Production of anti-BCMA CAR T cells

Abstract

The present invention provides improved anti-BCMA CAR T cell compositions and methods for producing anti-BCMA CAR T cell therapies. More specifically, the present invention relates to improved methods for producing anti-BCMA CAR T cells that produce stronger, longer-lasting, and more effective adoptive T cell immunotherapy. In certain embodiments, the cells are produced from subjects with multiple myeloma or lymphoma.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 944,485, filed December 6, 2019, and U.S. Provisional Application No. 62 / 830,004, filed April 5, 2019, each of which is incorporated herein by reference in its entirety.

[0003] Statement regarding sequence listing

[0004] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The text file containing the sequence listing is named BLBD_118_02WO_ST25.txt. The text file is 7KB, was created on March 27, 2020, and was submitted electronically via EFS-Web at the same time as this specification is submitted. Background Art Technical Field

[0006] The present invention relates to improved anti-BCMA CAR T cell compositions and methods for producing anti-BCMA CAR T cells. More specifically, the present invention relates to improved methods for producing anti-BCMA CAR T cells that produce stronger, longer-lasting, and more effective adoptive T cell immunotherapy.

[0007] Existing technology

[0008] Adoptive immunotherapy is the transfer of T lymphocytes to a subject to provide a therapy for a disease. Adoptive immunotherapy has an unrealized potential for treating a variety of diseases including cancer, infectious diseases, autoimmune diseases, inflammatory diseases and immunodeficiency. However, most, if not all, adoptive immunotherapy strategies require T cell activation and amplification steps to produce clinically effective therapeutic doses of T cells. Current technologies for producing therapeutic doses of T cells (including engineered T cells) are still limited by cumbersome T cell manufacturing processes. For example, T cell amplification often requires labor-intensive and expensive cloning, and / or multiple rounds of activation / amplification to achieve treatment-related T cell numbers. In addition, existing T cell activation / amplification methods are typically coupled with substantial T cell differentiation and typically result in short-term effects, including short-term survival and lack of persistence and in vivo amplification of transferred T cells. More recent manufacturing methods have produced stronger and more durable T cells, but these cells are still prone to exhaustion and loss of effector immune cell function.

[0009] There remains an unmet need for improvements in T cell manufacturing and for stronger and longer-lasting T cell therapies. Summary of the Invention

[0010] The present invention generally provides adoptive T cell immunotherapies with improved efficacy and durability and methods for their preparation.

[0011] In various embodiments, a cGMP-manufactured population of anti-B cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cells is provided, the population comprising at least 10% CD27 + Anti-BCMA CAR T cells.

[0012] In certain embodiments, the population comprises at least 15% CD27 + Anti-BCMA CAR T cells.

[0013] In certain embodiments, the population comprises at least 20% CD27 + Anti-BCMA CAR T cells.

[0014] In some embodiments, the population comprises at least 25% CD27 + Anti-BCMA CAR T cells.

[0015] In further embodiments, the population comprises at least 30% CD27 + Anti-BCMA CAR T cells.

[0016] In certain embodiments, CD27 + Anti-BCMA CAR T cells are LEF1 + and / or TCF1 + Anti-BCMA CAR T cells.

[0017] In another embodiment, CD27 + Anti-BCMA CAR T cells are LEF1 + and TCF1 + Anti-BCMA CAR T cells. In various embodiments, the cGMP manufactured anti-BCMA CAR T cell population comprises at least 10% LEF1 + and / or CCR7 + and TCF1 + Anti-BCMA CAR T cells.

[0018] In some embodiments, the population comprises at least 15% LEF1 + and / or CCR7 + and TCF1 + Anti-BCMA CART cells.

[0019] In certain embodiments, the population comprises at least 20% LEF1+ and / or CCR7 + and TCF1 + Anti-BCMA CART cells.

[0020] In some embodiments, the population comprises at least 25% LEF1 + and / or CCR7 + and TCF1 + Anti-BCMA CART cells.

[0021] In further embodiments, the population comprises at least 30% LEF1 + and / or CCR7 + and TCF1 + Anti-BCMA CART cells.

[0022] In another embodiment, LEF1 + and / or CCR7 + and / or TCF1 + Anti-BCMA CAR T cells are CD27 + Anti-BCMA CAR T cells.

[0023] In some embodiments, LEF1 + and / or CCR7 + and / or TCF1 + Anti-BCMA CAR T cells are LEF1 + CCR7 + TCF1 + CD27 + Anti-BCMA CAR T cells.

[0024] In some embodiments, CD27 + and / or LEF1 + and / or CCR7 + and TCF1 + Anti-BCMA CAR T cells contain CD4 + Anti-BCMA CAR T cells.

[0025] In certain embodiments, CD27 + and / or LEF1 + and / or CCR7 + and TCF1 + Anti-BCMA CAR T cells contain CD8 + Anti-BCMA CAR T cells.

[0026] In certain embodiments, CD27 + and / or LEF1 +and / or CCR7 + and TCF1 + Anti-BCMA CAR T cells contain CD4+ and CD8 + Anti-BCMA CAR T cells.

[0027] In certain embodiments, the cells are produced from a subject with multiple myeloma or lymphoma.

[0028] In certain embodiments, the cells are produced from a subject with relapsed / refractory multiple myeloma.

[0029] In some embodiments, the cell comprises a lentivirus comprising a polynucleotide encoding an anti-BCMA CAR.

[0030] In a specific embodiment, the BCMA CAR comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0031] In further embodiments, the anti-BCMA CAR is encoded by the polynucleotide sequence set forth in SEQ ID NO: 2.

[0032] In certain embodiments, the cells are autologous.

[0033] In certain embodiments, the cells are cryopreserved.

[0034] In certain embodiments, the cells are formulated for administration to a subject having multiple myeloma or lymphoma.

[0035] In some embodiments, human anti-B cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cells are contacted with a phosphatidylinositol 3-kinase (PI3K) inhibitor ex vivo for about 5 days to about 7 days, wherein gene expression of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A, and CCL5, or (ii) CCL1, NR4A2, ATF3, CCL5, and WNT5B is at least 1.5-fold or at least 2-fold higher in the anti-BCMA CAR T cells than in anti-BCMA CAR T cells contacted with the PI3K inhibitor ex vivo for about 10 days.

[0036] In certain embodiments, human anti-B cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cells are contacted with a phosphatidylinositol 3-kinase (PI3K) inhibitor ex vivo for about 5 to about 7 days, wherein gene expression of one, two, three, four, five, six, seven, eight, nine, or all of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2, and WDR62, or (ii) NKD2 and NQO1 is at least 1.5-fold or at least 2-fold lower in the anti-BCMA CAR T cells than in anti-BCMA CAR T cells contacted with the PI3K inhibitor ex vivo for about 10 days.

[0037] In further embodiments, human anti-B cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cells are provided that have been contacted with a phosphatidylinositol-3 kinase (PI3K) inhibitor ex vivo for about 5 days to about 7 days; wherein in the anti-BCMA CAR T cells, the gene expression of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A and CCL5 or (ii) each of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of CCL1, NR4A2, ATF3, CCL5 and WNT5B is at least 1.5-fold or at least 2-fold higher and (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR1 is expressed in the anti-BCMA CAR T cells as compared to the anti-BCMA CAR T cells contacted with the PI3K inhibitor ex vivo for about 10 days. The gene expression of one, two, three, four, five, six, seven, eight, nine or all of (i) ILDR2, ATAD3, NKD2 and WDR62 is at least 1.5-fold or at least 2-fold lower.

[0038] In certain embodiments, the CD4+ anti-BCMA CAR T cells have a central memory T cell (TCM)-like phenotype.

[0039] In further embodiments, the CD8+ anti-BCMA CAR T cells have a stem cell memory T cell (TSCM)-like phenotype.

[0040] In certain embodiments, CD4 + Anti-BCMA CAR T cells have a TCM-like phenotype and CD8 + Anti-BCMA CAR T cells have a TSCM-like phenotype.

[0041] In some embodiments, the cells are produced from a subject with multiple myeloma or lymphoma.

[0042] In certain embodiments, the cells are produced from a subject with relapsed / refractory multiple myeloma.

[0043] In certain embodiments, the cell comprises a lentivirus comprising a polynucleotide encoding an anti-BCMA CAR.

[0044] In a specific embodiment, the BCMA CAR comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0045] In a specific embodiment, the anti-BCMA CAR is encoded by the polynucleotide sequence set forth in SEQ ID NO: 2.

[0046] In certain embodiments, the cells are autologous.

[0047] In certain embodiments, the cells are cryopreserved.

[0048] In certain embodiments, the cells are formulated for administration to a subject having multiple myeloma or lymphoma.

[0049] In further embodiments, the PI3K inhibitor is ZSTK474.

[0050] In a specific embodiment, a pharmaceutical composition is provided comprising a physiologically acceptable excipient and a therapeutically effective amount of the anti-BCMA CAR T cells contemplated herein.

[0051] In some embodiments, the therapeutically effective amount of the anti-BCMA CAR T cells is at least about 5.0×10 7 anti-BCMA CAR T cells.

[0052] In certain embodiments, the therapeutically effective amount of the anti-BCMA CAR T cells is at least about 15.0×10 7 anti-BCMA CAR T cells.

[0053] In certain embodiments, wherein the therapeutically effective amount is at least about 45.0×10 7 anti-BCMA CAR T cells.

[0054] In certain embodiments, the therapeutically effective amount is at least about 80.0×10 7 anti-BCMA CAR T cells.

[0055] In further embodiments, the composition is formulated in a solution comprising 50:50 PlasmaLyte A:CryoStorCS10.

[0056] In certain embodiments, a method of treating a subject having multiple myeloma or lymphoma with a composition contemplated herein is provided.

[0057] In certain embodiments, the subject has relapsed / refractory multiple myeloma.

[0058] In various embodiments, a method for producing anti-BCMA CAR T cells is provided, the method comprising: activating a T cell population and stimulating the T cell population to proliferate; transducing the T cells with a lentiviral vector encoding an anti-BCMA CAR comprising the amino acid sequence set forth in SEQ ID NO: 1; culturing the transduced T cells to proliferate for a period of about 5 days to about 7 days; wherein the previous steps are performed in the presence of a PI3K inhibitor, and wherein the lentiviral vector encoding an anti-BCMA CAR comprising the amino acid sequence set forth in SEQ ID NO: 1 is transduced into the cultured T cells. The expression of genes of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A and CCL5 or (ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of CCL1, NR4A2, ATF3, CCL5 and WNT5B is at least 1.5-fold or at least two-fold higher than that in T cells transduced with a lentiviral vector of a CAR and cultured for a period of about 10 days.

[0059] In certain embodiments, a method for producing an anti-BCMA CAR T cell is provided, the method comprising: activating a T cell population and stimulating proliferation of the T cell population; transducing the T cells with a lentiviral vector encoding an anti-BCMA CAR comprising the amino acid sequence set forth in SEQ ID NO: 1; culturing the transduced T cells to proliferate for a period of about 5 days to about 7 days; wherein the aforementioned steps are performed in the presence of a PI3K inhibitor, and wherein in the cultured T cells, gene expression of one, two, three, four, five, six, seven, eight, nine, or all of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2, and WDR62, or (ii) NKD2 and NQO1 is at least 1.5-fold or at least two-fold lower than in T cells transduced with a lentiviral vector encoding an anti-BCMA CAR comprising the amino acid sequence set forth in SEQ ID NO: 1 and cultured for expansion for a period of about 10 days.

[0060] In various embodiments, a method for producing anti-BCMA CAR T cells is provided, the method comprising: activating a T cell population and stimulating the proliferation of the T cell population; transducing the T cells with a lentiviral vector encoding an anti-BCMA CAR comprising the amino acid sequence listed in SEQ ID NO: 1; culturing the transduced T cells to proliferate for a period of about 5 days to about 7 days; wherein the aforementioned steps are performed in the presence of a PI3K inhibitor, and wherein the cultured T cells are activated in combination with the lentiviral vector encoding an anti-BCMA CAR comprising the amino acid sequence listed in SEQ ID NO: 1. In some embodiments, the present invention relates to a method for treating a T cell that is transduced with a lentiviral vector containing a CAR and cultured for expansion for a period of about 10 days, wherein the expression of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A and CCL5 or (ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of the genes is at least 1.5-fold or at least two-fold higher, and the expression of (i) 1, 2, 3, 4, 5, 6, 7, 8, 9 or all of the genes is at least 1.5-fold or at least two-fold lower.

