EA047474B1 - IMPROVED METHOD OF PRODUCING T CELLS - Google Patents

Description

Cross-references to related applications

This application claims the benefit of U.S. Provisional Patent Application No. 62/768,579 filed November 16, 2018, the entire contents of which are incorporated herein by reference.

1. Field of technology

The present invention relates to the field of medicine, in particular to the production of cellular methods for the treatment of cancer.

2. State of the art

Immunotherapy based on T cells expressing an artificial receptor, such as a chimeric antigen receptor or T cell receptor (TCR), polypeptides that target T cells to a specific tumor-associated antigen and provide primary and general costimulatory signaling to activate and enhance T cell proliferation, is becoming an increasingly promising treatment option, especially for patients who have exhausted other treatment options. However, for autologous CAR T cell-based therapy, cell production remains labor-intensive and time-consuming. As a result, there is a need in the art for improved methods for producing CAR T cells that will reduce the time and cost of obtaining such cells. The present document provides such an improved method.

3. Brief description of the essence of the invention

In a first aspect, the present invention provides methods for producing cells obtained from blood using a method comprising membrane filtration and ammonium chloride potassium buffer (ACH) to separate the cells from other blood components. In a first embodiment, the present invention provides a method for producing cells from peripheral blood mononuclear cells (PBMCs) of a subject from whom a blood sample is obtained, comprising the steps of: (a) obtaining blood cells from the blood sample, such as using leukapheresis or blood collection, to obtain peripheral blood mononuclear cells (PBMCs); (b) optionally freezing the blood cells and obtaining PBMCs after thawing, or optionally obtaining and freezing the PBMCs and then thawing before subsequent steps; (c) isolating the PBMCs using a membrane filtration system and ammonium chloride potassium buffer (ACH); (d) washing the PBMCs by centrifugation; (e) optionally cryopreserving the PBMCs; (f) optionally thawing the PBMCs if the PBMCs have been cryopreserved (e); (g) washing the PBMCs using a membrane filtration system; (h) producing cells from the PBMCs; and (i) washing the cells using a membrane filtration system. In a particular embodiment, steps (a)-(h) of the method are performed in order. In a particular embodiment, the cells are chimeric antigen receptor (CAR) T cells (CAR T cells).

In another embodiment, provided herein is a method for producing chimeric antigen receptor (CAR) T cells (CAR T cells) from peripheral blood mononuclear cells (PBMCs) of a subject from whom a blood sample is obtained, comprising the steps of: (a) obtaining PBMCs from the blood sample; (b) isolating the PBMCs that have been obtained from the blood sample using a membrane filtration system and ammonium chloride potassium buffer (ACH); (c) washing the PBMCs by centrifugation; (d) optionally cryopreserving the PBMCs; (e) optionally thawing the PBMCs cryopreserved in step (d); (f) washing the PBMCs using a membrane filtration system; (g) producing CAR T cells from the PBMCs of step (f); and (h) washing the CAR T cells of step (g) using a membrane filtration system. In a particular embodiment, steps (a)-(h) in the said method are carried out in order.

In another embodiment, provided herein is a method for producing chimeric antigen receptor (CAR) T cells (CAR T cells) from peripheral blood mononuclear cells (PBMCs) of a subject from whom a blood sample is obtained, comprising the steps of: (a) isolating PBMCs obtained from the blood sample of the subject, wherein said isolation is carried out using a membrane filtration system and ammonium chloride potassium buffer (ACH); (b) washing the PBMCs by centrifugation; (c) optionally cryopreserving the PBMCs; (d) optionally thawing the PBMCs cryopreserved in step (c); (e) washing the PBMCs using a membrane filtration system; (f) producing CAR T cells from the PBMCs of step (f); and (g) washing the CAR T cells of step (f) using a membrane filtration system. In a particular embodiment, steps (a)-(g) in the said method are performed in order.

In another embodiment, provided herein is a method for producing cells from peripheral blood mononuclear cells (PBMCs) from a blood sample of a subject, comprising the steps of: (a) isolating PBMCs from a blood sample of the subject, wherein the blood sample is a leukapheresis blood sample or a blood sample obtained by blood collection, using a membrane filtration system and ammonium chloride potassium buffer (ACH); (b) optionally cryopreserving the PBMCs of step (a); (c) optionally thawing the PBMCs from

- 1 047474 dii (b); (d) washing the thawed PBMCs of step (c) using a membrane filtration system; (e) producing cells from the PBMCs of step (d); and (f) washing the cells of step (e) using a membrane filtration system. In a particular embodiment, steps (a)-(f) of the method are performed in order. In a particular embodiment, the cells are chimeric antigen receptor (CAR)-expressing T cells (CAR T cells).

In another embodiment, provided herein is a method for producing cells from peripheral blood mononuclear cells (PBMCs) from a blood sample of a subject, comprising the following steps: (a) isolating PBMCs from a blood sample of the subject, wherein the blood sample is a leukapheresis blood sample or a blood sample obtained by blood collection, using a membrane filtration system and ammonium chloride potassium buffer (ACH); (b) cryopreserving the PBMCs of step (a); (c) thawing the PBMCs of step (b); (d) washing the thawed PBMCs of step (c) using a membrane filtration system; (e) producing cells from the PBMCs of step (d); and (f) washing the cells of step (e) using a membrane filtration system. In a particular embodiment, steps (a)-(f) of said method are performed in order. In a specific embodiment, the cells are chimeric antigen receptor (CAR)-expressing T cells (CAR T cells).

In another embodiment, provided herein is a method for producing cells from peripheral blood mononuclear cells (PBMCs) from a blood sample of a subject, comprising the following steps: (a) isolating PBMCs from the blood sample of the subject using a membrane filtration system and ammonium chloride potassium buffer (ACH); (b) optionally cryopreserving and thawing the PBMCs of step (a); (c) optionally washing the thawed PBMCs of step (b) using a membrane filtration system; (d) producing cells from the PBMCs; and (e) washing the cells using the membrane filtration system. In a specific embodiment, steps (a)-(e) of the method are performed in order. In a specific embodiment, the blood sample is a leukapheresis blood sample. In a specific embodiment, the cells are chimeric antigen receptor (CAR) expressing T cells (CAR T cells).

In another embodiment, provided herein is a method for producing cells from peripheral blood mononuclear cells (PBMCs) from a blood sample of a subject, comprising the following steps: (a) isolating PBMCs from a blood sample of the subject using a membrane filtration system and ammonium chloride potassium buffer (ACH); (b) cryopreserving and thawing the PBMCs of step (a); (c) washing the thawed PBMCs of step (b) using a membrane filtration system; (d) producing cells from the PBMCs; and (e) washing the cells using the membrane filtration system. In a specific embodiment, steps (a)-(e) of the method are performed in order. In a specific embodiment, the blood sample is a leukapheresis blood sample. In a specific embodiment, the cells are chimeric antigen receptor (CAR) expressing T cells (CAR T cells).

In particular embodiments of any of the above embodiments, the cells are T cells (T lymphocytes), natural killer cells (NK cells), or dendritic cells. In particular embodiments, the T cells are cytotoxic T lymphocytes (CTL), CD4' T cells, CD8' T cells, or central memory T cells ( _Tcm ). In particular embodiments, the cells, e.g., T cells, have been genetically modified to express a polypeptide, e.g., a chimeric receptor. In more particular embodiments, the chimeric receptor is a T cell receptor (TCR) or a chimeric antigen receptor. In more particular embodiments, the cells in any of the above methods are chimeric antigen receptor-expressing T cells (CAR T cells).

