Improved t cell manufacturing process

Abstract

To provide a method of manufacturing chimeric antigen receptor-expressing T cells (CAR T cells) from peripheral blood mononuclear cells (PBMCs) from a subject from which a blood sample is obtained.SOLUTION: The manufacturing method comprises a step (a) of obtaining the PBMCs from the blood sample; a step (b) of isolating the PBMCs obtained from the blood sample, by using a membrane filtration system and ammonium chloride-potassium buffer; a step (c) of washing the PBMCs via centrifugation; a step (d) of optionally cryopreserving the PBMCs; a step (e) of optionally thawing the PBMCs cryopreserved in the step (d); a step (f) of washing the PBMCs using a membrane filtration system; a step (g) of manufacturing the CAR T cells from the PBMCs from the step (f); and a step (h) of washing the CAR T cells from the step (g) by using a membrane filtration system.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 768,57, filed November 16, 2018. No. 9, which is incorporated herein by reference in its entirety. will be done.

[0002] The present invention relates to the medical field, in particular to the production of cellular therapeutic agents for the treatment of cancer. [Background technology]

[0003] Artificial receptors, for example, target T cells to specific tumor-associated antigens, activate T cells, and provides primary and generally costimulatory signaling to increase proliferation of on T cells expressing a chimeric antigen receptor or T cell receptor (TCR), a polypeptide Based on this, immunotherapy is an increasingly promising treatment, especially for patients who have exhausted other treatment options. However, in the case of autologous CAR T cell therapy, cell manufacturing remains As a result, there is a great deal of effort in the art to reduce the time and expense of producing such cells. Improved methods for producing CAR T cells are needed to reduce Such methods are provided herein. Summary of the Invention

[0004] In a first aspect, the present invention provides a method for membrane filtration and isolation of cells from other blood components. from blood using a method incorporating ammonium chloride-potassium chloride (ACK) buffer. In a first embodiment, a method for producing a cell that can be obtained by the method described herein is provided. A method for producing cells from peripheral blood mononuclear cells (PBMCs) derived from a subject from whom a blood sample is obtained. The method includes: (a) removing hemocytes from a blood sample, for example using leukoreduction or blood sampling; (b) to obtain peripheral blood mononuclear cells (PBMCs); (b) optionally, to freeze and thaw blood cells, Obtain BMCs or optionally obtain and freeze PBMCs prior to further steps (c) Thawing the P using a membrane filtration system and ammonium chloride-potassium (ACK) buffer (d) isolating the PBMCs; (e) optionally, separating the PBMCs. (f) if the PBMCs were cryopreserved in step (e), optionally (g) washing PBMCs using a membrane filtration system; (h) preparing cells from PBMCs. (i) washing the cells using a membrane filtration system. Steps (a) through (h) of the method are carried out in sequence. In certain embodiments, the cells are The target cells are T cells that express a CAR T cell (CAR T cell).

[0005] In another embodiment, the present invention relates to a method for the preparation of a method for the preparation of a blood sample comprising the steps of: A method for producing chimeric antigen receptor (CAR)-expressing T cells (CAR T cells) from PBMCs (a) obtaining PBMCs from a blood sample; (b) detecting the presence of PBMCs in a membrane filtration system and a chloride ion exchange membrane; PBMCs obtained from blood samples were isolated using ammonium-potassium (ACK) buffer. (c) washing the PBMCs by centrifugation; (d) optionally cryopreserving the PBMCs. (e) optionally thawing the PBMCs cryopreserved in step (d); (f) using a membrane filtration system. (g) preparing CAR T cells from the PBMCs of step (f); h) washing the CAR T cells of step (g) using a membrane filtration system. In an embodiment, steps (a) to (h) of the method are carried out in sequence.

[0006] In another embodiment, the present invention relates to a method for the preparation of a method for the preparation of a blood sample comprising the steps of: A method for producing chimeric antigen receptor (CAR)-expressing T cells (CAR T cells) from PBMCs The present invention provides a method for isolating and in situ detecting PBMCs obtained from a blood sample from a subject, the method comprising: (a) isolating and in situ detecting PBMCs obtained from a blood sample from a subject; In this case, the isolation uses a membrane filtration system and ammonium chloride-potassium (ACK) buffer. (b) washing the PBMCs by centrifugation; (c) optionally cryopreserving the PBMCs; d) optionally thawing the PBMCs cryopreserved in step (c); (e) using a membrane filtration system. (f) preparing CAR T cells from the PBMCs of step (f); (g) Washing the CAR T cells of step (f) using a membrane filtration system. In this embodiment, steps (a) to (g) of the method are carried out in order.

[0007] In another embodiment, the present invention relates to a method for the detection of peripheral blood mononuclear cells (PBMCs) from a blood sample from a subject. The present invention provides a method for producing cells from PBMs (proliferative mesenchymal stem cells), the method comprising: (a) extracting PBMs from a blood sample from a subject; C., in which case the blood sample is a leukoreduced blood sample or a membrane filtration system and The blood sample was derived from blood collection using ammonium chloride-potassium (ACK) buffer. (b) optionally, cryopreserving the PBMCs of step (a); (c) optionally, (d) thawing the PBMCs of step (c); and (d) washing the thawed PBMCs of step (c) using a membrane filtration system. (e) preparing cells from the PBMCs of step (d); (f) using a membrane filtration system to prepare cells from the PBMCs of step ( In certain embodiments, steps (a) to (f) of the method further comprise washing the cells of step (a) to (f). In certain embodiments, the cells express a chimeric antigen receptor (CAR). The T cells that stimulate this reaction are called CAR T cells.

[0008] In another embodiment, the present invention relates to a method for the detection of peripheral blood mononuclear cells (PBMCs) from a blood sample from a subject. The present invention provides a method for producing cells from PBMs (proliferative mesenchymal stem cells), the method comprising: (a) extracting PBMs from a blood sample from a subject; C., in which case the blood sample is a leukoreduced blood sample or a membrane filtration system and The blood sample was derived from blood collection using ammonium chloride-potassium (ACK) buffer. (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) washing the thawed PBMCs of step (d); (f) preparing cells from the PBMCs; and (f) washing the cells of step (e) using a membrane filtration system. In certain embodiments, steps (a) to (f) of the method are carried out in sequence. In this embodiment, the cells are T cells expressing chimeric antigen receptors (CARs) (CAR T cells). (cell).

[0009] In another embodiment, the present invention relates to a method for the detection of peripheral blood mononuclear cells (PBMCs) from a blood sample from a subject. The present invention provides a method for producing cells from a culture medium comprising: (a) a membrane filtration system and ammonium chloride; - Potassium (ACK) buffer is used to isolate PBMCs from a blood sample from a subject; b) optionally, cryopreserving and thawing the PBMCs of step (a); (c) optionally, subjecting the PBMCs to membrane filtration; (d) preparing cells from the PBMCs using a perfusion system; (e) washing the cells using a membrane filtration system. Steps (a) through (e) of the method are carried out in sequence. In certain embodiments, the blood sample is a white blood cell sample. In certain embodiments, the cells are chimeric antigen receptor (CAR)-expressing cells. ) expressing T cells (CAR T cells).

[0010] In another embodiment, the present invention relates to a method for the detection of peripheral blood mononuclear cells (PBMCs) from a blood sample from a subject. The present invention provides a method for producing cells from a culture medium comprising: (a) a membrane filtration system and ammonium chloride; - isolating PBMCs from a blood sample from a subject using potassium (ACK) buffer; b) cryopreserving and thawing the PBMCs from step (a); (c) filtering the PBMCs from step (b) using a membrane filtration system; (d) washing thawed PBMCs; (e) preparing cells from PBMCs using a membrane filtration system; In certain embodiments, steps (a)-(e) of the method include washing the cells using a In certain embodiments, the blood sample is a leukoreduced blood sample. In certain embodiments, the cells are chimeric antigen receptor (CAR)-expressing T cells (CARs). RT cells).