[0061] In various embodiments, a method for producing anti-BCMA CAR T cells is provided, comprising: activating a T cell population and stimulating the proliferation of the T cell population; transducing the T cells with a lentiviral vector encoding an anti-BCMA CAR comprising the amino acid sequence set forth in SEQ ID NO: 1; culturing the transduced T cells to proliferate for a period of about 5 days to about 7 days; wherein the aforementioned steps are performed in the presence of a PI3K inhibitor, and wherein the proliferated cells are CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + .

[0062] In certain embodiments, the anti-BCMA CAR T cells comprise at least 10% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

[0063] In further embodiments, the anti-BCMA CAR T cells comprise at least 15% CD27 + and / or LEF1 +and / or CCR7 + and / or TCF1 + T cells.

[0064] In some embodiments, the anti-BCMA CAR T cells comprise at least 20% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

[0065] In some embodiments, the anti-BCMA CAR T cells comprise at least 25% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

[0066] In certain embodiments, the anti-BCMA CAR T cells comprise at least 30% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

[0067] In another embodiment, CD27 + LEF1 + and / or CCR7 + and / or TCF1 + .

[0068] In further embodiments, CD27 + LEF1 + and CCR7 + and TCF1 + .

[0069] In certain embodiments, CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + Anti-BCMA CAR T cells contain CD4 + Anti-BCMA CAR T cells.

[0070] In certain embodiments, CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + Anti-BCMA CAR T cells contain CD8 +Anti-BCMA CAR T cells.

[0071] In another embodiment, CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + Anti-BCMA CAR T cells contain CD4+ and CD8 + Anti-BCMA CAR T cells.

[0072] In certain embodiments, the T cells are autologous.

[0073] In additional embodiments, the method further comprises isolating peripheral blood mononuclear cells (PBMCs) as a source of the T cells.

[0074] In some embodiments, PBMCs are isolated from a subject with multiple myeloma or lymphoma.

[0075] In certain embodiments, the subject has relapsed / refractory multiple myeloma.

[0076] In certain embodiments, the method further comprises cryopreserving the PBMCs prior to activation and stimulation.

[0077] In further embodiments, the T cells are cryopreserved in expanded culture.

[0078] In further embodiments, T cells are activated and stimulated to proliferate for about 18 hours to about 24 hours.

[0079] In certain embodiments, activation of T cells comprises contacting the T cells with an anti-CD3 antibody or antigen-binding fragment thereof.

[0080] In certain embodiments, the anti-CD3 antibody or antigen-binding fragment thereof is soluble.

[0081] In additional embodiments, the anti-CD3 antibody or antigen-binding fragment thereof is bound to a surface.

[0082] In some embodiments, the surface is a bead, optionally a paramagnetic bead.

[0083] In further embodiments, stimulation of T cells comprises contacting the T cells with an anti-CD28 antibody or antigen-binding fragment thereof.

[0084] In certain embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is soluble.

[0085] In additional embodiments, the anti-CD28 antibody or antigen-binding fragment thereof is bound to a surface.

[0086] In some embodiments, the surface is a bead, optionally a paramagnetic bead, optionally the paramagnetic bead is bound to the anti-CD3 antibody or antigen-binding fragment thereof.

[0087] In certain embodiments, the cells are transduced with an HIV-1 derived lentiviral vector.

[0088] In some embodiments, the anti-BCMA CAR is encoded by the polynucleotide sequence set forth in SEQ ID NO: 2.

[0089] In further embodiments, the PI3K inhibitor is ZSTK474.

[0090] In various embodiments, a method for increasing CD4 + TCM-like anti-BCMACAR T cells and CD8 + A method of producing TSCM-like anti-BCMA CAR T cells, comprising contacting anti-BCMA CAR T cells with a PI3K inhibitor ex vivo for about 5 days to about 7 days, wherein CD4 T cells are expressed in the anti-BCMA CAR T cells as compared to anti-BCMA CAR T cells contacted with the PI3K inhibitor ex vivo for about 10 days. + TCM-like anti-BCMA CAR T cells and CD8 + The number of TSCM-like anti-BCMA CAR T cells was at least two-fold higher.

[0091] In certain embodiments, the anti-BCMA CAR T cells comprise at least 10% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

[0092] In other embodiments, the anti-BCMA CAR T cells comprise at least 15% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

[0093] In some embodiments, the anti-BCMA CAR T cells comprise at least 20% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

[0094] In certain embodiments, the anti-BCMA CAR T cells comprise at least 25% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

[0095] In further embodiments, the anti-BCMA CAR T cells comprise at least 30% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

[0096] In certain embodiments, the T cells are autologous.

[0097] In certain embodiments, the method further comprises isolating peripheral blood mononuclear cells (PBMCs) as a source of the T cells.

[0098] In additional embodiments, PBMCs are isolated from a subject with multiple myeloma or lymphoma.

[0099] In some embodiments, the subject has relapsed / refractory multiple myeloma.

[0100] In further embodiments, the anti-BCMA CAR T cells comprise an HIV-1 derived lentiviral vector.

[0101] In a specific embodiment, the BCMA CAR comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0102] In additional embodiments, the anti-BCMA CAR is encoded by the polynucleotide sequence set forth in SEQ ID NO: 2.

[0103] In some embodiments, a pharmaceutical composition is provided comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of the anti-BCMA CAR T cells contemplated herein.

[0104] In certain embodiments, a pharmaceutical composition is provided comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of a CD4 + TCM anti-BCMA CAR T cells and CD8 + TSCM anti-BCMA CAR T cells.

[0105] In certain embodiments, a method of treating a subject having multiple myeloma or lymphoma is provided, the method comprising administering a composition contemplated herein.

[0106] In further embodiments, the subject has relapsed / refractory multiple myeloma.

[0107] In various embodiments, a method is provided for increasing gene expression of each of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A and CCL5 or (ii) CCL1, NR4A2, ATF3, CCL5 and WNT5B in an anti-BCMA CAR T cell, comprising contacting the anti-BCMA CAR T cell with a PI3K inhibitor ex vivo for about 5 days to about 7 days, wherein gene expression of each of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A and CCL5 or (ii) CCL1, NR4A2, ATF3, CCL5 and WNT5B is at least 1.5-fold higher in the anti-BCMA CAR T cell than in anti-BCMA CAR T cells contacted with the PI3K inhibitor ex vivo for about 10 days.

[0108] In certain embodiments, a method for reducing gene expression of each of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2, and WDR62, or (ii) NKD2 and NQO1 in an anti-BCMA CAR T cell is provided, comprising contacting the anti-BCMA CAR T cell with a PI3K inhibitor ex vivo for about 5 days to about 7 days, wherein gene expression of each of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2, and WDR62, or (ii) NKD2 and NQO1 is at least 1.5-fold lower in the anti-BCMA CAR T cell than in anti-BCMA CAR T cells contacted with the PI3K inhibitor ex vivo for about 10 days.

[0109] In certain embodiments, a method is provided for increasing gene expression of each of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A, and CCL5, or (ii) CCL1, NR4A2, ATF3, CCL5, and WNT5B, and decreasing gene expression of each of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR22, ILDR2, ATAD3, NKD2, and WDR62, or (ii) NKD2 and NQO1 in anti-BCMA CAR T cells, comprising contacting the anti-BCMA CAR T cells with a PI3K inhibitor ex vivo for about 5 days to about 7 days, wherein the expression of PI3K in the anti-BCMA CAR T cells is increased compared to that in the anti-BCMA CAR T cells contacted with the PI3K inhibitor ex vivo for about 10 days. In T cells, the gene expression of each of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A, and CCL5 or (ii) CCL1, NR4A2, ATF3, CCL5, and WNT5B is at least 1.5-fold higher and the gene expression of each of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2, and WDR62 or (ii) NKD2 and NQO1 is at least 1.5-fold lower.

[0110] In some embodiments, a method for increasing the therapeutic efficacy of anti-BCMA CAR T cells is provided, comprising contacting anti-BCMA CAR T cells with a PI3K inhibitor ex vivo for about 5 days to about 7 days, wherein gene expression of each of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A, and CCL5, or (ii) CCL1, NR4A2, ATF3, CCL5, and WNT5B is increased by at least 1.5-fold in the anti-BCMA CAR T cells and in anti-BCMA CAR T cells contacted with the PI3K inhibitor ex vivo for about 10 days, indicating increased therapeutic efficacy.

[0111] In certain embodiments, a method for increasing the therapeutic efficacy of anti-BCMA CAR T cells is provided, comprising contacting anti-BCMA CAR T cells with a PI3K inhibitor ex vivo for about 5 days to about 7 days, wherein gene expression of each of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2, and WDR62, or (ii) NKD2 and NQO1 is reduced in the anti-BCMA CAR T cells and in anti-BCMA CAR T cells contacted with the PI3K inhibitor ex vivo for about 10 days, at least 1.5-fold lower, indicating increased therapeutic efficacy.

[0112] In some embodiments, a method for increasing the therapeutic efficacy of an anti-BCMA CAR T cell is provided, comprising contacting an anti-BCMA CAR T cell with a PI3K inhibitor ex vivo for about 5 days to about 7 days, wherein gene expression of each of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A, and CCL5 or (ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of CCL1, NR4A2, ATF3, CCL5, and WNT5B is increased by at least 1.5-fold and gene expression of each of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR3, or (ii) NQO2, CCNA3, IL17F, EMP1, SNHG19, PRR3, or (iii) NQO3, CCNA3, IL17F, EMP1, SNHG19, PRR3, or (iv) NQO3, CCNA3, IL17F, EMP1, SNHG19, PRR3, or (v) NQO3, CCNA3, IL17F, EMP1, SNHG19, PRR3, or (v) NQO3, CCNA3, IL17F, EMP1, SNHG19, PRR3, or (vi ... A decrease in gene expression of each of (i) ILDR2, ATAD3, NKD2, and WDR62, or (ii) NKD2 and NQO1 by at least 1.5-fold is indicative of increased therapeutic efficacy.

[0113] In certain embodiments, the anti-BCMA CAR T cells are from a subject with multiple myeloma or lymphoma.

[0114] In additional embodiments, the anti-BCMA CAR T cells are from a subject with relapsed / refractory multiple myeloma.

[0115] In certain embodiments, the anti-BCMA CAR T cell comprises an HIV-1-derived lentiviral vector comprising a polynucleotide encoding the anti-BCMA CAR.

[0116] In a specific embodiment, the BCMA CAR comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0117] In further embodiments, the anti-BCMA CAR is encoded by the polynucleotide sequence set forth in SEQ ID NO: 2.

[0118] In some embodiments, the anti-BCMA CAR T cells are autologous.

[0119] In certain embodiments, the PI3K inhibitor is ZSTK474. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Figure 1 The length of T cell culture with PI3K inhibitors is shown to regulate T cell phenotype. Five multiple myeloma PBMC batches are used to manufacture anti-BCMA CAR T cells in the absence of PI3K inhibitors, or after transduction with a lentiviral transduction encoding anti-BCMACAR, PI3K inhibitors are used to culture 7 days or 10 days. T cells are stained with anti-human antibodies for CD3, CD62L, CCR7 and CD45RA at day 7 and day 10 and analyzed by flow cytometry. Each dot plot on gated live CD3+ lymphocytes is shown.

[0121] Figure 2 T cells showed a stronger phenotype after 7 days of culture with PI3K inhibitors compared to 10 days of culture. Five multiple myeloma PBMC batches were used to make anti-BCMA CAR T cells in the presence of PI3K inhibitors for 7 or 10 days. T cells were stained with anti-human antibodies for CCR7, CD25, CD28, CD122, ICOS, CD45RO, CD57 and TIM3 on days 7 and 10 and analyzed by CyTOF. Each dot plot on gated live CD3+ lymphocytes was analyzed.