In particular embodiments of any of the above embodiments, at least one or all of the membrane filtration systems are a tangential flow filtration system. In particular embodiments of any of the above embodiments, at least one or all of the membrane filtration systems may be a rotary membrane filtration system. In a particular embodiment of any of the above embodiments, the membrane filtration system is a rotary membrane filtration system, such as a LOVO automated cell processing system. In another particular embodiment of any of the above embodiments, the AHC buffer comprises 50-300 mmol ammonium chloride, 5-25 mmol potassium carbonate, and 0.05-0.25 mmol sodium edetate. In a more particular embodiment, the AHC buffer comprises 150 mmol ammonium chloride, 10 mmol potassium carbonate, and 0.1 mmol sodium edetate. In another specific embodiment of any of the above embodiments, said method improves the reduction of platelets and red blood cells in PBMCs by 18-36% compared to the same method using density gradient centrifugation instead of each application of spin membrane filtration. In another specific embodiment of any of the above embodiments, said method improves the reduction of platelets and

- 2,047,474 red blood cells in said PBMCs by 10-50%, 15-45%, 20-40%, 20-35%, 20-30%, 25-35%, 25-30%, 25-40% or 30-35% compared to the same method using density gradient centrifugation instead of each application of spin membrane filtration. In another specific embodiment of any of the above embodiments, said method improves the recovery of cells, such as CAR T cells, after T cell production by 17-36% compared to the same method using density gradient centrifugation instead of each application of spin membrane filtration. In another specific embodiment of any of the above embodiments, said method improves the recovery of cells, such as CAR T cells, after T cell production by about 10-50%, 15-45%, 20-40%, 20-35%, 20-30%, 25-35%, 25-30%, 25-40%, or 30-35% compared to the same method using density gradient centrifugation instead of each use of spin membrane filtration.

4. Detailed description of the essence of the invention

4.1 Improved cell production method - a review.

The present document provides a method for producing cells that includes an improved method for producing cells. The production method began with the isolation of autologous peripheral blood mononuclear cells (PBMCs) from a sample obtained by leukapheresis or from a sample obtained by drawing blood from a patient.

The manufacturing process began with the isolation of PBMCs from a leukapheresis sample or from a patient blood draw sample. Autologous PBMCs were isolated from a leukapheresis sample or from a patient blood draw sample using a membrane filtration system, such as a spin membrane filtration system, such as one with a pore size of 3.6 μm, to remove platelets and cellular debris. After removal of platelets and cellular debris, ammonium chloride potassium buffer (AKB) was used to lyse the red blood cells. The PBMCs were then separated from the AKB buffer solution and washed by centrifugation. This combination of spin membrane filtration followed by AKB RBC lysis represents an initial improvement over an earlier process that utilized density gradient centrifugation (Cell-Saver 5+ (CS5+; Haemonetics, Braintree, MA)).

After washing, the isolated PBMCs are resuspended and then incorporated into a suitable solution, such as Cryostor. Alternatively, T cells (or NK cells or dendritic cells) can be isolated from the PBMCs prior to incorporation into such a solution. In the case of T cells, magnetic beads such as Dynabeads coated with anti-CD3 antibodies, anti-CD4 antibodies or anti-CD8 antibodies can be used to isolate the T cells. The resulting PBMC solutions or isolated cells can then be frozen using a programmable cryofreezer, stored and released. This step can be followed by a cell culture step. For example, a 37°C water bath can be used on the day designated as day 0 to thaw cryopreserved PBMCs or isolated cells before washing and resuspending the cells in a culture medium suitable for growing such cells using a membrane filtration system, such as a spin membrane filtration system or centrifugation. Preferred T cell growth medium (TCGM) contains 93% (v/v) X-VIVO-15 cell culture medium (Lonza), 10 mM HEPES, 2 mM GlutaMAX™, 5% (v/v) human AB serum, and 100 IU/mL recombinant human interleukin-2 (rhIL-2) (see Hollyman et al, J. Immunother. 2009, 32:169-180).

The culture can be initiated and activated by seeding isolated PBMCs into a gas permeable cell differentiation bag at a concentration of 1*10 ⁶ PBMCs/ml in a medium such as TCGM with the addition of activation reagents (e.g. for T cells, Dynabeads coated with anti-CD3 antibodies and anti-CD28 antibodies). The cells can then be incubated at 37°C, preferably in air with 5% CO2. In the case of CAR T cells, such incubation can be continued until transduction of the T cells with a CAR-expressing vector. T cell activation can be measured by an increase in cell size (cell blasting) and cell clustering. Cell size (an indicator of activation) can be monitored after initiation of culture using, for example, the Coulter method.

To produce CAR T cells, transduction with a CAR expression vector can be accomplished by diluting a specified volume of vector in a medium, such as TCGM, which is then added to the cell culture. The volume of vector can be selected based on the number of PBMCs plated on day 0, the viral titer of the vector lot used, and the target multiplicity of infection (MOI). The target MOI in certain embodiments is vector lot specific and is selected to achieve comparable results from lot to lot. The cells are then incubated at 37°C with 5% CO2.

Following transduction with the vector, the culture is incubated and allowed to grow for a period of time and at the target seeding density. The culture can then be reseeded, for example, into gas-permeable cell expansion bags or another culture medium.

- 3 047474 a bioreactor bag such as the WAVE™ Bioreactor and incubate.

Then, for example, on day 10, the cells were harvested and washed using a membrane filtration system, such as a rotary membrane filtration system, and then the culture was transferred aseptically using a closed system into a bag for processing into a finished drug product. This represents a third improvement over the previous original method, which used the CS5+ device for the final wash.

For any of the above steps using a rotating membrane filter, the preferred device is the LOVO automated cell processing system (Fresenius Kabi).

In a specific embodiment, provided herein is a method for producing CAR T cells from peripheral blood mononuclear cells (PBMCs) of a subject from whom a blood sample is obtained, comprising the steps of: (a) obtaining blood cells from the blood sample, such as using leukapheresis or blood draw to obtain peripheral blood mononuclear cells (PBMCs); (b) optionally freezing the blood cells and obtaining PBMCs after thawing, or optionally obtaining and freezing the PBMCs and then thawing before subsequent steps; (c) isolating the PBMCs using a membrane filtration system, such as a spin membrane filtration system and ammonium chloride potassium buffer (ACH); (d) washing the PBMCs by centrifugation; (e) optionally cryopreserving the PBMCs; (f) optionally thawing the PBMCs if the PBMCs are cryopreserved in step (e); (g) washing the PBMCs using a membrane filtration system, such as a rotating membrane filtration system; (h) producing cells from the PBMCs; and (i) washing the cells using a membrane filtration system, such as a rotating membrane filtration system. In a particular embodiment, steps (a)-(d) and (g)-(i) of the method are performed in order. In a particular embodiment, steps (a)-(i) of the method are performed in order.

In another embodiment, provided herein is a method for producing chimeric antigen receptor (CAR) T cells (CAR T cells) from peripheral blood mononuclear cells (PBMCs) of a subject from whom a blood sample is obtained, comprising the steps of: (a) obtaining PBMCs from the blood sample; (b) isolating the PBMCs that have been obtained from the blood sample using a membrane filtration system and ammonium chloride potassium buffer (ACH); (c) washing the PBMCs by centrifugation; (d) optionally cryopreserving the PBMCs; (e) optionally thawing the PBMCs cryopreserved in step (d); (f) washing the PBMCs using a membrane filtration system; (g) producing CAR T cells from the PBMCs of step (f); and (h) washing the CAR T cells of step (g) using a membrane filtration system. In a particular embodiment, steps (a)-(h) in the said method are carried out in order.