[0011] In certain embodiments of any of the above embodiments, the cell is a T cell (T lymphocyte). In certain embodiments, the T cells are selected from the group consisting of T cells, natural killer cells (NK cells), and dendritic cells. The cells may be cytotoxic T lymphocytes (CTLs), CD4+ T cells, CD8+ T cells, or T cells. Central Memory (T cm ) cells. In certain embodiments, the cells, e.g., T cells, are The polypeptide is genetically engineered to express a chimeric receptor. In certain embodiments, the chimeric receptor is a T cell receptor (TCR) or a chimeric antigen receptor. In a more specific embodiment, the cell of any of the above methods is a chimeric antigen receptor-expressing T cells (CAR T cells).

[0012] In certain embodiments of any of the above embodiments, at least one of the membrane filtration system(s) One or all of the above embodiments are tangential flow filtration systems. In certain embodiments, at least one or all of the membrane filtration system(s) comprises a spinning It may be a membrane filtration system. In certain embodiments of any of the above embodiments, the membrane The filtration system is a spinning membrane filtration system such as the LOVO automated cell processing system. In another specific embodiment of any of the above embodiments, the ACK buffer comprises 50 to 300 mM of ammonium chloride, 5 to 25 mM of potassium carbonate, and 0.05 to 0.25 mM of ED In a more specific embodiment, the ACK buffer contains 150 mM sodium TA. Contains ammonium chloride, 10 mM potassium carbonate, and 0.1 mM sodium EDTA. In another particular embodiment of any of the above embodiments, the method further comprises the steps of: Compared to the same method using density gradient centrifugation instead of each use of blend filtration, Improves the reduction of platelets and red blood cells from BMC by 18% to 36%. In another particular embodiment, the method further comprises the steps of: The platelets and red blood cells from the PBMCs were significantly increased in comparison to the same method using density gradient centrifugation. Reduce blood cell counts by 10%-50%, 15%-45%, 20%-40%, 20%-35%, 20 %~30%, 25%~35%, 25%~30%, 25%~40%, or 30~35% improvement In another particular embodiment of any of the above embodiments, the method further comprises: Compared to the same method using density gradient centrifugation instead of each use of membrane filtration, T The recovery of cells after cell production, such as CAR T cells, is improved by 17% to 36%. In another particular embodiment of any of the embodiments, the method further comprises the steps of spinning membrane filtration. Compared to the same method using density gradient centrifugation instead of each use, cells after T cell production For example, CAR T cell recovery is 10% to 50%, 15% to 45%, 20% to 40%, or 2 0%~35%, 20%~30%, 25%~35%, 25%~30%, 25%~40%, or 30-35% improvement. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] 4.1. Improved Cell Production Processes – Overview The present invention relates to a cell manufacturing process incorporating an improved cell production process. The manufacturing process involves the production of autologous peripheral blood monoclonal antibodies from patients' leukapheresis or blood collection. Start by isolating primary PBMCs.

[0014] The manufacturing process involves isolating PBMCs from the patient's leukapheresis or blood draw. For example, a membrane filtration system, e.g., pore size 3 mm, is used to remove platelets and cell debris. 0.6 μm spinning membrane filtration system from leukapheresis or collection Autologous PBMCs are isolated from blood. After removing platelets and cell debris, they are diluted with ammonium chloride. Red blood cells are lysed using acetylcholine (ACK) buffer. PBMCs are then soaked in ACK buffer. Separate from the buffer and wash by centrifugation. Spinning membrane filtration followed by R This combination of ACK lysis of BCs was performed using density gradient centrifugation (Cell-Saver 5+ (CS5+; Haemonetics, Braintree, Massachusetts) This represents the first improvement over the previous process.

[0015] After washing, the isolated PBMCs are resuspended and then resuspended in an appropriate solution, e.g., Cryostor Alternatively, the T cells (or NK cells) may be pretreated prior to being formulated in such a solution. For T cells, anti-CD3, anti-CD4, anti-CD5, anti-CD6, anti-CD7, anti-CD8, anti-CD9, anti-CD10, anti-CD11, anti-CD12, anti-CD13, anti-CD14, anti-CD15, anti-CD16, anti-CD17, anti-CD18, anti-CD19, anti-CD20, anti-CD21, anti-CD22, anti-CD19, anti-CD2 4) or magnetic beads such as Dynabeads coated with anti-CD8 antibodies. The formulated PBMCs or isolated cells may then be used to isolate cells. Use a controlled rate freezer The cells may then be frozen, stored, and shipped. This step may be followed by a cell culture step. For example, a 37°C water bath is defined as day 0, and cryopreserved PBMCs or isolated The cells are thawed and then centrifuged using a membrane filtration system, e.g., a spinning membrane filtration system, or centrifugation. The cells may then be washed and resuspended in a growth medium suitable for the growth of such cells. The new T cell growth medium (TCGM) is a mixture of 93% (v / v) cell culture medium X-VIVO-15 (Lonza), 10mM HEPES, 2mM GlutaMAX™, 5%(v / v) human AB serum, and 100 IU / ml recombinant human interleukin 2 (rhIL -2) (Hollyman et al, J Immunother 2009, 32:169-180).

[0016] Isolated PBMCs were incubated with activating reagents (e.g., anti-CD3 and anti-CD28 for T cells). 1x1 in medium supplemented with 1x1000 ng / ml of TCGM (coated Dynabeads) 0 6Culture the cells by seeding them into gas-permeable cell differentiation bags at a concentration of 100 PBMC / mL. The cells are then incubated at 37° C., preferably in an air containing 5% CO2. In the case of CAR T cells, such incubation may be performed in air. The transduction may be continued until the T cells are transduced with the CAR-expressing vector. Activation is measured by an increase in cell size (cell blasting) and cell clustering. Cell size (an indicator of activation) can be determined, for example, using the Coulter method. Alternatively, monitoring may be performed after the start of culture.

[0017] For the production of CAR T cells, transduction with a CAR expression vector requires the administration of a specific amount of vector. The vector is diluted in a medium, such as TCGM, which is then added to the cell culture. The amount of vector may be determined by the number of PBMCs seeded on day 0, the vector used, The selection may be based on the virus titer of the vector lot and the multiplicity of infection (MOI) of the target. The target MOI in certain embodiments is vector lot specific and can vary between batches. The cells were then incubated at 37°C, 5% CO2 for 1 h at 4°C for 2 h. Welcome to the new home!

[0018] After transduction with the vector, the cultures are incubated and plated at the target seeding density for a period of time. The cultures are then grown in, for example, gas permeable cell growth bags or WAVE (commercially available) The cells may then be reseeded into other culture bags, such as a target bioreactor, and incubated.

[0019] Thereafter, for example on day 10, the cells are harvested and filtered using a membrane filtration system, for example a spinning membrane filtration system. A closed system is used to clean the product using a filtration system and then formulate it into the final drug product. Aseptically transfer the cultures into the processing bags using the CS5+ instrument for this final wash. This represents a third improvement over the previous baseline process which performed

[0020] For any of the above processes using a spinning membrane filter, a preferred The device was a LOVO automated cell processing system (Fresenius Kabi).

[0021] In certain embodiments, the present invention relates to a method for the detection of peripheral blood mononuclear cells (PBMCs) from a subject from whom a blood sample is obtained. The present invention provides a method for producing CAR T cells from PBMCs, the method comprising: (a) administering to a subject a subject, e.g., a subject, subject to a pulmonary bypass cytology test, Obtain peripheral blood mononuclear cells (PBMCs) by obtaining blood cells from blood samples using cytopheresis or blood sampling. (b) optionally freezing blood cells and obtaining PBMCs after thawing, or optionally (c) membrane filtration systems, e.g., spinning PBM was prepared using a phosphate membrane filtration system and ammonium chloride-potassium (ACK) buffer. (d) washing the PBMCs by centrifugation; (e) optionally freezing the PBMCs. (f) if the PBMCs were cryopreserved in step (e), optionally (g) thawing the PBMCs using a membrane filtration system, e.g., a spinning membrane filtration system; (h) preparing cells from PBMCs; (i) using a membrane filtration system, e.g., a spinning membrane filtration system. In certain embodiments, steps (a) to (d) and In certain embodiments, steps (a) to (i) of the method are carried out in sequence. ) are executed in order.

