200 μL for cell counting. Count the TILs using appropriate standard operating procedures and an automated cell counting device. Record the count.

Repeat the process for all batches of feeder cells tested.

Results and Acceptance Criteria

result

The gamma irradiation dose was sufficient to impair feeder cell replication. All batches were expected to meet the evaluation criteria, and a reduction in the total number of surviving feeder cells remaining on day 7 of REP culture was also demonstrated compared to day 0.

All feeding batches are expected to meet the evaluation criteria of 100-fold increase in TIL growth on day 7 of REP culture.

The expected counts on day 14 of the feeding control flasks continued the non-proliferative trend seen on day 7.

Acceptance Standards

For each batch of feeder cells, each replicate of the TIL line tested met the following acceptance criteria.

The acceptance standard is 2 times, as described below (see Table 27 below).

Table 27: Acceptance Criteria

Detection Acceptance Standards Irradiated MNC/Incomplete Replication Function No growth was observed on days 7 and 14. TIL amplification <![CDATA[Each TIL population should be expanded at least 100-fold (minimum 1.3 × 10⁷ live cells)]]>

To assess whether the irradiation dose was sufficient to induce replication dysfunction in MNC feeder cells when cultured in the presence of 30 ng/mL OKT3 antibody and 3000 IU/mL IL-2. Replication dysfunction was assessed by the total number of viable cells (TVC) determined by automated cell counting on days 7 and 14 of the regeneration period (REP).

The acceptance criterion is "no growth," meaning that the total number of live cells does not increase from the initial number of live cells introduced into the culture on day 0 of the REP on days 7 and 14.

The ability of feeder cells to support TIL expansion was evaluated. TIL growth was measured by the fold increase in viable cells from day 0 of REP to day 7 of REP. On day 7, TIL culture expansion to a minimum of 100-fold (i.e., greater than 100 times the total number of viable TIL cells introduced into culture on day 0 of REP) was measured by automated cell counting.

Contingency Testing for MNC Feeding Batches That Do Not Meet Acceptance Standards.

If an MNC feeder cell batch does not meet any of the above acceptance criteria, the following steps will be taken to retest the batch to rule out simple experimenter error as the cause.

If a batch has two or more remaining satellite test vials, the batch must be retested. If a batch has one or no remaining satellite test vials, the batch is deemed non-compliant according to the acceptance criteria listed above.

For the batches in question and the control batch to be considered acceptable, they must meet the above acceptance criteria. Once these criteria are met, the batch will be released for use.

Example 10: A qualified single batch of peripheral blood mononuclear cells irradiated with gamma rays

This embodiment describes a novel, simplified procedure for qualifying a single batch of gamma-irradiated peripheral blood mononuclear cells (PBMCs) for use as allogeneic feeder cells in the exemplary methods described herein. This embodiment provides a protocol for evaluating batches of irradiated PBMC cells for clinical TIL production batches. Each batch of irradiated PBMCs is prepared from a single donor. In over 100 identification protocols, it has been shown that, in all cases, batches of irradiated PBMCs from the SDBB (San Diego Blood Bank) can achieve >100-fold TIL expansion by day 7 of the regeneration period (REP). This improved identification protocol is intended for batches of irradiated donor PBMCs from the SDBB, which must still be tested to verify that the received gamma irradiation dose is sufficient to impair their replication function. Once they are demonstrated to remain impaired in replication over a 14-day period, the donor PBMC batch is considered "qualified" for use in clinical TIL production batches.

background

For the current standard REP for TILs, growth-arrested PBMCs irradiated with gamma irradiation are required. Membrane receptors on PBMCs bind to anti-CD3 (clonal OKT3) antibodies and cross-link with TILs in the culture, thereby stimulating TIL expansion. PBMC batches are prepared from whole blood leukocytes collected from various donors. Under GMP conditions, the whole blood leukocyte apheresis products are centrifuged, washed, irradiated, and cryopreserved on a Ficoll-Hypaque.

Importantly, patients receiving TIL therapy should not be infused with live PBMCs, as this could lead to graft-versus-host disease (GVHD). Therefore, by administering cellular γ-irradiation to arrest the growth of donor PBMCs, double-strand DNA breaks are caused, and the PBMCs lose cell viability upon reculture.

Evaluation criteria

The evaluation criterion for irradiated PBMC batches is that they have incomplete replication function.

Experimental setup

Using upright T25 tissue culture flasks, the culture batches were tested in mini-REP format, as if they were co-cultured with TIL. A control batch: A batch of irradiated PBMCs proven to meet the criteria in 7.2.1 was tested together with the test batches as a control. For each batch of irradiated donor PBMCs tested, the tests were repeated in flasks.

Experimental protocol

Day 0

Prepare approximately 90 mL of CM2 medium for each batch of donor PBMCs to be tested. Keep CM2 warm in a 37°C water bath. Thaw aliquots of 6 × ^10⁶ IU/mL IL-2. Return the CM2 medium to the BSC, wipe with 70% EtOH, and then place it in a hood. For each batch of PBMCs to be tested, transfer approximately 60 mL of CM2 to a separate sterile bottle. Add IL-2 from the thawed 6 × ^10⁶ IU/mL stock solution to this medium to a final concentration of 3000 IU/mL. Label this bottle “CM2/IL2” (or similar) to distinguish it from any unreplenished CM2.

Preparation of OKT3

Remove the anti-CD3 (OKT3) stock solution from the 4°C freezer and place it in BSC. Use OKT3 at a final concentration of 30 ng/mL in mini-REP medium. Prepare a 10 μg/mL anti-CD3 (OKT3) working solution using the 1 mg/mL stock solution. Store in the freezer until needed.

For each PBMC batch tested, prepare 150 μL of anti-CD3 (OKT3) stock solution diluted 1:100. For example, to test four PBMC batches at once, prepare 600 μL of 10 μg/mL anti-CD3 (OKT3) by adding 6 μL of 1 mg/mL stock solution to 594 μL of CM2 supplemented with 3000 IU/mL IL-2.

Prepare flask

Add 19 ml of CM2/IL-2 to each labeled T25 flask. When preparing cells, place the flasks in a 37°C, humidified, 5% _CO2 incubator.

Preparation of irradiated PBMC

Remove the vials of the PBMC batch to be tested from LN ₂ storage. Place them at -80°C or on dry ice before thawing. For each batch to be thawed, place 30 mL of CM2 (without IL-2 replenishment) into a 50 mL conical tube. Label each tube with the different batch number of the PBMC to be thawed. Cap the tube tightly and place it in a 37°C water bath before use. If necessary, return the 50 mL conical tube to the BSC, wipe with 70% EtOH, and then place it in a hood.

Remove the PBMC vials from the freezer and thaw them in a float rack at 37°C. Thaw until a small amount of ice remains in the vials. Using a sterile transfer pipette, immediately transfer the contents of the vials to a 30 mL CM2 vial in a 50 mL conical tube. Remove approximately 1 mL of culture medium from the tube to rinse the vial; return the rinse solution to the 50 mL conical tube. Cap tightly and gently rotate to wash the cells.

Centrifuge at 400×g for 5 minutes at room temperature. Aspirate the supernatant and resuspend the cell pellet in 1 mL of warm CM2/IL-2 using a 1000 μl pipette tip. Alternatively, resuspend the cell pellet by dragging a capped tube along an empty tube rack before adding culture medium. After resuspending the cell pellet, adjust the volume to 4 mL with CM2/IL-2 medium. Record the volume.

Use an automated cell counter to take a small aliquot (e.g., 100 μL) for cell counting. Count in duplicate according to the specific automated cell counter SOP. PBMCs usually need to be diluted before cell counting. A starting dilution of 1:10 is recommended, but this may vary depending on the type of cell counter used. Record the count.

Using CM2/IL-2 medium, adjust the PBMC concentration to 1.3 × ^10⁷ cells/mL according to step 7.4.15.2. Mix thoroughly by gentle rotation or by gently aspirating with a serum pipette.

Set up the culture flask

Return two labeled T25 flasks to the BSC from the tissue culture incubator. Return the 10 μg/mL anti-CD3/OKT3 vial to the BSC. Add 1 mL of 1.3 × ^10⁷ PBMC cell suspension to each flask. Add 60 μl of 10 μg/mL anti-CD3/OKT3 to each flask. Return the capped flasks to the tissue culture incubator and incubate undisturbed for 14 days. Return the anti-CD3/OKT3 vial to the refrigerator until the next batch is needed. Repeat this process for each batch of PBMCs to be evaluated.

On day 14, the non-proliferation of PBMCs was measured.

Return the duplicated T25 flasks to the BSC. For each flask, using a new 10 mL serum pipette, remove approximately 17 mL from each flask, then carefully pull up the remaining culture medium to measure the volume remaining in the flask. Record the volume.

Use the same serum pipette to mix the sample by blowing up and down.

Take 200 μL of sample from each flask and count the cells. Use an automated cell counter to count the cells. Repeat these steps 7.4.26–7.4.31 for each batch of PBMCs being evaluated.

Results and Acceptance Criteria

result

The gamma irradiation dose was sufficient to impair feeder cell replication. All batches were expected to meet the evaluation criteria and demonstrate a reduction in the total number of surviving feeder cells remaining on day 14 of REP culture compared to day 0.

Acceptance Standards

Each irradiated donor PBMC batch met the following acceptance criteria: "No growth"—meaning that the total number of viable cells on day 14 was less than the initial number of viable cells cultured on day 0 of the REP.

Emergency testing of PBMC batches that do not meet acceptance standards

If an irradiated donor PBMC batch does not meet the above acceptance criteria, the following steps are taken to retest the batch to rule out simple experimenter error as the cause of failure. If the batch has more than two remaining accessory vials, then the batch will be retested. If the batch has one remaining or no remaining accessory vials, then the batch fails according to the acceptance criteria in Section 10.2 above.

To be considered acceptable, both the control batch and two copies of the relevant batch from the PBMC batch that underwent emergency testing met the acceptance criteria. Once this criterion was met, the batch was released for use.

Example 11: Preparation of IL-2 stock solution

This embodiment describes a process for dissolving purified lyophilized recombinant human interleukin-2 into a stock sample suitable for further tissue culture protocols, including all those described in this application and embodiments, including those involving the use of rhIL-2.

step

Prepare a 0.2% acetic acid solution (HAc). Add 29 mL of sterile water to a 50 mL conical tube. Add 1 mL of 1N acetic acid to the 50 mL conical tube. Mix by inverting the tube 2-3 times. Filter the sterilized HAc solution using a Sterelip filter.

Prepare 1% HSA in PBS. Add 4 mL of 25% HSA stock solution to 96 mL of PBS in a 150 mL sterile filter. Filter the solution. Store at 4°C. Record the amount of rhIL-2 prepared in each vial.

Prepare a stock solution of rhIL-2 (final concentration 6 × ^10⁶ IU/mL). Each batch of rhIL-2 is different and information should be found in the manufacturer's Certificate of Analysis (COA), such as: 1) the mass of rhIL-2 per vial (mg), 2) the specific activity of rhIL-2 (IU/mg), and 3) the recommended reconstitution volume (mL) of 0.2% HAc.

The required volume of 1% HSA for a batch of rhIL-2 is calculated using the following equation:

For example, according to CellGenix rhIL-2 batch 10200121COA, the specific activity of a 1 mg vial is × ^10⁶ lU/mg. It is recommended to redissolve rhIL-2 in 2 mL of 0.2% HAc.

Wipe the rubber stopper of the IL-2 vial with alcohol. Using a 16G needle attached to a 3mL syringe, inject the recommended volume of 0.2% HAc into the vial. When withdrawing the needle, be careful not to move the stopper. Invert the vial three times and rotate until all powder is dissolved. Carefully remove the stopper and place it on the alcohol wipe. Add the calculated volume of 1% HSA to the vial.

Storage of rhIL-2 solution. For short-term storage (<72 hours), store vials at 4°C. For long-term storage (>72 hours), aliquot the vials into smaller volumes and store in cryovials at -20°C until ready for use. Avoid repeated freezing/thawing. Expiry date is 6 months after preparation date. The Rh-IL-2 label includes the supplier and catalog number, lot number, expiry date, operator name, concentration, and volume of aliquots.

Example 12: Preparation of culture media for PREP and REP processes

This embodiment describes a procedure for preparing a tissue culture medium for protocols involving the culture of 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 culture medium can be used to prepare any TILs described in this application and the embodiments.

Preparation of CM1

Remove the following reagents from the refrigerator and heat them in a 37°C water bath: (RPMI 1640, human AB serum, 200 mL M glutamine). Prepare CM1 medium according to Table 28 below by adding each component to the top of a 0.2 μm filter unit suitable for the volume to be filtered. Store at 4°C.

Table 28: Preparation of CM1

Element Final concentration Final volume 500mL Final volume IL RPMI1640 NA 450mL 900mL Human AB serum, heat inactivated 10% 50mL 100mL 200mM L-glutamine 2 mM 5mL 10mL 55mM BME 55μM 0.5mL 1mL 50 mg/mL Gentamicin Sulfate 50 μg/mL 0.5mL 1mL

On the day of use, preheat the required amount of CM1 in a 37°C water bath and add 6000 IU/mL IL-2.

Other supplements - as needed, see Table 29.

Table 29: Other supplements for CM1 (as needed)

Preparation of CM2

Following the requirements in Section 7.3 above, remove the prepared CM1 from the refrigerator or prepare fresh CM1. Remove the prepared CM1 from the refrigerator and prepare the required amount of CM2 by mixing the prepared CM1 with an equal volume in a sterile culture medium bottle. On the day of use, add 3000 IU/mL IL-2 to the CM2 culture medium. On the day of use, prepare a sufficient amount of CM2 with 3000 IU/mL IL-2. Label the CM2 culture medium bottle with the name, the preparer's initials, the date of filtration/preparation, and a two-week shelf life. Store at 4°C until needed for tissue culture.

Preparation of CM3

Prepare CM3 on the day it is needed. CM3 is prepared the same as the culture medium, supplemented with 3000 IU/mL IL-2 on the day of use. Prepare sufficient CM3 for the experiment by adding the IL-2 stock solution directly to the AIM-V bottle or bag. Shake gently. Immediately after adding AIM-V, label the bottle “3000 IU/mL IL-2”. If there is excess CM3, store it in a bottle at 4°C and label it with the culture medium name, the preparer's initials, the date of preparation, and its expiration date (7 days after preparation). After storing at 4°C for 7 days, discard the culture medium supplemented with IL-2.

Preparation of CM4

CM4 is the same as CM3, except it is supplemented with 2 mM GlutaMAX™ (final concentration). For every 1 L of CM3, add 10 mL of 200 mM GlutaMAX ^™ . Prepare sufficient CM4 for the experiment by adding the IL-2 stock solution and GlutaMAX ^™ stock solution directly to the AIM-V bottle or bag. Shake gently. Immediately after adding AIM-V, label the bottle “3000 IL/mL IL-2 and GlutaMAX”. If there is excess CM4, store it in a bottle at 4°C, labeling it with the culture medium name “GlutaMAX” and its expiration date (7 days after preparation). After storing at 4°C for 7 days, discard the culture medium supplemented with IL-2.

Example 13: Evaluation of serum-free culture medium used in process 2A

This embodiment provides data evaluating the efficacy of serum-free media as alternatives to the standard CM1, CM2, and CM4 media currently used in process 2A. This study assessed the efficacy of available serum-free media (SFM) and serum-free alternatives as substitutes in three phases.

Phase 1: The efficacy of TIL amplification using standard serum-free medium versus CTS Optimizer or Prime T CDM or Xvivo-20 serum-free medium (with or without serum substitutes or platelet lysis buffer) was compared (n=3).

Phase 2: Candidate serum-free culture medium conditions were tested in a mini-scale 2A process using G-Rex 5M (n=3).

Background Information

Although the current combination of media used in Pre-REP and Post-REP cultures has proven effective, REP failure may occur with AIM-V. If an effective serum-free alternative is identified, the process in CMO will be more straightforward and simpler by reducing the number of media types used from three to one. Furthermore, SFM reduces the chance of incidental disease by eliminating the use of human serum. This example provides data supporting the use of serum-free media in the 2A process.

Table 30: Abbreviations

μL

CM1,2,4 Complete culture medium 1, 2, 4

CTS OpTimizer SFM Cell Therapy System OpTimizer serum-free culture medium

g gram

Hr Hour

IFU User Manual

IL-2 (Interleukin-2 cytokine)

Min

mL

℃ Celsius

PreREP (Pre-Rapid Amplification Protocol)

REP (Rapid Amplification Protocol)

RT (Room temperature)

SR serum alternative

TIL (tumor-infiltrating lymphocytes)

Experimental Design

As described in LAB-008, begin Pre-REP and REP. Figure 55 shows an overview of these three experimental phases.

As shown in Figure 55, this project aims to test serum-free culture medium and supplements in two steps.

Step 1: Select a serum-free culture medium supplier. Set up pre-REP and post-REP to simulate the 2A process in G-Rex 24-well plates. Initiate PreREP by culturing each fragment/well of a G-Rex 24-well plate in triplicate or quadruplicate for each condition. Start REP on day 11 by culturing 4 × 10e5 TIL/well in G-Rex 24-well plates; aliquot on day 16; harvest on day 22. Use CTS OpTimizer, X-Vivo 20, and Prime T-CDM as potential serum-free medium alternatives for use in PreREP and REP. Add CTS immune SR serum alternative (Lifetech) or platelet lysate serum (SDBB) at 3% to SFM. Plan to detect at least 3 tumors in both pre-REP and post-REP for each condition to simulate the 2A process.

Step 2: Further testing of the identified candidates was conducted in a mini-scale 2A process according to protocol (TP-17-007). In short, pre-REP was initiated by culturing 2 fragments/G-Rex 5M flasks in triplicate under each condition. REP was started on day 11 using 2×10e6/G-Rex 5M flasks, aliquoted on day 16, and harvested on day 22.

Note: In one experiment, several tumors were treated and set up to measure multiple parameters.

observe

When comparing the serum-free culture medium used in process 2A with the standard culture medium, equivalent or statistically better cell growth results were observed.

Compared with TILs grown in the standard medium used in process 2A, TILs grown in serum-free medium showed similar phenotypes, IFN-γ production, and metabolite analysis.

result

The efficacy of serum-free culture medium on pre-REP and post-REP TIL amplification was investigated.

CTS Optimizer + SR (serum substitute) showed enhanced pre-REP TIL amplification and comparable REPTIL amplification. CTS OpTimizer, X-Vivo 20, and Prime T-CDM with or without 3% CTS Immune CTS SR were tested against standard conditions. In M1079 and L4026, the CTS Optimizer + CSR condition showed significantly enhanced pre-REP TIL amplification compared to standard conditions (CM1, CM2, CM4) (p<0.05). Conversely, CTS Optimizer without CSR did not aid in pre-REP TIL amplification (Appendices-1,2,3). In two of the three tumors tested, CTS Optimizer + CSR showed comparable TIL amplification in PostREP. Significant variation was generated in both pre-REP and post-REP under X-Vivo 20 and Prime T-CDM conditions, while CTS Optimizer was relatively consistent across the four replicates. Furthermore, compared to the standard, platelet lysis buffer with added SFM did not enhance pre-REP and post-REP TIL amplification. These findings suggest that a serum alternative is certainly needed to provide growth comparable to our standard, and CTS Optimizer + CSR is a potential candidate.

Candidate conditions were tested in the G-Rex 5M mini.

Phenotypic analysis of post-REP TIL. See Table 31 below.

Table 31: CD8 differences using CTS OpTimizer (CD8 skewing)

Comparability of interferon-γ

Interferon-γ ELISA (Quantikine). The production of IFN-γ was detected using the Quantikine ELISA kit from the R&D system. Compared to our standard conditions, CTS+SR produced a considerable amount of IFN-γ.

Example 14: The T cell growth factor mixture IL-2/IL-15/IL-21 enhanced the proliferation and effector function of tumor-infiltrating T cells.

Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes (TILs) has demonstrated clinical efficacy in patients with metastatic melanoma and cervical cancer. In some studies, better clinical outcomes have been positively correlated with the total number of cells infused and/or the percentage of CD8+ T cells. Most current manufacturing protocols utilize only IL-2 to promote TIL growth. Protocols using IL-15 and IL-21 to enhance lymphocyte expansion have been reported. This study describes the positive effects of adding IL-15 and IL-21 to a recently implemented second-generation IL-2-TIL protocol in clinical practice.

Materials and methods

The process of TIL generation involves a pre-rapid expansion protocol (pre-REP), in which tumor fragments of 1–3 ^mm³ are placed in a culture medium containing IL-2. During pre-REP, TILs migrate from the tumor fragments and expand in response to IL-2 stimulation.

To further stimulate TIL growth, TILs were expanded through a secondary culture phase called the Rapid Expansion Protocol (REP), which includes irradiated PBMC feeder cells, IL-2, and anti-CD3. In this study, a shortened pre-REP and REP expansion protocol was developed to expand TILs while maintaining the phenotypic and functional properties of the final TIL product.

This shortened TIL production protocol was used to evaluate the effects of IL-2 alone versus the IL-2/IL-15/IL-21 combination. TILs generated from colorectal cancer, melanoma, cervical cancer, triple-negative breast cancer, lung cancer, and renal cell carcinoma tumors were compared between the two culture protocols. At the end of pre-REP, the amplification, phenotype, function (CD107a+ and IFNγ), and TCR Vβ repertoire of the cultured TILs were assessed.

Pre-REP cultures were initiated using the standard IL-2 (600 IU/mL) protocol, or using a protocol that included IL-15 (180 IU/mL) and IL-21 (IU/mL) in addition to IL-2. Cell expansion was assessed at the end of pre-REP. Cultures were classified as enhanced compared to IL-2 expansion if overall growth was increased by at least 20%. This article presents phenotypic and functional studies of melanoma and lung tumors. See Table 32 below.

Table 32: Enhanced amplification during pre-REP with IL-2/IL-15/IL-21 in multiple indications

These data demonstrate that, compared with IL-2 alone, culturing TILs with IL-2/IL15/IL-21 resulted in increased TIL product yields, in addition to differences in the phenotype and function of lung tumors.

The triple mixture's effect on TIL amplification is indication-specific, with low-yield tumors benefiting the most.

Treatment with NSCLC TIL products increased the CD8+/CD4+ T cell ratio.

As assessed by CD107a expression levels in melanoma and NSCLC, the addition of IL-15 and IL-21 to IL-2 appeared to enhance T cell activity.

The data provided herein suggest that TIL amplification using a shorter, more robust process (such as the 2A process described in this application and other embodiments) can be adapted to include a mixture of IL-2/IL-15/IL-21 cytokines, thereby providing a means to further promote TIL amplification, particularly in certain indications.

Experiments are underway to further evaluate the impact of IL-2/IL-15/IL-21 on TIL function.

Further experiments will evaluate the role of the triple mixture during REP (first amplification).

These observations are particularly relevant to the optimization and standardization of TIL culture protocols required for large-scale TIL production, which offer broad applicability and availability for mainstream anticancer therapies.

Example 15: Evaluation of allogeneic feeder cell:TIL ratio range of 100:1 to 25:1

This study examined TIL proliferation at allogeneic feeder cell ratios of 25:1 and 50:1, relative to the control (currently used in process 1C with a 100:1 allogeneic feeder cell ratio to TIL).

A study published by the Surgical Division of the National Cancer Institute (NCI) showed that the optimal activation threshold for TILs in G-Rex 100 flasks at the start of REP ⁽¹⁾ was 5 × ^10⁶ allogeneic feeder cells/ ^cm² . This was validated by mathematical modeling, and the same model predicted that using a feeder cell layer optimized for cell-to-cell contact per unit area, the ratio of allogeneic feeder cells to TILs could be reduced to 25:1 with minimal impact on TIL activation and expansion.

This study determined the optimal density of feeder cells per unit area at the start of REP and validated the effective range of allogeneic feeder ratios required at the start of REP to reduce and normalize the number of feeder cells used per clinical batch. The study also validated the ability to start REP with fewer than 200 × ^10⁶ TILs co-cultured with a fixed number of feeder cells.

The volume of AT cells (diameter 10 μm): V = (4/3) ^πr³ = 523.6 ^μm³

BG-REX 100(M) cylinder, 40 μm in height (4 cells): V = (4/3) ^πr³ = 4 × ^{10¹² μm³}

C. Number of cells required to fill cylinder B: 4 × ^10¹² μm³ ^{/ 523.6 μm³ =} 7.6 × 10⁸ ^μm³ * 0.64 = 4.86 × ^10⁸

D. The optimal number of cells to activate in 4D space: 4.86 × 10⁸ ^{/ 24} = 20.25 × ^10⁶

E. Number of feeder cells and TILs in G-REX 500: TILs: 100 × ^10⁶ , Feeder cells: 2.5 × ^10⁹

Equation 1: Provides an approximation of the number of mononuclear cells required to activate TILs in an icosahedral geometry within a cylinder with a base of 100 ^cm² . Experimental results for T cell threshold activation (~5 × ^10⁸ ) were calculated, which are very similar to the NCI experimental data ⁽¹⁾ . (C) The multiplier (0.64) is the random packing density of the equivalent spheres calculated by Jaeger and Nagel in 1992 ⁽²⁾ . (D) The divisor 24 is the number of equivalent spheres that can contact similar objects in 4-dimensional space, the “Newton number” ⁽³⁾ .

References

⁽¹⁾ Jin, Jianjian, et.al., Simplified Method of the Growth of Human TumorInfiltrating Lymphocytes (TIL) in Gas-Permeable Flasks to Numbers Needed for Patient Treatment. J Immunother. 2012 Apr; 35(3):283–292.

⁽²⁾ Jaeger HM,Nagel SR.Physics of the granular state.Science.1992 Mar20;255(5051):1523-31.

⁽³⁾ ORMusin(2003)."The problem of the twenty-five spheres".Russ.Math.Surv.58(4):794–795.

Example 16: Production of cryopreserved TIL cells for therapy using a closed system

This embodiment describes the cGMP production of Iovance Biotherapeutics, Inc.'s TIL cell therapy process in G-Rex flasks according to current Good Tissue Practices and current Good Manufacturing Practices. The material will be manufactured from commercial materials in accordance with US FDA Good Manufacturing Practices (21 CFR Parts 210, 211, 1270, and 1271) and ICH Q7 standards applicable to Phase I.

A process overview is provided in Table 33 below.

Table 33: Process Overview

In this embodiment, unless otherwise stated, 1.0 mL/L is assumed to be 1.0 g/kg. Upon opening, the following tests will be performed at 2°C to 8°C: human serum, type AB (HI) Gemini, 1 month; 2-mercaptoethanol, 1 month. Gentamicin sulfate (50 mg/mL stock solution) can be stored at room temperature for 1 month. Bags containing 10 L of AIM-V medium should only be heated once at room temperature and should not be left out for more than 24 hours before use. During the day 22 harvest, TILs can be harvested from G-Rex 500MCS flasks using two Gatherex ^™ instruments.

Day 0, prepare CM1 culture medium.

Prepare RPMI 1640 medium. In a BSC, using an appropriately sized pipette, take 100.0 mL of RPMI 1640 medium from 1000 mL and place it into an appropriately sized container labeled “waste”.

In BSC, add the reagents to the RPMI 1640 culture medium bottles. Add the following reagents to the RPMI 1640 culture medium bottles as shown in the table below. Record the volume added. Volume added per bottle: heat-inactivated human AB serum (100.0 mL); GlutaMax (10.0 mL); Gentamicin sulfate 50 mg/mL (1.0 mL); 2-mercaptoethanol (1.0 mL).

Cap the RPMI 1640 culture medium bottle and rotate the bottle to ensure thorough mixing. Filter the RPMI 1640 medium from step 8.1.6 through a 1L 0.22-micron filter unit. Label the filtered medium. Aseptically cover the filtered medium with the label containing the following information.

Thaw a 1.1 mL aliquot of IL-2 (6 × ^10⁶ IU/mL) (BR71424) until all ice has melted. Record the IL-2 batch number and expiration date. Transfer the IL-2 stock solution to the culture medium. In BSC, transfer 1.0 mL of the IL-2 stock solution to the CM1 Day 0 culture medium bottle prepared in step 8.1.8. Add one bottle of CM1 Day 0 culture medium and 1.0 mL of IL-2 (6 × ^10⁶ IU/mL). Cap the bottle and rotate it to mix the culture medium containing IL-2. Relabel as "Complete Culture Medium CM1 Day 0".

Using an appropriately sized pipette, dispense 20.0 mL of culture medium into a 50 mL conical tube. In the BSC, transfer 25.0 mL of “Complete CM1 Day 0 Culture Medium” (prepared in step 8.1.13) into a 50 mL conical tube. Label the tube “Tissue Piece”. Aseptically transfer the medium into the BSC via the G-Rex100MCS (W3013130). In the BSC, close all clamps on the G-Rex100MCS, leaving the exhaust filter clamp open. Connect the red tubing of the G-Rex100MCS flask to the larger diameter end of the relay pump fluid transfer kit (W3009497) via the Luer connector. Place the Baxa pump next to the BSC. Remove the pump tubing portion of the relay pump fluid transfer kit from the BSC and install it in the relay pump. Inside the BSC, remove and discard the syringe from the Pumped Liquid Dispensing System (PLDS) (W3012720).

Connect the PLDS pipette to the smaller diameter end of the relay pump fluid transfer kit via the Luer connector. Place the pipette tip in the "Complete CM1 Day 0 Medium" for aspiration. Open all clamps between the medium and the G-Rex100MCS. Pump the Complete CM1 medium into the G-Rex100MCS flask. Set the pump speed to "High" and "9", and then pump all the Complete CM1 Day 0 medium into the G-Rex100MCS flask. After transferring all the medium, clean the tubing and stop pumping.

Disconnect the pump from the flask. Ensure all clamps on the flasks are closed except for the vent filter. Remove the relay pump fluid transfer kit from the red culture medium line and place a red cap (W3012845) on the red culture medium line. Remove the G-Rex100MCS flask from the BSC and heat-seal the red cap on the red line near the terminal Luer connector. Label the G-Rex100MCS flask with the "Day 0" label provided by QA. Label the sample with the following "Day 0" label. Incubator parameters: 37.0±2.0℃; _CO2 percentage: 5.0±1.5% _CO2 .

Place the 50mL conical tube in an incubator and heat for ≥30 minutes.

Day 0, preparation of tumor washing culture medium.

Add gentamicin to the HBSS. In BSC, add 5.0 mL of gentamicin (W3009832 or W3012735) to one 500 mL HBSS medium (W3013128) bottle. Record the volume. Add to each bottle: HBSS (500.0 mL); gentamicin sulfate 50 mg/mL (5.0 mL). Mix the reagents thoroughly. Filter the gentamicin-containing HBSS prepared in step 8.2.1 through a 1 L 0.22 μm filter unit (W1218810). Aseptically cap the filtered medium and label it with the following information.

Day 0, Tumor Treatment

Obtain tumor specimens and immediately transfer them to a 2°C to 8°C kit for processing and recording tumor information. Label three 50mL conical tubes: the first is labeled "Forceps," the second "Scalpel," and the third "Fresh Tumor Washing Medium." Label five × 100mm culture dishes "Wash 1," "Wash 2," "Wash 3," "Hold," and "Poor." Label one six-well plate "Good Intermediate Fragment."

Using an appropriately sized pipette, transfer 5.0 mL of “Tumor Washing Medium” to each well of a 6-well plate to obtain good intermediate tumor fragments (total 30.0 mL). Using an appropriately sized pipette, transfer 50.0 mL of the “Tumor Washing Medium” prepared in step 8.2.4 to each of the “Wash 1,” “Wash 2,” “Wash 3,” and “Hold” 100 mm culture dishes (total 200.0 mL). Using an appropriately sized pipette, transfer 20.0 mL of the “Tumor Washing Medium” prepared in step 8.2.4 to each of the 50 mL Erlenmeyer flasks (total 60.0 mL). Aseptically remove the caps from both 6-well plates. Use the caps for the selected tumor fragments. Aseptically deliver the tumor to the BSC. Record the treatment start time.

Tumor Washing 1: Using forceps, remove the tumor from the specimen bottle and transfer it to "Washing 1". Gently wash the tumor with forceps and record the time. According to the sample plan, transfer 20.0 mL (or the available volume) of solution from the tumor specimen bottle to a 50 mL Erlenmeyer flask. Label and store the collected bioload sample at 2°C to 8°C until submission for testing.

Tumor Washing 2: Using a new set of forceps, remove the tumor from the "Washing 1" dish and transfer it to the "Washing 2" dish. Gently agitate with the forceps for ≥3 minutes to wash the tumor specimen, then let it stand. Record the time.

Using a pipette, drop 4 individual drops of tumor wash culture medium from the conical flask into each of the 6 circles on the top flap (2 flaps) of the 6-well plate. Place one additional drop in two circles, for a total of 50 drops.

Tumor Washing 3: Using forceps, remove the tumor from the "Washing 2" dish and transfer it to the "Washing 3" dish. Gently agitate the tumor specimen with forceps and let it stand for ≥3 minutes. Record the time.

Place the ruler under the 150mm petri dish lid. Using forceps, aseptically transfer the tumor specimen onto the 150mm dissecting dish lid. Arrange all tumor samples from beginning to end, recording the approximate total length and number of fragments. Assess the tumor for necrotic tissue/fatty tissue. Assess whether >30% of the entire tumor area is necrotic tissue and/or fatty tissue; if so, ensure the tumor size is appropriate. Assess whether <30% of the entire tumor area is necrotic tissue or fatty tissue; if so, proceed.

Cleaning and dissection. If the tumor is large and >30% of the external tissue is observed to be necrotic/fat, a "cleaning and dissection" is performed by removing the necrotic/fatty tissue using a combination of scalpel and/or forceps, while preserving the internal structure of the tumor. To maintain the internal structure of the tumor, only vertical cutting pressure is used. No scalpel sawing motion is used.

Using a combination of scalpel and/or forceps, cut the tumor sample into uniform, appropriately sized fragments (up to 6 intermediate fragments). To preserve the internal structure of the tumor, use only vertical cutting pressure. Do not use a sawing motion with the scalpel. Ensure that the undissected intermediate fragments are completely immersed in the "tumor washing medium". Transfer each intermediate fragment to a "preservation" dish.

One intermediate fragment is processed at a time, cutting the tumor fragment in the dissecting dish into small pieces of approximately 3mm × 3mm × 3mm to minimize the amount of hemorrhagic, necrotic, and/or fatty tissue in each piece. Only vertical cutting pressure is used to preserve the internal structure of the tumor. No scalpel or sawing motion is employed.

Select up to eight (8) tumor fragments without hemorrhage, necrotic tissue, and/or adipose tissue. Use a ruler as a reference. Continue dissection until eight good fragments are obtained, or dissect an entire intermediate fragment. Transfer each selected fragment to one of the drops of "tumor washing medium".

After selecting up to eight (8) small pieces from the intermediate fragments, the residue of the intermediate fragments is placed into a new single well of the “good intermediate fragment” 6-well plate.

If the required tissue remains, select additional good tumor fragments from the "Good Intermediate Fragments" 6-well plate to fill the droplet to a maximum of 50. Record the total number of anatomical fragments produced.

Remove the 50mL conical tube containing the tissue fragment from the incubator. Ensure the conical tube is heated for ≥30 minutes. Place the 50mL conical tissue fragment into the BSC, ensuring that the sterility of the open-processed surface is not compromised.

Using a pipette, scalpel, forceps, or a combination thereof, transfer the selected 50 best tumor fragments from the appropriate culture dish lid into a 50 mL conical tube for "tissue fragments". If a tumor fragment falls off during transfer and the desired tissue is preserved, additional fragments from the wells of good tumor intermediate fragments should be added. Record the number of fragments.

Remove the G-Rex100MCS containing culture medium from the incubator. Aseptically transfer the G-Rex100MCS flask into the BSC. When transferring the flask, do not hold it by the lid or bottom. Transfer the flask by holding it by the side. In the BSC, lift the lid of the G-Rex100MCS flask, ensuring the internal tubing remains sterile. Rotate the conical tube containing the tumor fragment to suspend the tumor fragment, and quickly pour the contents into the G-Rex100MCS flask. Ensure the tumor fragment is evenly distributed on the membrane of the flask. If necessary, gently shake the flask back and forth to evenly distribute the tumor fragment. Record the number of tumor fragments on the membrane at the bottom of the container and observe the number of tumor fragments floating in the container. Note: If the number of fragments inoculated does not equal the number collected, contact area management and the documentation in Section 10.0.

G-Rex100MCS was incubated with the following parameters: G-Rex flask incubation; temperature LED display: 37.0℃ ± 2.0℃; _CO2 percentage: 5.0% ± 1.5% _CO2 . Calculations were performed to determine the appropriate time to remove the G-Rex100MCS from the incubator on day 11. Calculations: Incubation time; Lower limit = Incubation time + 252 hours; Upper limit = Incubation time + 276 hours.

Day 11 - Culture Medium Preparation

Monitor the incubator. Incubator parameters: Temperature LED display: 37.0℃±2.0℃; _CO2 percentage: 5.0±1.5% _CO2 . Heat 3 x 1000 mL RPMI 1640 medium (W3013112) bottles and 3 x 1000 mL AIM-V (W3009501) bottles in the incubator for ≥30 minutes. Record the time. Medium: RPMI 1640 and AIM-V. Place another 1 x 1000 mL AIM-V medium (W3009501) bottle at room temperature for future use.

When the time is up, remove the RPMI 1640 medium. Record the end of incubation time as described in step 8.4.4. Ensure the medium is heated for ≥30 minutes. In the BSC, take 100.0 mL from each of three preheated 1000 mL RPMI 1640 medium bottles and place them into appropriately sized containers labeled “Waste.” In the BSC, add the following reagents to each of the three RPMI 1640 medium bottles and record the volume added to each bottle: GemCell human serum, heat-inactivated type AB (100.0 mL), GlutaMax (10.0 mL), Gentamicin sulfate, 50 mg/mL (1.0 mL), 2-mercaptoethanol (1.0 mL).

Cap the bottles and rotate to ensure thorough mixing of the reagents. Filter each bottle of culture medium through a separate 1L 0.22-micron filter. Aseptically cap the filtered culture medium and label each bottle “CM1 Day 11 Culture Medium”. Thaw 3 × 1.1 mL aliquots of IL-2 (6 × ^10⁶ IU/mL) (BR71424) until all ice is thawed. Record the IL-2 batch number and expiration date.

Remove three vials of AIM-V medium from the incubator. Record the end of incubation time. Ensure the medium has been heated for ≥30 minutes. Using a micropipette, add 3.0 mL of thawed IL-2 to a 1 L vial of preheated AIM-V medium. After adding IL-2, rinse the micropipette tip with medium. Use a new sterile micropipette tip for each aliquot. Record the total volume added. Label the vial “IL-2 containing AIM-V”. Aseptically transfer the 10 L Labtainer bag and relay pump transfer kit to the BSC. Close all tubing on the 10 L Labtainer bag. Connect the larger diameter tubing end of the relay pump transfer kit to the center female port of the 10 L Labtainer bag using a Luer lock connection.

Install the Baxa pump next to the BSC. Connect the transfer kit tubing through the Baxa pump. Set the Baxa pump to "High" and "9". Remove the syringe from the Pumped Liquid Dispensing System (PLDS) and discard it. Ensure the sterility of the PLDS pipette is not compromised.

Connect the PLDS pipette to the smaller diameter end of the relay pump fluid transfer kit via the Luer connector. Place the pipette tip into the AIM-V culture medium bottle containing IL-2 (prepared in step 8.4.13) for aspiration. Open all clamps between the culture medium bottle and the 10L Labtainer.