In another embodiment, provided herein is a method for producing chimeric antigen receptor (CAR) T cells (CAR T cells) from peripheral blood mononuclear cells (PBMCs) of a subject from whom a blood sample is obtained, comprising the steps of: (a) isolating PBMCs obtained from the blood sample of the subject, wherein said isolation is carried out using a membrane filtration system and ammonium chloride potassium buffer (ACH); (b) washing the PBMCs by centrifugation; (c) optionally cryopreserving the PBMCs; (d) optionally thawing the PBMCs cryopreserved in step (c); (e) washing the PBMCs using a membrane filtration system; (f) producing CAR T cells from the PBMCs of step (f); and (g) washing the CAR T cells of step (f) using a membrane filtration system. In a particular embodiment, steps (a)-(g) in the said method are performed in order.

In another embodiment, provided herein is a method for producing cells from peripheral blood mononuclear cells (PBMCs) from a blood sample of a subject, comprising the following steps: (a) isolating PBMCs from a blood sample of the subject, wherein the blood sample is a leukapheresis blood sample or a blood sample obtained by blood collection, using a membrane filtration system and ammonium chloride potassium buffer (ACH); (b) optionally cryopreserving the PBMCs of step (a); (c) optionally thawing the PBMCs of step (b); (d) washing the thawed PBMCs of step (c) using a membrane filtration system; (e) producing cells from the PBMCs of step (d); and (f) washing the cells of step (e) using a membrane filtration system. In a particular embodiment, steps (a)-(f) of said method are performed in order. In a specific embodiment, the cells are chimeric antigen receptor (CAR)-expressing T cells (CAR T cells).

In another embodiment, the present document provides a method for producing cells from peripheral blood mononuclear cells (PBMCs) from a blood sample of a subject, comprising the steps of: (a) isolating PBMCs from a blood sample of the subject, wherein the blood sample is a leukapheresis blood sample or a blood sample obtained by drawing blood

- 4 047 474 vi, using a membrane filtration system and ammonium chloride potassium buffer (ACH); (b) cryopreserving PBMCs of step (a); (c) thawing the PBMCs of step (b); (d) washing the thawed PBMCs of step (c) using a membrane filtration system; (e) producing cells from the PBMCs of step (d); and (f) washing the cells of step (e) using a membrane filtration system. In a particular embodiment, steps (a)-(f) of the method are performed in order. In a particular embodiment, the cells are chimeric antigen receptor (CAR)-expressing T cells (CAR T cells).

In another embodiment, provided herein is a method for producing cells from peripheral blood mononuclear cells (PBMCs) from a blood sample of a subject, comprising the following steps: (a) isolating PBMCs from the blood sample of the subject using a membrane filtration system and ammonium chloride potassium buffer (ACH); (b) optionally cryopreserving and thawing the PBMCs of step (a); (c) optionally washing the thawed PBMCs of step (b) using a membrane filtration system; (d) producing cells from the PBMCs; and (e) washing the cells using the membrane filtration system. In a particular embodiment, the blood sample is a leukapheresis blood sample. In a particular embodiment, steps (a)-(e) of the method are performed in order.

In another embodiment, provided herein is a method for producing cells from peripheral blood mononuclear cells (PBMCs) from a blood sample of a subject, comprising the following steps: (a) isolating PBMCs from a blood sample of the subject using a membrane filtration system and ammonium chloride potassium buffer (ACH); (b) cryopreserving and thawing the PBMCs of step (a); (c) washing the thawed PBMCs of step (b) using a membrane filtration system; (d) producing cells from the PBMCs; and (e) washing the cells using the membrane filtration system. In a particular embodiment, the blood sample is a leukapheresis blood sample. In a particular embodiment, steps (a)-(e) of the method are performed in order.

In a specific embodiment, provided herein is a method of obtaining peripheral blood mononuclear cells (PBMCs) from a subject from whom a blood sample is obtained for the production of chimeric antigen receptor (CAR) T cells (CAR T cells), comprising the steps of: (a) obtaining blood cells from the blood sample, such as using leukapheresis or blood draw to obtain peripheral blood mononuclear cells (PBMCs); (b) optionally freezing the blood cells and obtaining the PBMCs after thawing, or optionally obtaining and freezing the PBMCs and then thawing before subsequent steps; (c) isolating the PBMCs using a membrane filtration system, such as a spin membrane filtration system and ammonium chloride potassium buffer (ACH); (d) washing the PBMCs by centrifugation; (e) optionally cryopreserving the PBMCs; (f) optionally thawing the PBMCs if the PBMCs are cryopreserved in step (e); and (g) washing the PBMCs using a membrane filtration system, such as a rotating membrane filtration system. In a particular embodiment, steps (a) to (d) are performed in order. In a particular embodiment, steps (a) to (g) of the method are performed in order.

In another embodiment, provided herein is a method for obtaining peripheral blood mononuclear cells (PBMCs) from a subject from whom a blood sample is obtained for the production of chimeric antigen receptor (CAR) T cells (CAR T cells), comprising the steps of: (a) obtaining PBMCs from the blood sample; (b) isolating PBMCs that have been obtained from the blood sample using a membrane filtration system and ammonium chloride potassium buffer (ACH); (c) washing the PBMCs by centrifugation; (d) optionally cryopreserving the PBMCs; (e) optionally thawing the PBMCs cryopreserved in step (d); and (f) washing the PBMCs using a membrane filtration system. In a particular embodiment, steps (a)-(f) of the method are performed in order.

In another embodiment, provided herein is a method for producing peripheral blood mononuclear cells (PBMCs) from a subject from whom a blood sample is obtained for the production of chimeric antigen receptor (CAR) T cells (CAR T cells), comprising the steps of: (a) isolating PBMCs obtained from the blood sample from the subject, wherein said isolating is done using a membrane filtration system and ammonium chloride potassium buffer (ACH); (b) washing the PBMCs by centrifugation; (c) optionally cryopreserving the PBMCs; (d) optionally thawing the PBMCs cryopreserved in step (c); and (e) washing the PBMCs using a membrane filtration system. In a particular embodiment, steps (a)-(e) of said method are performed in order.

In another embodiment, the present document provides a method for obtaining peripheral blood mononuclear cells (PBMCs) from a subject from whom a blood sample is obtained for the production of chimeric antigen receptor (CAR) expressing T cells (CAR T cells), comprising the steps of: (a) isolating PBMCs from a blood sample of the subject, wherein the blood sample is subjected to blood sample leukapheresis or obtained by drawing blood using a system

- 5 047474 membrane filtration and ammonium chloride potassium buffer (ACH); (b) optionally cryopreserving the PBMCs of step (a); (c) optionally thawing the PBMCs of step (b); and (d) washing the thawed PBMCs of step (c) using a membrane filtration system. In a particular embodiment, steps (a)-(d) of the method are performed in order.

In another embodiment, provided herein is a method for obtaining peripheral blood mononuclear cells (PBMCs) from a subject from whom a blood sample is obtained for the production of chimeric antigen receptor (CAR) T cells (CAR T cells), comprising the steps of: (a) isolating PBMCs from a blood sample of the subject, wherein the blood sample is subjected to blood sample leukapheresis or obtained by drawing blood using a membrane filtration system and ammonium chloride potassium buffer (ACH); (b) cryopreserving the PBMCs of step (a); (c) thawing the PBMCs of step (b); and (d) washing the thawed PBMCs of step (c) using a membrane filtration system. In a particular embodiment, steps (a)-(d) of the method are performed in order.

In another embodiment, provided herein is a method for obtaining peripheral blood mononuclear cells (PBMCs) from a subject from whom a blood sample is obtained for the production of chimeric antigen receptor (CAR) T cells (CAR T cells), comprising the steps of: (a) isolating peripheral blood mononuclear cells (PBMCs) from the subject's blood sample using a membrane filtration system and ammonium chloride potassium buffer (ACH); (b) optionally cryopreserving and thawing the PBMCs of step (a); and (c) optionally washing the thawed PBMCs of step (b) using a membrane filtration system. In a particular embodiment, the blood sample is a leukapheresis blood sample. In a particular embodiment, steps (a)-(c) of the method are performed in order.