[0022] In another embodiment, the present invention relates to a method for the preparation of a method for the preparation of a blood sample comprising the steps of: A method for producing chimeric antigen receptor (CAR)-expressing T cells (CAR T cells) from PBMCs (a) obtaining PBMCs from a blood sample; (b) detecting the presence of PBMCs in a membrane filtration system and a chloride ion exchange membrane; PBMCs obtained from blood samples were isolated using ammonium-potassium (ACK) buffer. (c) washing the PBMCs by centrifugation; (d) optionally cryopreserving the PBMCs. (e) optionally thawing the PBMCs cryopreserved in step (d); (f) using a membrane filtration system. (g) preparing CAR T cells from the PBMCs of step (f); h) washing the CAR T cells of step (g) using a membrane filtration system. In an embodiment, steps (a) to (h) of the method are carried out in sequence.

[0023] In another embodiment, the present invention relates to a method for the preparation of a method for the preparation of a blood sample comprising the steps of: A method for producing chimeric antigen receptor (CAR)-expressing T cells (CAR T cells) from PBMCs The present invention provides a method for isolating and in situ detecting PBMCs obtained from a blood sample from a subject, the method comprising: (a) isolating and in situ detecting PBMCs obtained from a blood sample from a subject; In this case, the isolation uses a membrane filtration system and ammonium chloride-potassium (ACK) buffer. (b) washing the PBMCs by centrifugation; (c) optionally cryopreserving the PBMCs; d) optionally thawing the PBMCs cryopreserved in step (c); (e) using a membrane filtration system. (f) preparing CAR T cells from the PBMCs of step (f); (g) Washing the CAR T cells of step (f) using a membrane filtration system. In this embodiment, steps (a) to (g) of the method are carried out in order.

[0024] In another embodiment, the present invention relates to a method for the detection of peripheral blood mononuclear cells (PBMCs) from a blood sample from a subject. The present invention provides a method for producing cells from PBMs (proliferative mesenchymal stem cells), the method comprising: (a) extracting PBMs from a blood sample from a subject; C., in which case the blood sample is a leukoreduced blood sample or a membrane filtration system and The blood sample was derived from blood collection using ammonium chloride-potassium (ACK) buffer. (b) optionally, cryopreserving the PBMCs of step (a); (c) optionally, (d) thawing the PBMCs of step (c); and (d) washing the thawed PBMCs of step (c) using a membrane filtration system. (e) preparing cells from the PBMCs of step (d); (f) using a membrane filtration system to prepare cells from the PBMCs of step ( In certain embodiments, steps (a) to (f) of the method further comprise washing the cells of step (a) to (f). In certain embodiments, the cells express a chimeric antigen receptor (CAR). The T cells that stimulate this reaction are called CAR T cells.

[0025] In another embodiment, the present invention relates to a method for the detection of peripheral blood mononuclear cells (PBMCs) from a blood sample from a subject. The present invention provides a method for producing cells from PBMs (proliferative mesenchymal stem cells), the method comprising: (a) extracting PBMs from a blood sample from a subject; C., in which case the blood sample is a leukoreduced blood sample or a membrane filtration system and The blood sample was derived from blood collection using ammonium chloride-potassium (ACK) buffer. (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) washing the thawed PBMCs of step (d); (f) preparing cells from the PBMCs; and (f) washing the cells of step (e) using a membrane filtration system. In certain embodiments, steps (a) to (f) of the method are carried out in sequence. In this embodiment, the cells are T cells expressing chimeric antigen receptors (CARs) (CAR T cells). (cell).

[0026] In another embodiment, the present invention relates to a method for the detection of peripheral blood mononuclear cells (PBMCs) from a blood sample from a subject. The present invention provides a method for producing cells from a culture medium comprising: (a) a membrane filtration system and ammonium chloride; - Potassium (ACK) buffer is used to isolate PBMCs from a blood sample from a subject; b) optionally, cryopreserving and thawing the PBMCs of step (a); (c) optionally, subjecting the PBMCs to membrane filtration; (d) preparing cells from the PBMCs using a perfusion system; (e) washing the cells using a membrane filtration system. The sample is a leukoreduced blood sample. In certain embodiments, step (a) of the method Execute ~(e) in order.

[0027] In another embodiment, the present invention relates to a method for the detection of peripheral blood mononuclear cells (PBMCs) from a blood sample from a subject. The present invention provides a method for producing cells from a culture medium comprising: (a) a membrane filtration system and ammonium chloride; - isolating PBMCs from a blood sample from a subject using potassium (ACK) buffer; b) cryopreserving and thawing the PBMCs from step (a); (c) filtering the PBMCs from step (b) using a membrane filtration system; (d) washing thawed PBMCs; (e) preparing cells from PBMCs using a membrane filtration system; In certain embodiments, the blood sample is leukoreduced using In a particular embodiment, steps (a) to (e) of the method are carried out in sequence. do.

[0028] In certain embodiments, chimeric antigen receptor (CAR)-expressing T cells (TCA) are used herein. To produce AR T cells, peripheral blood mononuclear cells (PBMCs) are collected from subjects from whom blood samples are obtained. (a) preparing a blood sample using, for example, leukoreduction or blood sampling, (b) obtaining peripheral blood mononuclear cells (PBMCs) from the fluid sample; and (b) optionally freezing the blood cells. Alternatively, PBMCs can be obtained and frozen and then frozen. (c) membrane filtration systems, such as spinning membrane filtration systems and chloride nitrification systems; (d) centrifugation to isolate PBMCs; (e) optionally, cryopreserving the PBMCs; (f) removing the PBMCs from step (e) using the P (g) optionally thawing PBMCs if they have been cryopreserved; For example, the method includes washing the PBMCs using a spinning membrane filtration system. In certain embodiments, steps (a) to (d) are carried out in sequence. Execute a) to (g) in order.

[0029] In another embodiment, the present invention relates to chimeric antigen receptor (CAR)-expressing T cells (CARs). To produce RT cells, peripheral blood mononuclear cells (PBMCs) are isolated from subjects from whom blood samples are obtained. The present invention provides a method for preparing a PBMC comprising: (a) obtaining PBMCs from a blood sample; and (b) subjecting the PBMC to membrane filtration. The system and ammonium chloride-potassium (ACK) buffer were used to obtain the α-amino acid salts from blood samples. (c) isolating the PBMCs; (d) optionally washing the PBMCs by centrifugation; (e) optionally thawing the PBMCs cryopreserved in step (d); (f) In certain embodiments, the method further comprises washing the PBMCs using a membrane filtration system. Steps (a) to (f) are carried out in order.

[0030] In another embodiment, the present invention relates to chimeric antigen receptor (CAR)-expressing T cells (CARs). To produce RT cells, peripheral blood mononuclear cells (PBMCs) are isolated from subjects from whom blood samples are obtained. The present invention provides a method for preparing a method for isolating PBMCs obtained from a blood sample from a subject, the method comprising: (a) isolating PBMCs obtained from a blood sample from a subject; In this case, the isolation involves a membrane filtration system and an ammonium-potassium chloride (ACK) buffer. (b) washing the PBMCs by centrifugation; (c) optionally freezing the PBMCs. (d) optionally thawing the PBMCs cryopreserved in step (c); (e) membrane filtration. In certain embodiments, the method further comprises washing the PBMCs using a filtration system. Execute a)~(e) in order.

[0031] In another embodiment, the present invention relates to chimeric antigen receptor (CAR)-expressing T cells (CARs). To produce RT cells, peripheral blood mononuclear cells (PBMCs) are isolated from subjects from whom blood samples are obtained. The present invention provides a method for preparing a PBMC comprising: (a) isolating PBMCs from a blood sample from a subject; In the case of, the blood sample is a leukoreduced blood sample or a membrane filtration system and ammonium chloride. (b) a blood sample derived from blood collection using an acetylcholine-potassium (ACK) buffer; (c) optionally, cryopreserving the PBMCs of step (b); (d) washing the thawed PBMCs of step (c) using a membrane filtration system. In certain embodiments, steps (a) to (d) of the method are carried out in sequence.