[0122] Figure 3 showed that T cells cultured for 7 days in the presence of PI3K inhibitors showed an increase in CD27 + Enrichment in T cells. Anti-BCMA CAR T cells were made using five multiple myeloma PBMC batches in the presence of a PI3K inhibitor. T cells were stained with anti-human antibodies for CD4, CD8, and CD27 on days 7 and 10 and analyzed by CyTOF. VISNE diagrams show CD27 gating expression in different cell populations.

[0123] Figure 4A -B shows that T cells showed a stronger phenotype after 7 days of culture with PI3K inhibitors compared to 10 days of culture. Five batches of multiple myeloma PBMCs were used to make anti-BCMA CAR T cells in the presence of PI3K inhibitors for 7 or 10 days. T cells were treated with antibodies against (1) CCR7, CD25, CD28, HLA-DR and TIM3 ( Figure 4A ) or CD45RO, CD57, CD70, CD244 and PD-1 ( Figure 4B ) and analyzed by CyTOF. VISNE diagrams show the expression of different T cell phenotypic markers in 7-day culture (upper row) and 10-day culture (lower row). The gated group represents CD27 + cell.

[0124] Figure 5 Shown is the expression of CD27 T cells grown in the presence of PI3K inhibitors for 10 days compared to T cells grown in the presence of PI3K inhibitors for 7 days. + Anti-BCMA CAR T cells were produced using five batches of multiple myeloma PBMCs in the presence of a PI3K inhibitor for 7 or 10 days. CD27 was identified by VISNE analysis. + T cells were stained with anti-human antibodies against CD28, ICOS, HLA-DR, CD25, and TIM3 on days 7 and 10, and CD4 T cells (top) and CD8 + T cells (bottom) were analyzed by CyTOF.

[0125] Figure 6 Differential gene expression due to the duration of anti-BCMA CAR T cell manufacturing is shown. Anti-BCMA CAR T cells were manufactured using multiple myeloma PBMC batches for 7 days (1) or 10 days (13) in the absence of a PI3K inhibitor or for 7 days (10) or 10 days (6) in the presence of a PI3K inhibitor. RNA was extracted from T cells and transcriptional profiles were analyzed using Nanostring immunology plots. A heat map of the top 50 differentially expressed genes between manufacturing conditions is shown.

[0126] Figure 7 The efficacy of anti-BCMA CAR T cells produced in the presence of PI3K inhibitors for 7 days was shown to be increased compared to anti-BCMA CAR T cells produced in the presence of PI3K inhibitors for 10 days. Healthy donor PBMCs were activated, transduced with a lentiviral vector encoding anti-BCMA CAR, and expanded for 6 days (7-day process) or 9 days (10-day process) in the presence of IL-2 and PI3K inhibitors. 10 days before adoptive cell therapy, NSG mice were injected intravenously with 2×10 6 Firefly luciferase-labeled Daudi tumor cells were injected into mice with 2.5, 5, or 10 × 10 6 anti-BCMA CAR + T cells or T cells transduced with vehicle. Tumor burden was monitored by luminescence.

[0127] Figure 8 T cells made in the presence of PI3K were shown to be enriched for CD27 + CD4 + TCM-like cells and CD27 + CD8 + TSCM-like cells. Anti-BCMA CART cells manufactured from multiple myeloma PBMC batches in the presence of PI3K inhibitors were stained with a panel of ~36 T cell phenotyping antibodies and analyzed with CyTOF. Naive T cells (T naive), central memory T cells (TCM), effector memory T cells (EM), effector T cells (TEff) and stem cell memory T cells (TSCM) are shown. The data presented show that according to CD27 + The % of enriched cells relative to T cell subsets was analyzed for each DP batch.

[0128] Figure 9 The FlowSOM analysis is shown. Figure 9 CD8 + T cell data. FlowSOM identified 20 different T cell clusters. Three major classes of T cells were identified based on cluster 4 (enriched in memory T cell markers - favorable) and cluster 5 (enriched in effector T cell markers - less favorable). %CD27 is shown. + CD8 + T cells, manufacturing methods, and clinical responses in subjects treated with anti-BCMA CAR T cells.

[0129] Figure 10 Figure 2 shows differential gene expression analysis of anti-BCMA CAR T cells manufactured from multiple myeloma cell batches using either a 7-day or 10-day PI3K manufacturing process. RNA was extracted from 12 batches of anti-BCMA CAR T cells and transcriptomes were analyzed using Nanostring immunology panels. A heatmap of the top 25 differentially expressed genes between the 7-day and 10-day manufacturing processes is shown. % CD27 is shown. + T cells, manufacturing methods, and clinical responses in subjects treated with anti-BCMA CAR T cells.

[0130] Figure 11A A volcano plot of cyTOF-stained T cell populations in durable responders versus non-durable responders for an anti-BCMA CAR T cell drug product is shown. The plot shows that the most significant differences in cell composition between durable responders and non-durable responders are naive and stem cell memory T cells. The generalized linear model coefficients are shown on the X-axis, and the p-values ​​are shown on the Y-axis.

[0131] Figure 11BFigure 3 shows the ratio of CD4 TSCM (upper panel) and CD8 TSCM (lower panel) in the anti-BCMA CAR T cell drug product compared with durable responders and non-durable responders. TSCM cells are enriched in the drug product of patients with durable responses.

[0132] Figure 12A Figure 3 shows box plots of the proportion of LEF-1 expression in CD4 (upper left panel) and CD8 (upper middle panel) T cells in anti-BCMA CAR T cell drug products compared to durable responders and non-durable responders, as determined by CyTOF. The proportion of LEF-1-expressing cells and LEF-1 gene expression were increased in durable responders compared to non-durable responders, indicating enrichment of early memory T cells in these drug products.

[0133] Figure 12A Correlation between LEF-1 gene expression in the drug product and patient sBCMA levels two months after treatment with anti-BCMA CAR T cells is shown. These data suggest an association between the early memory phenotype in the drug product and the depth of treatment response.

[0134] Figure 13 Shown are the percentages of live CD3+ cells expressing CCR7 (upper left panel), LEF1 (upper middle panel), and CD57 (upper right panel) in PBMCs and anti-BCMA CAR T cells (DP) as determined by CyTOF. Figure 13 Further shown are the expression of CCR7 (on the y-axis) compared to the maximum vector copy number (VCN) on CD3+ cells extracted from whole blood at different time points after anti-BCMA CAR T cell infusion determined by PCR on the x-axis. Figure 13 , lower left), LEF-1( Figure 13 , lower middle panel) and CD57 ( Figure 13 , lower right panel) of the percentage of CD3+ viable cells.

[0135] Figure 14 The percentage of live CD3+ cells expressing CD57 (a marker of senescence), LEF-1, CCR7, and CD27 (memory cells) is shown in a cluster heat map. The data were grouped using average linkage hierarchical clustering, and the top three clusters were associated with patient clinical responses at 6 months as determined by the cluster dendrogram.

[0136] A brief description of sequence identifiers

[0137] SEQ ID NO: 1 shows the amino acid sequence of the anti-BCMA CAR.

[0138] SEQ ID NO: 2 shows the polynucleotide sequence encoding the anti-BCMA CAR.

[0139] In the aforementioned sequences, if X is present, it refers to any amino acid or the absence of a certain amino acid. Specific implementation plan

[0140] A. Overview

[0141] The present invention generally relates to improved methods for manufacturing T cell compositions. Although T cell therapies are more common than they were 5 years ago, the obstacles faced by these therapies still exist, notably weak or suboptimal efficacy. The present manufacturing method provides a solution that greatly increases the efficacy of cell therapy products such as CAR T cell products. Without wishing to be bound by any particular theory, the inventors have unexpectedly found that reducing the duration of T cell manufacturing using PI3K inhibitors enables further improvements in reducing cell doses and increasing cell efficacy and persistence compared to manufacturing processes using longer duration PI3K inhibitors. Surprisingly, the improved drug product manufactured using a shorter PI3K inhibitor-based process is enriched for CD27 + CD8 + Stem cell memory T cells (TSCM) and CD27 + CD4 + In certain embodiments, the improved drug product manufactured using a shorter PI3K inhibitor-based process is enriched for CD27 + 、LEF1 + and / or TCF1 + The cells produced are capable of subsequently differentiating and providing long-lasting immune effector cell function.

[0142] Drug product phenotyping and gene expression analysis also allow clinicians to determine the likelihood of how a particular drug product will perform. The enriched T cells also contain increased gene expression of one or more of the following: nuclear receptor subfamily 4 group A member 2 (NR4A2), CD229 (LY9), Lin-7 homolog A (LIN7A), Wingless type MMTV integration site family member 5B (WNT5B), B cell CLL / lymphoma 6 (BCL6), early growth response protein 1 (EGR1), early growth response protein 2 (EGR2), activating transcription factor 3 (ATF3), CC motif chemokine 1 (CCL1), interleukin 1A (IL-1A), and CC motif chemokine 2 (CCL3). factor 5 (CCL5); and decreased gene expression of one or more of the following: NAD(P)H quinone dehydrogenase 1 (NQO1), cyclin A1 (CCNA1), interleukin 17F (IL17F), epithelial membrane protein 1 (EMP1), small nuclear RNA host gene 19 (SNHG19), proline-rich protein 22 (PRR22), immunoglobulin-like domain-containing receptor 2 (ILDR2), ATPase family AAA domain-containing protein 3 (ATAD3), naked epidermis homolog 2 (NKD2), and WD repeat domain 62 (WDR62).

[0143] In certain embodiments, the enriched T cells comprise increased gene expression of one or more of: CCL1, NR4A2, ATF3, CCL5, and WNT5B; and decreased gene expression of one or more of: NQO1 and NKD2.

[0144] In various embodiments, a method for manufacturing T cells is provided, which increases the effectiveness of adoptive cell therapy. In a specific preferred embodiment, in the presence of phosphatidylinositol -3 kinases (PI3K) inhibitors (e.g., ZSTK474 (CASNO.475110-96-4)), engineered CAR T cell compositions are manufactured under conditions sufficient to increase the effectiveness of engineered cells, for a certain period of time. In a preferred embodiment, T cells are activated and stimulated (about 24 hours, 18 hours -24 hours) in the presence of PI3K inhibitors, lentiviral transduction (about 24 hours, 18 hours -24 hours) is used for the polynucleotides encoding CAR in the presence of PI3K inhibitors, and amplified in the presence of PI3K inhibitors for about 4 days or about 6 days (e.g., 6 days or 8 days, respectively).

[0145] In various embodiments, the five-day T cell manufacturing process includes activating and stimulating T cells in the presence of a PI3K inhibitor (approximately 24 hours, 18 hours-24 hours), transducing the cells with a lentivirus comprising a polynucleotide encoding a CAR in the presence of a PI3K inhibitor (approximately 24 hours, 18 hours-24 hours), and expanding the cells in the presence of a PI3K inhibitor for approximately 4 days (e.g., 6 days total).

[0146] In various embodiments, the seven-day T cell manufacturing process includes activating and stimulating T cells in the presence of a PI3K inhibitor (approximately 24 hours, 18 hours-24 hours), transducing the cells with a lentivirus comprising a polynucleotide encoding a CAR in the presence of a PI3K inhibitor (approximately 24 hours, 18 hours-24 hours), and expanding the cells in the presence of a PI3K inhibitor for approximately 6 days (e.g., 8 days total).

[0147] In certain embodiments, methods of increasing the expression of T cell activation or potency genes and / or decreasing the expression of T cell differentiation or exhaustion genes are contemplated. The manufactured T cell compositions contemplated herein can be used to treat, prevent, or ameliorate at least one symptom of cancer, such as a hematological malignancy.

[0148] In various embodiments, it is contemplated that CD27 produced in the presence of a PI3K inhibitor + A composition of enriched anti-B cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cells manufactured under current good manufacturing practice (cGMP). In certain embodiments, a shorter 5-day or 7-day manufacturing process produces CD27 + 、LEF1 + 、CCR7 + and / or TCF1 + Enriched clusters of anti-BCMACAR T cells.

[0149] In various embodiments, it is contemplated that CD27 + Enriched CD8 + TSCM-like T cells and CD27 + Anti-BCMA CAR T cell composition enriched for CD4+ TCM-like T cells.