In another embodiment, provided herein is a method for obtaining peripheral blood mononuclear cells (PBMCs) from a subject from whom a blood sample is obtained for the production of chimeric antigen receptor (CAR) T cells (CAR T cells), comprising the steps of: (a) isolating peripheral blood mononuclear cells (PBMCs) from the subject's blood sample using a membrane filtration system and ammonium chloride potassium buffer (ACH); (b) cryopreserving and thawing the PBMCs of step (a); and (c) washing the thawed PBMCs of step (b) using the membrane filtration system. In a particular embodiment, the blood sample is a leukapheresis blood sample. In a particular embodiment, steps (a)-(c) of the method are performed in order.

For any of the above embodiments, the T cells, NK cells, or dendritic cells can be isolated from the PBMCs prior to being formulated into solution. In more specific embodiments, the cells in any of the above methods are chimeric antigen receptor expressing T cells (CAR T cells). In the case of T cells, magnetic beads such as Dynabeads coated with anti-CD3 antibodies, anti-CD4 antibodies, or anti-CD8 antibodies can be used to isolate the T cells.

Various aspects of the production method are discussed in more detail below.

4.2. Cryopreservation.

PBMCs or cells, such as T cells, subjected to a cell production method, such as a CAR T cell production method, as described herein, may be cryopreserved. As used herein, cryopreservation means preserving cells by cooling to sub-zero temperatures, such as about or below the boiling point of liquid nitrogen, or -196°C. At sub-zero temperatures, cryoprotectants are preferably used to prevent damage to the preserved cells due to freezing at low temperatures or warming to room temperature. Cryoprotectants that can be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, Nature, 1959; 183:13941395; Ashwood-Smith, Nature, 1961; 190:1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, Ann. N.Y. Acad. Sci., 1960; 85:576), and polyethyleneglycol (Sloviter and Ravdin, Nature, 1962; 196:48). The preferred cooling rate is ~1 to 3°C/min. After at least 2 h, the T cells reach -80°C and can be placed directly in liquid nitrogen (-196°C) for permanent storage, such as in long-term cryogenic storage tanks.

4.3. Initiation and stimulation of culture.

T cells, such as unmodified T cells or T cells expressing CD3 and CD28 or comprising a polypeptide comprising a CD3Z signaling domain and a CD28 costimulatory domain, can be expanded using antibodies to CD3 and CD28, such as antibodies attached to beads; see, e.g., U.S. Patent Nos. 5,948,893; 6,534,055; 6,352,694; 6,692,964; 6,887,466; and 6,905,681. Magnetic beads, such as Dynabeads, coated with anti-CD3 antibodies and anti-CD28 antibodies can be used to expand T cells.

4.4 Chimeric antigen receptors.

In certain embodiments, the PBMCs, such as immune cells, more particularly,

- 6 047474

T cells obtained by performing the method described herein express one or more chimeric antigen receptors (CARs) on their surface. Typically, CARs comprise an intracellular domain of a first protein (e.g., an antigen-binding protein), a transmembrane domain, and an intracellular signaling domain, e.g., a primary signaling domain, and optionally one or more costimulatory domains. In preferred embodiments, once the extracellular domain binds to a target protein, such as a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA), a signal is generated via the intracellular signaling domain that activates an immune cell, e.g., to target and kill the cell expressing the target protein.

Extracellular domains: The extracellular domains of CARs bind to an antigen of interest. In some embodiments, the extracellular domain of a CAR comprises a receptor or a portion of a receptor that binds to said antigen. In some embodiments, the extracellular domain comprises or is an antibody or an antigen-binding portion thereof. In particular embodiments, the extracellular domain comprises or is a single-chain Fv (scFv) domain. A single-chain Fv domain can comprise, for example, a VL linked to a VH via a flexible linker, wherein V _L and V _H are antibodies that bind to said antigen.

In some embodiments, the antigen recognized by the extracellular domain of the polypeptide described herein is a tumor-associated antigen (TAA) or tumor-specific antigen (TSA). In various specific embodiments, the tumor-associated antigen or tumor-specific antigen is, but is not limited to, Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, B-cell maturation antigen (BCMA), epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-24-associated antigen (MAGE), CD19, CD22, CD27, CD30, CD34, CD45, CD70, CD99, CD117, EGFRvIII (epidermal growth factor variant III), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T-cell receptor alternative reading frame protein gamma), Trp-p8, STEAPI (six-transmembrane prostatic epithelial antigen 1), chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), acute cystic disease fluid protein (GCDFP-15), HMB-45 antigen, melanoma antigen-A (MART-I), myo-DI, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, dimeric form of pyruvate kinase isoenzyme type M2 (tumor M2-PK), abnormal ras protein, or abnormal p53 protein. In some other embodiments, the TAA or TSA recognized by the extracellular domain of the CAR is integrin αγβ3 (CD61), galactin, or Ral-B.

In some embodiments, the TAA or TSA recognized by the extracellular domain of the CAR is a cancer testicular antigen (CT), such as BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ES0-1, NY-SAR-35, OY-TES-1, SPANXBI, SPA17, SSX, SYCPI, or TPTE.

In some other embodiments, the TAA or TSA recognized by the extracellular domain of the CAR is a carbohydrate or ganglioside, such as fuc-GMI, GM2 (oncofetal antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, and the like.

In some other embodiments, the TAA or TSA recognized by the extracellular domain of the CAR is alpha-actinin-4, Bage-l, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA 125, CA 15-3 (CA 27.29\ BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-l, dek-can fusion protein, EBNA, EF2, Epstein-Barr virus antigens, ETV6 fusion protein -AML1, HLA-A2, HLA-All, hsp70-2, KIAA0205, Mart2, Mum-1, 2 and 3, neo-PAP, class I myosin, OS-9, fusion protein pml-RARo, PTPRK, K-ras, N-ras, trisophosphate isomerase, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1/Lage -2, SP17, SSX-2, TRP2-Int2, gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, MAGE-l, MAGE-3, RAGE, GAGE-l, GAGE-2, p15(58) , RAGE, SCP-1, Hom/Mel-40, PRAME, p53, HRa, HER-2/neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HPV) antigens E6 and E7 ), TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, 13-catenin, Mum -1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, C0-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-C0-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP or TPS.

In a specific embodiment, the tumor-associated antigen or tumor-specific antigen is a tumor antigen associated with AML as described in S. Anguille et al., Leukemia (2012), 26, 2186-2196.

Other tumor-associated and tumor-specific antigens are known in the art.

In a specific embodiment, wherein the antigen is BCMA, the chimeric

- 7 047474 antigen receptor is BCMA02 (see Chekmasova et al., Blood, 126:3094 (2015)). In a more specific embodiment, the CAR T cell expressing BCMA02 is LB2121 or LB21217.

The prior art provides receptors, antibodies and scFv that bind to TSA and TAA, suitable for constructing chimeric antigen receptors, as well as nucleotide sequences that encode them.