Using a PLDS, transfer the prepared preheated AIM-V medium containing IL-2, along with two additional AIM-V bottles, into a 10L Labtainer bag. Add three bottles of filtered CM1 Day 11 medium. After adding the last bottle, remove the tubing from the bag connection. Note: Stop the pump between each addition of medium. Remove the PLDS from the transfer kit and cap the Luer connector of the tubing in the BSC with a red cap. Gently knead the bag to mix. Affix the following information to the medium bag: Expiry date is 24 hours from the preparation date.

Connect a 60 mL syringe to the available female port of the "Complete CM2 Day 11 Culture Medium" bag. Discard 20.0 mL of culture medium into a 50 mL conical tube. Place a red cap on the female port of the "Complete CM2 Day 11 Culture Medium" bag. Label the bag and store the culture medium sample at 2°C to 8°C until submission for testing. Heat-seal the red cap on the transfer kit tubing near the red cap. Leave the transfer kit on the bag.

In the BSC, add 4.5 mL of AIM-V medium labeled "For Cell Counting Dilution" and batch number to four 15 mL conical tubes. Label the tubes with batch number and tube number (1-4). Label the four cryopreservation tubes "Feder Cells" and tube number (1-4). Transfer all remaining 2-mercaptoethanol, GlutaMax, and human serum from the BSC to 2°C to 8°C.

On the outside of the BSC, weld the 1L transfer pack to the transfer kit on the prepared "Complete CM2 Day 11 Culture Medium" bag. Label the transfer pack "Feeder Cell CM2 Culture Medium" and the batch number. Make a mark on the tubing of the 1L transfer pack a few inches from the bag. Place the empty transfer pack on a balance until the tubing reaches the marked point. Tare the balance, leaving the empty transfer pack on the balance.

Set the Baxa pump to "Medium" and "4". Pump 500.0 ± 5.0 mL of the "Complete CM2 Day 11" medium prepared in step 8.4.22 into the "Cell CM2 Medium" transfer pack. Weigh and record the volume of complete CM2 medium added to the transfer pack.

Once filled, heat-seal the tubing. Separate the CM2 Day 11 medium bag with the transfer kit from the feeder cell medium transfer pack, keeping the weld facing the 1L transfer pack. Place the prepared “Complete CM2 Day 11 Medium” in an incubator until used.

Day 11 - TIL Harvest

Incubator parameters: Temperature LED display: 37.0℃±2.0℃; _CO2 percentage: 5.0±1.5% _CO2 . Before removing the G-Rex100MCS from the incubator, check to ensure compliance with the culture parameters. The lower limit is the same as above.

Record the time of removal from the incubator. Carefully remove the G-Rex100MCS from the incubator, ensuring that all clamps are closed except for the large filter tubing. Record the processing start time.

Label the 300mL transfer pack as “TIL suspension”. Transfer the TIL suspension from the aseptically welded gravity blood filter (single line). Place the 300mL transfer pack on a balance and record the dry weight. Label the 1L transfer pack as “supernatant”.

Aseptically weld the red culture medium removal line from the G-Rex100MCS to the "Supernatant" transfer pack. Aseptically weld the clear cell removal line from the G-Rex100MCS to one of the two spike lines on top of the blood filter connected to the "TIL Suspension" transfer pack. Place the G-Rex100MCS on the left side of the GatheRex and the "Supernatant" and "TIL Suspension" transfer packs on the right side of the GatheRex.

Attach the red culture medium extraction line from the G-Rex100MCS to the top clip (marked with a red line) and tubing on the GatheRex. Attach the clear harvest line from the G-Rex100MCS to the bottom clip (marked with a blue line) and tubing on the GatheRex. Connect the gas line from the GatheRex to the sterile filter of the G-Rex100MCS flask. Before removing the supernatant from the G-Rex100MCS flask, ensure all clips on the cell extraction line are closed. Transfer approximately 900 mL of culture supernatant from the G-Rex100MCS to a 1 L transfer bag. Visually inspect the G-Rex100MCS flask to ensure it is level and the culture medium has been reduced to the end of the aspirator tube.

After removing the supernatant, close all clamps to the red tubing.

Tap the flask firmly and rotate the culture medium to release the cells. Check the flask to ensure all cells have been separated. Note: If cells are not separated, contact area management. Tilt the flask away from the collection tube to allow the tumor fragment to settle along the edge. Slowly tilt the flask toward the collection tube, leaving the fragment on the other side of the flask. If the cell collection pipette is not at the junction of the wall and bottom membrane, tapping the flask at a 450-degree angle is usually sufficient to correctly position the pipette.

Release all clamps leading to the TIL suspension transfer pack. Using GatheRex, transfer the cell suspension through a blood filter into the 300 mL transfer pack. Keep the edges slanted until all cells and culture medium have been collected. Examine the membrane for adherent cells. Rinse the bottom of the G-Rex 100 MCS. Cover approximately 1/4 of the gas exchange membrane with rinse culture medium. Ensure all clamps are closed. Heat-seal the TIL suspension transfer pack (following procedure note 5.12) as close to the weld as possible to keep the manifold length approximately the same. Heat-seal the “supernatant” transfer pack. Maintain sufficient weld line. Record the weight of the TIL suspension transfer pack and calculate the volume of the cell suspension.

Solder the 4-inch plasma transfer kit to the "supernatant" transfer pack, keeping the Luer connector attached to the 4-inch plasma transfer kit, and transfer to the BSC. Solder the 4-inch plasma transfer kit to the 300 mL "TIL suspension" transfer pack, keeping the Luer connector attached to the 4-inch plasma transfer kit, and then transfer to the BSC.

Draw approximately 20.0 mL of supernatant from the 1 L “Supernatant” transfer pack and add it to a sterile 50 mL conical tube labeled “Bac-T”. Using an appropriately sized syringe, remove 1.0 mL of sample from the 50 mL conical-marked Bac-T and inoculate it into an anaerobic flask.

Label four cryovials with vial numbers (1-4). Using a separate 3 mL syringe, extract 4 x 1.0 mL cell counting samples from the TIL suspension transfer pack using the Luer connector and place them into their respective cryovials. Place a red cap (W3012845) on the line. Keep the TIL transfer pack in an incubator until needed. Perform cell counting and calculations. Perform an initial undiluted cell count. If no dilution is required, set "Sample [μL]" = 200 and "Dilution [μL]" = 0.

Record cell counts and TIL counts. If the total number of viable TIL cells is less than 5 × ^10⁶ , proceed to "Day 11 G-Rex Filling and Seeding". If the total number of viable TIL cells is greater than 5 × ^10⁶ , continue with "Flow Cytometry Calculation".

Flow cytometry calculation

If the total number of viable TIL cells is ≥4.0 × ^10⁷ , calculate the volume of 1.0 × ^10⁷ cells to be obtained from the flow cytometry sample. Total number of viable cells required for flow cytometry: 1.0 × ^10⁷ cells. Cell volume required for flow cytometry: viable cell concentration divided by 1.0 × ^10⁷ cells.

If applicable: Recalculate the total number of viable cells and volumetric flow rate. Calculate the remaining total number of viable cells and remaining volume after removing the cell count sample below.

TIL cryopreservation of samples

If applicable: Calculate the volume required for cryopreservation. Calculate the cell volume required to obtain 1.0 × ^10⁷ cryopreserved cells.

Table 534: Calculations for cryopreservation

If applicable: Remove the sample for cryopreservation. Take the calculated volume from the TIL suspension transfer kit. Place it into an appropriately sized conical tube and label it "Cryopreservation Sample 1 × ^10⁷ Cells," along with the date and batch number. Place a red cap (W3012845) on the TIL suspension transfer kit.

Centrifuge “Cryopreserved Sample 110 ⁷ Cells” according to the following parameters: speed: 350×g, time: 10:00 min, temperature: ambient, braking: full (9); acceleration: full speed (9).

Add CS-10. Aseptically aspirate the supernatant from the BSC. Gently tap the bottom of the tube to resuspend the cells in the remaining liquid. Add CS-10. Slowly add 0.5 mL of CS-10. Record the volume added. Fill cryopreservation vials to approximately 0.5 mL.

Day 11 - Feeder Cells

Obtain three bags of feeder cells from an LN2 freezer, each with at least two different batch numbers. Store the cells on dry ice until ready to thaw. Record the feeder cell information. Confirm that at least two different batches of feeder cells have been obtained. Place the feeder cell bags into individual zip-top bags according to the batch numbers and thaw in a 37.0°C ± 2.0°C water bath or cell warmer for approximately 3 to 5 minutes, or until the ice has just disappeared.

Prepare the cell feeder filaments. Solder the 4S-4M60 to the CC2 Cell Connect (W3012820), replacing the single tip of the Cell Connect device with the four tips of the 4S-4M60 manifold. Solder as needed.

Additional culture medium transfer pack. Solder the "Cell Feeder CM2 Culture Medium" transfer pack to the CC2 Luer interface. Attach the bag to the side of the harness via the needle-free injection port. Transfer the component containing complete CM2 Day 11 culture medium to the BSC.

Combine the thawed feeder cells. In the BSC, aspirate 10 mL of air into a 100 mL syringe. Replace the 60 mL syringe on the CC2 with this. Before removing the cap, wipe each port on the feeder cell bag with an alcohol pad. Insert the three tips of the CC2 into the three feeder cell bags. Maintain constant pressure while rotating the tips in one direction. Ensure not to puncture the sides of the ports. Open the stopcock valve to open the tubing removed from the feeder cell bags and close the tubing leading to the needleless injection port. Aspirate the contents of the feeder cell bags into the syringe. Immediately drain all three bags. After draining the feeder cell bags, clamp the tubing on the feeder cell bags while maintaining syringe pressure. Do not detach the syringe from the tubing. Record the total volume of feeder cells in the syringe.

Add feeder cells to the transfer bag. Turn the stopcock valve to close the tubing to the feeder cell bag and open the tubing to the culture medium transfer bag. Ensure the tubing to the culture medium transfer bag is not loose. Add the feeder cells from the syringe to the "Feeder Cell CM2 Culture Medium" transfer bag. Clamp the tubing of the transfer bag containing the feeder cells, leaving the syringe on the tubing. Press the bag to mix the combined feeder cells in the transfer bag. Label the bag "Feeder Cell Suspension".

Calculate the total volume of the feeder cell suspension. Remove the cell counting samples. Using a separate 3 mL syringe, extract 4 × 1.0 mL of cell counting sample from the feeder cell suspension transfer pack using the needle-free injection port. Aliquot each sample into labeled cryovials.

Cell counting and calculations were performed using NC-200 and process note 5.14. Cell counting samples were diluted by adding 0.5 mL of cell suspension to 4.5 mL of AIM-V medium labeled "For Cell Counting Dilution". This produced a 1:10 dilution.

Record the cell count and sample volume. If the total number of viable cells is less than 5 × ^10⁹ , continue. If the total number of viable cells is ≥ 5 × ^10⁹ , proceed as described above to obtain a higher cell count. Obtain additional feeder cells as needed, adding them to the transfer pack as described above. Calculate the volume of feeder cell suspension required to obtain 5 × ^10⁹ viable feeder cells. Calculate the volume of excess feeder cells to be removed. Round down to the nearest integer.

Remove excess feeder cells. In a new 100mL syringe, aspirate 10mL of air and connect the syringe to the tubing. Open the tubing of the "Feder Cell Suspension" transfer kit. Using the syringe, aspirate the calculated feeder cell volume, then transfer another 10.0mL from the transfer kit into the 100mL syringe. After removing the feeder cell volume, close the tubing leading to the feeder cell suspension transfer kit. Do not remove the final syringe. Replace the syringe with a new one when it is full. Multiple syringes can be used to remove the total volume. Aspirate 10mL of air into each new syringe. Record the total volume of feeder cells removed (including the additional 10mL).

Add OKT3. In the BSC, using a 1.0 mL syringe and a 16G needle, draw 0.15 mL of OKT3. Aseptically remove the needle from the syringe and connect the syringe to the needleless injection port. Inject the OKT3. Open the stopcock valve to enter the "feeder cell suspension" transfer pack and add 10 mL of previously removed feeder cells to flush the OKT3. Invert the syringe and push air through to clear the tubing leading to the feeder cell suspension transfer pack. Leave the remaining feeder cell suspension in the syringe. Close all clamps and then remove the wiring harness from the BSC. Heat-seal the feeder cell suspension transfer pack, leaving enough tubing for soldering.

Day 11: G-Rex filling and injection

Set up the G-Rex500MCS. Remove the G-Rex500MCS from the packaging and inspect the flask for any cracks or kinks. Ensure all Luer connections and seals are tightened. Close all clamps on the G-Rex500MCS lines except the vent filter line. Draw a line at the 4.5L mark using a marker. Remove the "Complete CM2 Day 11 Medium" from the incubator.

Prepare the pumping medium. Solder the red tubing of the G-Rex500MCS to the relay pump transfer kit connected to the Complete CM2 Day 11 medium. Hang the "Complete CM2 Day 11 Medium" bag on the infusion stand. Feed the pump tubing through the Baxa pump. Pump the medium into the G-Rex500MCS. Set the Baxa pump to "High" and "9". Pump 4.5L of medium into the G-Rex500MCS, filling to the 4.5L mark on the flask. Heat-seal the red tubing of the G-Rex500MCS near the solder joint. Label the flask "Day 11". Solder the feeder cell: suspension transfer pack to the flask. Aseptically solder the red tubing of the G-Rex500MCS to the "feeder cell suspension" transfer pack.

Add feeder cells to the G-Rex500MCS. Open all clamps between the feeder cell suspension and the G-Rex500MCS, and add the feeder cell suspension to the flask via gravity feed. Heat-seal the red tubing near the feeder cell suspension. Weld the TIL suspension transfer pack to the flask. Aseptically weld the red tubing of the G-Rex500MCS to the TIL suspension transfer pack.

Add TIL to the G-Rex500MCS. Open all clamps between the TIL suspension and the G-Rex500MCS and add the TIL suspension to the flask via gravity feed. Heat-seal the red tubing near the weld to remove the TIL suspension bag.

Incubate the G-Rex500MCS. Check that all clamps on the G-Rex500MCS, except for the large filter line, are closed, and place it in the incubator. Incubator parameters: Temperature LED display: 37.0℃±2.0℃, _CO2 percentage: 5.0±1.5% _CO2 .

Calculate the incubation window. Perform calculations to determine the correct time to remove G-Rex500MCS from the incubator on day 16. Lower limit: Incubation time + 108 hours. Upper limit: Incubation time + 132 hours.

Day 11, excessive TIL frozen.

Freeze any excess TIL vials. Record and verify the total number of vials placed in the Controlled Rate Freezer (CRF). Once freezing is complete, transfer the vials from the CRF to appropriate storage containers.

On day 16, culture medium was prepared.

Preheat AIM-V medium. At least 12 hours before use, remove three 10L CTS AIM-V medium bags from 2°C to 8°C and store them at room temperature, protected from light. Label each bag. Record the start time and date of preheating. Ensure all bags are preheated for 12 to 24 hours.

Connect the larger diameter end of the fluid pump transfer kit to one of the female ports on the 10L Labtainer bag using the Luer interface connector. Set the 10L Labtainer for supernatant, labeled "Supernatant". Before removing it from the BSC, ensure all clamps are closed.

Thaw 5 x 1.1 mL aliquots of IL-2 (6 × ^10⁶ IU/mL) (BR71424) from each CTS AIM V culture bag until all ice has melted. Aliquot 100.0 mL of Glutamax into appropriately sized receiving containers. Record the volume added to each receiving container and label each container “GlutaMax”.

Add IL-2 to GlutaMax. Using a micropipette, add 5.0 mL of IL-2 to each GlutaMax receiver. Ensure the pipette tip is rinsed as per Instructions 5.18, using a new pipette tip for each additional mL. Record the volume added to each GlutaMax receiver, labeling each receiver “GlutaMax + IL-2” and the receiver number.

Prepare the CTS AIM V culture medium bag for formulation. Ensure the CTS AIM V 10L culture medium bag (W3012717) is preheated at room temperature and kept in the dark for 12 to 24 hours before use. Record the end of incubation time. In the BSC, secure the sealing clip to the 4-inch plasma transfer kit and connect it to the bag using the spike port. Maintain constant pressure while rotating the spike in one direction. Ensure not to puncture the side of the port. Connect the larger diameter end of the relay pump fluid transfer kit to the 4-inch plasma transfer kit via the Luer interface.

Install the Baxa pump next to the BSC. Remove the pump tubing section of the relay pump fluid transfer kit from the BSC and install it into the relay pump.

Prepare the culture medium. In the BSC, remove and discard the syringe from the Pumped Liquid Dispensing System (PLDS). Ensure the sterility of the PLDS pipette is not compromised. Connect the PLDS pipette to the smaller diameter end of the relay pump fluid transfer kit via the Luer interface connection, placing the pipette tip in the "GlutaMax+IL-2" prepared for aspiration. Open all clamps between the receiver and the 10L bag.

Pump GlutaMax+IL-2 into the bag. Set the pump speed to "Medium" and "3" and pump all of the GlutaMax+IL-2 into the 10L CTS AIM V culture medium bag. Once no solution remains, clean the tubing and stop the pump. Record the volume of GlutaMax containing IL-2 added to each of the AIM V bags below.

Remove the PLDS. Ensure all clamps are closed, and remove the PLDS pipette from the relay pump fluid transfer kit. Remove the relay pump fluid transfer kit and replace the red cap on the 4-inch plasma transfer kit.

Label each prepared “Complete CM4 Day 16 Culture Medium” bag.

According to the sample plan, remove and retain the culture medium. Using a 30 mL syringe, remove 20.0 mL of “Complete CM4 Day 16 Culture Medium” by connecting the syringe to the 4-inch plasma transfer kit, and then add the sample to a 50 mL conical tube. After removing the syringe, ensure that the 4-inch plasma transfer kit is clamped or capped with the red cap.

Connect the new relay pump fluid transfer kit. Connect the larger diameter end of the new fluid pump transfer kit to the 4-inch plasma transfer kit connected to the "Complete CM4 Day 16 Culture Medium" bag. Label with the sample plan inventory label and store the culture medium sample at 2°C to 8°C until submission for testing.

Monitor the incubator. If applicable, monitor the other prepared bags. Incubator parameters: Temperature LED display: 37.0±2.0℃, _CO2 percentage: 5.0±1.5% _CO2 .

Heat the complete CM4 Day 16 culture medium. Heat the first bag of complete CM4 Day 16 culture medium in an incubator for ≥30 minutes until usable. If applicable, heat the other bags.

Prepare the dilution buffer. In BSC, add 4.5 mL of AIM-V medium labeled "For Cell Counting Dilution" to each 4 x 15 mL conical tube. Label the conical tubes. Label 4 cryovials.

Day 16, REP repackaging

Monitoring incubator. Incubator parameters: Temperature LED display: 37.0±2.0℃, _CO2 percentage: 5.0±1.5% CO2 _.

Remove the G-Rex500MCS from the incubator. Before removing the G-Rex500MCS from the incubator, perform the following checks to ensure that the culture parameters are met: upper limit, lower limit, and removal time. Remove the G-Rex500MCS from the incubator.

Heat-seal the 1L transfer package (W3006645), leaving approximately 12 inches of tubing. Label the 1L transfer package “TIL Suspension”. Place the 1L transfer package, including the entire tubing, on a scale and record the dry weight.

GatheRex Setup. Aseptically solder the red culture medium extraction line from the G-Rex500MCS to the relay pump transfer kit on the prepared 10L Labtainer bag "supernatant". Aseptically solder the clear cell extraction line from the G-Rex500MCS to the prepared TIL suspension transfer kit. Place the G-Rex500MCS flask on the left side of the GatheRex. Place the supernatant lab container bag and TIL suspension transfer kit on the right side. Install the red culture medium extraction line from the G-Rex500MCS to the top clip (marked with a red line), and install the tubing on the GatheRex. Install the clear harvest line from the G-Rex500MCS to the bottom clip (marked with a blue line), and install the tubing on the GatheRex. Connect the GatheRex gas line to the sterile filter of the G-Rex500 MCS. Note: Before removing the supernatant from the G-Rex500MCS, ensure that all clips on the cell extraction line are closed.