[0150] In various embodiments, it is contemplated that LEF1 produced in the presence of a PI3K inhibitor + and / or CCR7 + and / or TCF1 + A composition manufactured in accordance with current Good Manufacturing Practice (cGMP) of enriched anti-B cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cells. In certain embodiments, the enriched cluster is also CD27 +Anti-BCMA CAR T cells.

[0151] In various embodiments, it is contemplated that CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + Enriched CD8 + TSCM-like T cells and CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + Enriched CD4 + Anti-BCMACAR T cell composition of TCM-like T cells.

[0152] Thus, the methods and compositions contemplated herein represent significant improvements over existing adoptive cellular immunotherapies.

[0153] Techniques for recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification, and related techniques and procedures can generally be performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology that are cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (updated July 2008 by John Wiley and Sons); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, GreenePub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Editors: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach, Warren Strober 2001 John Wiley&Sons, NY, NY); Real-Time PCR: Current Technology and Applications, edited by Julie Logan, Kirstin Edwards and Nick Saunders, 2009, Caister Academic Press, Norfolk, UK; Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Oligonucleotide Synthesis (N. Gait ed., 1984); Nucleic Acid The Hybridization (B. Hames & S. Higgins eds., 1985); Transcription and Translation (B. Hames & S. Higgins eds., 1984); Animal Cell Culture (R. Freshney ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Next-Generation Genome Sequencing (Janitz, 2008 Wiley-VCH); PCR Protocols (Methods in Molecular Biology) (Park ed., 3rd ed., 2010 Humana Press); Immobilized Cells And Enzymes (IRL Press, 1986); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P.Calos, ed., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Roitt, Essential Immunology, 6th ed., (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (Q. E. Colligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds., 1991); Annual Review of Immunology; and monographs in journals such as Advances in Immunology.

[0154] B. Definition

[0155] Before describing the present disclosure in greater detail, it may be helpful to understand the present disclosure by providing definitions of certain terms used herein.

[0156] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to the methods and materials described herein can be used to practice or test specific embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of this disclosure, the following terms are defined below.

[0157] The articles "a," "an," and "the" are used herein to refer to one or more than one (ie, to at least one) grammatical object of the article. For example, "an element" means one element or more than one element.

[0158] As used herein, the terms "about" or "approximately" refer to a value, level, value, quantity, frequency, percentage, size, size, amount, weight, or length that varies by up to 30%, 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a reference value, level, value, quantity, frequency, percentage, size, size, amount, weight, or length. In specific embodiments, when preceding a numerical value, the terms "about" or "approximately" indicate that value plus or minus a range of 15%, 10%, 5%, or 1%.

[0159] As used herein, the term "substantially" refers to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, "substantially the same" refers to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that produces an effect, such as a physiological effect, that is about the same as a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0160] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises", and "comprising" should be understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or any other group of steps or elements. "Consisting of" is intended to include and be limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are essential or required, and that no other elements can be present. "Consisting essentially of" is intended to include any element listed after the phrase and is limited to other elements that do not interfere with or affect the activity or action described in the disclosure regarding the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but no other elements are optional and may or may not be present, depending on whether they affect the activity or action of the listed elements.

[0161] Reference throughout this specification to "an embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "another embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the aforementioned phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0162] As used herein, the term "T cell manufacturing" or "method of manufacturing T cells" or equivalent terms refers to a method of producing a therapeutic composition of T cells, which manufacturing method can include one or more or all of the following steps: harvesting, stimulation, activation, transduction, and expansion. In a preferred embodiment, expansion is no more than 5 to 7 days after transduction. A five-day T cell manufacturing process includes activation and stimulation on day 0, transduction on day 1, and expansion until the end of day 5. A seven-day T cell manufacturing process includes activation and stimulation on day 0, transduction on day 1, and expansion until the end of day 7. A 10-day T cell manufacturing process includes activation and stimulation on day 0, transduction on day 1, and expansion until the end of day 10. In a preferred embodiment, the T cell manufacturing method includes the use of PI3K throughout the manufacturing process.

[0163] As used herein, the term "PI3K inhibitor" refers to a small organic molecule that binds to and inhibits at least one activity of PI3K. PI3K proteins can be divided into three categories: Class 1 PI3K, Class 2 PI3K, and Class 3 PI3K. Class 1 PI3K exists as a heterodimer that is composed of one of four p110 catalytic subunits (p110α, p110β, p110δ, and p110γ) and one of two regulatory subunit families. In certain embodiments, the PI3K inhibitor exhibits selectivity for one or more isoforms of the Class 1 PI3K inhibitor (i.e., selectivity for one or more of p110α, p110β, p110δ, and p110γ, as well as p110α, p110β, p110δ, and p110γ). In certain embodiments, the PI3K inhibitor will not exhibit isoform selectivity and will be considered a "pan-PI3K inhibitor."

[0164] The terms "T cell" or "T lymphocyte" are art-recognized and are intended to include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell can be a T helper (Th) cell, such as a T helper 1 (Th1) or T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4 +T cells)CD4 + T cells, cytotoxic T cells (CTL; ​​CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells or any other T cell subset. Preferably, the manufactured T cells are enriched for CD27 + T cells, CD27 + CD4 + T cells and / or CD27 + CD8 + In certain preferred embodiments, the T cells produced are enriched for LEF1 + and / or CCR7 + and / or TCF1 + T cells and / or LEF1 + and / or CCR7 + and / or TCF1 + CD4 + T cells and / or LEF1 + and / or CCR7 + and / or TCF1 + CD8 + In certain preferred embodiments, the T cells produced are enriched for CD27 + LEF1 + and / or CCR7 + and / or TCF1 + T cells and / or CD27 + LEF1 + and / or CCR7 + and / or TCF1 + CD4 + T cells and / or CD27 + LEF1 + and / or CCR7 + and / or TCF1 + CD8 + More preferably, the manufactured T cells are enriched in stem cell memory T cells (TSCM) and central memory T cells (TCM).

[0165] "Potent T cells" and "young T cells" are used interchangeably in certain embodiments and refer to a T cell phenotype in which the T cells are capable of proliferation and are accompanied by a decrease in differentiation. In certain embodiments, the young T cells have the phenotype of naive T cells, TSCM, or TCM. In various embodiments, the manufacturing methods contemplated herein produce stronger T cells, such as naive T cells, TSCM, or TCM. In certain embodiments, the young T cells comprise one or more or all of the following biomarkers that are enriched: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, CD95, CD45RO, and CD38.

[0166] As used herein, the term "proliferation" refers to an increase in cell division (either symmetrical or asymmetrical cell division). In certain embodiments, "proliferation" refers to symmetrical or asymmetrical division of T cells. "Increased proliferation" occurs when the number of cells in a treated sample increases compared to cells in an untreated sample.

[0167] As used herein, the term "differentiation" refers to a process that reduces the potency or proliferation of a cell or moves the cell to a more developmentally restricted state. In certain embodiments, the differentiated T cells acquire immune effector cell function.

[0168] An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, induction of ADCC and / or CDC). Illustrative immune effector cells contemplated herein are T lymphocytes, particularly cytotoxic T cells (CTLs; CD8 + T cells), TILs, and helper T cells (HTL; CD4 + cell).

[0169] "Modified T cells" refer to T cells that have been modified by introducing a polynucleotide encoding a CAR as contemplated herein. Modified T cells include genetically modified and non-genetically modified (e.g., episomal or extrachromosomal).

[0170] As used herein, the term "genetic engineering" or "genetic modification" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material of a cell.

[0171] The terms "genetically modified cells," "modified cells," and "redirected cells" are used interchangeably.

[0172] As used herein, the term "gene therapy" refers to the introduction of additional genetic material into the total genetic material in the cell in the form of DNA or RNA to restore, correct or change the expression of a gene or to achieve the purpose of expressing a therapeutic polypeptide (e.g., TCR or CAR) and / or one or more cytokines. In a specific embodiment, T cells are modified to express CAR without modifying the genome of the cell, for example, by introducing an additional vector expressing TCR or CAR into the cell.

[0173] The term "ex vivo" generally refers to an activity that occurs outside an organism, such as an artificial environment outside an organism, preferably an artificial environment with minimal natural condition changes, in or on living tissue. In specific embodiments, an "ex vivo" procedure involves obtaining living cells or tissues from an organism and in a laboratory setting, usually cultivating or regulating under aseptic conditions, and depending on the circumstances, typically lasting several hours or up to about 24 hours, but including up to 48 or 72 hours. In certain embodiments, these tissues or cells can be collected and frozen, and thawed later to carry out ex vivo processing. Tissue culture experiments or procedures that continue to exceed several days using living cells or tissues are typically considered "in vitro," but in certain embodiments, this term can be used interchangeably with ex vivo.

[0174] The term "in vivo" generally refers to activities that occur inside an organism, such as cell self-renewal and cell expansion. In one embodiment, the term "in vivo expansion" refers to the ability of a cell population to increase in number in vivo.

[0175] The term "stimulation" refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, including but not limited to signal transduction via the TCR / CD3 complex.

[0176] "Stimulatory molecule" refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand.

[0177] As used herein, "stimulatory ligand" means a ligand that, when present on an antigen presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner on a T cell (referred to herein as a "stimulatory molecule"), thereby mediating a primary response of the T cell (including but not limited to activation, initiation of an immune response, proliferation, etc.). Stimulatory ligands include, but are not limited to, CD3 ligands (e.g., anti-CD3 antibodies) and CD2 ligands (e.g., anti-CD2 antibodies), and peptides (e.g., CMV, HPV, EBV peptides).

[0178] The term "activated" refers to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. In a specific embodiment, activation can also be associated with induced cytokine production and detectable effector functions. The term "activated T cells" refers in particular to T cells that are proliferating. The signal generated by the TCR alone is not sufficient to fully activate the T cells and one or more secondary or costimulatory signals are also required. Therefore, T cell activation comprises a primary stimulation signal and one or more secondary costimulatory signals generated by the TCR / CD3 complex. Costimulation can be demonstrated by the proliferation and / or cytokine production of T cells that have received the primary activation signal, such as by the CD3 / TCR complex or by the stimulation of CD2.

[0179] A "co-stimulatory signal" refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, results in T cell proliferation, cytokine production, and / or up- or down-regulation of specific molecules (eg, CD28).

[0180] "Costimulatory ligand" refers to a molecule that binds to a costimulatory molecule. A costimulatory ligand can be soluble or provided on a surface. A "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand (e.g., an anti-CD28 antibody).

[0181] As used herein, "autologous" refers to cells from the same subject. As used herein, "allogeneic" refers to cells from the same species that are genetically different from the comparison cells. As used herein, "isogenic" refers to cells from a different subject that are genetically identical to the comparison cells. As used herein, "xenogeneic" refers to cells from a different species than the comparison cells. In preferred embodiments, the cells produced by the methods contemplated herein are autologous.

[0182] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits symptoms of cancer that can be treated with gene therapy vectors, cell-based therapeutics, and methods disclosed elsewhere herein. Suitable subjects (e.g., patients) include experimental animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and domestic animals or pets (such as cats or dogs). Non-human primates and preferably human patients are included. Typical subjects include human patients who have, have been diagnosed as having, or are at risk of having cancer.

[0183] As used herein, the term "patient" refers to a subject who has been diagnosed with a particular indication that can be treated with the gene therapy vectors, cell-based therapeutics, and methods disclosed elsewhere herein.

[0184] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition and can even include a minimal reduction in one or more measurable markers of the disease or condition being treated (e.g., cancer). Optionally, treatment can involve alleviation or remission of the disease or condition, or delay in progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition or its associated symptoms.

[0185] As used herein, "prevent" and similar words such as "prevented" or "preventing" refer to actions used to prevent, inhibit, or reduce the likelihood of the occurrence or recurrence of a disease or condition (e.g., cancer). Prevention also refers to delaying the onset or recurrence of a disease or condition or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar words also encompass reducing the intensity, effects, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0186] As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide without control and may invade nearby tissues.

[0187] As used herein, the term "malignant" refers to a group of cancer cells that show one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., invasion and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via the lymph or blood). As used herein, the term "metastasis" refers to the spread of cancer from one part of the body to another. Tumors formed by cells that have spread are called "metastatic tumors" or "metastasis". Metastatic tumors contain cells similar to those in the original (primary) tumor.