In some specific embodiments, the antigen recognized by the extracellular domain of the chimeric antigen receptor is an antigen that is not typically considered a TSA or TAA, but which is nonetheless associated with tumor cells or tumor-induced damage. In some embodiments, the antigen is, for example, a growth factor, cytokine, or interleukin, for example, a growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis. Such growth factors, cytokines, or interleukins can include, for example, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8). Tumors can also create a hypoxic environment local to the tumor. Thus, in other specific embodiments, the antigen is a hypoxia-associated factor, such as HIF-1cl HIF-Ιβ, HIF-2cl HIF-2e, HIF-3a, or HIF-3β. Tumors can also cause localized damage to normal tissue by causing the release of molecules known as pathogen-associated molecular pattern molecules (DAMPs; also known as alarmins). Thus, in some other specific embodiments, the antigen is a DAMP, such as heat shock protein, high mobility group chromatin-associated protein 1 (HMGB 1), S100A8 (MRP8, calgranulin A), S100A9 (MRP14, calgranulin B), serum amyloid A (SAA), or may be deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.

Transmembrane domain: In some embodiments, the extracellular domain of the CAR is connected to a transmembrane domain of a polypeptide by a linker, spacer, or loop polypeptide sequence, such as a sequence from CD28 or a sequence from CTLA4. The transmembrane domain may be derived from or derived from a transmembrane domain of any transmembrane protein and may include all or a portion of such a transmembrane domain. In particular embodiments, the transmembrane domain may be derived from or derived from, for example, CD8, CD16, a cytokine receptor, and an interleukin receptor or a growth factor receptor, or the like.

Intracellular signaling domains: In specific embodiments, the intracellular domain of a CAR is or comprises an intracellular domain or motif of a protein that is expressed on or near the surface of T cells and initiates the activation and/or proliferation of said T cells. Such a domain or motif is capable of transmitting a primary antigen-binding signal that is necessary for the activation of T lymphocytes in response to antigen binding to the extracellular portion of the CAR. Typically, this domain or motif comprises or is an ITAM (immunoreceptor tyrosine-based activation motif). Polypeptides containing ITAMs suitable for CARs include, for example, the CD3 zeta chain (CD3Z) or ITAM-containing portions thereof. In a specific embodiment, the intracellular domain is or comprises a CD3Z intracellular signaling domain; the CD3Z intracellular signaling domain may be referred to as the primary signaling domain. In specific embodiments, the intracellular domain (primary signaling domain) is derived from a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit, or an IL-2 receptor subunit.

In some embodiments, the CAR further comprises one or more costimulatory domains or motifs, such as as part of an intracellular domain of the polypeptide. The one or more costimulatory domains or motifs may be or may comprise one or more of a CD27 polypeptide costimulatory sequence or domain, a CD28 polypeptide costimulatory sequence or domain, an OX40 (CD134) polypeptide costimulatory sequence or domain, a 4-1BB (CD137) polypeptide costimulatory sequence or domain, or an inducible T cell (ICOS) polypeptide costimulatory sequence or domain, or another costimulatory domain or motif, or any combination thereof.

CARs may also comprise a T cell survival motif. The T cell survival motif may be any polypeptide sequence or motif that facilitates the survival of T lymphocytes following stimulation with an antigen. In some embodiments, the T cell survival motif is or is derived from CD3, CD28, the IL-7 receptor intracellular signaling domain (IL-7R), the IL-12 receptor intracellular signaling domain, the IL-15 receptor intracellular signaling domain, the IL-21 receptor intracellular signaling domain, or the transforming growth factor β receptor intracellular signaling domain (TGFe).

4.5. T cells (T lymphocytes).

T cells obtained by the methods presented herein may provide

- 8 047474 be naive T lymphocytes or major histocompatibility complex (MHC) antigen-restricted T lymphocytes. In some embodiments, the T lymphocytes are tumor infiltrating lymphocytes (TILs). In certain embodiments, the T cells are cytotoxic T cells (cytotoxic T lymphocytes or CTLs), CD4+ T cells, CD8+ T cells, effector T cells (TEFFs), or central memory T cells (TCMs).

T cells may be NKT cells (natural killer T cells), which are CD1d-restricted T cells that express the T cell receptor (TCR). Unlike conventional T cells, which detect peptide antigens presented by conventional major histocompatibility complex (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d, a non-classical MHC molecule. Two types of NKT cells are currently recognized. Invariant or type I NKT cells express a very limited TCR repertoire - the canonical α chain (Vo24-Jn18 in humans) associated with a limited repertoire of β chains (V[311 in humans). A second population of NKT cells, termed non-classical or non-invariant type II NKT cells, exhibit more heterogeneous use of the TCR αβ. Adaptive or invariant (type I) NKT cells can be identified by the expression of at least one or more of the following TCR markers: Vo24-Jn18, Vb11, CD1d, CD3, CD4, CD8, GalCer. CD161, and/or CD56.

The T cells obtained by the methods described herein may be genetically modified T cells, for example, they may be modified to express a polypeptide such as a T cell receptor (TCR) or a chimeric antigen receptor (CAR), such as one of the CARs described above in section 4.4. The modified immune cells, such as T cells, are preferably autologous to the individual to which the modified immune cells are to be administered. In some other embodiments, the modified immune cells are preferably allogeneic to the individual to which the modified immune cells are to be administered. When allogeneic T cells are used to produce the modified T cells, T cells are preferably selected that reduce the likelihood of graft versus host disease (GVHD) in the individual. For example, in some embodiments, virus-specific T cells are selected to produce the modified T cells; Such T cells are expected to have a significantly reduced native ability to bind to and thus be activated by any recipient antigens. In some embodiments, recipient-mediated rejection of allogeneic T cells can be reduced by co-administering to the host one or more immunosuppressive agents, such as cyclosporine, tacrolimus, sirolimus, cyclophosphamide, or the like.

Modified immune cells, such as modified T cells, may optionally contain a suicide gene or a safety switch that allows substantially all of the modified immune cells to be killed when needed. For example, modified T cells in certain embodiments may contain the HSV thymidine kinase (HSV-TK) gene, which causes the modified T cells to die when exposed to ganciclovir. In another embodiment, the modified T cells contain an inducible caspase, such as inducible caspase 9 (iCaspase9), such as a fusion protein between caspase 9 and human FK506 binding protein, which allows dimerization using a specific pharmaceutical small molecule. See Straathof et al., Blood, 105(11):4247-4254 (2005).

4.6. CAR T cell production.

In certain embodiments, any of the CARs can be administered to any of the cells described herein using any of the methods described herein. In a particular embodiment, the method comprises the following: activating cells, such as T cells, expanding the cells, transducing the cells using a vector, such as a lentiviral vector, encoding the CAR. In a particular embodiment, cells, such as T cells, are activated using anti-CD3 antibodies and anti-CD28 antibodies. In particular embodiments, the anti-CD3 antibodies and anti-CD28 antibodies are immobilized by binding to Fc receptors on endogenous antigen-presenting cells, such as monocytes and dendritic cells. In particular embodiments, cells, such as T cells, are expanded using a static bag and/or expansion via a WAVE bioreactor (GE Healthcare Life Sciences).

4.7. Dendritic cells.

Dendritic cells (DCs) obtained by the methods disclosed herein can be identified as follows. In the immature state, DCs can be characterized by low levels of MHC proteins and B7 costimulatory molecules, as well as the ability to conduct phagocytosis and pinocytosis, and an absence of the surface molecules CD83 and CD25. In the mature state, they can be characterized by an altered pattern of cell surface proteins, where the surface expression of some or all of the following molecules is increased: CD25, CD40, CD70, CD80, CD83, CD86, and MHC proteins. Mature DCs differ from immature DCs in that the former are more immunostimulatory, typically retain the ability to migrate into draining lymphatics, and are more likely to migrate to the lymph nodes.

- 9 047474 nodes in vivo and represent increasingly endogenously expressed and exogenous antigen in the context of MHC. Under physiological conditions, only mature DCs are able to activate naive T cells.