[0032] In another embodiment, the present invention relates to a method for the detection of peripheral blood mononuclear cells (PBMCs) from a blood sample from a subject. The present invention provides a method for producing cells from PBMs (proliferative mesenchymal stem cells), the method comprising: (a) extracting PBMs from a blood sample from a subject; C., in which case the blood sample is a leukoreduced blood sample or a membrane filtration system and The blood sample was derived from blood collection using ammonium chloride-potassium (ACK) buffer. (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. In an embodiment, steps (a) to (d) of the method are carried out in sequence.

[0033] In another embodiment, the present invention relates to chimeric antigen receptor (CAR)-expressing T cells (CARs). To produce RT cells, peripheral blood mononuclear cells (PBMCs) are isolated from subjects from whom blood samples are obtained. The method includes: (a) providing a membrane filtration system and ammonium-potassium chloride (A isolating peripheral blood mononuclear cells (PBMCs) from a blood sample from the subject using CK) buffer; (b) optionally, cryopreserving and thawing the PBMCs of step (a); (c) optionally, Washing the thawed PBMCs of step (b) using a filtration system. In certain embodiments, the blood sample is a leukoreduced blood sample. Steps (a) to (c) are carried out in this order.

[0034] In another embodiment, the present invention relates to chimeric antigen receptor (CAR)-expressing T cells (CARs). To produce RT cells, peripheral blood mononuclear cells (PBMCs) are isolated from subjects from whom blood samples are obtained. The method includes: (a) providing a membrane filtration system and ammonium-potassium chloride (A isolating peripheral blood mononuclear cells (PBMCs) from a blood sample from the subject using CK) buffer; (b) cryopreserving and thawing the PBMCs from step (a); (c) filtering the PBMCs from step (a) using a membrane filtration system; b) washing the thawed PBMCs. In certain embodiments, the blood sample is In a particular embodiment, steps (a) to (c) of the method are Execute in order.

[0035] For any of the above embodiments, the T cells, NK cells or Alternatively, dendritic cells may be isolated from PBMCs. The cells in this method are chimeric antigen receptor expressing T cells (CAR T cells). In this case, Dynabeads coated with anti-CD3, anti-CD4, or anti-CD8 antibodies were used. Magnetic beads such as may be used to isolate T cells.

[0036] Various aspects of the manufacturing process are discussed in detail below.

[0037] 4.2. Cryopreservation PBs undergoing the cell manufacturing process described herein, e.g., the CAR T cell manufacturing process. MCs or cells, e.g., T cells, may be cryopreserved. "Cryopreservation" means storing food at a temperature below freezing, for example, at the boiling point of liquid nitrogen, i.e., about -196°C or refers to the preservation of cells by cooling to just below that temperature. Or use at subzero temperatures to prevent damage to frozen cells due to warming to room temperature. Preferred cytoprotective agents that can be used include dimethyl sulfoxide (DMSO) (Love lock and Bishop,Nature,1959;183:1394-139 5;Ashwood-Smith,Nature,1961;190:1204-120 5), glycerol, polyvinylpyrrolidone (Rinfret, Ann.NYAca d. Sci., 1960;85:576), and polyethylene glycol (Slovit and Ravdin, Nature, 1962;196:48) However, the cooling rate is not limited thereto. A preferred cooling rate is about 1°C to 3°C per minute. After 24 hours, the T cells are allowed to reach a temperature of -80°C and stored permanently in a long-term cryogenic storage vessel. It can be placed directly into liquid nitrogen (-196°C) to freeze.

[0038] 4.3. Culture initiation and stimulation T cells, e.g., unmodified T cells, or T cells expressing CD3 and CD28, or A polypeptide comprising a D3ζ signaling domain and a CD28 costimulatory domain The cells are amplified using antibodies against CD3 and CD28, e.g., antibodies bound to beads. See, e.g., U.S. Patents 5,948,893 and 6,534,055. No. 5, No. 6,352,694, No. 6,692,964, No. 6,887,466 See US Pat. Nos. 6,905,681 and 6,905,681. Magnetic beads such as coated Dynabeads may be used for T cell expansion.

[0039] 4.4. Chimeric Antigen Receptors In certain embodiments, PBMCs produced during the practice of the methods described herein, such as immune cells, Virus cells, more specifically T cells, express one or more chimeric antigen receptors (CAs) on their surface. Generally, the CAR expresses a first protein (e.g., an antigen-binding protein R). ), e.g., the extracellular domain, transmembrane domain, and intracellular signaling domain from A primary signaling domain and, optionally, one or more costimulatory domains. In certain embodiments, the extracellular domain is a tumor-associated antigen (TAA) or a tumor-specific antigen (TTA). When it binds to a target protein such as ribosomal protein (SA), it transmits a signal via its intracellular signaling domain. This activates immune cells, e.g., cells expressing the target protein. are targeted and killed.

[0040] Extracellular domain: The extracellular domain of the CAR binds to an antigen of interest. In this case, the extracellular domain of the CAR contains a receptor, or a portion of a receptor, that binds to the antigen. In certain embodiments, the extracellular domain comprises an antibody or an antigen-binding portion thereof, or an antibody or antigen-binding portion thereof. In certain embodiments, the extracellular domain is It comprises or is a single chain Fv (scFv) domain. Single chain Fv domains can be, for example, linked by a flexible linker. H V connected to L Including See above V L and V H is derived from an antibody that binds to said antigen.

[0041] In certain embodiments, the polypeptides described herein are recognized by their extracellular domains. The antigens that are used are tumor-associated antigens (TAA) or tumor-specific antigens (TSA). In certain embodiments, the tumor-associated or tumor-specific antigen is, but is not limited to, Her2, Prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (C EA), 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, CD2 7, CD30, CD34, CD45, CD70, CD99, CD117, EGFRvII I (epidermal growth factor variant III), mesothelin, PAP (prostatic acid phosphatase), Prostein, TARP (T cell receptor gamma alternative reading frame protein), Tr p-p8, STEAPI (six-transmembrane epithelial antigen 1 of the prostate), chromogranin, cytochrome P450 latin, desmin, glial fibrillary acidic protein (GFAP), total cystic disease fluid protein Protein (GCDFP-15), HMB-45 antigen, protein Melan-A (by T lymphocytes) melanoma antigen recognized by mitogen-activated receptor 1 (MART-I), myo-D1, muscle specific actin (M SA), neurofilament, neuron-specific enolase (NSE), placental alkali phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, dimeric form pyruvate kinase isoenzyme M2 type (tumor M2-PK), abnormal ras protein In certain other embodiments, the extracellular The TAA or TSA recognized by the domain is integrin αvβ3 (CD61) , galactin, or Ral-B.

[0042] In certain embodiments, the TAA or TSA recognized by the extracellular domain of the CAR are cancer / testis (CT) antigens, e.g., BAGE, CAGE, CTAGE, FATE, G AGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ES0-1, NY-SAR-35, OY-TES-1, SPANXBI , SPA17, SSX, SYCPI, or TPTE.

[0043] In certain other embodiments, the TAA or T recognized by the extracellular domain of the CAR SA can be used to bind carbohydrates or gangliosides, e.g., fuc-GM1, GM2 (oncofetal antigen Gen-Immunogenic-1, OFA-I-1); GD2 (OFA-I-2), GM3, GD3, etc. It is.

[0044] In certain other embodiments, the TAA or T recognized by the extracellular domain of the CAR SA inhibits α-actinin-4, Bage-1, BCR-ABL, and Bcr-Abl fusion proteins. Protein, β-catenin, CA125, CA15-3 (CA27.29\BCAA), CA 195, CA242, CA-50, CAM43, Casp-8, CDC27, CDK4, cdkn2a, CEA, coa-l, dek-can fusion protein, EBNA, EF2 , Epstein-Barr virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA-All, hsp70-2, KIAA0205, Mart2, Mum-1, 2, and and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein , PTPRK, K-ras, N-ras, triosephosphate isomerase, Gage3, 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-1, MAGE-3 , RAGE, GAGE-l, GAGE-2, p15(58), RAGE, SCP-1, H om / Mel-40, PRAME, p53, HRas, HER-2 / neu, E2A-P RL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HP V) Antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, T AG-72-4, CA19-9, CA72-4, CAM17.1, NuMa, K-ras , 13-catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD6 8\KP1, C0-029, FGF-5, G250, Ga733(EpCAM), HTg p-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY- C0-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TL P, or TPS.