[0188] As used herein, the term "benign" or "non-malignant" refers to a tumor that can grow larger but does not spread to other parts of the body. Benign tumors are self-limited and usually do not invade or metastasize.

[0189] "Cancer cell" or "tumor cell" refers to a single cell of a cancerous growth or tissue. A tumor generally refers to a swelling or lesion formed by an abnormal growth of cells, which can be benign, pre-malignant, or malignant. Most cancers form tumors, but some cancers, such as leukemias, do not necessarily form tumors. For those cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of tumors.

[0190] "Enhancement" or "promotion" or "increase" or "amplification" generally refers to that the compositions encompassed herein can produce, induce or cause a greater physiological response (i.e., downstream effect) compared to the response caused by a vehicle or control molecule / composition. A measurable physiological response can include an increase in T cell expansion, activation, persistence and / or cancer cell death and killing ability, as well as other aspects apparent from the understanding of the art and the description herein. An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase of 1.1 times, 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times or more (e.g., 500 times, 1000 times) (including all integers and decimal points therebetween and above 1, such as 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by a vehicle or control composition.

[0191] "Reduced" or "diminished" or "lessened" or "reduced" or "mitigated" generally refers to the ability of the compositions encompassed herein to produce, elicit or induce less of a response (i.e., a physiological response) than the response elicited by a vehicle or control molecule / composition. A "reduced" or "reduced" amount is generally a "statistically significant" amount and can include a decrease of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) compared to the response elicited by a vehicle or control composition.

[0192] "Maintain" or "maintain" or "maintenance" or "no change" or "no substantial change" or "no substantial decrease" generally refers to the ability of the compositions contemplated herein to produce, elicit or cause a similar physiological response (i.e., downstream effect) in a cell as compared to the response elicited by vehicle or a control molecule / composition. A comparable response is one that is not significantly different or measurably different from a reference response.

[0193] "Antigen (Ag)" refers to a compound, composition or substance that can stimulate antibody production or T cell response in an animal, including a composition (such as a composition comprising a tumor-specific protein) that is injected or absorbed into an animal. Antigens react with products having specific humoral or cellular immunity, including products induced by heterologous antigens such as disclosed antigens. "Target antigen" or "target antigen of interest" is an antigen to which the CAR binding domain contemplated herein is designed to bind.

[0194] "Epitope" or "antigenic determinant" refers to the region of an antigen to which a binding agent binds.

[0195] Unless otherwise indicated, "polypeptide", "polypeptide fragment", "peptide" and "protein" are used interchangeably and are in accordance with the conventional meaning, i.e., an amino acid sequence. The polypeptide is not limited to a specific length, for example, it may comprise a full-length protein sequence or a fragment of a full-length protein, and may include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc., as well as other naturally occurring and non-naturally occurring modifications known in the art. The polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. The polypeptides contemplated herein explicitly encompass the CARs of the present disclosure, or sequences having deletions, additions and / or substitutions of one or more amino acids compared to the CARs disclosed herein. In certain embodiments, the term "polypeptide" also includes variants, fragments and fusion polypeptides.

[0196] As used herein, "isolated peptide" or "isolated polypeptide" or the like refers to a peptide or polypeptide molecule that has been isolated and / or purified in vitro from its cellular environment and from association with other components of the cell, i.e., the peptide or polypeptide molecule is not significantly associated with substances in the body. Similarly, "isolated cells" refer to cells that have been obtained from a tissue or organ in vivo and are substantially free of extracellular matrix.

[0197] The difference between a polypeptide variant and a naturally occurring polypeptide may be one or more substitutions, deletions, additions and / or insertions. Such variants may be naturally occurring or may be generated synthetically, for example, by modifying one or more of the above-mentioned polypeptide sequences. For example, in certain embodiments, it may be desirable to improve the binding affinity and / or other biological properties of CAR by introducing one or more substitutions, deletions, additions and / or insertions into the binding domain, hinge, TM domain, co-stimulatory signaling domain or primary signaling domain of the CAR polypeptide. Preferably, the polypeptides of the present invention include polypeptides having at least about 65%, 70%, 75%, 85%, 90%, 95%, 98% or 99% amino acid identity thereto.

[0198] Polypeptides include "polypeptide fragments." Polypeptide fragments are fragments of biologically active polypeptides that can be monomeric or polymeric and have amino-terminal deletions, carboxyl-terminal deletions, and / or internal deletions or substitutions of naturally occurring or recombinantly produced polypeptides. In certain embodiments, a polypeptide fragment can comprise an amino acid chain that is at least 5 to about 500 amino acids long. It will be appreciated that in certain embodiments, a fragment is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length.

[0199] Fusion polypeptides and fusion proteins refer to polypeptides having at least two, three, four, five, six, seven, eight, nine, or ten or more polypeptide segments.

[0200] As used herein, the term "polynucleotide" or "nucleic acid" refers to messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive strand RNA (RNA ( + )), negative strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA), or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. Preferably, the polynucleotides of the present invention include polynucleotides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing), or variants thereof, typically wherein the variant retains at least one biological activity of the reference sequence. In various illustrative embodiments, the present invention contemplates, in part, polynucleotides comprising expression vectors, viral vectors, and transfer plasmids, as well as compositions and cells comprising the polynucleotides.

[0201] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from its naturally occurring sequences on either side, e.g., a DNA fragment that has been removed from the sequences that normally surround it. An "isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature and have been made by the hand of man.

[0202] "Control elements" or "regulatory sequences" present in an expression vector are those untranslated regions of the vector—origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Shine Dalgarno sequence or Kozak sequence), introns, polyadenylation sequence, 5' and 3' untranslated regions—that interact with host cell proteins to effect transcription and translation. These elements can vary in their strength and specificity. Depending on the vector system and host utilized, a variety of suitable transcription and translation elements can be used, including ubiquitous promoters and inducible promoters.

[0203] "Endogenous" control sequences are sequences naturally associated with a given gene in the genome. "Exogenous" control sequences are sequences that have been placed in juxtaposition with a gene by genetic manipulation (i.e., molecular biology techniques) so that transcription of the gene is directed by the associated enhancer / promoter. "Heterologous" control sequences are exogenous sequences that are derived from a different species than the cell being genetically manipulated.

[0204] As used herein, the term "promoter" refers to a recognition site for a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In a specific embodiment, a promoter that functions in mammalian cells includes an AT-rich region approximately 25 to 30 bases upstream of the start transcription site and / or another sequence found 70 to 80 bases upstream of the start of transcription, i.e., a CNCAAT region in which N can be any nucleotide.

[0205] The term "enhancer" refers to a segment of DNA that contains sequences that can provide enhanced transcription and, in some cases, can function independently of its orientation relative to another control sequence. An enhancer can function synergistically or additively with a promoter and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA that contains sequences that can provide both promoter and enhancer functions.

[0206] The term "operably linked" refers to a juxtaposition in which the described components are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter and / or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0207] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule.

[0208] Additional definitions are set forth throughout this disclosure.

[0209] CT cell production method

[0210] The T cells manufactured by the methods contemplated herein provide improved adoptive immunotherapy compositions. The present invention contemplates a 5- to 7-day T cell manufacturing process using a PI3K inhibitor that produces stronger T cells than the existing 10-day T cell manufacturing process using such inhibitors. Without wishing to be bound by any particular theory, it is believed that the T cell compositions manufactured by the methods contemplated herein, such as anti-BCMA CAR T cells, contain an increased number of (enriched) stronger T cell populations. In a specific embodiment, the 5- to 7-day manufacturing method contemplated herein produces CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + In certain embodiments, the 5- to 7-day manufacturing methods contemplated herein produce CD27 + and LEF1 + and / or CCR7 + and / or TCF1 + In certain embodiments, the 5- to 7-day manufacturing methods contemplated herein produce CD27 + and LEF1 + and CCR7 + and / or TCF1 + In certain embodiments, the 5- to 7-day manufacturing methods contemplated herein produce CD27 + and LEF1 + and CCR7 + and TCF1 + In certain embodiments, the 5- to 7-day manufacturing methods contemplated herein produce CD27 + CD8 + Stem cell memory T cells (TSCM) and CD27 + CD4 + In certain embodiments, the 5- to 7-day manufacturing methods contemplated herein produce LEF1 + CD8 + Stem cell memory T cells (TSCM) and LEF1 + CD4 + In certain embodiments, the 5- to 7-day manufacturing methods contemplated herein produce CD27 + LEF1 + CD8 + Stem cell memory T cells (TSCM) and CD27 + LEF1 + CD4+ In certain embodiments, the 5- to 7-day manufacturing methods contemplated herein produce CD27 + LEF1 + CCR7 + CD8 + Stem cell memory T cells (TSCM) and CD27 + LEF1 + CCR7 + CD4 + In certain embodiments, the 5- to 7-day manufacturing methods contemplated herein produce CD27 + LEF1 + TCF1 + CD8 + Stem cell memory T cells (TSCM) and CD27 + LEF1 + TCF1 + CD4 + In certain embodiments, the 5- to 7-day manufacturing methods contemplated herein produce CD27 + LEF1 + CCR7 + TCF1 + CD8 + Stem cell memory T cells (TSCM) and CD27 + LEF1 + CCR7 + TCF1 + CD4 + The researchers also found that the enriched clusters of central memory T cells (TCM) were enriched. Furthermore, T cells manufactured using a 5- to 7-day process with a PI3K inhibitor contained differential gene expression signatures compared to T cells manufactured using a 10-day process with a PI3K inhibitor. Adoptive cell therapies containing these enriched cell populations, such as CAR T-cell therapy, allow clinicians to reduce cell doses and increase cell potency and persistence without compromising the efficacy of the therapy.

[0211] In various embodiments, the method for manufacturing T cells comprises activating a T cell population and stimulating the proliferation of the T cell population; transducing the T cells with a viral vector comprising a polynucleotide encoding a CAR; and culturing the transduced T cells to proliferate for a period of about 4 days to about 6 days; wherein all method steps are performed in the presence of a PI3K inhibitor.

[0212] Illustrative examples of PI3K inhibitors suitable for use in specific embodiments of the T cell manufacturing methods contemplated herein include, but are not limited to, BKM120 (a class 1 PI3K inhibitor, Novartis), XL147 (a class 1 PI3K inhibitor, Exelixis), (a pan-PI3K inhibitor, GlaxoSmithKline), and PX-866 (a class 1 PI3K inhibitor; p110α, p110β, and p110γ isoforms, Oncothyreon). Other illustrative examples of selective PI3K inhibitors include, but are not limited to, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, ZSTK474, and IPI-145. Further illustrative examples of pan-PI3K inhibitors include, but are not limited to, BEZ235, LY294002, GSK1059615, TG100713, and GDC-0941.

[0213] In the most preferred embodiment contemplated herein, the manufacturing method utilizes the PI3K inhibitor ZSTK474 (CAS NO. 475110-96-4).

[0214] In various embodiments, the PI3K inhibitor is used throughout the manufacturing process at a concentration of at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 500 nM, at least 1 μM, at least 10 μM, at least 50 μM, at least 100 μM, or at least 1 M.

[0215] In a preferred embodiment, the PI3K inhibitor is used at a concentration of about 1 μM throughout the manufacturing process.

[0216] T cells can be obtained from many sources, including but not limited to peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments, any number of techniques known to the skilled artisan such as sedimentation (e.g., FICOLL) can be used. TM T cells are obtained by isolation) from a unit of blood collected from a subject.

[0217] In certain embodiments, PBMCs are used as a source of T cells in the T cell production methods contemplated herein. + 、CD8 + or CD4 + and CD8 + The T lymphocyte population is a heterogeneous population of T cells and may include other mononuclear cells, such as monocytes, B cells, NK cells, and NKT cells.