4.8. Natural killer cells.

Natural killer (NK) cells obtained by the methods described herein can be defined by the expression of CD56 or CD16 and the absence of a T cell receptor (CD3). NK cells can be adaptive NK cells or memory NK cells, these terms are interchangeable, and they refer to a subset of NK cells that are phenotypically CD3 ^- and CD56 +, express NKG2C and CD57 and optionally CD16, but lack expression of one or more of the following: PLZF, SYK, FceRy and EAT-2. The isolated subsets of CD56+ NK cells produced by the methods provided herein may include expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and/or inhibiting KIR, NKG2A and DNAM-1. CD56' may be implicit or explicit expression. NK cells produced by the methods described herein may be genetically modified NK cells, for example, they may be modified to express a polypeptide such as a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

4.9 Expression vectors and cell transduction.

Typically, polynucleotide sequences that express the chimeric antigen receptor are transferred into a cell (e.g., T cells or NK cells) in a polynucleotide vector. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, since they provide for long-term stable integration of the transgene and its propagation in daughter cells. Expression of natural or synthetic nucleic acids encoding CARs can be achieved by operably linking a nucleic acid encoding a CAR polypeptide or portions thereof to a promoter and incorporating the construct into an expression vector suitable for expression in a eukaryotic cell, such as a T cell.

The expression vector can generally be delivered to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other texts on virology and molecular biology. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, poxviruses, herpes simplex virus I, and the like. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

Typically, a suitable vector comprises an origin of replication that is functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01/96584; WO 01/29058 and U.S. Patent No. 6,326,193).

A number of viral systems have been developed for gene transfer into mammalian cells. For example, the gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells ex vivo. Alternatively, adenoviral vectors can be used.

The vector may contain a promoter and optionally one or more promoter elements, such as enhancers, and regulate the frequency of transcription initiation. These are typically located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often variable so that promoter function is maintained when the elements are inverted or moved relative to each other. In the thymidine kinase (TK) promoter, for example, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. The cytomegalovirus (CMV) immediate early promoter sequence can be used to drive CAR expression. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation factor growth factor-1a (EF-Iu). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian early virus 40 (SV40) promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Moreover, the present invention should not be limited to the use of constitutive promoters. Inducible promoters may also be used, including, but not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, or the tetracycline promoter.

To assess the expression of a CAR polypeptide on a CAR T cell, an expression vector introduced into the cell is

- 10 047474 may also contain a selectable marker gene, a reporter gene, or both to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected with viral vectors. The selectable marker may be on a separate polynucleotide and co-transfected into a T cell along with a vector encoding a CAR. The selectable markers and reporter genes may be flanked by appropriate regulatory sequences to ensure their expression in the host cell. Suitable selectable markers include, for example, antibiotic resistance genes such as neo, etc.

Reporter genes can be used to identify potentially transfected cells and to assess the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present or expressed in the recipient organism or tissue and that encodes a polypeptide whose expression results in some readily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at an appropriate time after introduction of the polynucleotide encoding it into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000, FEBS Letters, 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or purchased commercially. In general, the construct with the minimal 5' flanking region that exhibits the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be linked to a reporter gene and used to assess the ability of compounds to modulate promoter-activated transcription.

Methods for introducing and expressing genes into eukaryotic cells, such as T cells, are known in the art. In the context of an expression vector, the vector can be readily introduced into the T cell by any method known in the art. Physical methods for introducing a polynucleotide, such as a vector encoding a CAR, into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing a polynucleotide into a host cell is calcium phosphate transfection.

5. Examples

5.1 Example 1: Improved method for producing CAR T cells.

5.1.1. Original method.

The original CAR T cell production process was developed for BCMA-targeted chimeric antigen receptor (CAR)-expressing T cells (see Hollyman et al., J. Immunother. 2009; 32:169–180). The process parameters, such as PBMC isolation, T cell activation, transduction, and expansion, were initially developed and optimized using a small-scale T-flask process. Once the process parameters were established and optimized, the process was scaled up for clinical production. The process began with leukapheresis followed by isolation of peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation using a Cell-Saver 5+ (CS5+; Haemonetics, Braintree, Massachusetts); washings prior to culturing using batch centrifugation of the resulting PBMC culture in TCGM-HABS (T cell growth medium containing X-VIVO-15™ chemically defined hematopoietic cell medium (Lonza, Basel, Switzerland) without phenol red, supplemented with 5% v/v human AB serum, 2 mM GlutaMAX™ (Gibco), 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Thermo Fisher Scientific), and 300 IU/mL recombinant human interleukin-2 (rhIL2)). The same medium supplemented with 1% sodium pyruvate and 1% minimal essential vitamins was also tested and found to provide an equivalent number of population doublings in eight days. For simplicity, TCGM-HABS was chosen for the method. After comparison with additional media, the concentration of rhIL2 decreased from 300 to 100 IU/ml.

T cells in PBMCs were then activated using soluble anti-CD3 and anti-CD28 antibodies immobilized by binding to Fc receptors on endogenous antigen-presenting cells such as monocytes and dendritic cells. T cell activation was confirmed by cell size determination; resting T cells have a volume of ~180-200 fL, whereas highly activated T cells expand in size to >500 fL. The activation step was followed by expansion in a static bag, followed by expansion in a WAVE bioreactor (GE Healthcare Life Sciences). Expansion was followed by transduction of the expanded T cells with a lentiviral vector encoding a BCMA-targeting CAR. Based on studies using a range of MOIs from 0.625 to 40 and monitoring population doubling, cell size, and anti-BCMA CAR expression, a range of 10-30 MOI was used for the primary clinical production route.

- 11 047474 va. The resulting cells were collected and washed with a post-collection wash using a CD5+ device

However, the initial steps in the original method, from leukapheresis to T cell activation, required some labor-intensive steps, in particular the initial PBMC isolation and gradient centrifugation-dependent wash were modified to improve efficiency.

5.1.2. Improved method.

T cell homogeneity is key to the consistency of CAR T cell drug production. To this end, it was necessary to improve the original method by introducing a LOVO-AHC isolation step, a LOVO initial wash before culture, and a LOVO final wash step instead of the CS5+ device-dependent steps in the original method.

The method for isolating LOVO-AHC PBMCs involved two steps performed sequentially: a wash using the LOVO Automated Cell Processor (Fresenius Kabi, Lake Zurich, IL; referred to herein as LOVO) followed by incubation with AHC (ammonium chloride potassium buffer) lysis buffer. AHC buffer is 150 mM _NH4Cl , 10 mM KHCO3, and 0.1 mM sodium edetate in water (e.g., deionized water). The LOVO wash step utilized the LOVO Automated Cell Processor, which has a rotating membrane filtration system designed to efficiently remove platelets and cellular debris while preserving PBMC populations in the leukapheresis feed. The wash step was performed in a closed, pre-sterilized, disposable LOVO kit, which includes a cell separation device (a rotating membrane filtration system) to facilitate the removal of platelets, cell debris, and plasma.

After the LOVO wash step, the remaining cell suspension was added with AHC buffer to lyse the red blood cells. This step was performed in a transfer bag with a cell contact surface made of polyvinyl chloride. The red blood cell-depleted PBMCs were then separated from the AHC buffer solution and washed by centrifugation.

The isolated and washed PBMCs were then optionally cryopreserved (if they needed to be transported prior to use) using standard cryopreservation and controlled rate freezing techniques. The cells were thawed in a 37°C bath immediately prior to subsequent use. In three sets of the study, the cryopreserved cells produced adequate numbers of T cells and, following production of CAR ^- T cells, CAR ⁺ T cells.