[0045] In certain embodiments, the tumor-associated or tumor-specific antigen is S. Anguille et al, Leukemia (2012), 26, 2186-2196 It is an AML-associated tumor antigen.

[0046] Other tumor-associated and tumor-specific antigens are known to those skilled in the art.

[0047] In certain embodiments where the antigen is BCMA, the chimeric antigen receptor is BCMA02 ( See Chekmasova et al., Blood 126:3094 (2015). In a more specific embodiment, the BCMA02-expressing CAR T cells are b2121 or bb21217.

[0048] Receptors, antibodies, and antibodies that bind to TSAs and TAAs useful for constructing chimeric antigen receptors and scFv are known in the art, and the nucleotide sequences encoding them are also available in the art. It is known in the art.

[0049] In certain embodiments, the antigen recognized by the extracellular domain of the chimeric antigen receptor is Not generally considered to be a TSA or TAA, but still associated with tumor cells or an antigen associated with damage caused by a tumor. For example, the antigen may be, for example, a growth factor, a cytokine or an interleukin, e.g., a circulating A growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis. Such growth factors, cytokines, or interleukins may 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) may also be included. Tumors may also be affected by tumor-localized hypoxic environments. Thus, in other particular embodiments, the antigen is a hypoxia-associated factor, e.g. For example, HIF-1α, HIF-1β, HIF-2α, HIF-2β, HIF-3α, or Tumors also cause localized damage to normal tissues, resulting in damage-related The release of molecules known as deoxyribonucleic acid (DAMPs; also known as alarmins) Thus, in other particular embodiments, the antigen is a DAMP. , e.g., heat shock proteins, chromatin-associated proteins high mobility group box 1 (H MGB 1), S100A8 (MRP8, calgranulin A), S100A9 (MRP 14, calgranulin B), serum amyloid A (SAA) or deoxyribonucleic acid (DEA). It may be bonucleases, adenosine triphosphate, uric acid, or heparin sulfate.

[0050] Transmembrane domain: In certain embodiments, the extracellular domain of the CAR is bound to a linker, spacer, or hinge polypeptide sequence, e.g., a sequence derived from CD28 or a sequence derived from CTLA4. The transmembrane domain is optionally linked to the transmembrane domain of the polypeptide by a sequence similar to that described above. may be obtained or derived from the transmembrane domain of a transmembrane protein of It may include all or part of such a transmembrane domain. Domains include, for example, CD8, CD16, cytokine receptors, and interleukin receptors. The polypeptide may be obtained or derived from a receptor, a growth factor receptor, or the like.

[0051] Intracellular signaling domain: In certain embodiments, the intracellular domain of the CAR is It is an intracellular domain or motif of a protein expressed on or near the surface of a cell. or containing the same, resulting in activation and / or proliferation of said T cells. The domain or motif is a T lymphocyte specific antigen binding domain that is expressed in response to binding of an antigen to the extracellular portion of the CAR. It is capable of transmitting the primary antigen-binding signal required for activation of the target cell. The main or motif contains an ITAM (immunoreceptor tyrosine-based activation motif) or is an ITAM. Suitable ITAM-containing polypeptides for CAR include, for example, ZetaCD3 In certain embodiments, the intracellular domain comprises the CD3 chain (CD3ζ) or an ITAM-containing portion thereof. The intracellular signaling domain is or includes the CD3ζ intracellular signaling domain; The CD3ζ transduction domain is sometimes called the primary signaling domain. In certain embodiments, the intracellular domain (primary signaling domain) is a lymphocyte receptor a mitochondrial chain, a TCR / CD3 complex protein, an Fc receptor subunit, or an IL-2 receptor It is derived from the body subunit.

[0052] In certain embodiments, the CAR further comprises, for example, a portion of the intracellular domain of the polypeptide. Contains one or more costimulatory domains or motifs as part of one or more costimulatory domains Alternatively, the motif may be a costimulatory CD27 polypeptide sequence or domain, a costimulatory CD 28 Polypeptide sequences or domains, costimulatory OX40 (CD134) polypeptides Sequence or domain, costimulatory 4-1BB (CD137) polypeptide sequence or domain Main or costimulatory induced T cell costimulatory (ICOS) polypeptide sequence or domain the main or other costimulatory domain or motif, or any combination thereof The embodiment may be or may include one or more of the following:

[0053] CARs can also include a T cell survival motif. T cell survival motifs enhance the ability of the CAR to stimulate antigens. The polypeptide may be any polypeptide sequence or motif that promotes survival of T lymphocytes following stimulation. In certain embodiments, the T cell survival motif is a cellular motif of CD3, CD28, IL-7 receptor. Intracellular signaling domain of IL-7R, IL-12 receptor The intracellular signaling domain of the IL-15 receptor and the intracellular signaling domain of the IL-21 receptor The transduction domain, or intracellular signaling domain, of the transforming growth factor beta (TGFβ) receptor Mainly or derived from them.

[0054] 4.5.T cells (T lymphocytes) The T cells produced by the methods provided herein may be derived from naive T lymphocytes or MH lymphocytes. In certain embodiments, the T lymphocytes are tumor infiltrating lymphocytes. In certain embodiments, the T cells are cytotoxic T cells (cytotoxic T lymphocytes, or CTLs), CD4+ T cells, CD8+ T cells, T effector (T EF F ) cells, or T central memory (T CM ) cells.

[0055] The T cell may be an NKT cell (natural killer T cell), which is a T cell receptor Refers to CD1d-restricted T cells that express the TCR (TCR) and conventional major histocompatibility complex (MHC) Unlike conventional T cells, which detect peptide antigens presented by molecules, NKT cells It recognizes lipid antigens presented by the non-classical MHC molecule CD1d. Three types of NKT cells have been recognized. Invariant or type I NKT cells are highly restricted. A limited TCR repertoire, i.e., a limited range of β chains (Vβ11 in humans) They express the canonical α chain (Vα24-Jα18 in humans) that is responsible for the non-classical or non-inverting A second population of NKT cells, termed type II NKT cells, is more heterogeneous and expresses TCRα Adaptive or invariant (type I) NKT cells express the following markers: They may be identified by expression of at least one of the following: TCR Vα24-Jα18; Vb11, CD1d, CD3, CD4, CD8, αGalCer, CD161 and / or is CD56.

[0056] The T cells produced by the methods described herein may be genetically modified T cells. For example, a T cell receptor (TCR) or a chimeric antigen receptor (CAR), for example Modified to express one of the CAR polypeptides described in Section 4.4 above. The modified immune cells, e.g., T cells, are preferably administered In certain other embodiments, the modified immune cells are When allogeneic T cells are used to prepare modified T cells, In this case, T cells that would reduce the likelihood of graft-versus-host disease (GVHD) in an individual are selected. For example, in certain embodiments, the virus-specific T cells are Such T cells are capable of expressing natural T cells against any recipient antigen. The binding ability of natural IL-1 receptors is significantly reduced, and therefore they are thought to be activated by them. In certain embodiments, recipient-mediated rejection of allogeneic T cells is achieved by one or more immunosuppressants. Antibiotics, such as cyclosporine, tacrolimus, sirolimus, and cyclophosphamide It can be reduced by co-administration to the host.

[0057] The modified immune cells, e.g., modified T cells, optionally include substantially all of the modified immune cells. Optionally, the cells may contain a "suicide gene" or "safety switch" that allows the death of all For example, the modified T cells, in certain embodiments, upon contact with ganciclovir, The virus may contain the HSV thymidine kinase gene (HSV-TK), which causes the death of the virus. In embodiments, the modified T cells express an inducible caspase, such as inducible caspase 9 (iCa spase9), for example, allows dimerization of caspase 9 with certain small molecule drugs. The protein contains a fusion protein with the human FK506 binding protein, which binds to the FK506 receptor. See, e.g., Wang et al., Blood 105(11):4247-4254 (2005). I want to.