Reduce the volume of the G-Rex500MCS. Following SOP-01777, transfer approximately 4.5 L of culture supernatant from the G-Rex500MCS to a 10 L Labtainer. Visually inspect the G-Rex500MCS to ensure the flask level and culture medium are reduced to the end of the pipette.

Prepare the flask for TIL harvesting. After removing the supernatant, close all clamps to the red tubing.

Begin TIL harvesting. Record the start time of TIL harvesting. Tap the flask firmly and rotate the culture medium to release the cells. Check the flask to ensure all cells have been separated. Tilt the flask to ensure the tube is at the edge of the flask. If the cell collection tube is not at the junction of the wall and bottom membrane, tapping the flask at a 450-degree angle is usually sufficient to properly position the tube.

TIL Harvest. Release all clamps leading to the TIL suspension transfer bag. Transfer the cell suspension into the TIL suspension transfer bag using GatheRex. Note: Ensure the slanted edges are maintained until all cells and culture medium have been collected. Check for adherent cells on the membrane.

Rinse the membrane in the flask. Rinse the bottom of the G-Rex500MCS. Cover about 1/4 of the gas exchange membrane with rinse medium. Close the clamps on the G-Rex500MCS. Make sure all clamps on the G-Rex500MCS are closed.

Heat-seal. Heat-seal the transfer package containing the TIL as close to the weld as possible to keep the total tube length approximately the same. Heat-seal the 10L Labtainer containing the supernatant and send it to the BSC for sample collection.

Record the weight of the transfer pack containing the cell suspension and calculate the volume of suspension. The prepared transfer pack is used for sample removal. Solder the 4-inch plasma transfer kit to the TIL suspension transfer pack from above, with the mother Luer interface end as close to the bag as possible.

Remove the test sample from the cell supernatant. In the BSC, using a mother Luer port and an appropriately sized syringe, remove 10.0 mL of supernatant from a 10 L laboratory container. Place it into a 15 mL conical tube, label it "BacT," and retain this tube for BacT sample preparation. Using another syringe, remove 10.0 mL of supernatant and place it into a 15 mL conical tube. Retain the tube for mycoplasma sample preparation. Label the tube "Mycoplasma Diluent." Seal the supernatant bag. Place a red cap on the Luer port to close the bag and remove it from the BSC.

Remove the cell counting samples. In the BSC, use a separate 3 mL syringe to remove 4 x 1.0 mL cell counting samples from the TIL suspension transfer pack using a Luer connector. Place the samples into the cryovials prepared above.

Remove the mycoplasma sample. Using a 3 mL syringe, remove 1.0 mL from the TIL suspension transfer pack and place it into the 15 mL cone-shaped label "Mycoplasma Diluent" prepared above. Label and store the mycoplasma sample at 2°C to 8°C until submission for testing.

Prepare the transfer kit for inoculation. In the BSC, connect the large-diameter tubing end of the relay pump fluid transfer kit to the Luer interface adapter on the transfer kit containing the TIL. Clamp the tubing near the transfer kit using hemostatic forceps. Place a red cap on the end of the transfer kit.

Place the TILs in an incubator. Remove the cell suspension from the BSC and place it in the incubator until needed. Record the time.

Cell counting was performed. Cell counting and calculation were performed using an NC-200. The cell counting sample was initially diluted by adding 0.5 mL of cell suspension to 4.5 mL of the prepared AIM-V medium, resulting in a 1:10 dilution.

Calculate the number of flasks used for subculture. Calculate the total number of flasks to be inoculated. Note: Round up the number of G-Rex500MCS flasks to a near-integer.

Table 35: Flask Calculations

The maximum number of G-Rex500MCS flasks to be inoculated is 5. If the calculated number of culture flasks exceeds 5, inoculate only 5 flasks using the full volume of available cell suspension.

Determine the number of additional culture medium bags required. Calculate the number of culture medium bags needed besides those prepared above. Round the required number of culture medium bags up to the nearest integer.

Table 36: Calculation of Culture Medium Bags

Prepare additional culture medium as needed. For every two G-Rex-500M flasks required as calculated above, prepare one 10L bag of "CM4 Day 16 Medium". While preparing and heating the additional medium, inoculate the first G-REX-500M flask.

Prepare additional culture medium bags as needed. Prepare and heat the calculated number of additional culture medium bags determined above.

Fill the G-Rex500MCS. Open the G-Rex500MCS on the benchtop and inspect the container for cracks or kinks in the tubing. Ensure all Luer connections and seals are tightened. Mark the 4500mL line on the outside of the flask with a marker. Close all clamps on the G-Rex500MCS except for all large filter clamps. Aseptically solder the red culture tubing from the G-Rex500MCS to the fluid transfer kit on the prepared culture bag.

Prepare the culture medium for pumping. Attach the "CM4 Day 16 Culture Medium" to the infusion pole. Feed the medium into the pump tubing using the Baxa pump.

Pump the culture medium into the G-Rex500MCS. Set the Baxa pump to "High" and "9", then pump 4500 mL of culture medium into the flask. Pump 4.5 L of "CM4 Day 16 Medium" into the G-Rex500MCS, filling the flask to the marked line. Once the 4.5 L of medium has been transferred, stop the pump.

Heat seal. Heat seal the red culture medium tubing of the G-Rex500MCS near the weld, then remove the culture medium bag.

Repeat the filling and sealing steps for each flask calculated above, once the culture medium is heated and ready for use. Using gravity filling with the pump above allows for the simultaneous filling of multiple flasks. Each bag of culture medium can only fill two flasks.

The recorded and labeled flasks are full. Each flask is labeled with a letter and "Day 16".

Incubate the flasks as needed. Place the flasks in the incubator while waiting for TIL inoculation. Record the total number of flasks filled.

Calculate the volume of cell suspension to be added. Calculate the target volume of TIL suspension to be added to the new G-Rex500MCS flask.

Table 637: Cell suspension volume

If there are more than 5 flasks, use the full volume of cell suspension to inoculate only 5 flasks.

Prepare the flasks for inoculation. Remove the G-Rex500MCS from step 8.10.70 of the incubator.

Prepare for pumping. Close all clamps on the G-Rex500MCS except for the large filter line. Feed the pump line through the Baxa pump.

Remove TIL from the incubator. Remove the TIL suspension transfer pack from the incubator and record the end time of incubation.

Prepare a cell suspension for inoculation. Aseptically weld the "TIL suspension" transfer package from above to the pump inlet line.

Place the TIL suspension bag on the scale. Using the Baxa pump set to "Low" and "2", fill the tubing from the TIL suspension bag to the weld. Place the scale on the scale.

Inoculate the flasks with TIL suspension. Set the Baxa pump to "Medium" and "5". Pump the calculated volume of TIL suspension into the flasks. Record the volume of TIL suspension added to each flask.

Heat seal. Heat seal the TIL suspension transfer package, leaving enough tubing to weld onto the next flask.

Fill the remaining flasks. Between each inoculated flask, ensure the TIL suspension transfer pack is mixed, and repeat the filling and sealing steps to inoculate all remaining flasks.

Monitor the incubators. If culture flasks must be dispensed between two incubators, ensure both incubators are monitored simultaneously. Incubator parameters: Temperature LED display: 37.0℃±2.0℃, _CO2 percentage: 5.0±1.5% _CO2 . Record the time each flask is placed in the incubator.

Calculate the incubation window. Perform the following calculations to determine the time range for removing G-Rex500MCS from the incubator on day 22. Lower limit: time + 132 hours; Upper limit: time + 156 hours.

On day 22, prepare the washing buffer.

Prepare a 10L Labtainer bag. In the BSC, connect the 4-inch plasma transfer kit to the 10L Labtainer bag via the Luer interface connection. Label the 10L Labtainer bag “Supernatant,” batch number, and initial/date. Close all clamps before transferring out of the BSC. Note: Prepare one 10L Labtainer bag for every two G-Rex 500MCS flasks to be harvested.

Solder the fluid transfer kit. Outside the BSC, close all clips on the 4S-4M60. Solder the relay fluid transfer kit to one of the male Luer interface terminals of the 4S-4M60.

Introduce Plasmalyte-A and 25% human albumin into the BSC. Integrate the 4S-4M60 and relay fluid transfer kit into the BSC.

Table 38: Components

Table 39: Plasmalyte-A

**Available for purchase from http://ecatalog.baxter.com/ecatalog/loadproduct.html?cid=20016&lid=10001&hid=20001&pid=821874.

Pump Plasmalyte into a 3000mL bag. Insert three Plasmalyte-A bags into the 4S-4M60 connector kit. Note: Wipe the port cap with an alcohol swab (W3009488) before removing. Caution: Maintain constant pressure while rotating the nozzle in one direction. Ensure not to puncture the side of the port. Connect the Origen 3000mL collection bag to the larger diameter end of the relay pump transfer kit via the Luer interface connection. Close the clamps on the unused tubing of the 3000mL Origen bag. Install the Baxa pump next to the BSC. Feed the transfer kit tubing through the Baxa pump located outside the BSC. Set the pump to "High" and "9". Open all clamps from Plasmalyte-A to the 3000mL Origen bag. Pump all Plasmalyte-A into the 3000mL Origen bag. After transferring all Plasmalyte-A, stop the pump. If necessary, remove air from the 3000ml Lorigen bag by reversing the pump and manipulating the bag's position. Close all clamps.

Remove the 3000mL bag from the relay pump fluid transfer kit via the Luer interface connection and place a red cap (W3012845) on the tubing leading to the bag.

Add 25% human albumin to a 25mL bag. Open the venting mini-tip. Ensure the blue cap is securely fastened without compromising the sterility of the tip. Insert the venting mini-tip into the septum of the 25% human albumin vial. Note: Ensure the sterility of the tip is not compromised. Repeat twice for a total of three (3) spiked 25% human albumin vials. Remove the blue cap from one venting mini-tip and connect a 60mL syringe to the 25% human serum albumin vial. Draw 60mL of 25% human serum albumin. You may need to use more than one vial of 25% human serum albumin. If necessary, disconnect the syringe from the venting mini-tip and connect it to the next venting mini-tip in the 25% human serum albumin vial. Once you have 60mL, remove the syringe from the venting mini-tip. Connect the syringe to the needleless inlet of a 3000mL Origen bag containing Plasmalyte-A. Add all 25% human albumin. Repeat to obtain a final volume of 120.0 mL of 25% human albumin. After adding all 25% human albumin, gently mix the bag. Label the bag “LOVO Wash Buffer” and indicate an expiration time of 24 hours.

Prepare the IL-2 dilution. Using a 10 mL syringe, dispense 5.0 mL of LOVO wash buffer through the needle-free syringe on the LOVO wash buffer bag. Add the LOVO wash buffer to a 50 mL conical tube and label it "IL-2 dilution".

Aliquot the CRF blank bag with LOVO wash buffer. Using a 100 mL syringe, draw 70.0 mL of LOVO wash buffer from the needleless injection port. Note: Wipe the needleless injection port with an alcohol pad before each use. Cap the syringe with the red cap and label it with "Blank Cryopreservation Bag" and the batch number. Note: Keep the syringe at room temperature until needed in step 8.14.3.

Complete the preparation of the wash buffer. Close all clamps on the LOVO wash buffer bag.

Thaw IL-2. Thaw one 1.1 mL IL-2 (6 × ^10⁶ IU/mL) container until all ice is thawed. Record the IL-2 batch number and expiration date. Note: Ensure the IL-2 label is affixed.

IL-2 preparation. Add 50 μL of IL-2 stock solution (6 × ^10⁶ IU/mL) to a 50 mL conical tube labeled “IL-2 dilution”.

IL-2 preparation. Relabel the conical tube with "IL-2 ^6x10⁴ ", date, batch number, and expiry date (24 hours). Cap and store at 2°C to 8°C.

Pre-freezing: Place 5 cryovials at 2°C to 8°C for pre-treatment in order to freeze the final product.

Prepare the cell counting dilution. In a BSC, add 4.5 mL of AIM-V medium (labeled with the batch number and "for cell counting dilution") to four separate 15 mL conical tubes and label the tubes.

Prepare for cell counting. Label four cryovials with tube numbers (1-4).

Day 22, TIL harvest

Incubator monitoring. Incubator parameters: Temperature LED display: 37℃±2.0℃, _CO2 percentage: 5%±1.5%.

Remove the G-Rex500MCS flask from the incubator. Before removing the G-Rex500MCS from the incubator, check the flask and confirm the culture parameters (culture time).

Prepare TIL collection bags. Label the 3000mL collection bags with "TIL Suspension", batch number, and initial/date.

Seal any additional connections. Near the end of each connection, heat-seal the two Luer connectors on the collection bag.

GatheRex Setup. Aseptically weld the red culture medium removal line from the G-Rex 500MCS (as per Procedure Note 5.11) to the prepared 10L Labtainer bag. Note: For use with multiple GatheRex devices, refer to Procedure Note 5.16. Aseptically weld the clear cell removal line from the G-Rex 500MCS to the prepared TIL suspension collection bag row (as per Procedure Note 5.11). Place the G-Rex 500MCS flask on the left side of the GatheRex. Place the supernatant Labtainer bag and the combined TIL suspension collection bag on the right side. Attach the red culture medium removal line from the G-Rex 500MCS to the top clamp (marked with a red line) and tubing of the GatheRex. Attach the clear cell harvest line from the G-Rex 500MCS to the bottom clamp (marked with a blue line) and tubing of the GatheRex. Connect the GatheRex gas line to the sterile filter of the G-Rex 500MCS. Before removing the supernatant from the G-Rex500MCS, ensure that all clamps on the cell removal line are closed.

Reduce volume. Transfer approximately 4.5 L of supernatant from the G-Rex500MCS to a supernatant bag. Visually inspect the G-Rex500MCS to ensure the flask is level and the culture medium has been reduced to the end of the aspirator tube. Repeat the steps as needed.

Prepare the flask for TIL harvesting. After removing the supernatant, close all clamps to the red tubing.

Begin collecting TILs. Record the start time of TIL harvest. Tap the flask firmly and rotate the culture medium to release the cells. Check the flask to ensure all cells have been separated. Place the 3000 mL TIL suspension collection bag on a dry cloth on a flat surface. Tilt the flask to ensure the tubing is at the edge of the flask. Note: If the cell collection tubing is not at the junction of the wall and bottom membranes, tapping the flask at a 45° angle is usually sufficient to properly position the tubing.

TIL Harvest. Loosen all clamps leading to the TIL suspension collection bag. Using GatheRex, transfer the TIL suspension into a 3000 mL collection bag. Note: Keep the edges tilted until all cells and culture medium are collected. Check the membrane for adherent cells.

Rinse the membrane in the flask. Rinse the bottom of the G-Rex500MCS. Cover about 1/4 of the gas exchange membrane with the rinse medium.

Close the clips on the G-Rex500MCS. Ensure all clips are closed.

Heat seal. Heat seal the collection bag containing TIL as close to the weld as possible to keep the main tube length approximately the same. Heat seal the supernatant bag.

Harvest the remaining G-Rex 500MCS flasks. Repeat the above steps to combine all TILs into the same collection bag. Replace the 10L supernatant bag after every second flask.

Prepare the LOVO source bag. Obtain a new 3000mL collection bag. Label it "LOVO Source Bag," batch number, and initial/date. Heat-seal the tubing of the "LOVO Source Bag," remove the female Luer connector, leaving enough wire for soldering.

Weigh the LOVO source bag. Place an appropriately sized plastic container on the scale and tare. Place the LOVO source bag (including the port and tubing) into the bin and record the dry weight.

Transfer the cell suspension to the LOVO source bag. Close all clamps on the 170μm gravity blood filter.

Transfer the cell suspension to the LOVO source bag. Aseptically weld the long end of the gravity blood filter to the LOVO source bag. Aseptically weld one of the two source lines of the filter to the "merged TIL suspension" collection bag. After welding, heat-seal any unused tubing on the filter to remove it. Open all necessary clamps and suspend the collection bag on the IV pole to elevate the TIL suspension, initiating gravity flow transfer of the TIL through the blood filter into the LOVO source bag. Gently rotate or knead the TIL suspension bag while draining to maintain even TIL suspension.

Close all clips. After transferring all TILs to the LOVO source bag, close all clips.

Heat seal. Heat seal as close as possible to the weld (according to process note 5.12) to remove the gravity blood filter.

Remove the cell counting samples. In the BSC, using a separate 3 mL syringe, remove 4 x 1.0 mL cell counting samples from the LOVO source bag using the needle-free injection port. Place the samples into the cryovials prepared in step 8.11.36.

Cell counting was performed. Cell counting and calculation were performed using an NC-200. Initially, the cell counting sample was diluted by adding 0.5 mL of cell suspension to 4.5 mL of the prepared AIM-V medium, resulting in a 1:10 dilution.

Record the cell count and sample volume. Calculate the total number of viable TIL cells. If the total number of viable cells is ≥1.5 x ^10⁹ , proceed. Calculate the total number of nucleated cells.

Prepare the mycoplasma dilution. In a BSC, take 10.0 mL from a supernatant bag through the Luer sample port and place it into a 15 mL Erlenmeyer flask. Label the 15 mL Erlenmeyer flask "Mycoplasma Dilution".

LOVO

Open LOVO and start the "TIL G-Rex Harvest" protocol, following the on-screen prompts. The buffer type is PlasmaLyte. Follow the LOVO touchscreen prompts.

Determine the target volume of the final product. Using the total nucleated cell count (TNC) value and the table below, determine and record the target volume of the final product (mL).

Table 40: Calculation of Final Product Volume

Follow the prompts on the LOVO touchscreen.

Insert the disposable reagent kit. Before inserting the disposable reagent kit, wipe the pressure sensor port with an alcohol wipe and then with a lint-free cloth. Insert the disposable reagent kit. Follow the on-screen instructions to install the disposable reagent kit.

Remove the filtrate bag. After filling with the standard LOVO disposable reagent kit, touch the "Next" button. The "Container Information and Location" screen will appear. Remove the filtrate bag from the scale.

Ensure the filtrate container is new and off-scale.

Enter the filtrate volume. Aseptically weld the LOVO auxiliary bag to the existing filter bag's male Luer connector line. Ensure all clamps are open and fluid passages are unobstructed. Touch the filter container capacity input field. The numeric keypad will appear. Enter the total new filtrate volume (5,000 mL). Touch the button to accept the input. Note: The estimated filtrate volume should not exceed 5,000 mL.

Place the filter container on the benchtop. Note: If the tube was removed from clamp F during welding, put it back in the clamp. Place the new filter container on the workbench. Do not hang the filter bag on scale #3. Scale #3 will be empty during this process.

Follow the prompts on the LOVO touchscreen after the filter container is replaced.

Ensure the kit is loaded correctly. A "Disposable Kit Dry Check" overlay will be displayed. Check that the kit is loaded correctly and all clamps are open. Check all tubing for kinks or other obstructions and correct them if necessary. Ensure the kit is installed correctly and check all LOVO clamps. Press the "Yes" button. All LOVO mechanical clamps will automatically close, and the "Check Disposable Kit Installation" screen will appear. LOVO has undergone a series of pressurization steps to check the kit.

Kit check results. If the kit check passes, proceed to the next step. *If not, a second kit check can be performed after the first check. *If not, check all tubing for kinks or other obstructions and correct them. *If not, ensure all Robert clamps are correctly installed. If the second check fails: contact the area applicator and prepare to install the new kit in Section 10.0. Repeat steps 8.13.23 to the required steps 8.13.30.

Connect PlasmaLyte. The "Connect Solution" screen will appear. The wash value will always be 3000 mL. Enter this value on the screen.

Aseptically weld a 3000mL PlasmaLyte bag to the tubing passing through clip 1. Hang the PlasmaLyte bag on bar IV and place both corner loops on the hooks.

Verify that PlasmaLyte is connected. Open all plastic clips. Verify that the solution volume entry is 3000 mL. Touch the "Next" button. The "Perfusion Single-Use Kit" overlay will appear. Confirm that PlasmaLyte is connected and that all solder joints and plastic clips on the tubing leading to the PlasmaLyte bag are open, then touch the "Yes" button.