[0218] In a preferred embodiment, the T cell manufacturing process begins with obtaining a PBMC source from the circulating blood of an individual by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction, and the cells are placed in a suitable buffer or medium for subsequent processing. Cells can be washed with PBS or with another suitable solution lacking calcium, magnesium and most (if not all) divalent cations. As will be understood by those of ordinary skill in the art, washing steps can be achieved by methods known to those skilled in the art, such as by using a semi-automatic circulation centrifuge. For example, Cobe 2991 cell processor, Baxter CytoMate etc. After washing, cells can be resuspended in various biocompatible buffers or other saline solutions with or without buffer. In certain embodiments, unwanted components in the apheresis sample can be removed and cells can be directly resuspended in culture medium. Methods for T cell manufacturing are disclosed in U.S. patent application No. 15 / 306,729, filed October 25, 2016, entitled "Improved Methods for Manufacturing Adoptive Cell Therapies"; U.S. patent application No. 15 / 316,792, filed December 6, 2016, entitled "Improved T Cell Compositions"; and U.S. patent application No. 16 / 060,184, filed June 7, 2018, entitled "Improved T Cell Compositions," each of which is incorporated herein by reference in its entirety. In certain embodiments, a cell population comprising T cells (e.g., PBMCs) is used in the manufacturing methods contemplated herein. In other embodiments, an isolated or purified T cell population is used in the manufacturing methods contemplated herein.

[0219] PBMCs can be treated to activate and stimulate the T cell population contained therein to achieve a sufficient therapeutic dose of the T cell composition. In certain embodiments, T cells may generally be activated and expanded using methods as described, for example, in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in its entirety.

[0220] In a preferred embodiment, T cells are activated and stimulated in the presence of a PI3K inhibitor (e.g., ZSTK474). The methods contemplated herein differ from existing methods in that only a single round of activation and stimulation is performed, whereas methods in the art routinely use two, three, four, or five or more rounds of activation and amplification.

[0221] T cell activation can be achieved by providing a primary stimulation signal through the T cell TCR / CD3 complex or via stimulation of the CD2 surface protein. The TCR / CD3 complex can be stimulated by contacting the T cell with an appropriate CD3 binding agent (e.g., a CD3 ligand or an anti-CD3 monoclonal antibody). Illustrative examples of CD3 antibodies include, but are not limited to, OKT3, G19-4, BC3, and 64.1. In addition to the primary stimulation signal provided by the TCR / CD3 complex or via CD2, the induction of T cell responses also requires a second costimulatory signal. In a specific embodiment, a CD28 binding agent can be used to provide a costimulatory signal. Illustrative examples of CD28 binding agents include, but are not limited to, natural CD28 ligands, e.g., natural ligands of CD28 (e.g., members of the B7 protein family, such as B7-1 (CD80) and B7-2 (CD86); and anti-CD28 monoclonal antibodies or fragments thereof that are capable of cross-linking CD28 molecules, e.g., monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10.

[0222] In a preferred embodiment, T cells are activated with soluble anti-CD3 antibodies and stimulated to proliferate with anti-CD28 antibodies. In a specific embodiment, the anti-CD3 antibodies and anti-CD8 antibodies are immobilized, tethered or bound to beads, such as paramagnetic beads, e.g., Dynabeads.

[0223] In certain embodiments, anti-CD3 antibodies and anti-CD8 antibodies are located on the cell surface. In a preferred embodiment, primary and costimulatory ligands, such as anti-CD3 antibodies and anti-CD28 antibodies, are presented on antigen presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.) present in the PBMC fraction.

[0224] In certain embodiments, T cells are activated and stimulated for about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours. In certain embodiments, T cells are activated and stimulated for about 24 hours.

[0225] In certain embodiments, T cells are activated and stimulated for about 16 hours to about 30 hours, about 16 hours to about 24 hours, about 18 hours to about 24 hours, or about 20 hours to about 24 hours.

[0226] In a preferred embodiment, cells that have undergone the activation and stimulation steps are transduced in the presence of a PI3K inhibitor (e.g., ZSTK474). Although the purpose of this step of the process is to transduce immune effector cells, other cells may be present and transduced, for example, CD4 T cells if PBMCs are used as the starting material. + 、CD8 + or CD4 + and CD8 + Cells and other mononuclear cells, such as monocytes, B cells, NK cells and NKT cells. In a preferred embodiment, activated and stimulated T cells are transduced with a viral vector comprising a polynucleotide encoding CAR. The illustrative examples of the viral vector systems suitable for use in the specific embodiments considered in a specific embodiment include but are not limited to adeno-associated virus (AAV) vectors, retroviral vectors (for example, lentiviral vectors), herpes simplex virus vectors, adenoviral vectors and vaccinia virus vectors.

[0227] In a preferred embodiment, cells are transduced with a lentivirus comprising a polynucleotide encoding CAR. As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Visna-Maedi virus (visna-maedi virus, VMV) virus; Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). In one embodiment, preferably based on HIV-1 vector backbone (i.e., HIV cis-acting sequence elements).

[0228] In various embodiments, the lentiviral vectors contemplated herein comprise a chimeric 5' long terminal repeat (LTR), such as a chimeric CMV / 5'LTR promoter and one or more or all of the following accessory elements: cPPT / FLAP (Zennou et al., 2000, Cell, 101: 173), a Psi (Ψ) packaging signal (Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109), an export element, such as an RRE (Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991. Cell, 101: 173). 58:423), a poly (A) sequence, optionally a WPRE (Zufferey et al., 1999, J. Virol., 73:2886) or HPRE (Huang et al., Mol. Cell. Biol., 5:3864), an insulator element, a selectable marker or a cell suicide gene, and a modified self-inactivating (SIN) 3′ LTR. A "self-inactivating" (SIN) vector refers to a replication-defective vector, such as a retroviral vector or a lentiviral vector, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. In specific embodiments, the lentiviral vector is pseudotyped with the vesicular stomatitis virus G-protein (VSV-G) envelope protein to enable the vector to infect a wide range of cells. In certain embodiments, the lentiviral vector is produced according to known methods. See, e.g., Kutner et al., BMC Biotechnol. 2009; 9: 10. doi: 10.1186 / 1472-6750-9-10; Kutner et al. Nat. Protoc. 2009; 4(4): 495–505. doi: 10.1038 / nprot.2009.22.

[0229] In certain embodiments, the cells are transduced for about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours after activation and stimulation. In certain embodiments, the cells are transduced for about 24 hours.

[0230] In specific embodiments, the cells are transduced about 16 hours to about 30 hours, about 16 hours to about 24 hours, about 18 hours to about 24 hours, or about 20 hours to about 24 hours after activation and stimulation.

[0231] In a preferred embodiment, after transduction, the cells are cultured in the presence of a PI3K inhibitor (e.g., ZSTK474) under conditions that promote the proliferation or expansion of immune effector cells such as T cells, CART cells, or anti-BCMA CART cells. Unexpectedly, the inventors found that very short proliferation or expansion cycles of 1, 2, 3, 4, 5, or 6 days (after transduction) produced CD27-rich cells. + Highly effective cell therapy products of cells, TCM and TSCM.

[0232] In certain embodiments, conditions suitable for T cell proliferation or expansion culture include culturing cells in an appropriate culture medium (e.g., minimal essential medium or RPMI medium 1640 or X-vivo 15, (Lonza)) and one or more factors necessary for proliferation and viability, including but not limited to serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α, or any other additives suitable for cell growth known to those skilled in the art. Further illustrative examples of cell culture media include, but are not limited to, RPMI 1640, Clicks, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 5 and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in an amount sufficient to allow T cell growth and expansion. Illustrative examples of other additives for T cell expansion include, but are not limited to, surfactants, plasma protein powder (plasmanate), pH buffers (such as HEPES), and reducing agents (such as N-acetyl-cysteine ​​and 2-mercaptoethanol).

[0233] In a preferred embodiment, T cells are cultured in T cell growth medium (TCGM) to proliferate or expand for 1, 2, 3, 4, 5 or 6 days, wherein the growth medium is prepared with X-VIVO 15 supplemented with 10 mM HEPES, 2 mM GlutaMax and 5% human AB serum. In a preferred embodiment, the manufacturing process is carried out in the presence of one or more cytokines, preferably IL-2, IL-7 and / or IL-15, and more preferably IL-2.

[0234] In certain embodiments, the cell proliferation or expansion phase is carried out for about 1 day to about 6 days, about 2 days to about 6 days, about 3 days to about 6 days, or about 4 days to about 6 days. In preferred embodiments, the cell proliferation or expansion phase is carried out for about 4 days to about 6 days.

[0235] In certain embodiments, the cell proliferation or expansion phase is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days. In a preferred embodiment, the cell proliferation or expansion phase is about 4 days. In a specific preferred embodiment, the cell proliferation or expansion phase is about 6 days.

[0236] In various embodiments, T cell compositions are manufactured in the presence of one or more inhibitors of the PI3K pathway. The inhibitors can target one or more activities or a single activity in the pathway. Without wishing to be bound by any particular theory, it is contemplated that during the stimulation, activation, and / or expansion phases of the manufacturing process, treating or contacting T cells with one or more inhibitors of the PI3K pathway preferentially increases young T cells, thereby producing superior therapeutic T cell compositions.

[0237] In various embodiments, a method of manufacturing CAR T cells comprises activating a T cell population and stimulating proliferation of the T cell population; transducing the T cells with a lentiviral vector comprising a polynucleotide encoding a CAR; and culturing the transduced T cells to proliferate for a period of about 4 days to about 6 days; wherein all method steps are performed in the presence of a PI3K inhibitor, and wherein the proliferated CAR T cells are enriched for TCM and TSC cells compared to a manufacturing process in which the transduced cells are cultured in the presence of a PI3K inhibitor for a period of about 9 days.

[0238] In certain embodiments, a method of manufacturing anti-BCMA CAR T cells comprising a proliferation or expansion culture for about 4 to about 6 days results in CD4 T cells with a TCM phenotype compared to a manufacturing process in which the transduced cells are cultured in the presence of a PI3K inhibitor for about 9 days. + T cells are enriched by about 1.5-fold, about 2.0-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold and have a CD8 T cell phenotype. + T cells are enriched by about 1.5-fold, about 2.0-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold.

[0239] In various embodiments, a method of manufacturing CAR T cells comprises activating a T cell population and stimulating proliferation of the T cell population; transducing the T cells with a lentiviral vector comprising a polynucleotide (e.g., SEQ ID NO: 2) encoding a CAR (e.g., an anti-BCMA CAR comprising the amino acid sequence set forth in SEQ ID NO: 1); and culturing the transduced T cells to proliferate for a period of about 4 days to about 6 days; wherein all method steps are performed in the presence of a PI3K inhibitor, and wherein the proliferated CAR T cells are enriched for CD27 compared to a manufacturing process in which the transduced cells are cultured in the presence of a PI3K inhibitor for a period of about 9 days. + cell.

[0240] In certain embodiments, a method of manufacturing anti-BCMA CAR T cells comprising a proliferation or expansion culture for about 4 to about 6 days results in a decrease in CD27 compared to a manufacturing process in which the transduced cells are cultured in the presence of a PI3K inhibitor for about 9 days. + T cells are enriched by about 1.5-fold, about 2.0-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold.

[0241] In certain embodiments, a method of manufacturing anti-BCMA CAR T cells comprising a proliferation or expansion culture for about 4 to about 6 days results in an enrichment or increase in the number of one or more T cells expressing CD27, CD25, CD127, TCF1, LEF1, CD28, and / or CCR7 of about 1.5-fold, about 2.0-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold compared to a manufacturing process in which the transduced cells are cultured in the presence of a PI3K inhibitor for about 9 days. ... A method for manufacturing CART cells, comprising a proliferation or expansion culture for about 4 days to about 6 days, resulting in an enrichment or increase in the number of one or more T cells expressing CD27, CD25, CD127, TCF1 and / or LEF1 and / or CCR7 by about 1.5 times, about 2.0 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times or about 5 times compared to a manufacturing process in which the transduced cells are cultured in a PI3K inhibitor for about 9 days. In a specific embodiment, a method for manufacturing anti-BCMA CAR T cells, comprising a proliferation or expansion culture for about 4 days to about 6 days, resulting in a reduction of the number of T cells expressing one or more of granzyme A, granzyme B, perforin, T-bet and EOMES by about 1.5 times, about 2.0 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4.5 times or about 5 times compared to a manufacturing process in which the transduced cells are cultured in a PI3K inhibitor for about 9 days.