[0058] 4.6.Manufacture of CAR T cells In certain embodiments, any of the CARs can be produced using any of the methods described herein. and can be introduced into any of the cells described herein. The method includes: activating a cell, e.g., a T cell, expanding the cell, and administering a vector encoding a CAR; For example, transducing cells using lentiviral vectors. In one embodiment, cells, such as T cells, are activated using anti-CD3 and anti-CD28 antibodies. In certain embodiments, anti-CD3 and anti-CD28 antibodies are administered to cells, such as monocytes and dendritic cells. In certain embodiments, the antibody is immobilized by binding to an Fc receptor on a peripheral antigen presenting cell. The cells, e.g., T cells, are expanded using static bags and / or WAVE biosynthesis. Using the Oreactor (GE Healthcare Life Sciences) To multiply.

[0059] Dendritic cells The dendritic cells (DCs) produced by the methods disclosed herein are identified as follows: In their immature state, DCs express low levels of MHC proteins and B7 costimulatory molecules. They are characterized by the absence of CD83 and CD25 surface molecules, and the ability to perform phagocytosis and pinocytosis. In the mature state, DCs are characterized by altered patterns of cell surface proteins. may have increased surface expression of some or all of the following molecules: CD25, CD4 0, CD70, CD80, CD83, CD86, and MHC proteins. "Mature" DCs Unlike “immature” DCs, the former are more active upon immune stimulation and typically Retains the ability to migrate to draining lymph nodes and has a more endogenous expression of MHC Under physiological conditions, "mature" DCs retain their ability to present antigens and foreign antigens. Only these antibodies can activate naive T cells.

[0060] Natural killer cells The natural killer (NK) cells produced by the methods provided herein are 56 or CD16 expression and the absence of the T cell receptor (CD3). K cells may be "adaptive NK cells" or "memory NK cells", and these terms are used interchangeably. Interchangeable, phenotypically CD3- and CD56+, NKG2C and CD57, and optionally the subset of NK cells that express CD16, but one or more of the following: The expression of: PLZF, SYK, FceRγ, and EAT-2 is lacking. The isolated subpopulation of CD56+ NK cells produced by the provided methods is characterized by CD16, NKG2C, CD57, NKG2D, NCR ligand, NKp30, NKp40, NKp 46, which may include expression of activating and / or inhibitory KIR, NKG2A and DNAM-1 CD56+ may be expressed weakly or strongly. The NK cells produced may be genetically modified NK cells, e.g., T cell receptor (T The recombinant human IL-16 vector is modified to express a polypeptide such as a chimeric antigen receptor (CR) or a chimeric antigen receptor (CAR). It may be possible.

[0061] 4.9. Expression Vectors and Cell Transfection Generally, a polynucleotide sequence expressing a chimeric antigen receptor is In a vector, the vector is introduced into a cell (e.g., a T cell or a NK cell), such as a lentivirus. Vectors derived from retroviruses have been shown to allow long-term, stable integration of transgenes. and allowing its propagation in progeny cells, making it suitable for achieving long-term gene transfer. Expression of a natural or synthetic nucleic acid encoding a CAR produces a CAR polypeptide. or a nucleic acid encoding a portion thereof is operably linked to a promoter and is then administered to a eukaryotic cell, e.g. This can be achieved by incorporating the construct into an expression vector suitable for expression in T cells.

[0062] The expression vector may generally be provided to the cell in the form of a viral vector. The vector technique is well known in the art and is described, for example, in Sambrook et al. 01,Molecular Cloning:A Laboratory Manual ,Cold Spring Harbor Laboratory,New York) and other virology and molecular biology manuals. The viruses that are useful for this purpose are retroviruses, adenoviruses, adeno-associated viruses, and hepatitis B viruses. Examples include pesviruses, lentiviruses, poxviruses, and herpes simplex virus I. For example, but not limited to, U.S. Patent Nos. 5,350,674 and 5, See US Pat. No. 585,362.

[0063] Generally, a suitable vector will contain an origin of replication, a promoter, and / or a promoter sequence that are functional in at least one organism. A motor sequence, convenient restriction endonuclease sites, and one or more selectable markers. (e.g., WO01 / 96584; WO01 / 29058; and U.S. Pat. No. 6,333,663). No. 26,193).

[0064] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, a selected gene can be inserted into a vector using techniques known in the art. The recombinant virus can then be isolated. Alternatively, the vector can be delivered to the cells of a subject ex vivo using an adenovirus. Vectors may also be used.

[0065] The vector comprises a promoter and, optionally, one or more promoter elements, e.g. They may contain enhancers, which regulate the frequency of transcription initiation. It is located in the region 30 to 110 bp upstream of the initiation site, but most promoters Recently, it has been shown that promoter elements also contain functional elements downstream. are flexible enough to maintain promoter function even when elements are inverted or moved relative to one another For example, the thymidine kinase (tk) promoter requires 100 s of transcriptional activity before its activity begins to decline. Promoter elements can be spaced 50 bp apart. A CMMV promoter sequence may be used to drive expression of the CAR. The motor sequence regulates high levels of any polynucleotide sequence operably linked to it. A suitable promoter is a strong constitutive promoter sequence capable of driving expression. Another example is elongation growth factor-1α (EF-1α). SV40 early promoter, mouse mammary tumor virus (MMTV), human immune Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) Promoter, MoMuLV Promoter -, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Ruth sarcoma virus promoters, as well as human gene promoters, e.g., However, the actin promoter, myosin promoter, hemoglobin promoter, and and other constitutive promoters, including but not limited to the creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Metallothionein promoter, glucocorticoid promoter, progestin promoter, These include, but are not limited to, the tetracycline promoter, the tetracycline promoter, or the tetracycline promoter. Inducible promoters may also be used.

[0066] To assess the expression of the CAR polypeptide on the CAR T cells, an expression vector is introduced into the cells. The vector may also contain either a selectable marker gene or a reporter gene. Include both and transfect or infect via a viral vector. This makes it easier to identify and select cells expressing the gene from a cell population of interest. A selectable marker is incorporated into a separate polynucleotide to encode the CAR. The reporter gene may be co-transfected into T cells with a selectable marker or reporter gene. The gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo. .

[0067] Reporter genes identify potentially transfected cells and identify the location of regulatory sequences. Generally, reporter genes can be used to assess the functionality of a recipient organism. or are absent or not expressed in tissues, and their expression is limited to some easily detectable A gene encoding a polypeptide that has a detectable property, e.g., an enzymatic activity. At a suitable time after the gene encoding the polypeptide is introduced into the recipient cell. At this point, expression of the reporter gene is assayed. acetyltransferase, β-galactosidase, chloramphenicol acetyltransferase, The gene encoding the secreted alkaline phosphatase or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters rs 479:79-82). Suitable expression systems are well known and can be prepared using known techniques. Generally, the highest level of expression of the reporter gene is achieved by using a plasmid p53. Constructs having a minimal 5' flanking region exhibiting the following are identified as promoters: The promoter region is linked to a reporter gene and used to detect promoter-driven transcription. Agents may be evaluated for their ability to modulate.