Observe PlasmaLyte moving. Disposable kit perfusion begins, displaying the "Perfusion of Disposable Kit" screen. Visually observe PlasmaLyte moving through the tubing connected to the PlasmaLyte bag. If no fluid is moving, press the "Pause" button on the screen to determine if the clamps or solder are still closed. After resolving the issue, press the "Continue" button on the screen to continue using the disposable kit. Follow the LOVO touchscreen prompts.

Attach the source container to the tubing. Aseptically weld the LOVO source bag prepared in step 8.12.31 to the tubing passing through the S-clamp, following process note 5.11. It may be necessary to remove the tubing from the clamp. Note: If removed, ensure the source tubing is replaced in the S-clamp.

Hang the source container. Hang the source container on bar IV, with both corner bag loops on the hooks. Do not hang the source on scale #1. Open all clips to the source bag.

Connect the verified source container. Touch the Next button. The perfusion source coverage is displayed. Confirm that the source is connected to the disposable kit and that all solder and plastic clamps on the tubing leading to the source are open. Touch the Yes button.

Confirm that PlasmaLyte is moving. Begin source infusion; the source infusion screen will appear. Visually observe PlasmaLyte moving through the tubing connected to the source bag. If no fluid is moving, press the "Pause" button on the screen to check if the clamps or welds are still closed. Once the problem is resolved, press the "Continue" button on the screen to resume source infusion.

Startup screen. Once the source filling is successful, the "Startup Process" screen will be displayed. Press the Start button. Immediately after pressing the Start button, the "Pre-wash Cycle 1" pause screen will appear.

Invert the processing bag. Remove the processing bag from the #2 scale (or remove the tubing from the top port tubing guide plate in the process), and manually invert it to allow the washing buffer added during the single-use kit perfusion step to cover all the inner surfaces of the bag. Reattach the processing bag to the #2 scale (label on the bag facing left). Replace the top tube if it has been removed.

Invert the source bag. Before pressing the "Start" button, mix the source bag, but do not remove it from the IV electrode, by rubbing the corners of the bag and gently agitating the cells to form a homogeneous cell suspension. Press the Resume button. LOVO will begin processing the liquid in the source bag, displaying the Wash Cycle 1 screen.

Source flush paused. The "Source Flushing Paused" screen will appear once the discharge source container is empty and LOVO has added wash buffer to the wash source bag. Without removing the source bag from the IV stand, gently press the corners to mix thoroughly. Press Resume.

Pause the bag during mixing. To prepare the cells for passage through the vortex mixer again, dilute the treatment bag with wash buffer. After adding the wash buffer to the treatment bag, LOVO will automatically pause and display the "Mix Treatment Bag" pause screen. Without removing the bag from the scale, gently squeeze the bag to thoroughly mix the product. Press Continue.

Pause at the corners during the kneading process. When the treatment bag is empty, add washing buffer to the bottom of the treatment bag to rinse it. After adding the rinsing solution, LOVO will automatically pause and display the "Knead IP Corner" pause screen. Do not remove the bag from scale #2 while the treatment bag is still suspended on scale #2. Knead the corners of the bag to suspend any remaining cells. Ensure the bag is not swinging on the scale, then press the "Continue" button.

Wait for the "Remove Product" screen to appear. The "Remove Product" screen will appear when the LOVO process is complete. While this screen is displayed, you can manipulate all the bags on the LOVO kit. Note: You cannot touch any bag until the "Remove Product" screen is displayed.

Remove the retention bag. Place a hemostatic agent very close to the tubing near the port of the retention bag to prevent cell suspension from settling into the tubing. Heat seal under the hemostat (as per process note 5.12), ensuring sufficient wire count for welding in step 8.13.48. Remove the retention bag.

Prepare a retention bag for formulation. Weld the mother Luer locking end of the 4-inch plasma transfer kit to the retention bag. Transfer the retention bag.

Remove the product. Follow the instructions on the "Remove Product" screen. Close all clamps on the LOVO kit to prevent fluid movement.

Remove the product. Touch the Next button. Open all LOVO mechanical clips; the "Removal Kit" screen will appear.

Record the data. Follow the instructions on the "Disassembly Kit" screen. Touch the "Next" button. Close all LOVO mechanical clamps; the results overview screen will appear. Record the data from the results overview screen. Close all pump and filter supports. When prompted by LOVO, remove the kit. All recorded times are recorded directly from LOVO.

Final formulation and filler

Target Volume/Bag Calculation. From the DDD table below, select the number of CS750 bags to be filled, the target filling volume per bag, the volume to be removed from each bag for retention, and the final target volume per bag corresponding to the LOVO retention volume above.

Table 41: Target Volume/Bag Calculation

Prepare the CRF blank. Calculate the volumes of CS-10 and LOVO wash buffer to prepare the blank bag.

Table 42: Calculated Volume

Prepare the CRF blank. Outside the BSC, using the LOVO wash buffer syringe prepared above, add the calculated volume to an empty CS750 bag via the Luer connector. Note: Aseptic handling is not required for the blank CS750 bag formulation. Using an appropriately sized syringe, add the calculated CS-10 volume to the same CS750 bag as described above. Place a red cap on the CS750 bag. Expel as much air as possible from the CS-750 bag. Heat-seal the CS750 bag as close to the bag as possible, and remove the tubing. Label the CS750 bag with "CRF Blank," the batch number, and the first letter/date. Place the CRF blank on a cold storage bag until it is placed in the CRF.

Calculate the required IL-2 volume. Calculate the volume of IL-2 to be added to the final product.

Table 43: Calculation of IL-2 volume

Assemble the connection device. Aseptically solder the 4S-4M60 to the CC2 Cell Connect, replacing the single nozzle of the Cell Connect device with the four nozzles of the 4S-4M60 manifold.

Assemble the connected equipment. Aseptically solder the CS750 cryopreservation bag to the prepared wiring harness, replacing one of the four Luer interface male terminals (E) with each bag. Solder the CS-10 bag (as per process note 5.11) to the nozzle of the 4S-4M60. Store the CS-10 bag between two ice packs at 2°C to 8°C.

Prepare TIL using IL-2. Using an appropriately sized syringe, extract the determined amount of IL-2 from the “IL-2 6x10⁴” aliquot sample. Connect the syringe to the prepared retention bag via the Luer connector and inject the IL-2. Clean the tubing by pushing air from the syringe into the tubing.

Label the TIL bag "Prepared". Close the clip on the transfer kit, label the bag "Prepared TIL", and then remove it from the BSC.

Add the prepared TIL bag to the device. After adding IL-2, solder the prepared TIL bag to the remaining nozzle on the device.

Add CS10. Feed the assembled device with the prepared TIL, CS-750 bag, and CS-10 into the BSC. Note: The CS-10 bag and all CS-750 bags are placed between two pre-set cold bags at 2°C–8°C. The prepared TIL bag is not placed on the cold bags. Ensure all clamps on the device are closed. Turn the stopcock valve to close the syringe.

Switch syringes. Draw approximately 10 mL of air into a 100 mL syringe and replace the 60 mL syringe on the device.

Add CS10. Unscrew the stopcock valve to close the tubing leading to the CS750 bag. Open the clamp on the CS-10 bag and pull the calculated volume into the syringe. Note: Multiple syringes will be used to add the appropriate volume of CS-10. Close the clamp on the CS-10 bag, open the clamp on the prepared TIL bag, and add CS-10. Add the first 10.0 mL of CS10 at a rate of approximately 10.0 mL/min. Add the remaining CS-10 at a rate of approximately 1.0 mL/s. Note: Use multiple syringes to add the appropriate amount of CS-10. Record the time. Note: The target time from the first addition of CS-10 to the start of freezing is 30 minutes. Record the volume of CS10 added each time and the total volume added. Close all clamps on the CS10 bag.

Prepare the CS-750 bag. Turn the stopcock valve to open the syringe. Open the clamp of the prepared TIL bag, stopping the suspension before it reaches the stopcock valve. Close the clamp of the prepared TIL bag. Turn the stopcock valve to open the CS-750 final product bag. Using a new syringe, remove as much air as possible from the CS-750 final product bag by aspiration. Clamp the bag while maintaining syringe plunger pressure. Draw approximately 20 mL of air into a new 100 mL syringe and connect it to the device. Note: Each CS-750 final product bag should be placed between two cold bags to keep the prepared TIL suspension at a low temperature.

Dispense cells. Turn the stopcock valve to close the line to the final product bag. Pull the calculated volume from the prepared TIL bag into the syringe. Note: Multiple syringes can be used to obtain the correct volume. Turn the stopcock valve to close the line to the prepared TIL bag. Add cells to the final product bag one at a time. Record the cell volume in each CS750 bag above. Purge the tubing with air from the syringe to ensure the cells are evenly distributed at the tip of the nozzle. Close the clamp on the full bag. Repeat the steps for each final product bag, gently mixing the prepared TIL bag between bags. Record the TIL volume in each final product bag below.

Expel air from the final product bag and remove the retention solution. After filling the last final product bag, close all clamps. Draw 10 mL of air into a new 100 mL syringe and replace the syringe on the device. Work one bag at a time, removing all air from each product bag and adding the determined product volume to be retained. Note: After removing the sample volume, invert the syringe and purge the tubing leading to the top port of the product bag with air. After removing the retention volume and air, clamp the tubing onto the bag.

Record the retained volume taken from each bag.

Add the retention. Add the retention to a 50 mL conical tube and label it "Retention" and the batch number. Repeat once for each bag.

Prepare the final product for cryopreservation. Using hemostatic forceps, clamp the tubing close to the bag. Remove the red cap Luer connector from the syringe and the device containing the syringe. Remove the device from the BSC. Heat seal at F (according to process note 5.12), and remove the empty retention bag and CS-10 bag. Note: Retain the Luer connector for the syringe on the device. Discard the empty retention bag and CS-10 bag.

Label the final product bag. A sample final product label is attached below.

Prepare the final product for cryopreservation. Place the cryopreservation bags on ice packs or at 2°C to 8°C until cryopreservation.

Remove the cell counting sample. Using an appropriately sized pipette, remove the 2.0 mL of the sample removed above and place it into a 15 mL conical tube for cell counting.

Perform cell counting. Use the NC-200 to perform cell counting and calculations. Note: Dilute only one sample to an appropriate dilution to verify that the dilution is sufficient. Dilute other samples to appropriate dilution factors and then perform counting. Record the sample volume for cell counting. Note: If no dilution is required, "Sample [μL]" = 200, "Dilution [μL]" = 0. Determine the mean viable cell concentration and the viability of the cell counts performed.

Calculate the flow cytometry sample. Perform calculations to ensure sufficient cell concentration for flow cytometry sampling.

Table 44: Calculation of Cell Concentration by Flow Cytometry

Calculate IFN-γ. Calculate the sample to ensure sufficient cell concentration for IFN-γ.

Heat sealing. After determining the sample volume, heat seal the final product packaging bag, bringing it as close to the packaging bag as possible for removal from the equipment.

Table 745: Sample Labelling and Collection

For mycoplasma samples, add the volume of the cell suspension prepared above to a 15 mL conical tube labeled "Mycoplasma Diluent". Aseptic and BacT-free. Test sampling. In a BSC, using an appropriately sized syringe, remove 1.0 mL of sample from the retained cell suspension collected above and inoculate into an anaerobic bottle. Repeat the above steps for an aerobic bottle.

Label and store the samples. Label all samples with the sample plan inventory label and store them securely until transfer. Proceed to the next step to freeze-store the final product and samples.

Final product cryopreservation

Prepare the controlled-rate freezer. Verify that the CRF has been set up before freezing. Record the CRF device. Perform cryopreservation.

Set up the CRF probe. Puncture the septum in the CRF blank bag. Insert the temperature probe into the 6 mL sample vial.

Place the final product and samples in the CRF. Place the blank bag in the pretreatment box and transfer it to approximately the center of the CRF rack. Transfer the final product box to the CRF sample rack and the sample vials to the CRF vial rack. Transfer the product rack and sample vial rack to the CRF. Record the time of product transfer to the CRF and the reaction chamber temperature.

Determine the time required to reach 4℃±1.5℃ and begin CRF operation. Start operation after the chamber temperature reaches 4℃±1.5℃. Record the time.

Complete, store. Stop CRF after operation is complete. Remove the box and vial from CRF. Transfer the box and vial to gas phase LN ₂ for storage.

Example 17: Generating a TIL product rich in tumor-specific T cells with enhanced therapeutic activity

Objective: To produce TIL products rich in tumor antigen-specific T cells with enhanced therapeutic activity.

background:

T cells associated with malignant lesions are often dysfunctional and unable to control/prevent tumor growth (Schietinger et al., Immunity 2016). All references in this embodiment are incorporated herein by reference in their entirety for all purposes.

Tumor-infiltrating lymphocytes (TILs) can be extracted, activated, and proliferated ex vivo, and induced effective antitumor responses upon re-infusion in vivo (Rosenberg et al., Clin Cancer Res 2011). Adoptive cell transfer, first demonstrated in patients with metastatic melanoma, is being tested in the histology of other solid tumors. Clinical activity of TIL therapy against melanoma, head and neck cancer, and cervical cancer has been reported (SITC, 2017). Therefore, tumor-specific TILs can be rescued from the suppressive tumor microenvironment and reregulated and/or expanded to sufficient numbers to effectively target tumors.

Retrospective analysis of TIL clinical trials has shown that, compared to effector and effector memory T cells, poorly differentiated tumor-reactive T cells with strong proliferative and survival capabilities confer superior antitumor efficacy, and that next-generation TIL products should always contain elevated levels of poorly differentiated T cells (Klebanoff et al., J Immunother 2012).

T memory stem cells (TSCMs) are early progenitor cells of central memory T cells that have experienced antigens; TSCMs exhibit defining stem cell long-term survival, self-renewal, and pluripotency; therefore, TSCMs are considered the most ideal method for generating effective TIL products. Compared with other T cell subsets in adoptive cell transfer mouse models, TSCMs have shown enhanced antitumor activity (Gattinoni et al., Nat Med 2009, 2011; Gattinoni, Nature Rev. Cancer, 2012; Cieri et al., Blood 2013).

A strategy of favoring TIL compositions with a high proportion of TSCM is needed to ensure optimal antitumor activity.

Reprogramming tools developed using induced pluripotent stem cell (iPSC) technology can revitalize antigen-exposed T cells (Nishimura et al., Cell Stem Cell 2012; Vizcardo et al., Cell Stem Cell 2012). This approach requires further in vitro differentiation of iPSCs into a population of T cells best suited for tumor fighting, making it a lengthy two-step process that easily restricts the composition of the original T cell receptor (TCR). See alos, Stewart et al., 538:183-192 (2016) Regarding in vivo and in vivo delivery strategies for intracellular delivery of materials.

Signaling pathways including Wnt, NOTCH, and Myb have been shown to support the direct generation of TSCM-like cells from naive T cells and/or antigen-experienced T cells (Gattinoni et al., Nat Med 2009; Kondo et al., Nat Comm. 2017; Gautam et al., SITC 2017).

Cell fate reprogramming requires transient exposure to appropriate transcription factors (TFs). In the case of TILs, this exposure needs to target the majority of T cells to preserve the tumor-derived TCR repertoire.

SQZ-free microfluidic platforms represent an advanced intracellular delivery strategy; they have demonstrated the ability to deliver proteins, including transcription factors, to a variety of primary human cells, including T cells (Sharei et al., PNAS 2013; and Sharei et al., PLOS ONE 2015 and Greisbeck et al., The J of Immunology, Vol. 195, 2015). See also International Patent Publications WO2013/059343A1, WO2017/008063A1 and WO2017/123663A1, all of which are incorporated herein by reference in their entirety. The methods described in International Patent Publications WO2013/059343A1, WO2017/008063A1, and WO2017/123663A1 can be used in conjunction with this invention to expose TIL populations to transcription factors (TFs) and/or other methods capable of inducing transient protein expression; wherein the TFs and/or other molecules capable of inducing transient protein expression provide an increase in tumor antigen expression in the TIL populations and/or an increase in the number of tumor antigen-specific T cells, thereby resulting in reprogrammed TIL populations having higher immunity and improved therapeutic efficacy compared to unreprogrammed TIL populations.

Strategy:

It is proposed to use SQZ intracellular TF protein delivery technology to compare the ability of various reprogramming strategies to generate TIL products from tumor antigen-specific T cells with enhanced therapeutic activity.

The work plan includes the following key points/issues:

1. Tumor type

■Melanoma

2. Optimal delivery conditions

■SQZ Human T Cell Optimization Program + Other Conditions

■ Monitor delivery efficiency

3. Choosing a TF

■TCF-1

■NOTCH1/2ICD

■MYB

■+/- previous iPSC mix used for "full" reprogramming?

4. Activation/Amplification Phase Targeting TIL

■REP Day 0

■Other?

5. Dynamics of Reprogramming

■Days 7, 11, 14, 18…

6. TIL Subgroup Targets

■Initially a large quantity

■ The subgroups (TCM, TEM, TEFF, TEMRA) were sorted for later comparison.

7. Requirements for other factors in the culture medium

■IL7

■RSPO3/WNT3A

■Other?

8. Reading

■ Analyze Pre-REP and Post-REP TIL amplification phenotypes using (modified?) panels 1, 2, and 3.

■Post-REP TIL effector function assessment (detection of cytokine and/or activation marker production)

■Post-REP TIL Tumor Reactivity Assessment (Autologous Tumor Cell Co-culture Assay)

■ TCR library analysis (flow cytometry and/or RNA-seq)

■ Live cell metabolism assays, such as Seahorse

Other considerations

1. Production of protein TF

2. Tumor acquisition

3. Logistics (Cell Transport)

Table 46: Process

Example 18: Cryopreservation Method

This embodiment describes a method for cryopreserving the prepared TIL using a CryoMed Controlled Rate Freezer, model 7454 (Thermo Scientific), employing the simplified, closed procedure described in Example 16.

The equipment used is as follows: aluminum box holder (compatible with CS750 cryopreservation bags), cryopreservation boxes for 750mL bags, low-pressure (22psi) liquid nitrogen tank, refrigerator, thermocouple sensor (strip bag), and CryoStore CS750 cryopreservation bags (OriGenScientific).

The freezing process provides a 0.5°C rate from nucleation to -20°C and a 1°C/min cooling rate to the final temperature of -80°C. The program parameters are as follows: Step 1: Wait at 4°C; Step 2: 1.0°C/min (sample temperature) to -4°C; Step 3: 20.0°C/min (room temperature) to -45°C; Step 4: 10.0°C/min (room temperature) to -10.0°C; Step 5: 0.5°C/min (room temperature) to -20°C; Step 6: 1.0°C/min (sample temperature) to -80°C.

The procedure description of this embodiment is provided in conjunction with the method of Embodiment 16.

Example 19: Method for generating TIL products rich in tumor antigen-specific T cells with enhanced therapeutic activity

Phase 1a: Obtain a validated sd-RNAfm to achieve efficient and specific silencing.

The initial phase will involve obtaining a validated sd-RNAfm to effectively and specifically silence the following three genes: PD-1 (also known as PDCD1), TIM3, and CBLB.

Phase 1b: Identifying sequences that effectively silence LAG3 and CISH

Up to 20 sd-RNAs will be designed targeting novel genes. Gene silencing of exogenous targets in HeLa cells and endogenous targets in activated primary T cells will be evaluated. One or two lead compounds will be screened for each target gene (including PD-1 and LAG-3) that reduces expression levels by more than 80%. Fully improved versions of the selected sd-RNAs will be generated.

It is expected that one or two LAG3 and CISH-specific sd-RNAfm will be generated. Preferably, two target RNAs per target gene will be generated.

Phase 2: Validate sdRNA-mediated gene silencing in pre-REP TIL.

Up to six frozen melanoma/other pre-REP lines will be used to validate sdRNA targets (including PD-1, TIM3, CBLB, LAG3, and CISH). Conditions will be measured for sdRNA concentration, thawing time, repeat/sequential delivery, and culture conditions. Readings will be the percentage of gene silencing, assessed, such as by flow cytometry and/or qPCR. The effect of RNA delivery on the persistence of TIL growth and silencing over time will be assessed by amplifying TILs.