Party of the International Criminal Code Total content of produced T-cells % CAR ⁺ Total CAR ⁺ T cell count 3101 1.32x1010 29.4% 3.88x109 3167 6.05x109 22.5% 1.36x109 3219 5.76x109 23.8% 1.37x109

After thawing, PBMCs underwent cell culture, which included culture initiation and stimulation with anti-CD3 and anti-CD28 antibodies (day 0); T cell transduction with a lentiviral vector carrying the coding sequences for the chimeric antigen receptor to BCMA (day 1); cell counting and passage into gas-permeable cell culture bags (days 2-5); cell counting and re-passaging in the WAVE™ Bioreactor (days 6-9); subsequent cell harvest and final wash using the LOVO device (day 10).

Thus, the final steps of the improved LOVO-AHC process are: leukapheresis;

allocation of LOVO-AHK MKPK;

washing of PBMCs by centrifugation;

cryopreservation of microbial cells (if transportation is necessary);

defrosting the microplastics (if transportation is necessary);

PBMC washing using LOVO T cell activation, cell expansion and collection;

rinsing using LOVO.

The improved method had advantages over the original method, including closed process steps (reduced likelihood of contamination); increased reliability and reproducibility of the PBMC preparations; reduced drug production time; and reduced overall process complexity.

5.2. Example 2. Research of the advantages of an improved method.

Comparison of the original extraction method and the LOVO-AHK extraction method. This example shows that the improved method is superior to the original method in some parameters.

A feasibility study comparing the original C.Ь5+PBMC isolation method with the LOVO-AHC PBMC isolation method was performed with leukapheresis units from 3 healthy donors and 2 subjects with multiple myeloma (MM). Following parallel PBMC isolation using the original and LOVO AHC methods, the isolated PBMC were subjected to

- 12 047474 cryopreserved and processed using the same cell culture, DP composition and cryopreservation methods. As summarized below, the viability and phenotypic composition of PBMCs, PBMC CD4+ and CD8+ T cell subsets, cell culture characterization and drug release assay results indicated that the LOVO-AHC isolation method is a possible replacement for the CS5+ PBMC isolation procedure.

Isolation and extraction of PBMCs.

The PBMC isolation step is designed to remove red blood cells (RBC) and platelets (PLT) from the leukapheresis feedstock. As shown in Table 1, the updated PBMC isolation method provided more extensive and consistent RBC depletion and PLT depletion, thereby improving the quality of PBMC for cell culture initiation.

Table 1

Comparison of baseline or post-modification RBC/WBS and PLT/WBS

Donor party Removal of RBCs from PBMCs Removing PLT from MKPK Original method (RBC/ WBC) Updated method (RBC/ WBC) The difference between initial and subsequent changes Original method (PLT/WBC) Updated Method (PLT/WBC) The difference between initial and subsequent changes 1 4 1 -3 18 1 -17 2 5 2 -3 28 1 -27 3 4 2 -2 19 1 -18 4 4 2 -2 36 0 -36 5 2 1 -1 13 0 -13 Average value 4 2 -2 23 1 -22

Values = percentage of total cells.

Since the two PBMC production methods used different PBMC isolation devices, the cell yields obtained in the PBMC isolation step were evaluated and compared. As shown in Table 2, the PBMC recovery yields in the original and revised methods were very similar, with an average difference of -2% between the pre- and post-change.

Table 2

Comparison of PBMC extraction

Donor party Isolation and extraction of PBMC Original method (%) Updated method (%) The difference between initial and subsequent changes 1 82 86 4 2 70 73 2 3 87 78 -9 4 61 59 -2 5 73 69 -4 Average value 75 73 -2

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Thus, the improved method showed isolation and recovery of PBMCs and removal of RBCs; and superior removal of platelets compared to the original method.

Characterization of cell culture during production process - comparison of T cell growth and peak T cell activation.

The results of cell culture characterization are presented in Table 3. T cells obtained from the original and updated methods demonstrated comparable growth kinetics, as evidenced by similar T cell growth rates (the average difference in T cell growth before and after the change is -1 population doubling) and activation profile (the average day of peak activation before and after the change in cell culture is the same).

Table 3

T cell growth and peak T cell activation in days in cell culture

Car T cell batch to BCMA T cell growth (PDL of T cells at collection) Time of peak activation (Day in cell culture) Original method Updated method The difference between initial and subsequent changes Original method Updated method The difference between initial and subsequent changes 1 8 7 -1 Day 3 Day 3 Day 0 2 11 11 0 Day 4 Day 4 Day 0 3 7 7 0 Day 5 Day 4 Day -1 4 9 8 -1 Day 4 Day 4 Day 0 5 9 8 -1 Day 3 Day 4 Day 1 Average value 9 8 -1 Day 4 Day 4 Day 0

The results of the characterization of medicinal products during the production process - extraction after washing after collection.

Since the two drug production methods used different devices for the wash step after day 10 of harvest, the cell yield obtained in the post-harvest wash step was evaluated and compared. As shown in Table 4, the post-harvest wash recovery of drug (CAR T cells) harvest obtained from the updated method was significantly improved compared to the original method, with an average increase of 30% observed.

Table 4

Extraction after washing during collection

Batch ID MKPK Extraction after washing during collection Original method (%) Updated method (%) The difference between initial and subsequent changes 1 63 93 30 2 77 94 17 3 63 101* 38 4 71 97 26 5 52 88 36 Average value 65 95 30

* The value exceeds 100 due to measurement error.

- 14 047474

Thus, the improved method shows significantly improved CAR T cell recovery compared to the original method.

5.3. Example 3. Comparative studies of the advantages of methods.

This example demonstrates that the improved method is comparable to the original method in other respects.

The original and improved PBMC isolation methods were compared and determined to yield equivalent results as follows. The conditions for method change are summarized in Table 5. For each healthy donor, the leukapheresis unit was split equally between the original CS5+ device and the updated LOVO-AHC PBMC isolation methods. Following the PBMC isolation step, PBMC batches were either cryopreserved or used to initiate cell culture without cryopreservation. Prior to cell culture initiation, PBMCs were washed by intermittent centrifugation or the LOVO cell processor. Gas permeable cell culture bags were used throughout the cell culture period. On day 10 post-harvest, the drug was washed with either the CS5+ device or the LOVO cell processor. The formulated drug was frozen in bags for preclinical in vivo comparability assessment and post-thaw stability analysis or in vials for in vitro comparability assessment.

Table 5

Conditions for changing the method for assessing comparability

Condition ID Description Conditions of the method Stage of the method: Obtaining the MKPK Stage of the method: Preparation and cryopreservation of medicinal products Action: Isolation of the MCPC Action: Washing before cell culture initiation Action: Rinse after collection 1 Original method Ficoll Density Centrifugation with CS5+ Centrifugation of series Device CS5+ 2 Updated method The LOVOАХК method N/A (part of the LOVOАХК method) in the absence of the PBMC retention step Device for processing LOVO cells in the presence of the PBMC retention step with freezing LOVO Cell Processing Device

A comparability study was performed with five healthy donor leukapheresis units from which ten lots of BCMA CAR T cells were derived (before and after the change in route in each case).

Each of the leukapheresis starting units was split and processed for PBMC isolation in parallel with the original and updated methods. Three of the five batches of PBMC were cryopreserved followed by thawing and cell culture initiation, while the remaining two batches of PBMC were immediately processed for cell culture initiation without freezing. The comparability of cryopreserved (PBMC retention step) and fresh (PBMC retention step) PBMC in the BCMA CAR T cell production process has been previously established. Following cell culture, the original and updated methods continued with drug harvest and processing steps, including appropriate cell wash methods. In-process drug samples and cryopreserved samples generated using the original and updated methods were evaluated according to the comparative assay plan described below.

Comparative analytical tests.

The results of the comparison of the viability of the PBMCs are presented in Table 6. There was no consistent difference in viability, with the average difference before and after the change being +2%.