[0242] In various embodiments, a method of manufacturing an anti-BCMA CAR T cell comprises activating a T cell population and stimulating proliferation of the T cell population; transducing the T cells with a lentiviral vector comprising a polynucleotide encoding an anti-BCMA CAR comprising the amino acid sequence set forth in SEQ ID NO: 1 (e.g., SEQ ID NO: 2); and culturing the transduced T cells to proliferate for a period of about 4 days to about 6 days; wherein all method steps are performed in the presence of a PI3K inhibitor, and wherein the proliferated T cells are enriched for CD27 compared to a manufacturing process in which the transduced cells are cultured in the presence of a PI3K inhibitor for a period of about 9 days. + CD4 + TCM and CD27 + CD8 +TSCM cells.

[0243] In certain embodiments, a method of manufacturing anti-BCMA CAR T cells comprising a proliferation or expansion culture for about 4 to about 6 days results in CD27 TCM cells having a TCM phenotype compared to a manufacturing process in which the transduced cells are cultured in the presence of a PI3K inhibitor for about 9 days. + CD4 + T cells are enriched for CD27 by about 1.5-fold, about 2.0-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold and have a TSC M phenotype. + CD8 + T cells are enriched by about 1.5-fold, about 2.0-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold.

[0244] In various embodiments, a method of manufacturing an anti-BCMA CAR T cell comprises activating a T cell population and stimulating proliferation of the T cell population; transducing the T cells with a lentiviral vector comprising a polynucleotide encoding an anti-BCMA CAR comprising the amino acid sequence set forth in SEQ ID NO: 1 (e.g., SEQ ID NO: 2); and culturing the transduced T cells to proliferate for a period of about 4 days to about 6 days; wherein all method steps are performed in the presence of a PI3K inhibitor, and wherein the proliferated T cells are enriched for CD27 compared to a manufacturing process in which the transduced cells are cultured in the presence of a PI3K inhibitor for a period of about 9 days. + and / or LEF1 + and / or CCR7 + and / or TCF1 + CD4 + TCM and CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + CD8 + TSCM cells.

[0245] In certain embodiments, a method of manufacturing anti-BCMA CAR T cells comprising a proliferation or expansion culture for about 4 to about 6 days results in CD27 TCM cells having a TCM phenotype compared to a manufacturing process in which the transduced cells are cultured in the presence of a PI3K inhibitor for about 9 days. + and / or LEF1 + and / or CCR7 + and / or TCF1 + CD4 + T cells are enriched for CD27 by about 1.5-fold, about 2.0-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold and have a TSC M phenotype.+ and / or LEF1 + and / or CCR7 + and / or TCF1 + CD8 + T cells are enriched by about 1.5-fold, about 2.0-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, or about 5-fold.

[0246] In various embodiments, a method of making an anti-BCMA CAR T cell comprises activating a T cell population and stimulating the proliferation of the T cell population; transducing the T cell with a lentiviral vector comprising a polynucleotide encoding an anti-BCMA CAR comprising the amino acid sequence set forth in SEQ ID NO: 1 (e.g., SEQ ID NO: 2); and culturing the transduced T cell to proliferate for a period of about 4 days to about 6 days; wherein all method steps are performed in the presence of a PI3K inhibitor, and wherein the gene expression signature of the proliferating T cell has an enrichment for or increased expression of one or more or all of the following: nuclear receptor subfamily 4 group A member 2 (NR4A2), CD229 (LY9), Lin-7 homolog A (LIN7A), Wingless MMTV integration site family member 5B (WNT5B), B cell CLL / lymphoma 6 (BCL6), early growth factor receptor IL-63A1 (IL-63A1), IL-63A2 (IL-63A2 ... responsive protein 1 (EGR1), early growth response protein 2 (EGR2), activating transcription factor 3 (ATF3), CC motif chemokine 1 (CCL1), interleukin 1A (IL-1A), and CC motif chemokine 5 (CCL5); and decreased gene expression of one or more or all of the following: NAD(P)H quinone dehydrogenase 1 (NQO1), cyclin A1 (CCNA1), interleukin 17F (IL17F), epithelial membrane protein 1 (EMP1), small nuclear RNA host gene 19 (SNHG19), proline-rich protein 22 (PRR 22), immunoglobulin-like domain-containing receptor 2 (ILDR2), ATPase family AAA domain-containing protein 3 (ATAD3), naked epidermal homolog 2 (NKD2), and WD repeat domain 62 (WDR62).

[0247] In various embodiments, a method of making an anti-BCMA CAR T cell comprises activating a T cell population and stimulating proliferation of the T cell population; transducing the T cells with a lentiviral vector comprising a polynucleotide encoding an anti-BCMA CAR comprising the amino acid sequence set forth in SEQ ID NO: 1 (e.g., SEQ ID NO: 2); and culturing the transduced T cells to proliferate for a period of about 4 days to about 6 days; wherein all method steps are performed in the presence of a PI3K inhibitor, and wherein the gene expression signature of the proliferating T cells has enrichment or increased expression of CCL1, NR4A2, ATF3, CCL5, and WNT5B and decreased expression of NKD2 and NQO1.

[0248] "Gene expression" refers to the relative expression level and / or expression pattern of genes in a biological sample, i.e., a T cell population (e.g., anti-BCMA CAR T cells) manufactured in the presence or absence of a PI3K inhibitor or manufactured for different time lengths in the presence of a PI3K inhibitor. Gene expression can be measured at the level of cDNA, RNA, mRNA, or a combination thereof. The method for measuring gene expression includes but is not limited to quantitative real-time PCR, high-density oligonucleotide arrays, Nanostring transcriptome analysis, or RNA sequencing (RNA-Seq).

[0249] In certain embodiments, T cells (including CAR T cells, e.g., anti-BCMA CAR T cells) manufactured using the seven-day manufacturing method using the PI3K inhibitors contemplated herein are characterized by an increase in expression of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A, and CCL5, or (ii) CCL1, NR4A2, ATF3, CCL5, and WNT5B by at least 1.5-fold or at least 2-fold, compared to T cells manufactured using the 10-day manufacturing process contemplated herein. Using a PI3K inhibitor, T cells manufactured using the seven-day process are also characterized by a unique gene expression signature, in which the expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all 11 of the signature genes selected from the group consisting of: NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A and CCL5 is increased by at least 1.5-fold or at least 2-fold compared to T cells manufactured using a 10-day process using a PI3K inhibitor.

[0250] In certain embodiments, T cells (including CAR T cells, e.g., anti-BCMA CAR T cells) manufactured using the seven-day manufacturing method using the PI3K inhibitors contemplated herein are characterized by reduced expression of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2, and WDR62, or (ii) NKD2 and NQO1 by at least 1.5-fold or at least 2-fold, compared to T cells manufactured using the 10-day manufacturing process contemplated herein. Using a PI3K inhibitor, T cells manufactured using the seven-day process are also characterized by a unique gene expression signature, in which the expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or all 10 of the signature genes selected from the group consisting of: NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2 and WDR62 is reduced by at least 1.5-fold or at least 2-fold compared to T cells manufactured using a 10-day process using a PI3K inhibitor.

[0251] In certain embodiments, T cells (including CAR T cells, e.g., anti-BCMA CAR T cells) manufactured using the seven-day manufacturing method using the PI3K inhibitors contemplated herein are characterized by an increase in expression of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A, and CCL5, or (ii) CCL1, NR4A2, ATF3, CCL5, and WNT5B by at least 1.5-fold or at least 2-fold; and a decrease in expression of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2, and WDR62, or (ii) NKD2 and NQO1 by at least 1.5-fold or at least 2-fold, compared to T cells manufactured using the 10-day manufacturing process contemplated herein. Using a PI3K inhibitor, T cells manufactured using the seven-day process are also characterized by a unique gene expression signature, in which the expression of one, two, three, four, five, six, seven, eight, nine, ten, or all eleven of the signature genes selected from the group consisting of NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A, and CCL5 is increased by at least 1.5-fold or at least 2-fold, and the expression of one, two, three, four, five, six, seven, eight, nine, or all ten of the signature genes selected from the group consisting of NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2, and WDR62 is decreased by at least 1.5-fold or at least 2-fold compared to T cells manufactured using a 10-day process using a PI3K inhibitor.

[0252] In various embodiments, the method of making an anti-BCMA CAR T cell comprises activating a T cell population and stimulating the proliferation of the T cell population; transducing the T cell with a lentiviral vector comprising a polynucleotide encoding an anti-BCMA CAR comprising the amino acid sequence set forth in SEQ ID NO: 1 (e.g., SEQ ID NO: 2); NO:2); and culturing the transduced T cells to proliferate for a period of about 4 days to about 6 days; wherein all method steps are performed in the presence of a PI3K inhibitor, and wherein the proliferated T cells are enriched for TCM and TSC cells and wherein the gene expression of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A and CCL5 or (ii) CCL1, NR4A2, ATF3, CCL5 and WNT5B is at least 1.5-fold higher in the cultured T cells for a period of about 4 days to about 6 days compared to the T cells for a period of about 9 days.

[0253] In various embodiments, a method of making an anti-BCMA CAR T cell comprises activating a T cell population and stimulating the proliferation of the T cell population; transducing the T cells with a lentiviral vector comprising a polynucleotide encoding an anti-BCMA CAR comprising the amino acid sequence set forth in SEQ ID NO: 1 (e.g., SEQ ID NO: 2); and culturing the transduced T cells to proliferate for a period of about 4 days to about 6 days; wherein all method steps are performed in the presence of a PI3K inhibitor, and wherein the proliferated T cells are enriched for TCM, TSC M cells, and wherein the gene expression of one, two, three, four, five, six, seven, eight, nine, or all of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2, and WDR62, or (ii) NKD2 and NQO1, is at least 1.5-fold lower in cultured T cells proliferated for a period of about 4 days to about 6 days compared to T cells cultured for a period of about 9 days.

[0254] In various embodiments, a method of making an anti-BCMA CAR T cell comprises activating a T cell population and stimulating the T cell population to proliferate; transducing the T cells with a lentiviral vector comprising a polynucleotide encoding an anti-BCMA CAR comprising the amino acid sequence set forth in SEQ ID NO: 1 (e.g., SEQ ID NO: 2); and culturing the transduced T cells to proliferate for a period of about 4 days to about 6 days; wherein all method steps are performed in the presence of a PI3K inhibitor, and wherein the proliferated T cells are enriched for TCM, TSC-1 cells, and wherein, in the cultured T cells cultured for a period of about 4 days to about 6 days, (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A, and CCL2 are expressed compared to T cells cultured for a period of about 9 days. 5 or (ii) at least 1.5-fold higher gene expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of CCL1, NR4A2, ATF3, CCL5, and WNT5B, and at least 1.5-fold lower gene expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or all of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR22, ILDR2, ATAD3, NKD2, and WDR62 or (ii) NKD2 and NQO1.

[0255] The manufacturing method contemplated herein may also include cryopreservation of PBMC and / or cryopreservation of the T cell composition manufactured before the manufacturing process begins. The cryopreservation of adoptive cell therapy allows for storage, testing, transportation and release of therapeutic agents for human subjects. T cells are cryopreserved so that the cells remain viable after thawing. When necessary, the cryopreserved cells can be thawed, grown and expanded to obtain more such cells. As used herein, "cryopreservation" refers to preserving cells by cooling to subzero temperatures, such as (usually) 77K or -196°C (the boiling point of liquid nitrogen). Cryoprotectants are often used at subzero temperatures to prevent cell preservation from being damaged due to freezing at low temperatures or warming to room temperature. Cryoprotectants and optimal cooling rates can prevent cell damage. Cryoprotectants that can be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, Nature, 1959; 183: 1394-1395; Ashwood-Smith, Nature, 1961; 190: 1204-1205), glycerol, polyvinyl pyrrolidine (Rinfret, Ann. NY Acad. Sci., 1960; 85: 576), polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48), and CryoStor CS10, CryoStor CS5, and CryoStor CS2. In a preferred embodiment, the manufactured T cells are formulated in a solution comprising 50:50 PlasmaLyte A:CryoStor CS10. The preferred cooling rate is 1°C / min to 3°C / min. After at least two hours, the T cells have reached a temperature of -80°C and can be placed directly into liquid nitrogen (-196°C) for permanent storage, such as in long-term cryogenic storage containers.