It is expected that 80% silencing will be achieved using the lead compound 24 hours after REP harvest. The total cell count will be less than 10% of the untreated control.

Phase 3: Implement sdRNA-mediated gene silencing in process 2A.

Gene silencing will be optimized in fresh TIL formulations at 3 to 6 study scales. Conditions will be measured, including sdRNA concentration, time, repeat/sequential delivery, and culture conditions. Readings will be the percentage of gene silencing in post-REP TILs, assessed by flow cytometry and/or qPCR. Flow cytometry will be used to assess the effects of gene silencing on TIL phenotype and function. Optional rescue experiments and/or gene expression analyses will be performed to validate the specificity of the effects (e.g., the extent and impact of potential off-target silencing). At least two pairs of target/sdRNAs will be selected for further work. TIL phenotype will then be characterized. Non-specific and specific TIL activities comparable to or higher than control TILs will be evaluated to determine the optimal target/sdRNA.

Phase 4: Implement the optimized silencing scheme for full-scale TIL preparation.

Each target gene will undergo one industrial-scale TIL preparation. In addition to the products required for release, TIL products with new properties defined in Phase 3 will also be developed.

Example 20: Exemplary sd-RNA Preparation and Use

sd-RNA design

Approximately 2 to 20 sd-RNA sequences targeting a given target will be generated. In some cases, sd-RNA sequences will be screened based on a screening algorithm (commercially available from Advirna LLC, Worcester, SM, USA), designed based on functional screening of over 500 sd-RNA sequences. Regression analysis will be used to establish the correlation between the frequency of specific nucleotides in the sd-RNA duplex and modifications at any specific position and their function in gene repression assays. Selected sequences will be commercially synthesized at a scale of 0.2 μmol (e.g., by TriLink Biotechnologies) and dissolved in sterile RNase-free, DNase-free water for injection (commercially available from CalBiochem, 4.86505). The duplexes can be annealed by heating at 95°C for 5 minutes and then gradually cooling to room temperature.

Direct sdRNA delivery (passive ingestion)

Oligonucleotides (including the sd-RNA targeting genes described herein) can be diluted in serum-free medium and added in triplicate to 96-well plates. Cells can be seeded in suitable medium containing reduced FBS (in wells containing compounds pre-diluted to the specified fraction). HeLa cells can be transfected at 10,000 cells/well in EMEM medium containing 3% FBS. Primary human T cells (AllCells, CA) can be cultured in complete AIM-V (Gibco) medium containing 500 IU/mL IL2 (ProSpec). Prior to transfection, cells can be activated for at least 4 days with anti-CD3/CD28 Dynabeads (Gibco, 11131) according to the manufacturer's instructions. Unless otherwise specified, T cells can be transfected at 100,000 cells/well in 5% FBS without removing Dynabeads. To determine transfection efficiency, fluorescent images of live cells transfected with Cy3-conjugated sd-RNA were obtained using an Olympus BX-60 microscope. Nuclear staining was obtained by adding Hoechst33342 (molecular probe, H1398) to the transfected cells for 30 minutes to process the images.

Identification of lead sd-RNA compounds

This protocol can be used to identify the lead compound described in Example 18. A luciferase reporter plasmid can be constructed by inserting the PDCD1 targeting region into the Renilla luciferase sequence downstream of the psiCheck2 plasmid (Promega, C8021). For comparison, a previously validated MAP4K4 sd-RNA sequence can be inserted as a positive control.

For screening, HeLa cells were transfected with a cloned plasmid using Fugene HD (Promega, E2311) according to the manufacturer's instructions. In short, cells were seeded at 2.5 × ^10⁶ cells/10 ^cm² 390-cell dish in antibiotic-free EMEM (ATCC, 30-2003) medium, then transfected with a plasmid at a FugenE:DNA ratio of 2.5:1 for 6 hours. Cells were incubated for 16–18 hours, washed three times with PBS, trypsin-digested, and seeded into 96-well plates containing a pre-diluted sd-RNA compound at a final concentration of 1 μM sd-RNA/10,000 cells/100 μl EMEM containing 3% FBS. Cells were treated with sd-RNA for 48 hours to promote passive cellular uptake of the compound, and lysed with Glo lysis buffer (Promega, E266A) to detect the expression of Renida luciferase and firefly luciferase. For this purpose, 20 μl aliquots of each lysate were added to opaque 96-well plates and mixed with Matthews (Sea Kidney) assay buffer 59 397 or firefly luciferase 398 assay buffer (25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT, 2 mM ATP, 15 mM 399K2PO4, pH 7.8, and 1 mM D-luciferin). Substrate D-luciferin (Promega, E1605) and h-coelenterin (NanoLight, 301) were added immediately before use. Luminescence was measured on a SpectraMax i3 (Molecular Devices), normalized (Sea Kidney/Firefly), and expressed as a percentage of the untreated control.

Quantification of mRNA by qPCR

Total RNA can be isolated from transfected cells using the PureLink™ Pro96 Purification Kit (Invitrogen, 12173-011A) as recommended by the manufacturer. Untransfected (NT) cell dilutions at 1:5 and 1:25 can be prepared to generate standard curves. Gene expression is analyzed in a one-step multiplex qPCR 407 by mixing 20–40 ng of purified RNA with Quanta qScript RT-qPCR ToughMix (VWR, 89236672) and Taqman probes – PDCD1-FAM (Taqman, Hs01550088_m1) and GAPDH-VIC (Applied Biosystems, 4326317E) in the same reaction. Samples can be amplified using the Quanta-recommended settings on a StepOnePlus qPCR instrument (Applied Biosystems). PDCD1 expression can be normalized relative to GAPDH to adjust for a standard curve, expressed as a percentage of untargeted control (NTC) transfected cells.

Cell viability assay

The amplified TILs of this invention can be transfected with various doses of sdRNA oligonucleotides for 72 hours. Cells are washed and incubated at 37°C for 1 hour with a 1:10 dilution of CellTiter-Blue reagent (Promega, G808A). The wells are then placed at room temperature, and fluorescence at 530 nm excitation/590 nm emission is recorded. The linear range can be confirmed by seeding four series of 2-fold cell dilutions under the same conditions and plotting fluorescence readings.

Tumor-infiltrating lymphocyte separation

Tumor-infiltrating lymphocytes can be prepared as described herein, for example as shown in Figures 8 and 14.

TIL amplification

TILs can be inoculated in flasks as described herein for the first and/or second amplification steps. sd-RNA can be added during the first amplification (e.g., step b), after the first amplification (e.g., during step C), before or during the second amplification (e.g., before or during step D, after step D and before harvest in step E, during or after harvest in step F, before or during final preparation and/or transfer to the infusion bag in step F, and before any optional cryopreservation step in step F). Furthermore, sd-RNA can be added after thawing from any cryopreservation step in step F.

thymidine incorporation assay

TIL samples can be harvested at any amplification step and seeded triplicate in CellGro medium supplemented with 2% human AB serum in 96-well plates (104,443 cells/well). After 1 hour, 1 μCi/well of [methyl-3444H]thymidine (PerkinElmer, Waltham, MA) can be added to each well, and the plates can be incubated for 4 hours. Cells can then be harvested, and the incorporation of ^3H -thymidine can be measured using a Trilux 1450 microBeta liquid scintillation counter (Wallac) to examine TIL growth.

IFN-γ secretion in cells treated with sd-RNA

Following the manufacturer's instructions, the Human IFN-γ ELISA Development Kit (Mabtech) can be used to measure IFN-γ production in the supernatant from stimulated T cells. TILs can be prepared as described herein and treated with, for example, 2 μM sdRNA for several days, in some cases, four days. After this period, the supernatant can be collected for ELISA analysis to determine the level of IFN-γ production.

TIL processing

TILs treated with sd-RNA can be used in the treatment of cancer patients as described in this article.

Example 21: Exemplary Electroporation Method

TILs were prepared according to any of the methods described herein. The following transfection methods were tested: lipofectin and lipofectamin lipofection, electroporation using a square-wave BTX ECM 830 instrument or Bio-Rad Gene Pulser II, exponentially decreasing wave electroporation using an Eppendorf Multiporator, and the Amaxa nuclear transfector. All methods were initially based on the manufacturer’s recommendations and were subject to potential modifications as needed. The Amaxa nuclear transfection protocol offered the highest transfection efficiency. The Amaxa procedure was optimized using one of three solutions (V, R, and T) and different combinations of eight electroporation procedures. See also the methods described in U.S. Patent Application No. 2016/0230188 and U.S. Patent No. 8,859,229, the entire contents of which are incorporated herein by reference. Electroporation was also performed according to the method described in Menger et al., Cancer Res, April 15, 2016, 76(8): 2087-93. See J. Biol. Chem., the disclosure of which is incorporated herein by reference using the Agile Pulse BTX system (Harvard Instruments). Electroporated cells can be expanded using pre-REP and REP methods described elsewhere herein. Electroporation methods known in the art, such as those described in U.S. Patent Nos. 6,010,613 and 6,078,490, the disclosures of which are incorporated herein by reference. Pulsed electroporation can be optimized using the methods described below or modifications thereof:

TILs can be electroporated (at a rate of approximately ^10⁶ cells/mL) in cuvettes containing approximately 20 μg of a GFP-encoding plasmid and a control plasmid pUC with a 0.4 cm gap, using various electroporation procedures or methods. Approximately 24 hours after electroporation, GFP expression in the electroporated cells is analyzed by flow cytometry to determine transfection efficiency. The minimum voltage required for plasmid electroporation in TILs has been previously reported in International Patent Application Publication No. WO2014/184744 and U.S. Patent Application Publication No. US2013/0315884A1, the disclosures of which are incorporated herein by reference.

Example 22: Transient transfection protocol for preparing TIL products rich in tumor antigen-specific T cells with enhanced therapeutic activity

The experiments in this embodiment will investigate the effects of two different TIL enhancement strategies. Strategy 1: Transient expression of IL-2 or membrane-bound form of IL-15 (mb-IL15). Strategy 2: NOTCH-mediated TIL reprogramming. In some embodiments, NOTCH-mediated reprogramming includes mRNA expression of the intracellular domain (ICD) of NOTCH1 or NOTCH2. In some embodiments, NOTCH-mediated reprogramming includes mRNA expression of the NOTCH ligand DLL1.

The types of tumors to be studied will be melanoma, lung tumors, sarcomas, and others.

TIL will be generated using the procedures described above (including, for example, those described in Example 16).

The RNA molecules will be delivered using, for example, the method described in Example 21 (see also the methods described in U.S. Patent Application No. 2016/0230188 and U.S. Patent No. 8,859,229, the entire contents of which are incorporated herein by reference).

Delivery conditions will be determined, including, for example, mRNA reagent validation, transient transfection and reprogramming time, electroporation time, compatibility with the TIL process used, efficiency (including transfection efficiency), and scalability.

The experimental readings will include TIL phenotype (flow cytometry), TIL effector function (cytokine production assay), TIL tumor responsiveness (autologous tumor cell co-culture assay), TCR library analysis (flow cytometry and/or RNA-seq), TIL metabolic status (live cell metabolism assay, such as Seahorse), or any other parameters described above.

Expected results:

Delivery conditions will be designed as needed to achieve high expression of the target protein in post-REP TILs. Frozen melanoma pre-REP samples will be used for screening conditions. GFP mRNA will be delivered as the reagent for optimizing the experiment. Conditions will be tested using several TIL activation methods and electroporation protocols. Flow cytometry will be used to assess cell viability and the percentage of GFP-positive cells relative to the non-electroplated control. Screening conditions will be confirmed in TIL formulations from 2 to 3 fresh, available tissues. In some embodiments, transfection efficiency (ET) can be determined by fluorescence-activated cell sorting (FACS) at approximately 3, 6, 9, 12, 15, and/or 18 hours post-transfection. In some experiments, transfectants may be further analyzed every 12 to 24 hours until GFP is undetectable. In some embodiments, cell viability can be determined by trypan blue exclusion. This protocol is expected to result in >80% viability and >70% transfection efficiency.

Human IL-2 and mb-binding IL-15 mRNA will be generated and its function will be assessed. Up to six TIL cultures from different tissues will be transfected using validated conditions. Readings will be for transfection efficiency, TIL phenotype, and TIL effector function. This protocol is expected to result in >80% viability, >70% transfection efficiency, a TIL phenotype comparable to or improved over control, and significantly enhanced TIL effector function.

Delivery conditions will be designed as needed to reprogram T cells. Frozen melanoma pre-REP samples stored at Iovance will be used to screen conditions. The delivery reagent will be NOTCH1 or 2ICD mRNA. Conditions will be tested for several TIL activation methods and electroporation time. Flow cytometry will be used to assess cell viability, transfection efficiency, and percentage of T memory stem cells (TSCM) relative to the non-electroplated control. Selected conditions will be confirmed using fresh TIL formulations from 2 to 3 available tissues. This protocol is expected to result in >80% viability, >70% transfection efficiency, and a significant increase in TSCM frequency.

Using the transfection conditions defined above, reprogramming experiments were performed on up to six TIL formulations from various tissues. Readings included transfection efficiency, TIL phenotype, TCR library, and TIL effector function. This protocol is expected to result in a significant increase in TSCM frequency, will allow for the preservation of the TCR library of the TSCM subset relative to the entire TIL population, and will allow for the preservation of effector function relative to control.

Example 23: Obtaining and Validating sd-RXRNA

The experiments in this example provide data on sd-rxRNA constructs for five target molecules (PDCD1, TIM3, CBLB, LAG3, and CISH).

Phase 1: Obtain sd-rxRNAs for 5 target molecules.

Phase 2: Validation of sd-rxRNA-mediated gene silencing in pre-REP TIL (8 weeks).

Up to six frozen melanoma/other pre-REP cell lines. Experimental conditions were determined.

reading:

• Silence %: Expected ≥ 80%

The persistence of silence

• TIL growth: Expected to be within 10% of the control.

• TIL function: Increased production of expected cytokines

Phase 3: Implement sd-rxRNA-mediated gene silencing in the Gen 2 and/or Gen 3 processes (3 months)

• Fresh TIL formulations in 3 to 6 study-scales

• Same reading as above

• TIL phenotype

• TIL tumor reactivity

• At least two pairs of target/sd-rxRNAs will be selected for further work.

Phase 4: Implementing an optimized silencing protocol for industrial-scale TIL formulations (8 weeks).

• One industrial-scale formulation for each target gene.

Basic principle:

In the TME, TIL expresses several inhibitory molecules that negatively regulate the function of its effectors.

TILs can recover function when cultured in vitro, but will encounter immunosuppression again after re-infusion.

Ensuring that the T-cell suppressor pathway remains silent for at least several days after re-infusion can improve the efficacy of TILs against ACT.

Self-delivered siRNA (sd-rxRNA) provides an effective method for knocking out T cell genes. See, for example, Ligtenberg et al., Mol. Therapy, 26(6):1482-1493 (2018).

Target:

TIL effector function is re-established by silencing the inhibitory pathway.

Strategy:

During the rapid amplification protocol, transient knockout of 1) PDCD1, 2) TIM3, 3) CBLB, 4) LAG3, and 5) CISH was performed using sd-rxRNA. Particular attention was paid to the transient knockout of PD1 using sd-rxRNA during the rapid amplification protocol.

program:

To validate sd-rxRNA-mediated gene silencing in REP TILs (KD efficiency and persistence; T cell viability).

The effects of gene silencing on TIL phenotype and function (tumor reactivity).

Results Overview

Adding targeted sd-rxRNA during REP resulted in the successful knockout (KD) of 3 out of 5 target genes, including more than 80% (>80%) of PD1 knockout.

PD1: >80%

• TIM3: Approximately 70%

• LAG3: Approximately 70%

CISH: Approximately 40%

• CBLB: Undetectable

PD1 KD (knockout) is associated with reduced viability. PD1 KD is associated with reduced TIL amplification.

Significant phenotypic alterations were associated with PD1 and TIM3 KD, indicating higher levels of activation (enhanced expression of CD25, CCR7, CD56, 4-1BB, and OX40). In particular, significant phenotypic alterations were associated with PD1 KD, indicating high levels of activation (enhanced expression of CD25, CCR7, 4-1BB, and OX40 compared to the NTC control).

In these experiments, none of the sd-rxRNAs resulted in increased cytokine secretion in response to restimulation (INFγ/IL-2/TNF-α). Specifically, exposure of TILs to PDCD1 sd-rxRNAs did not increase CD107a mobilization or cytokine secretion (INFγ/IL-2/TNF-α) in response to restimulation. See, for example, Figure 54.

PD1 KD (knockout) increases the in vitro killing ability of TIL (see, for example, Figure 49).

method:

Day 0: Pre-REP begins. Add culture medium + IL-2.

Day 11: REP begins with medium containing IL-2+PBMCs, thawed/fresh pre-REP+sd-rxRNA (i.e., the second amplification begins).

• Day 14: Change the culture medium + sd-rxRNA containing IL-2 (OKT3 and feeder cells (PBMC) are optional); however, use only IL-2 (e.g., sd-rxRNA in a medium containing IL-2).

Day 17: Change the culture medium + sd-rxRNA (add other sd-rxRNA) (e.g., sd-rxRNA in medium containing IL-2).

Day 21: Change the culture medium + sd-rxRNA (add other sd-rxRNA) (e.g., sd-rxRNA in medium containing IL-2).

Day 22: Harvest TIL as described above.

Cell counting and viability determination.

ο Determine KD (knockout) efficiency (Q-PCR, flow cytometry)

As described above, phenotypic determinations were performed to characterize TIL.

o Examine activation markers (CD107a, IFNγ) (see, for example, Figure 45, inhibition/depletion markers; and Figure 46, IFNγ).

As shown in Figure 40, the experiment was conducted on five types of tumors: melanoma, breast tumors, lung tumors, sarcomas, and ovarian tumors.

Example 24: A variant implementation of Example 23

A TIL amplification method combined with transient alteration of protein expression was described according to the method discussed in Example 23. This example provides implementations with further variations of the method described in Example 23.

In some embodiments of the above methods, the method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population may include:

(i) Obtaining the first TIL group from the tumor removed from the patient;

(ii) A second TIL population is generated by first expansion of the first TIL population by culturing the first TIL population in a cell culture medium containing IL-2 and optional OKT-3;

(iii) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the number of the third TIL population is at least 100 times greater than that of the second TIL population; wherein the second expansion is performed for at least 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; and

(iv) Between day 11 and day 21, expose the second and/or third TIL populations to transcription factors (TFs) and/or other molecules capable of transiently altering protein expression; wherein the TFs and/or other molecules capable of transiently altering protein expression provided alterations in tumor antigen expression and/or the number of tumor antigen-specific T cells in the therapeutic TIL populations; and

(v) Harvest the therapeutic TIL clusters obtained from step (iv) on or after day 22; and

(vi) Optionally, the TIL clusters harvested in step (v) may be transferred to an infusion bag.

In some variant embodiments, other molecules capable of transiently altering protein expression include sd-RNAs, including, but not limited to, sd-rxRNAs. In some embodiments, TILs are exposed to sd-RNAs on days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least two days of days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least three days of days 11, 14, 17, and/or 21. In some embodiments, TILs are exposed to sd-RNAs on all days of days 11, 14, 17, and 21. In some embodiments, the sd-RNA targets PD-1. In some embodiments, the sd-RNA sequence used in this invention shows a 70% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 75% reduction in target gene expression.

In some embodiments, the sd-RNA sequence used in this invention shows an 80% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 85% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 90% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 95% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 99% reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.5 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.75 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 1.0 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, when delivered at a concentration of about 1.25 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, when delivered at a concentration of about 1.5 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, when delivered at a concentration of about 1.75 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, when delivered at a concentration of about 2.0 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, when delivered at a concentration of about 2.25 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, when delivered at a concentration of about 2.5 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, when delivered at a concentration of about 2.75 μM, the sd-RNA sequence used in this invention shows reduced target gene expression. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sd-RNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to TIL medium can be determined before or after changing the medium.