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Table 6

Comparison of PBMC viability before and after changes in the method

Party of the International Criminal Code Viability of the MCPC Original method (%) Improved method (%) The difference between initial and subsequent changes 1 95 97 2 2 95 97 2 3 97 97 0 4 95 96 1 5 95 96 1 Average value 95 97 2

Comparison of the composition of the MCPC.

The results of the comparison of the composition of the PBMC are presented in Table 7 for leukocytes CD45+, CD3+CD56^-T cells, CD14+ monocytes and in Table 8 for CD19' B cells, CD3-CD56' NK cells and CD56 CD16' granulocytes, respectively. PBMC compositions were comparable, as the mean differences in the content of CD45+ leukocytes, CD3+CD56^-T cells, CD14+ monocytes, CD19+ B cells, and CD3-CD56+ NK cells were +1, +2, +1, -1, and -1%, respectively, before and after the change. No significant presence of CD56-CD16' granulocytes was observed in any of the methods (not detected). Overall, PBMC compositions were comparable in the original and updated methods.

Table 7

Composition of PBMC: CD45+ leukocytes, CD3+CD56 ^- T cells, CD14+ monocytes

Party CD45+ leukocytes (1)3+(1)56-1-cells CD14+ monocytes Gated on viable* cells Gated on CD45+ leukocyte Gated on CD45+ leukocytes Original method (%) Updated method (%) The difference between initial and subsequent changes Original method (%) Updated method (%) The difference between initial and subsequent changes Original method (%) Updated method (%) The difference between initial and subsequent changes 1 95 97 2 57 61 4 20 17 -3 2 98 97 -1 41 48 7 28 24 -4 3 96 96 0 41 36 -5 31 40 9 4 94 96 2 20 20 0 42 45 3 5 95 97 2 43 45 2 30 31 1 Average value 96 97 1 40 42 2 30 31 1

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Table 8 PBMC composition: CD19+ B cells, CD3-CD56+ NK cells, CD56-CD16+ granulocytes

Party of the International Criminal Code CD 19+ B cells CD3-CD56+ NK cells CD56-CD16+ granulocytes Gated on viable cells Gated on CD45+ leukocytes Gated on CD45+ leukocytes Original method (%) Updated method (%) The difference between initial and subsequent changes Original method (%) Updated method (%) The difference between initial and subsequent changes Original method (%) Updated method (%) The difference between initial and subsequent changes 1 7 9 2 10 9 -1 0 0 0 2 16 15 -1 8 7 -1 0 0 0 3 AND 6 -5 9 10 1 0 0 0 4 8 7 -1 21 21 0 0 0 0 5 7 6 -1 AND AND 0 1 0 -1 Average value 10 9 -1 12 AND -1 0 0 0

Comparison of CD4 ⁺ and CD8 ⁺ T cell expression in PBMCs.

The PBMC T cell population was further characterized for CD4 ⁺ and CD8 ⁺ expression. As shown in Table 9, the PBMC CD4 ⁺ /CD8 ⁺ T cell subset was comparable between the two methods, with the mean pre- and post-change difference for CD4 ⁺ and CD8 ⁺ cells in the T cell population being -3 and +3%, respectively.

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Table 9

CD4+ and CD8+ T cell subsets of PBMCs

Party of the International Criminal Code CD4+ T cells CD8+ T cells Gated on CD3+CD56-T- cells Gated on CD3+CD56-T- cells Original method (%) Updated method(%) The difference between initial and subsequent changes Original method (%) Updated method(%) The difference between initial and subsequent changes 1 63 60 -3 29 34 5 2 69 68 -1 27 28 1 3 68 66 -2 24 24 0 4 68 65 -3 26 28 2 5 50 45 -5 41 47 6 Average value 64 61 -3 29 32 3

Differences were observed between the leukocyte composition data of normal PBMC donors obtained to assess the comparability of changes in this method and the data of PBMCs from patients with multiple myeloma (MM) from a previous study. Compared with healthy donors, PBMCs from patients with MM contain fewer B cells and T cells and an increased number of monocytes.

In addition, the CD4+/CD8+ ratio in the T cell population was also different. Using the original method, the mean (N=5) percentages of B cells, T cells, and monocytes in healthy donor PBMC batches were 9.7±4.2%, 40.6±13.1%, and 30.0±7.8%, respectively. The mean (N=5) CD4+/CD8+ T cell ratio in healthy donor PBMC batches was 58.8%/32.7% T cells. Using the same original method to obtain PBMC, the mean (N=24) percentages of B cells, T cells, and monocytes in CRB-401 PBMC batches were 1.9±1.5%, 29.9±15.7%, and 56.4±19.4%, respectively. The mean (N=24) CD4+/CD8+ T cell ratio in CRB-401 PBMC batches was 40.8%/54.4% T cells. These observed differences in the two data sets were not due to method variation and were consistent with those reported in the scientific literature.

Thus, according to the above parameters, the improved method is comparable to the original method.

Claims (18)

1. A method for producing chimeric antigen receptor (CAR) T cells (CART cells) from peripheral blood mononuclear cells (PBMCs) of a subject from whom a blood sample is obtained, comprising the following steps: a) isolation of PBMC from a blood sample using a membrane filtration system and ammonium chloride potassium buffer (ACP), where PBMC are separated from the ACP buffer solution and washed by centrifugation; b) production of CAR-T cells from PBMCs; c) washing CAR-T cells using a membrane filtration system. 2. The method according to claim 1, wherein the blood sample is blood that has undergone leukapheresis. 3. The method according to claim 1 or 2, wherein the T cells are isolated from the isolated PBMCs, and the CAR-T cells are obtained from the T cells. 4. The method according to claim 3, wherein magnetic beads coated with antibodies to CD3, antibodies to CD4 or antibodies to CD8 are used to isolate T cells. 5. The method according to claim 3 or 4, wherein the isolated PBMCs or isolated T cells are cryopreserved. 6. The method according to any one of claims 3-5, wherein the isolated PBMCs or isolated T cells are cultured - 18 047474 after their cryopreservation. 7. The method according to claim 5 or 6, wherein the isolated PBMCs or isolated T cells are thawed, washed and resuspended in a nutrient medium suitable for growing such cells. 8. The method according to claim 7, wherein the thawed PBMCs or thawed T cells are washed using a membrane filtration system or centrifugation prior to the step of culturing the thawed PBMCs or thawed T cells. 9. The method according to any one of claims 1 to 8, characterized in that any or all membrane filtration systems are a rotating membrane filtration system. 10. The method according to claim 9, wherein the rotating membrane filtration system has a pore size of 3.6 µm. 11. The method according to any one of claims 1-10, characterized in that the AHC buffer contains 50-300 mmol of ammonium chloride, 5-25 mmol of potassium carbonate and 0.05-0.25 mmol of sodium edetate. 12. The method according to claim 11, characterized in that the said AHC buffer contains 150 mmol of ammonium chloride, 10 mmol of potassium carbonate and 0.1 mmol of sodium edetate. 13. The method according to any one of claims 1 to 12, wherein producing CAR T cells comprises activating, expanding and transducing T cells with a vector encoding the CAR. 14. The method according to claim 13, wherein the vector is a lentiviral vector. 15. The method according to any one of claims 1 to 14, wherein the PBMCs or T cells are activated by incubation in T cell growth medium supplemented with activation reagents prior to T cell transduction. 16. The method according to claim 15, wherein the activation comprises using antibodies to CD3 and antibodies to CD28. 17. The method according to any one of claims 13-16, wherein after transduction, incubation additionally occurs, allowing the cells to multiply. 18. The method according to any one of claims 13-17, wherein the CAR is a CAR to BCMA.