[0068] Methods for introducing and expressing genes in eukaryotic cells, such as T cells, are known in the art. With respect to expression vectors, the vectors can be delivered to T cells by any method known in the art. A vector encoding a polynucleotide, e.g., a CAR, can be easily introduced into the Physical methods for introducing the vector into host cells include calcium phosphate precipitation, lipofection, and Examples of techniques include microprojectile guns, particle guns, microinjection, and electroporation. Methods for producing vectors and / or cells containing exogenous nucleic acid are well known in the art. For example, Sambrook et al. (2001, Molecular Cloning g:A Laboratory Manual,Cold Spring Harbor For more information, see the FDA's FDA Applied Pharmacology Laboratory, New York. The preferred method for introducing the protein into cells is calcium phosphate transfection. . EXAMPLES

[0069] 5.1. Example 1: Improved CAR-T Cell Manufacturing Process 5.1.1. Baseline Process For BCMA-directed chimeric antigen receptor (CAR)-expressing T cells, baseline CAR We developed a T cell manufacturing process (Hollyman et al., J. Immuno Ther. 2009,32:169-180). Isolation of PBMCs, T cells The manufacturing process parameters, such as activation, transduction, and proliferation of T cells, were initially determined using small-scale T cells. Developed and optimized using a flask-based process. Process parameters were confirmed. After establishment and optimization, the process was scaled up for clinical manufacturing. , starting with leukapheresis followed by Cell-Saver 5+ (CS5+; Density gradient centrifugation using 100% ethanol (Haemonetics, Braintree, MA) Peripheral blood mononuclear cells (PBMCs) were isolated by separation; TCGM-HABS of the obtained PBMCs were Culture medium (chemically defined hematopoietic cell medium X-VIVO-15™ (Lonza, Barr (Salzburg, Switzerland), phenol red-free, 5% v / v human AB serum 2 mM GlutaMAX™ (Gibco), 10 mM HEPES (4-(2-hydroxyethyl) (Xyethyl)-1-piperazineethanesulfonic acid (Thermo Fisher Science entific), and 300 IU / mL recombinant human interleukin-2 (rhIL 2) Wash the preculture using batch centrifugation culture in T cell growth medium supplemented with The same medium further supplemented with 1% sodium pyruvate and 1% minimum essential vitamins was also tested. and found to produce the same number of population doublings in 8 days. For simplicity, we use TCGM-H ABS was selected for the process. After comparing with the additional medium, the concentration of rhIL2 was increased to 300I The dose was reduced from 100 IU / mL to 100 IU / mL.

[0070] They are then immobilized by binding to Fc receptors on endogenous antigen-presenting cells, such as monocytes and dendritic cells. Activation of T cells in PBMCs using soluble anti-CD3 and anti-CD28 antibodies T cell activation was confirmed by measuring cell size; The volume of the cells is approximately 180–200 fL, whereas the size of highly activated T cells is 5 The activation step was followed by growth in static bags and then WAVE Bio The cells were grown in a reactor (GE Healthcare Life Sciences). Following expansion, the expanded T cells are transfected with lentivirus encoding a BCMA-targeting CAR. The virus vector was transduced using a multiplicity of infection ranging from 0.625 to 40, with population doubling. Based on studies monitoring BCMA CAR expression, cell size, and expression of anti-BCMA CAR, A range of ~30 MOI was used for the basic clinical manufacturing process. The resulting cells were harvested and Subsequent washes were performed using a CD5+ device.

[0071] However, the baseline process from leukapheresis to T cell activation The first step involves several laborious steps, in particular the initial PBMC isolation and density gradient centrifugation. A washing step that relied on centrifugation was necessary, and such a step was modified to improve efficiency.

[0072] 5.1.2. Improved Processes Consistency in the T cell manufacturing process is key to consistency in the production of CAR T cell medicines. For this purpose, instead of the steps that rely on CS5+ equipment in the baseline process, , LOVO-ACK isolation process, LOVO pre-culture start washing, and final LOVO washing process By replacing it, we tried to improve the baseline process.

[0073] The LOVO-ACK PBMC isolation process is performed using the LOVO Automated Cell Processing System (Fre senius Kabi, Lake Zurich, Illinois; referred to herein as "LOVO" Washing with ammonium chloride-potassium chloride (ACK) lysis buffer followed by ACK lysis buffer The ACK buffer consists of two sequential steps of incubation. 150 mM NH4Cl, 10 mM KHCO3, and 0.1 mM NaCl in deionized water The LOVO washing process uses the LOVO automated cell processing system. This system preserves the PBMC population in the leukapheresis starting material while A spinning membrane filtration system designed to effectively remove platelets and cell debris. The cleaning steps are carried out in sealed, sterile, single-use LOVO disposable kits. The kit includes a cell separator that facilitates the removal of platelets, cell debris, and plasma. It is equipped with a pinning membrane filtration system.

[0074] After the Lovo washing step, add ACK buffer to the remaining cell suspension to lyse red blood cells. This step was carried out using a transfer buffer with a cell contact surface made of polyvinyl chloride. The RBC-depleted PBMCs were then separated from the ACK buffer and centrifuged. So I washed it.

[0075] The isolated and washed PBMCs are then optionally cryopreserved and frozen using standard cryopreservation techniques and rate limiting techniques. The cells were frozen using controlled freezing (if shipped prior to use). Cells were thawed in a 37° water bath. Across the three test lots, the cryopreserved cells were adequately The researchers then produced sufficient numbers of T cells, and after CAR T production, produced CAR+ T cells. [Table 1]

[0076] After thawing, the PBMCs were cultured and stimulated with anti-CD3 and anti-CD28 antibodies ( Day 0); Lentiviral vector carrying the coding sequence for the anti-BCMA chimeric antigen receptor T cell transduction with ELISA (day 1); counting and re-transfection of cells in gas-permeable cell culture bags Seeding (Days 2-5); Counting and Reseeding of Cells in WAVE™ Bioreactors (Days 6-9); followed by cell harvest and final washing using the LOVO device (Day 10) The cells were subjected to a cell culture process including:

[0077] Therefore, the final steps of the improved LOVO-ACK process are as follows: Leukapheresis; b.LOVO-ACK PBMC isolation; c. Washing of PBMCs by centrifugation; d. PBMC cryopreservation (if prior to shipping); e. PBMC thawing (if shipped); f.PBMC washing using LOVO gT cell activation, proliferation, and cell recovery; Cleaning with h.LOVO.

[0078] The improved process provides an advantage over the baseline process in the closed process step. Enhanced robustness and reproducibility of PBMC preparation (reducing the possibility of contamination) faster drug processing times; and reduced overall process complexity.

[0079] 5.2. Example 2: Improved Process Advantage Study Comparison of the Baseline and LOVO-ACK Isolation Processes

[0080] This example shows that for some parameters, the improved process is better than the baseline process. This shows that it is better than

[0081] Baseline CS5+ PBMC isolation process vs. LOVO-ACK PBMC isolation process A feasibility study comparing the IL-16 expression profiles of IL-16-positive and IL-16-negative ... The baseline process and LOVO PBMCs were isolated in parallel through the ACK process, and then the isolated PBMCs were cryopreserved. They were processed using the same cell culture, DP preparation, and cryopreservation methods. As summarized below: Additionally, we investigated the viability and phenotypic composition of PBMCs, as well as the PBMC CD4+ and CD8+ T cell subsets. The results of the LOVO-ACK isolation process were based on the results of the cell culture characterization and formulation release studies. Our results demonstrate that this process is a viable alternative to the CS5+ PBMC isolation procedure.

[0082] PBMC isolation and collection

[0083] The PBMC isolation process separates red blood cells (RBCs) and platelets ( As shown in Table 1, the updated PBMC isolation method is designed to remove PLTs. The method results in more extensive and consistent reduction in red blood cells (RBCs) and depletion of platelets (PLTs) and therefore improved the quality of PBMCs for cell culture initiation. Table 1 - Comparison of PBMC RBC / WBC and PLT / WBC before and after the change [Table 2] Value = percentage of total cells

[0084] The two PBMC processes used different equipment for PBMC isolation. The cell yields obtained from the process were evaluated and compared. The baseline and updated cell yields were as shown in Table 2. The PBMC recovery rates for the different processes were very similar, with a mean difference between pre- and post-change of -2.0 p.a. -cent points. Table 2 - Comparison of recovery rates of PBMC isolation [Table 3] Thus, the improved process involves the isolation and recovery of PBMCs and removal of RBCs; demonstrated superior platelet removal compared to the baseline process.

[0085] Characterization of in-process cell cultures - Comparison of T cell expansion and peak T cell activation

[0086] The results of the cell culture characterization are shown in Table 3. The extent of T cell proliferation (pre- and post-modification T cells) The average difference in proliferation (-1 population doubling) and activation properties (average difference between pre- and post-modification cell cultures) was As evidenced by the similarity of the baseline profiles (same peak activation date), T cells produced from the process and the updated process showed comparable proliferation rates. Table 3 - T cell expansion and peak T cell activation date in cell culture [Table 4]

[0087] In-process pharmaceutical characterization results – post-recovery cleaning recovery rate

[0088] The two pharmaceutical processes used different equipment for the cleaning step after the 10-day harvest. The cell yields obtained during the post-harvest washing steps were evaluated and compared. The recovery rate of the drug (CAR T cells) obtained from the process was 100%. A significant improvement over in-process was observed, with an average increase of 30 percentage points. Table 4 - Post-recovery cleaning recovery rate [Table 5] * Value exceeds 100 due to measurement error.