In some embodiments, the sd-RNA sequence used in this invention exhibits a 70% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 75% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 80% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 85% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 90% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 95% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 99% reduction in PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to the TIL medium can be determined before or after changing the medium.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 0.75 μM to about 3 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.0 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.5 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of 2 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of approximately 2 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some embodiments, the sd-RNA added on day 11 is added to a culture medium containing sd-RNA, IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, more sd-RNA is added on day 14. In some embodiments, more sd-RNA is added on day 17. In some embodiments, more sd-RNA is added on day 21. In some embodiments, the same concentration of sd-RNA is added on days 11, 14, 17, and 21. In some embodiments, different concentrations of sd-RNA are added on days 11, 14, 17, and/or 21. In some embodiments, the sd-RNA targets PD-1.

Example 25: A variant implementation of Example 23

A TIL amplification method combined with transient alteration of protein expression was described according to the method discussed in Example 23. This example provides implementations with further variations of the method described in Example 23.

In some embodiments of the above methods, the method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population may include:

(a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) A first amplification is performed by culturing a first TIL population in a cell culture medium containing IL-2 and optional OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 3 to 14 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (b) to step (c) occurs without opening the system;

(d) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (d) occurs without opening the system;

(e) Between day 11 and day 21, the second and/or third TIL groups were exposed to transcription factors (TFs) and/or other molecules capable of transiently altering protein expression; wherein the TFs and/or other molecules capable of transiently altering protein expression provided alterations in tumor antigen expression and/or the number of tumor antigen-specific T cells in the therapeutic TIL groups.

(f) On or after day 22, harvest the therapeutic TIL clusters obtained from step (d); wherein the transition from step (d) to step (e) occurs without opening the system; and

(g) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system.

In some variant embodiments, other molecules capable of transiently altering protein expression include sd-RNAs, including, but not limited to, sd-rxRNAs. In some embodiments, TILs are exposed to sd-RNAs on days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least two days of days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least three days of days 11, 14, 17, and/or 21. In some embodiments, TILs are exposed to sd-RNAs on all days of days 11, 14, 17, and 21. In some embodiments, the sd-RNA targets PD-1.

In some embodiments, the sd-RNA sequence used in this invention shows a 70% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 75% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 80% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 85% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 90% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 95% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 99% reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.5 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to TIL medium can be determined before or after changing the medium.

In some embodiments, the sd-RNA sequence used in this invention exhibits a 70% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 75% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 80% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 85% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 90% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 95% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 99% reduction in PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to the TIL medium can be determined before or after changing the medium.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least two days on days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least three days on days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for all days on days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 0.75 μM to about 3 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least two days on days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least three days on days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for all days on days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.0 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.5 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of 2 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of approximately 2 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some embodiments, the sd-RNA added on day 11 is added to a culture medium containing sd-RNA, IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, more sd-RNA is added on day 14. In some embodiments, more sd-RNA is added on day 17. In some embodiments, more sd-RNA is added on day 21. In some embodiments, the same concentration of sd-RNA is added on days 11, 14, 17, and 21. In some embodiments, different concentrations of sd-RNA are added on days 11, 14, 17, and/or 21. In some embodiments, the sd-RNA targets PD-1.

Example 26: A variant implementation of Example 23

A TIL amplification method combined with transient alteration of protein expression was described according to the method discussed in Example 23. This example provides implementations with further variations of the method described in Example 23.

In some embodiments of the above methods, a method for treating a subject suffering from cancer is provided, the method comprising administering expanded tumor-infiltrating lymphocytes (TILs), the method comprising:

(a) First TIL clusters are obtained by processing tumor samples obtained from patients into multiple tumor fragments and tumors removed by the subject;

(b) Adding tumor fragments to the closed system;

(c) A first amplification is performed by culturing a first TIL population in a cell culture medium containing IL-2 and optional OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 3 to 14 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (b) to step (c) occurs without opening the system;

(d) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion takes approximately 7 to 14 days to obtain the third TIL population, wherein the third TIL population is a therapeutic TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (d) occurs without opening the system;

(e) Between day 11 and day 21, the second and/or third TIL groups were exposed to transcription factors (TFs) and/or other molecules capable of transiently altering protein expression; wherein TFs and/or other molecules capable of transiently altering protein expression provided an increase in tumor antigen expression and/or an increase in the number of tumor antigen-specific T cells in the therapeutic TIL groups.

(f) Harvest the therapeutic TIL cluster obtained from step (d); wherein the transition from step (d) to step (e) occurs without opening the system; and

(g) The TIL clusters harvested in step (e) are transferred to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system;

(h) Optionally, the infusion bag containing the TIL clusters harvested from step (f) is cryopreserved using a cryopreservation method; and

(i) Administer the therapeutically effective dose of the third TIL group in the infusion bag of step (g) to the patient.

In some variant embodiments, other molecules capable of transiently altering protein expression include sd-RNAs, including, but not limited to, sd-rxRNAs. In some embodiments, TILs are exposed to sd-RNAs on days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least two days of days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least three days of days 11, 14, 17, and/or 21. In some embodiments, TILs are exposed to sd-RNAs on all days of days 11, 14, 17, and 21. In some embodiments, the sd-RNA targets PD-1.

In some embodiments, the sd-RNA sequence used in this invention shows a 70% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 75% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 80% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 85% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 90% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 95% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 99% reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.5 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to TIL medium can be determined before or after changing the medium.

In some embodiments, the sd-RNA sequence used in this invention exhibits a 70% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 75% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 80% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 85% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 90% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 95% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 99% reduction in PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to the TIL medium can be determined before or after changing the medium.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 0.75 μM to about 3 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.0 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.5 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of 2 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of approximately 2 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some embodiments, the sd-RNA added on day 11 is added to a culture medium containing sd-RNA, IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, more sd-RNA is added on day 14. In some embodiments, more sd-RNA is added on day 17. In some embodiments, more sd-RNA is added on day 21. In some embodiments, the same concentration of sd-RNA is added on days 11, 14, 17, and 21. In some embodiments, different concentrations of sd-RNA are added on days 11, 14, 17, and/or 21. In some embodiments, the sd-RNA targets PD-1.

Example 27: A variant implementation of Example 23

A TIL amplification method combined with transient alteration of protein expression was described according to the method discussed in Example 23. This example provides implementations with further variations of the method described in Example 23.

In some embodiments of the above method, an expanded TIL population for treating a subject with cancer can be provided, wherein the expanded TIL population is a third TIL population obtained by a method including the following steps:

(a) First TIL clusters are obtained by processing tumor samples obtained from patients into multiple tumor fragments and tumors removed by the subject;

(b) Adding tumor fragments to the closed system;

(c) A first amplification is performed by culturing a first TIL population in a cell culture medium containing IL-2 and optional OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 3 to 14 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (b) to step (c) occurs without opening the system;

(d) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 14 days to obtain the third TIL population; wherein the third TIL population is a therapeutic TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (d) occurs without opening the system;

(e) Between day 11 and day 21, the second and/or third TIL groups were exposed to transcription factors (TFs) and/or other molecules capable of transiently altering protein expression; wherein TFs and/or other molecules capable of transiently altering protein expression provided an increase in tumor antigen expression and/or an increase in the number of tumor antigen-specific T cells in the therapeutic TIL groups.

(f) Harvest the therapeutic TIL cluster obtained from step (d); wherein the transition from step (e) to step (f) occurs without opening the system; and

(g) The TIL cluster harvested in step (e) is transferred to an infusion bag; wherein the transfer from step (f) to step (g) occurs without opening the system; and

(h) Optionally, the infusion bag containing the TIL clusters harvested from step (f) is cryopreserved using a cryopreservation method.

In some variant embodiments, other molecules capable of transiently altering protein expression include sd-RNAs, including, but not limited to, sd-rxRNAs. In some embodiments, TILs are exposed to sd-RNAs on days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least two days of days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least three days of days 11, 14, 17, and/or 21. In some embodiments, TILs are exposed to sd-RNAs on all days of days 11, 14, 17, and 21. In some embodiments, the sd-RNA targets PD-1.

In some embodiments, the sd-RNA sequence used in this invention shows a 70% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 75% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 80% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 85% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 90% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 95% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 99% reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.5 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to TIL medium can be determined before or after changing the medium.

In some embodiments, the sd-RNA sequence used in this invention exhibits a 70% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 75% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 80% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 85% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 90% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 95% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 99% reduction in PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to the TIL medium can be determined before or after changing the medium.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 0.75 μM to about 3 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.0 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.5 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of 2 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of approximately 2 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some embodiments, the sd-RNA added on day 11 is added to a culture medium containing sd-RNA, IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, more sd-RNA is added on day 14. In some embodiments, more sd-RNA is added on day 17. In some embodiments, more sd-RNA is added on day 21. In some embodiments, the same concentration of sd-RNA is added on days 11, 14, 17, and 21. In some embodiments, different concentrations of sd-RNA are added on days 11, 14, 17, and/or 21. In some embodiments, the sd-RNA targets PD-1.

Example 28: A variant implementation of Example 23

A TIL amplification method combined with transient alteration of protein expression was described according to the method discussed in Example 23. This example provides implementations with further variations of the method described in Example 23.

In some embodiments of the above method, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population is provided, the method comprising:

(a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

(e) Let the second TIL group stand for about 1 day;

(f) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody and antigen-presenting cells (APC) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (f) occurs without opening the system;

(g) During any of steps (d), (e), and/or (f) (including days 11 to 21), the second TIL group is contacted with at least one sd-RNA; wherein the sd-RNA is added at the following concentrations: 0.1 μM sd-RNA, 0.5 μM sd-RNA, 0.75 μM sd-RNA, 1 μM sd-RNA, 1.25 μM sd-RNA, 1.5 μM sd-RNA, 2 μM sd-RNA, 5 μM sd-RNA, or 10 μM sd-RNA; wherein the sd-RNA is used to inhibit the expression of a molecule selected from PD-1, LAG-3, TIM-3, CISH, and CBLB, and combinations thereof;

(h) Optionally, a sterile electroporation step is performed on the second TIL group; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

(i) Harvest the therapeutic TIL clusters obtained from step (g) or (h), providing the harvested TIL clusters; wherein the transition from step (g) to step (i) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters; and

(j) The TIL cluster harvested in step (i) is transferred to the infusion bag; wherein the transfer from step (i) to step (j) occurs without opening the system.

In some variant embodiments, other molecules capable of transiently altering protein expression include sd-RNAs, including, but not limited to, sd-rxRNAs. In some embodiments, TILs are exposed to sd-RNAs on days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least two days of days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least three days of days 11, 14, 17, and/or 21. In some embodiments, TILs are exposed to sd-RNAs on all days of days 11, 14, 17, and 21. In some embodiments, the sd-RNA targets PD-1.

In some embodiments, the sd-RNA sequence used in this invention shows a 70% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 75% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 80% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 85% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 90% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 95% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 99% reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.5 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to TIL medium can be determined before or after changing the medium.

In some embodiments, the sd-RNA sequence used in this invention exhibits a 70% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 75% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 80% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 85% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 90% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 95% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 99% reduction in PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to the TIL medium can be determined before or after changing the medium.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 0.75 μM to about 3 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.0 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.5 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of 2 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of approximately 2 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some embodiments, the sd-RNA added on day 11 is added to a culture medium containing sd-RNA, IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, more sd-RNA is added on day 14. In some embodiments, more sd-RNA is added on day 17. In some embodiments, more sd-RNA is added on day 21. In some embodiments, the same concentration of sd-RNA is added on days 11, 14, 17, and 21. In some embodiments, different concentrations of sd-RNA are added on days 11, 14, 17, and/or 21. In some embodiments, the sd-RNA targets PD-1.

Example 29: A variant implementation of Example 23

A TIL amplification method combined with transient alteration of protein expression was described according to the method discussed in Example 23. This example provides implementations with further variations of the method described in Example 23.

In some embodiments of the above method, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population is provided, the method comprising:

(a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

(e) Let the second TIL group stand for about 1 day;

(f) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody and antigen-presenting cells (APC) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (f) occurs without opening the system;

(g) During any of steps (d), (e), and/or (f) (including the period from day 11 to day 21), the second TIL group is contacted with at least one sd-RNA; wherein the sd-RNA is added at a concentration of 2 μM; wherein the sd-RNA is used to inhibit the expression of a molecule selected from PD-1, LAG-3, TIM-3, CISH, and CBLB, and combinations thereof;

(h) Optionally, a sterile electroporation step is performed on the second TIL group; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

(i) Harvest the therapeutic TIL clusters obtained from step (g) or (h), providing the harvested TIL clusters; wherein the transition from step (g) to step (i) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters; and

(j) The TIL cluster harvested in step (i) is transferred to the infusion bag; wherein the transfer from step (i) to step (j) occurs without opening the system.

In some variant embodiments, other molecules capable of transiently altering protein expression include sd-RNAs, including, but not limited to, sd-rxRNAs. In some embodiments, TILs are exposed to sd-RNAs on days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least two days of days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least three days of days 11, 14, 17, and/or 21. In some embodiments, TILs are exposed to sd-RNAs on all days of days 11, 14, 17, and 21. In some embodiments, the sd-RNA targets PD-1.

In some embodiments, the sd-RNA sequence used in this invention shows a 70% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 75% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 80% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 85% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 90% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 95% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 99% reduction in target gene expression. In some embodiments, when delivered at a concentration of about 2.0 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In any of the foregoing embodiments, the specified concentration relative to TIL medium can be determined before or after changing the medium.

In some embodiments, the sd-RNA sequence used in this invention exhibits a 70% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 75% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 80% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 85% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 90% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 95% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 99% reduction in PD-1 expression. In some embodiments, when delivered at a concentration of approximately 2.0 μM, the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In any of the foregoing embodiments, the specified concentration relative to TIL medium can be determined before or after changing the culture medium.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 2 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 2 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 2.0 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.75 μM to about 2.25 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of 2 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of approximately 2 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some embodiments, the sd-RNA added on day 11 is added to a culture medium containing sd-RNA, IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, more sd-RNA is added on day 14. In some embodiments, more sd-RNA is added on day 17. In some embodiments, more sd-RNA is added on day 21. In some embodiments, the same concentration of sd-RNA is added on days 11, 14, 17, and 21. In some embodiments, different concentrations of sd-RNA are added on days 11, 14, 17, and/or 21, wherein the sd-RNA is added at an amount of approximately 2 μM for at least one day. In some embodiments, the sd-RNA targets PD-1.

Example 30: A variant implementation of Example 23

A TIL amplification method combined with transient alteration of protein expression was described according to the method discussed in Example 23. This example provides implementations with further variations of the method described in Example 23.

In some embodiments of the above method, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population is provided, the method comprising:

(a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

(e) Let the second TIL group stand for about 1 day;

(f) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody and antigen-presenting cells (APC) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (f) occurs without opening the system;

(g) During any of steps (d), (e), and/or (f) (including days 11 to 21), the second TIL group is contacted with at least one sd-RNA; wherein the sd-RNA is added at the following concentrations: 0.1 μM sd-RNA, 0.5 μM sd-RNA, 0.75 μM sd-RNA, 1 μM sd-RNA, 1.25 μM sd-RNA, 1.5 μM sd-RNA, 2 μM sd-RNA, 5 μM sd-RNA, or 10 μM sd-RNA; wherein the sd-RNA is used to inhibit the expression of a molecule selected from PD-1, LAG-3, TIM-3, CISH, and CBLB, and combinations thereof;

(h) Optionally, a sterile electroporation step is performed on the second TIL group; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

(i) Harvest the therapeutic TIL clusters obtained from step (g) or (h), and provide the harvested TIL clusters; wherein the transition from step (g) to step (i) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

(j) The TIL cluster harvested in step (i) is transferred to an infusion bag; wherein the transfer from step (i) to step (j) occurs without opening the system; and

(k) Cryopreservation of harvested TIL groups using dimethyl sulfoxide-based cryopreservation medium.

In some variant embodiments, other molecules capable of transiently altering protein expression include sd-RNAs, including, but not limited to, sd-rxRNAs. In some embodiments, TILs are exposed to sd-RNAs on days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least two days of days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least three days of days 11, 14, 17, and/or 21. In some embodiments, TILs are exposed to sd-RNAs on all days of days 11, 14, 17, and 21. In some embodiments, the sd-RNA targets PD-1.

In some embodiments, the sd-RNA sequence used in this invention shows a 70% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 75% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 80% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 85% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 90% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 95% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 99% reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.5 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to TIL medium can be determined before or after changing the medium.

In some embodiments, the sd-RNA sequence used in this invention exhibits a 70% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 75% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 80% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 85% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 90% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 95% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 99% reduction in PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to the TIL medium can be determined before or after changing the medium.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 0.75 μM to about 3 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.0 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.5 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of 2 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of approximately 2 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some embodiments, the sd-RNA added on day 11 is added to a culture medium containing sd-RNA, IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, more sd-RNA is added on day 14. In some embodiments, more sd-RNA is added on day 17. In some embodiments, more sd-RNA is added on day 21. In some embodiments, the same concentration of sd-RNA is added on days 11, 14, 17, and 21. In some embodiments, different concentrations of sd-RNA are added on days 11, 14, 17, and/or 21. In some embodiments, the sd-RNA targets PD-1.

Example 31: A variant implementation of Example 23

A TIL amplification method combined with transient alteration of protein expression was described according to the method discussed in Example 23. This example provides implementations with further variations of the method described in Example 23.

In some embodiments of the above method, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population is provided, the method comprising:

(a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Between day 11 and day 21, the first TIL group was contacted with at least one sd-RNA; wherein the sd-RNA was added at the following concentrations: 0.1 μM sd-RNA/10,000 TIL/100 μL medium, 0.5 μM sd-RNA/10,000 TIL/100 μL medium, 0.75 μM sd-RNA/10,000 TIL/100 μL medium, 1 μM sd-RNA/10,000 TIL/100 μL medium, 1.25 μM sd-RNA/1 0,000 TIL/100 μL of culture medium, 1.5 μM sd-RNA/10,000 TIL/100 μL of culture medium, 2 μM sd-RNA/10,000 TIL/100 μL of culture medium, 5 μM sd-RNA/10,000 TIL/100 μL of culture medium, or 10 μM sd-RNA/10,000 TIL/100 μL of culture medium; wherein, sd-RNA is used to inhibit the expression of the molecule, which is selected from PD-1, LAG-3, TIM-3, CISH, and CBLB and combinations thereof;

(e) Optionally, the first TIL group is subjected to a sterile electroporation step; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

(f) Adding OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (f) occurs without opening the system.

(g) Let the second TIL group stand for about 1 day;

(h) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (h) occurs without opening the system;

(i) Harvest the therapeutic TIL clusters obtained from step (h), providing the harvested TIL clusters; wherein the transition from step (h) to step (i) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

(j) The TIL cluster harvested in step (i) is transferred to an infusion bag; wherein the transfer from step (i) to step (j) occurs without opening the system; and

(k) Cryopreservation of harvested TIL groups using dimethyl sulfoxide-based cryopreservation medium.

In some variant embodiments, other molecules capable of transiently altering protein expression include sd-RNAs, including, but not limited to, sd-rxRNAs. In some embodiments, TILs are exposed to sd-RNAs on days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least two days of days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least three days of days 11, 14, 17, and/or 21. In some embodiments, TILs are exposed to sd-RNAs on all days of days 11, 14, 17, and 21. In some embodiments, the sd-RNA targets PD-1.

In some embodiments, the sd-RNA sequence used in this invention shows a 70% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 75% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 80% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 85% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 90% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 95% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 99% reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.5 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to TIL medium can be determined before or after changing the medium.

In some embodiments, the sd-RNA sequence used in this invention exhibits a 70% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 75% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 80% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 85% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 90% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 95% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 99% reduction in PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to the TIL medium can be determined before or after changing the medium.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 0.75 μM to about 3 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.0 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.5 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of 2 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of approximately 2 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some embodiments, the sd-RNA added on day 11 is added to a culture medium containing sd-RNA, IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, more sd-RNA is added on day 14. In some embodiments, more sd-RNA is added on day 17. In some embodiments, more sd-RNA is added on day 21. In some embodiments, the same concentration of sd-RNA is added on days 11, 14, 17, and 21. In some embodiments, different concentrations of sd-RNA are added on days 11, 14, 17, and/or 21. In some embodiments, the sd-RNA targets PD-1.

Example 32: A variant implementation of Example 23

A TIL amplification method combined with transient alteration of protein expression was described according to the method discussed in Example 23. This example provides implementations with further variations of the method described in Example 23.

In some embodiments of the above method, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population is provided, the method comprising:

(a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Between day 11 and day 21, the first TIL group was contacted with at least one sd-RNA; wherein the sd-RNA was added at the following concentrations: 0.1 μM sd-RNA/10,000 TIL, 0.5 μM sd-RNA/10,000 TIL, 0.75 μM sd-RNA/10,000 TIL, 1 μM sd-RNA/10,000 TIL, 1.25 μM sd-RNA/10,000 TIL. NA/10,000 TIL, 1.5 μM sd-RNA/10,000 TIL, 2 μM sd-RNA/10,000 TIL, 5 μM sd-RNA/10,000 TIL, or 10 μM sd-RNA/10,000 TIL; wherein, the sd-RNA is used to inhibit the expression of the molecule, which is selected from PD-1, LAG-3, TIM-3, CISH, and CBLB and combinations thereof;

(e) Optionally, the first TIL group is subjected to a sterile electroporation step; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

(f) Adding OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (f) occurs without opening the system.

(g) Let the second TIL group stand for about 1 day;

(h) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (g) to step (h) occurs without opening the system;

(i) Harvest the therapeutic TIL clusters obtained from step (h), providing the harvested TIL clusters; wherein the transition from step (h) to step (i) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

(j) The TIL cluster harvested in step (i) is transferred to an infusion bag; wherein the transfer from step (i) to step (j) occurs without opening the system; and

(k) Cryopreservation of harvested TIL groups using dimethyl sulfoxide-based cryopreservation medium.

In some variant embodiments, other molecules capable of transiently altering protein expression include sd-RNAs, including, but not limited to, sd-rxRNAs. In some embodiments, TILs are exposed to sd-RNAs on days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least two days of days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least three days of days 11, 14, 17, and/or 21. In some embodiments, TILs are exposed to sd-RNAs on all days of days 11, 14, 17, and 21. In some embodiments, the sd-RNA targets PD-1.

In some embodiments, the sd-RNA sequence used in this invention shows a 70% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 75% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 80% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 85% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 90% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 95% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 99% reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.5 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to TIL medium can be determined before or after changing the medium.

In some embodiments, the sd-RNA sequence used in this invention exhibits a 70% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 75% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 80% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 85% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 90% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 95% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 99% reduction in PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to the TIL medium can be determined before or after changing the medium.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 0.75 μM to about 3 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least two days on days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least three days on days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for all days on days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.0 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least two days on days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least three days on days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for all days on days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.5 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of 2 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of approximately 2 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some embodiments, the sd-RNA added on day 11 is added to a culture medium containing sd-RNA, IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, more sd-RNA is added on day 14. In some embodiments, more sd-RNA is added on day 17. In some embodiments, more sd-RNA is added on day 21. In some embodiments, the same concentration of sd-RNA is added on days 11, 14, 17, and 21. In some embodiments, different concentrations of sd-RNA are added on days 11, 14, 17, and/or 21. In some embodiments, the sd-RNA targets PD-1.

Example 33: A variant implementation of Example 23

A TIL amplification method combined with transient alteration of protein expression was described according to the method discussed in Example 23. This example provides implementations with further variations of the method described in Example 23.

In some embodiments of the above method, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population is provided, the method comprising:

(a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

(e) Let the second TIL group stand for about 1 day;

(f) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody and antigen-presenting cells (APC) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (f) occurs without opening the system;

(g) During any of steps (d), (e), and/or (f) (including day 11 to day 21), the second TIL group is contacted with at least one sd-RNA; wherein the sd-RNA is added at the following concentrations: 0.1 μM sd-RNA/10,000 TIL/100 μL medium, 0.5 μM sd-RNA/10,000 TIL/100 μL medium, 0.75 μM sd-RNA/10,000 TIL/100 μL medium, 1 μM sd-RNA/10,000 TIL/100 μL medium, 1.25 μM sd-RNA/10,000 TIL/100 μL medium, 1.5 μM sd-RNA/10,000 TIL/100 μL medium, 2 μM sd-RNA/10,000 TIL/100 μL medium, 5 μM sd-RNA/10,000 TIL/100 μL medium, or 10 μM sd-RNA/10,000 TIL/100 μL medium; wherein, sd-RNA is used to inhibit the expression of the molecule, which is selected from PD-1, LAG-3, TIM-3, CISH, and CBLB and combinations thereof;

(h) Optionally, a sterile electroporation step is performed on the second TIL group; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

(i) Harvest the therapeutic TIL clusters obtained from step (g) or (h), and provide the harvested TIL clusters; wherein the transition from step (g) to step (i) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

(j) The TIL cluster harvested in step (i) is transferred to an infusion bag; wherein the transfer from step (i) to step (j) occurs without opening the system; and

(k) Cryopreservation of harvested TIL groups using dimethyl sulfoxide-based cryopreservation medium.

In some variant embodiments, other molecules capable of transiently altering protein expression include sd-RNAs, including, but not limited to, sd-rxRNAs. In some embodiments, TILs are exposed to sd-RNAs on days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least two days of days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least three days of days 11, 14, 17, and/or 21. In some embodiments, TILs are exposed to sd-RNAs on all days of days 11, 14, 17, and 21. In some embodiments, the sd-RNA targets PD-1.

In some embodiments, the sd-RNA sequence used in this invention shows a 70% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 75% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 80% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 85% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 90% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 95% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 99% reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.5 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to TIL medium can be determined before or after changing the medium.

In some embodiments, the sd-RNA sequence used in this invention exhibits a 70% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 75% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 80% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 85% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 90% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 95% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 99% reduction in PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to the TIL medium can be determined before or after changing the medium.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 0.75 μM to about 3 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.0 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.5 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of 2 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of approximately 2 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some embodiments, the sd-RNA added on day 11 is added to a culture medium containing sd-RNA, IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, more sd-RNA is added on day 14. In some embodiments, more sd-RNA is added on day 17. In some embodiments, more sd-RNA is added on day 21. In some embodiments, the same concentration of sd-RNA is added on days 11, 14, 17, and 21. In some embodiments, different concentrations of sd-RNA are added on days 11, 14, 17, and/or 21. In some embodiments, the sd-RNA targets PD-1.

Example 34: A variant implementation of Example 23

A TIL amplification method combined with transient alteration of protein expression was described according to the method discussed in Example 23. This example provides implementations with further variations of the method described in Example 23.

In some embodiments of the above method, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population is provided, the method comprising:

(a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

(e) Let the second TIL group stand for about 1 day;

(f) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody and antigen-presenting cells (APC) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (c) to step (f) occurs without opening the system;

(g) During any of steps (d), (e), and/or (f) (including day 11 to day 21), the second TIL group is contacted with at least one sd-RNA; wherein the sd-RNA is added at the following concentrations: 0.1 μM sd-RNA/10,000 TIL/100 μL medium, 0.5 μM sd-RNA/10,000 TIL/100 μL medium, 0.75 μM sd-RNA/10,000 TIL/100 μL medium, 1 μM sd-RNA/10,000 TIL/100 μL medium, 1.25 μM sd-RNA/10,000 TIL/100 μL medium, 1.5 μM sd-RNA/10,000 TIL/100 μL medium, 2 μM sd-RNA/10,000 TIL/100 μL medium, 5 μM sd-RNA/10,000 TIL/100 μL medium, or 10 μM sd-RNA/10,000 TIL/100 μL medium; wherein, sd-RNA is used to inhibit the expression of the molecule, which is selected from PD-1, LAG-3, TIM-3, CISH, and CBLB and combinations thereof;

(h) Optionally, a sterile electroporation step is performed on the second TIL group; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

(i) Harvest the therapeutic TIL clusters obtained from step (g) or (h), and provide the harvested TIL clusters; wherein the transition from step (g) to step (i) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

(j) The TIL cluster harvested in step (i) is transferred to an infusion bag; wherein the transfer from step (i) to step (j) occurs without opening the system; and

(k) Cryopreservation of harvested TIL groups using dimethyl sulfoxide-based cryopreservation medium.

In some variant embodiments, other molecules capable of transiently altering protein expression include sd-RNAs, including, but not limited to, sd-rxRNAs. In some embodiments, TILs are exposed to sd-RNAs on days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least two days of days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least three days of days 11, 14, 17, and/or 21. In some embodiments, TILs are exposed to sd-RNAs on all days of days 11, 14, 17, and 21. In some embodiments, the sd-RNA targets PD-1.

In some embodiments, the sd-RNA sequence used in this invention shows a 70% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 75% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 80% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 85% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 90% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 95% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 99% reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.5 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to TIL medium can be determined before or after changing the medium.

In some embodiments, the sd-RNA sequence used in this invention exhibits a 70% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 75% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 80% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 85% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 90% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 95% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 99% reduction in PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to the TIL medium can be determined before or after changing the medium.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 0.75 μM to about 3 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.0 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.5 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of 2 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of approximately 2 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some embodiments, the sd-RNA added on day 11 is added to a culture medium containing sd-RNA, IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, more sd-RNA is added on day 14. In some embodiments, more sd-RNA is added on day 17. In some embodiments, more sd-RNA is added on day 21. In some embodiments, the same concentration of sd-RNA is added on days 11, 14, 17, and 21. In some embodiments, different concentrations of sd-RNA are added on days 11, 14, 17, and/or 21. In some embodiments, the sd-RNA targets PD-1.

Example 35: A variant implementation of Example 23

A TIL amplification method combined with transient alteration of protein expression was described according to the method discussed in Example 23. This example provides implementations with further variations of the method described in Example 23.

In some embodiments of the above method, a method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population is provided, the method comprising:

(a) Obtaining the first TIL group from the tumor removed from the patient by processing a tumor sample obtained from the patient into multiple tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) The first expansion was performed by culturing the first TIL population in a cell culture medium containing IL-2 and optionally containing a 4-1BB agonist antibody for about 2 to 5 days;

(d) Add OKT-3 to generate a second TIL population; wherein the first amplification is performed in a closed container providing a first ventilated surface area; wherein the first amplification is performed for approximately 1 to 3 days to obtain the second TIL population; wherein the number of the second TIL population is at least 50 times greater than the number of the first TIL population; wherein the transition from step (c) to step (d) occurs without opening the system.

(e) Let the second TIL group stand for about 1 day;

(f) A second expansion is performed by supplementing the cell culture medium of the second TIL population with additional IL-2, optional OKT-3 antibody, optional OX40 antibody, and antigen-presenting cells (APCs) to generate a third TIL population; wherein the second expansion is performed for approximately 7 to 11 days to obtain the third TIL population; wherein the second expansion is performed in a closed container providing a second breathable surface area; wherein the transition from step (e) to step (f) occurs without opening the system;

(g) During any of steps (d), (e), and/or (f) (including day 11 to day 21), the second TIL group is contacted with at least one sd-RNA; wherein the sd-RNA is added at the following concentrations: 0.1 μM sd-RNA/10,000 TIL, 0.5 μM sd-RNA/10,000 TIL, 0.75 μM sd-RNA/10,000 TIL, 1 μM sd-RNA/10,000 TIL. TIL, 1.25 μM sd-RNA/10,000 TIL, 1.5 μM sd-RNA/10,000 TIL, 2 μM sd-RNA/10,000 TIL, 5 μM sd-RNA/10,000 TIL or 10 μM sd-RNA/10,000 TIL; wherein, the sd-RNA is used to inhibit the expression of the molecule, which is selected from PD-1, LAG-3, TIM-3, CISH and CBLB and combinations thereof;

(h) Optionally, a sterile electroporation step is performed on the second TIL group; wherein the sterile electroporation step mediates the transfer of at least one sd-RNA;

(i) Harvest the therapeutic TIL clusters obtained from step (g) or (h), providing the harvested TIL clusters; wherein the transition from step (e) to step (h) occurs without opening the system; wherein the harvested TIL clusters are therapeutic TIL clusters;

(j) The TIL cluster harvested in step (i) is transferred to an infusion bag; wherein the transfer from step (h) to step (i) occurs without opening the system; and

(k) Cryopreservation of harvested TIL groups using dimethyl sulfoxide-based cryopreservation medium.

In some variant embodiments, other molecules capable of transiently altering protein expression include sd-RNAs, including, but not limited to, sd-rxRNAs. In some embodiments, TILs are exposed to sd-RNAs on days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least two days of days 11, 14, 17, and/or 21. In some variant embodiments, TILs are exposed to sd-RNAs on at least three days of days 11, 14, 17, and/or 21. In some embodiments, TILs are exposed to sd-RNAs on all days of days 11, 14, 17, and 21. In some embodiments, the sd-RNA targets PD-1.

In some embodiments, the sd-RNA sequence used in this invention shows a 70% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 75% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 80% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows an 85% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 90% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 95% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in this invention shows a 99% reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, when delivered at a concentration of about 0.5 μM, the sd-RNA sequence used in this invention shows a reduction in target gene expression. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention shows reduced target gene expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to TIL medium can be determined before or after changing the medium.

In some embodiments, the sd-RNA sequence used in this invention exhibits a 70% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 75% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 80% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits an 85% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 90% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 95% reduction in PD-1 expression. In some embodiments, the sd-RNA sequence used in this invention exhibits a 99% reduction in PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM to about 10 μM (in some embodiments, about 0.25 μM to about 4 μM), the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, when delivered at a concentration of about 0.25 μM, the sd-RNA sequence used in this invention exhibits reduced PD-1 expression. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 0.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 1.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits reduced PD-1 expression when delivered at a concentration of about 2.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 2.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.25 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.5 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 3.75 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 4.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 5.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 6.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 7.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 8.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 9.0 μM. In some embodiments, the sdRNA sequence used in this invention exhibits decreased PD-1 expression when delivered at a concentration of about 10.0 μM. In any of the foregoing embodiments, the specified concentration relative to the TIL medium can be determined before or after changing the medium.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 0.75 μM to about 3 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 0.75 μM to about 3 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.0 μM to about 2.5 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.0 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least two days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for at least three days during days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of about 1.5 μM to about 2.5 μM for all days during days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of about 1.5 μM to about 2.5 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some variant embodiments, TIL is exposed to sdRNA at a concentration of 2 μM for at least two days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for at least three days out of days 11, 14, 17, and/or 21. In some variant embodiments, TIL is exposed to sdRNA at a concentration of approximately 2 μM for all days out of days 11, 14, 17, and 21. In some variant embodiments, sdRNA is added at a concentration of approximately 2 μM when the culture medium is changed. In some embodiments, the sdRNA targets PD-1.

In some embodiments, the sd-RNA added on day 11 is added to a culture medium containing sd-RNA, IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and optionally more than one of IL-2, OKT3, and APC (including, for example, PBMC). In some embodiments, the sd-RNA added on day 14 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 17 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, the sd-RNA added on day 21 is added to a culture medium containing sd-RNA and IL-2. In some embodiments, more sd-RNA is added on day 14. In some embodiments, more sd-RNA is added on day 17. In some embodiments, more sd-RNA is added on day 21. In some embodiments, the same concentration of sd-RNA is added on days 11, 14, 17, and 21. In some embodiments, different concentrations of sd-RNA are added on days 11, 14, 17, and/or 21. In some embodiments, the sd-RNA targets PD-1.

The embodiments described above are provided to offer a complete disclosure and description of how to prepare and use the compositions, systems, and methods of the present invention to those skilled in the art, and are not intended to limit the inventors' view to their invention. Modifications to the above-described modes of implementation that are obvious to those skilled in the art are intended to fall within the scope of the appended claims. All patents and publications mentioned in this specification represent the technical level of those skilled in the art to which this invention pertains.

All headings and section names are for clarity and reference purposes only and are not intended to be limiting in any way. For example, those skilled in the art will understand that the effectiveness of appropriately combining various aspects from different headings and sections is appropriate in accordance with the spirit and scope of the invention described herein.

All references cited herein are incorporated herein by reference in their entirety, and for all purposes, to the extent that each individual publication or patent application is expressly and individually indicated to be incorporated herein by reference in its entirety for all purposes.

Many modifications and variations can be made to this application without departing from the spirit and scope of the invention, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are provided by way of example only, and this application is limited only by the appended claims and the full scope of their equivalents.

Claims (26)

1. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (a) Obtaining the first TIL group from the tumor removed from the patient; (b) The first TIL population was expanded by culturing the first TIL population in a cell culture medium containing IL-2 for about 3-14 days to generate the second TIL population; (c) A second expansion was performed by supplementing the culture medium of the second TIL population with additional IL-2, OKT-3, and antigen-presenting cells (APCs) to generate a third TIL population. This second expansion took approximately 7-14 days to obtain the third TIL population, which is a therapeutic TIL population; and (d) Harvesting therapeutic TILs; Wherein, the first TIL group is modified to achieve a transient change in the expression of one or more proteins before or during the first amplification in step (b), the second TIL group is modified to achieve a transient change in the expression of one or more proteins before or during the second amplification in step (c), or the third TIL group is modified to achieve a transient change in the expression of one or more proteins after the second amplification in step (c) and before step (d). 2. The method according to claim 1, wherein the transient change in expression increases the expression of one or more proteins. 3. The method according to claim 1, wherein the transient change in expression comprises inserting one or more nucleic acid molecules into a first TIL group, a second TIL group, or a third TIL group to alter the expression of one or more proteins. 4. The method according to claim 3, wherein nucleic acid insertion is achieved by electroporation. 5. The method according to claim 3, wherein the more than one nucleic acid molecule comprises more than one RNA molecule. 6. The method according to claim 5, wherein the one or more RNA molecules comprises one or more mRNA molecules and/or one or more siRNA molecules. 7. The method of claim 6, wherein nucleic acid insertion is achieved by microfluidic membrane disruption. 8. The method according to claim 6, wherein nucleic acid insertion is achieved by carrier-free microfluidic membrane disruption, wherein the carrier-free microfluidic membrane disruption mediates the transfer of one or more mRNA molecules and/or one or more siRNA molecules into the TIL. 9. The method according to claim 8, wherein the disruption of the carrier-free microfluidic membrane causes TIL deformation. 10. The method according to claim 8, wherein the one or more mRNA molecules encode membrane-bound IL-2 and membrane-bound IL-12. 11. The method according to claim 1, wherein the one or more proteins include one or more of IL-2, IL-7, IL-10, IL-12, IL-15 and IL-21. 12. The method according to claim 11, wherein the one or more proteins include one or more of IL-2, IL-15 and IL-21. 13. The method according to claim 12, wherein the one or more proteins include one or more of IL-15 and IL-21. 14. The method according to claim 12, wherein the one or more proteins include one or more of membrane-bound IL-2, membrane-bound IL-15, and membrane-bound IL-21. 15. The method of claim 14, wherein the one or more proteins comprise IL-15 in a membrane-bound form. 16. The method according to claim 11, wherein the one or more proteins comprise one or more of IL-2 and IL-12. 17. The method of claim 16, wherein the one or more proteins comprises IL-2. 18. The method of claim 16, wherein the one or more proteins comprises IL-12. 19. The method of claim 16, wherein the one or more proteins comprise one or more of membrane-bound IL-2 and membrane-bound IL-12. 20. The method of claim 1, wherein the first amplification is performed for approximately 3 to 11 days. 21. The method according to claim 20, further comprising the following steps: (e) The TIL clusters harvested in step (d) are transferred to an infusion bag. 22. The method of claim 1, wherein the second amplification is performed over a period of approximately 11 days. 23. The method of claim 1, wherein the first amplification and the second amplification are each performed separately over a period of approximately 11 days. 24. The method of claim 1, wherein steps (b) to (d) are performed over approximately 22 days. 25. The method according to claim 21, further comprising the following steps: (f) Cryopreservation of the infusion bag from step (e). 26. The method according to claim 1, wherein steps (b), (c) and (d) are performed in a closed system.