[0089] Therefore, the improved process is more efficient at treating CAR T cells compared to the baseline process. The results show a remarkably good recovery rate of .

[0090] 5.3. Example 3: Intercomparability study of improved processes This example demonstrates that the improved process is comparable to the baseline process with respect to other parameters. Indicates equality.

[0091] Compare the baseline and improved PBMC isolation processes and see the following equivalence: The process change conditions are outlined in Table 5. For each healthy donor, The leukapheresis unit was equipped with a baseline CS5+ device and an updated LOVO Following the PBMC isolation step, the PBMC lot was The cells were either cryopreserved or used to initiate cell cultures without cryopreservation. Prior to initiating culture, PBMCs were washed by batch centrifugation or with the LOVO cell processing device. Gas-permeable cell culture bags were used throughout the duration of cell culture. The recovered drug substance was washed in either the CS5+ device or the LOVO cell processor. The drugs were subjected to non-clinical in vivo intercomparison evaluation and post-thaw stability improvement during use. in bags for storage or in vials for in vitro intercomparability evaluation, The result was saved. Table 5 - Process change conditions for intercomparability assessment [Table 6]

[0092] An intercomparison study was conducted using five healthy donor leukapheresis units. From this, ten anti-BCMA CAR T cell lots were generated (each with methodological variation). (before and after the change).

[0093] The baseline and updated processes were run in parallel for PBMC isolation. Each of the initial leukapheresis units was divided and processed into five PBMC lots. Three of the PBs were cryopreserved and then thawed to initiate cell culture, while the remaining two PBs were MC lots were not frozen but were processed immediately to initiate cell culture. Cryopreserved (PBMC preservation step) PBMCs and fresh (P The comparability of PBMCs (without BMC retention step) was previously established. Incorporate cell washing methods for the baseline and updated processes. The recovery and drug processing processes were continued. In-process and cryopreserved pharmaceutical test samples produced during the process were analyzed using the following ratios: The evaluation was carried out according to a comparative analytical test plan.

[0094] Comparative Analysis Test

[0095] The results of a comparison of PBMC viability are shown in Table 6. There was no consistent difference in viability, and the survival rates were significantly higher before and after the change. The average difference after the change was +2 percentage points. Table 6 - Comparison of PBMC viability before and after the change [Table 7]

[0096] Comparison of PBMC composition

[0097] The results of comparing PBMC composition were: CD45+ leukocytes, CD3+CD56- T cells, CD14 + Monocytes are shown in Table 7, CD19+ B cells, CD3-CD56+ NK cells, and CD5 6-CD16+ granulocytes are shown in Table 8. CD45+ leukocytes, CD3+ D56-T cells, CD14+ monocytes, CD19+ B cells, and CD3-CD56+ NK cells The mean differences between before and after the change were +1, +2, +1, -1, and -1 percent, respectively. In both processes, CD56-CD16+ granulocytes (not detected) In summary, the composition of PBMCs was significantly different from the baseline process and the updated The results were consistent across the entire process. Table 7-PBMC composition: CD45+ white blood cells, CD3+CD56- T cells, CD14+ monocytes [Table 8] Table 8-PBMC composition: CD19+B cells, CD3-CD56+NK cells, CD56-C D16+ granulocytes [Table 9]

[0098] Comparison of expression of PBMC CD4+ and CD8+ T cells

[0099] PBMC T cell populations were further characterized for CD4+ and CD8+ expression. As shown in Table 9, the CD4+ / CD8+ subset distribution of PBMC T cells was classified into two groups. The mean pre- and post-change mean CD4+ and CD8+ T cell populations were comparable across processes. The average differences were -3 and +3 percentage points, respectively. Table 9 - PBMC CD4+ and CD8+ T cell subsets [Table 10]

[0100] Normal donor PBMC leukocyte composition generated for interoperability evaluation of this process modification There are discrepancies between our data and PBMC data from multiple myeloma (MM) patients derived from previous studies. Differences were observed in the numbers of B and T cells in PBMCs from MM patients compared to healthy donors. and the number of monocytes is increased.

[0101] Furthermore, the CD4+ / CD8+ ratios within the T cell population were also different. Using the mean (N=5) B cell, T cell, and monocyte counts from healthy donor PBMC lots, ) percentages were 9.7% ± 4.2%, 40.6% ± 13.1%, and 30. 0%±7.8%. Mean (N=5) CD4+ / CD The ratio of 8+ T cells was 58.8% / 32.7% of T cells. Using the C process, B cells, T cells, and monocytes from the CRB-401 PBMC lot were The mean (N=24) percentages of 5.7%, 56.4% ± 19.4%. N=24) The CD4+ / CD8+ T cell ratio was 40.8% / 54.4% of T cells. Such differences observed between the two data sets were independent of process changes. , consistent with those reported in the scientific literature.

[0102] Therefore, with the above parameters, the improved process is different from the baseline process. are equivalent.

Claims

1. 1. A method for producing chimeric antigen receptor (CAR)-expressing T cells (CAR T cells) from peripheral blood mononuclear cells (PBMCs) from a subject from whom a blood sample is obtained, comprising: (a) obtaining PBMCs from the blood sample; (b) isolating the PBMCs from the blood sample using a spinning membrane filtration system; (c) further isolating the PBMCs obtained from the blood sample using ammonium chloride-potassium (ACK) buffer; (d) washing the PBMCs from step (c) by centrifugation; (e) washing the PBMCs from step (d) using a spinning membrane filtration system; (f) producing CAR T cells from the PBMCs from step (e); and (g) washing the CAR T cells from step (f) using a membrane filtration system.

2. 2. The method of claim 1, further comprising the steps of: cryopreserving the PBMCs from step (d); and thereafter, prior to step (e), thawing the cryopreserved PBMCs.

3. 3. The method of claim 1 or 2, wherein the spinning membrane filtration system is a system having a pore size of 3.6 μm.

4. 4. The method of claim 1, wherein the ACK buffer comprises 50-300 mM ammonium chloride, 5-25 mM potassium carbonate, and 0.05-0.25 mM sodium EDTA.

5. The method of any one of claims 1 to 4, wherein the ACK buffer is 150 mM ammonium chloride, 10 mM potassium carbonate, and 0.1 mM sodium EDTA.

6. 6. The method of any one of claims 1 to 5, wherein producing CAR T cells from the PBMCs comprises activating, expanding and transducing the cells with a viral vector encoding the CAR.

7. 7. The method of claim 6, wherein the PBMCs are activated by incubation in T cell expansion medium supplemented with an activation agent until transduction of the T cells.

8. The method of claim 6 or 7, wherein the activation comprises the use of an anti-CD3 antibody and an anti-CD28 antibody.

9. The method of any one of claims 6 to 8, wherein after the transduction, the culture is incubated to allow the cells to grow.

10. The method according to any one of claims 6 to 9, wherein the viral vector is a lentiviral vector.

11. The method according to any one of claims 1 to 10, wherein the blood sample is a leukoreduced blood sample.

12. The method of any one of claims 1 to 11, further comprising isolating T cells from the isolated PBMCs and producing the CAR T cells from the T cells.

13. The method according to any one of claims 1 to 12, wherein the isolation of the T cells uses magnetic beads coated with anti-CD3, anti-CD4 or anti-CD8 antibodies.

14. The method according to any one of claims 1 to 13, wherein the isolated PBMCs or isolated T cells are cryopreserved.

15. The method according to any one of claims 1 to 14, wherein the isolated PBMCs or isolated T cells are cultured after their cryopreservation.

16. The method of any one of claims 1 to 15, wherein the isolated PBMCs or isolated T cells are thawed, washed and resuspended in a growth medium suitable for the growth of such cells.

17. 17. The method of claim 16, wherein the thawed PBMCs or thawed T cells are washed using the spinning membrane filtration system or centrifugation prior to cell culture of the thawed PBMCs or thawed T cells.