[0256] D. Chimeric Antigen Receptor

[0257] The method contemplated herein is used to manufacture stronger adoptive cell therapies that redirect the cytotoxicity of immune effector cells to cancer cells expressing target antigens. In preferred embodiments, the manufacturing method contemplated herein includes transducing activated and stimulated T cells with viral vectors encoding chimeric antigen receptors (CARs) to redirect immune effector cells.

[0258] CAR is the specificity of antibody for target antigen (for example, tumor antigen) and T cell receptor activation intracellular domain is combined to produce the molecule of the chimeric protein that shows specific anti-tumor cell immune activity.CAR considered herein includes signal peptide, the extracellular domain (also referred to as binding domain or antigen-specific binding domain) that is combined with specific target antigen, membrane spaning domain and one or more intracellular signal transduction domains.

[0259] In a specific embodiment, CAR includes an extracellular binding domain that specifically binds to a target polypeptide. In a specific embodiment, the extracellular binding domain comprises an antibody or an antigen-binding fragment thereof. In a preferred embodiment, the binding domain includes scFv. In another preferred embodiment, the binding domain includes one or more Camelidae VHH antibodies or single domain antibodies (sdAb).

[0260] In certain embodiments, the CAR comprises an extracellular domain that binds an antigen selected from the group consisting of: alpha folate receptor (FRα), alpha vβ6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule EPCAM, ephrin type A receptor 2 (EPHA2), fibroblast activation protein (FAP), Fc receptor-like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3), EGFR family including ErbB2 (HER2), IL-10R, IL-13R2, Kappa, cancer / testis antigen 2 (LAGE-1A), Lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, melanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC Class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), cancer / testis antigen 1 (NY-ESO-1), polysialic acid; placenta-specific 1 (PLAC1), antigen preferentially expressed in melanoma (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), synovial sarcoma, breakpoint X 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), trophoblast glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms tumor 1 (WT-1).

[0261] In preferred embodiments, the CAR comprises an extracellular domain that binds a B cell maturation antigen.

[0262] In certain embodiments, CAR includes a hinge domain. Illustrative hinge domains include, but are not limited to, hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8 α and CD4, which may be wild-type hinge regions from these molecules or may be altered. In preferred embodiments, CAR includes a CD8 α hinge region.

[0263] " transmembrane (TM) domain " of CAR fuses extracellular binding moiety and intracellular signal transduction domain and anchors CAR to the plasma membrane of immune effector cells.TM domain can be derived from natural, synthetic, semi-synthetic or recombinant sources.Illustrative TM domain can be derived from (i.e. at least including one or more transmembrane regions): α chain, β chain, γ chain or δ chain of T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, AMN and PDCD1.

[0264] In a preferred embodiment, CAR comprises a TM domain derived from CD8 α. In another embodiment, the CAR encompassed herein comprises a TM domain derived from CD8 α and a short oligopeptide or polypeptide linker preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length, connecting the TM domain of CAR and the intracellular signaling domain. Glycine-serine linkers provide particularly suitable linkers.

[0265] In preferred embodiments, the CAR includes an intracellular signaling domain that includes one or more "co-stimulatory signaling domains" and a "primary signaling domain."

[0266] The primary signaling domain, which acts in a stimulatory manner, may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0267] Illustrative examples of ITAM-containing primary signaling domains suitable for use in CARs encompassed within specific embodiments include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a specific preferred embodiment, the CAR comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling domain and the costimulatory signaling domain can be connected in series to the carboxyl terminus of the transmembrane domain in any order.

[0268] In certain embodiments, the CAR comprises one or more co-stimulatory signaling domains to enhance the efficacy and expansion of T cells expressing the CAR receptor.

[0269] Suitable for use in the CAR of the present invention include, but are not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, and ZAP70. In one embodiment, CAR comprises one or more costimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3 ζ primary signaling domain.

[0270] In a preferred embodiment, the CAR comprises a CD8α signal peptide; an extracellular domain that binds BCMA; a CD8α hinge and transmembrane domain; a CD137 costimulatory domain; and CD137; and a CD3ζ primary signaling domain. In a more preferred embodiment, the anti-BCMA CAR comprises the amino acid sequence set forth in SEQ ID NO: 1, and in an even more preferred embodiment, the anti-BCMA CAR comprises the polynucleotide sequence set forth in SEQ ID NO: 2.

[0271] E. Compositions and Formulations

[0272] The compositions contemplated herein comprise a therapeutically effective amount of CAR T cells. In a preferred embodiment, the compositions contemplated herein comprise a therapeutically effective amount of anti-BCMA CAR T cells. Compositions include, but are not limited to, pharmaceutical compositions. "Pharmaceutical composition" refers to a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution that is administered to cells or animals alone or in combination with one or more other therapeutic modalities. It should also be understood that, if desired, the composition can also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents. There is virtually no restriction on other components that may be included in the composition, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0273] In preferred embodiments, the compositions contemplated herein comprise cGMP manufactured populations of CAR T cells enriched in T cells expressing one or more of CD27, LEF1, and TCF1 on the cell surface. In preferred embodiments, the enriched populations of CAR T cells manufactured using a 5- to 7-day process in the presence of a PI3K inhibitor comprise at least 10% CD27. + , at least 15% CD27 + , at least 20% CD27 + , at least 25% CD27 + , at least 30% CD27 + , at least 35% CD27 + , at least 40% CD27 + , at least 45% CD27 + or at least 50% CD27 + In certain embodiments, the enriched clusters of CAR T cells produced using a 5- to 7-day process in the presence of a PI3K inhibitor contain at least 10% CD27 + 、LEF1 + and / or TCF1 + , at least 15% CD27 + 、LEF1 + and / or TCF1 + , at least 20% CD27 + 、LEF1 + and / or TCF1 + , at least 25% CD27 + 、LEF1 + and / or TCF1 + , at least 30% CD27 + 、LEF1 + and / or TCF1 + , at least 35% CD27 +、LEF1 + and / or TCF1 + , at least 40% CD27 + 、LEF1 + and / or TCF1 + , at least 45% CD27 + 、LEF1 + and / or TCF1 + , or at least 50% CD27 + 、LEF1 + and / or TCF1 + In certain embodiments, the enriched clusters of CAR T cells produced using a 5- to 7-day process in the presence of a PI3K inhibitor contain at least 10% CD27 + LEF1 + TCF1 + , at least 15% CD27 + LEF1 + TCF1 + , at least 20% CD27 + LEF1 + TCF1 + , at least 25% CD27 + LEF1 + TCF1 + , at least 30% CD27 + LEF1 + TCF1 + , at least 35% CD27 + LEF1 + TCF1 + , at least 40% CD27 + LEF1 + TCF1 + , at least 45% CD27 + LEF1 + TCF1 + or at least 50% CD27 + LEF1 + TCF1 + CAR T cells. In certain embodiments, the T cells are also CCR7 + .

[0274] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0275] As used herein, "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, vehicle, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent or emulsifier that has been approved by the U.S. Food and Drug Administration for use in humans or livestock. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; astragalus; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; and any other nontoxic, compatible substance employed in pharmaceutical formulations.

[0276] In certain embodiments, the composition comprises an amount, and more preferably a therapeutically effective amount, of the CAR-expressing T cells contemplated herein.

[0277] As used herein, the terms "amount" or "dose" refer to an "effective amount," "effective dose," "effective amount," or "effective dose" of CAR T cells sufficient to achieve beneficial or desired prophylactic or therapeutic results, including clinical results.

[0278] The "therapeutically effective amount" or "therapeutically effective dose" of CAR T cells is also a dosage in which any toxic or deleterious effects of CAR T cells (e.g., CRS) are exceeded by therapeutic beneficial effects. The term "therapeutically effective amount" includes the amount of an effective "treatment" subject (e.g., patient). In one embodiment, the therapeutically effective dose is the minimum effective dose (MED) of CAR T cells for treating multiple myeloma in a subject. In one embodiment, the therapeutically effective dose is the maximum tolerated dose (MTD) at which anti-BCMA CAR T cells do not cause unresolved CRS in a subject. In a preferred embodiment, a therapeutically effective amount of CAR T cells (e.g., anti-BCMA CAR T cells) manufactured using a 5-day or 7-day manufacturing process in the presence of a PI3K inhibitor is administered to a subject, wherein the amount of the cell is less than the amount of the cell required for achieving the same results using a PI3K inhibitor and a 10-day manufacturing process for CART cells.

[0279] In certain embodiments, the compositions are preferably formulated for parenteral administration, such as intravascular (intravenous or intraarterial) administration.In a preferred embodiment, the compositions contemplated herein are infused intravenously into the subject as a single dose.

[0280] In one embodiment, the CAR in the composition administered to the subject is + The amount of T cells is at least about 5.0 × 10 7 cells, at least about 15.0×10 7 cells, at least about 45.0×10 7 cells, at least about 80.0×10 7 cells or at least about 12.0 × 10 8 cells.

[0281] In one embodiment, the CAR in the composition administered to the subject is + The number of T cells is greater than about 5.0 × 10 7 cells, greater than approximately 15.0×10 7 cells, greater than approximately 45.0×10 7 cells, greater than about 80.0×10 7 cells or greater than approximately 12.0 × 10 8 cells.

[0282] In one embodiment, the CAR in the composition administered to the subject is + The amount of T cells ranged from approximately 5.0 × 10 7 cells to approximately 15.0×10 7 cells, ranging from about 5.0×10 7 cells to approximately 45.0×10 7 cells, ranging from about 5.0×10 7 cells to approximately 80.0×10 7 cells, or between about 5.0×10 7 cells to approximately 12.0×10 8 Between cells.

[0283] For the uses provided herein, the volume of cells is typically one liter or less, and can be 500 mL or less, or even 250 mL or 100 mL or less.

[0284] In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of CAR T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0285] Pharmaceutical compositions comprising a therapeutically effective dose of CAR T cells may comprise a buffer, such as neutral buffered saline, phosphate buffered saline, or the like; a carbohydrate, such as glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide or amino acid, such as glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative.

[0286] Liquid pharmaceutical compositions, whether they are solutions, suspensions or other similar forms, may include one or more of the following: sterile diluents such as water for injection, saline solutions (preferably physiological saline), Ringer's solution, isotonic sodium chloride, fixed oils that can serve as solvents or suspension media (such as synthetic mono- or diglycerides), polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral formulations can be placed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0287] In certain embodiments, the CAR T cell compositions contemplated herein are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects. In certain embodiments, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0288] Serum-free culture media have several advantages over serum-containing culture media, including simplified and better-defined compositions, reduced levels of contaminants, elimination of possible infectious agent sources, and reduced costs. In various embodiments, serum-free culture media are animal-free and can optionally be protein-free. Optionally, the culture media can contain biopharmaceutically acceptable recombinant proteins. "Animal-free" culture media refers to culture media whose compositions are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free culture media, and nutrients are obtained from synthetic, plant, or microbial sources. In contrast, "protein-free" culture media are defined as being substantially protein-free.

[0289] Illustrative examples of serum-free culture media used in certain embodiments include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0290] In a preferred embodiment, the composition comprising the CAR T cells contemplated herein is formulated in a solution comprising PlasmaLyte A.

[0291] In another preferred embodiment, the composition comprising the CAR T cells contemplated herein is formulated in a solution comprising a cryopreservation medium. For example, a cryopreservation medium with a cryopreservation agent can be used to maintain high cell viability after thawing. Illustrative examples of cryopreservation medium used in specific embodiments include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0292] In a more preferred embodiment, the composition comprising the CAR T cells contemplated herein is formulated in a solution comprising 50:50 PlasmaLyte A:CryoStor CS10.

[0293] F. Treatment Methods

[0294] The modified T cells produced by the methods contemplated herein provide improved adoptive immunotherapy for the treatment of various conditions, including but not limited to cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immunodeficiency. In a specific embodiment, the specificity of primary T cells is redirected to tumor or cancer cells by genetically modifying primary T cells with the CARs contemplated herein.

[0295] In certain embodiments, the CAR T cell compositions produced using the methods contemplated herein are used to treat solid tumors or cancers, including but not limited to liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.

[0296] In certain embodiments, the CAR T cell compositions made using the methods contemplated herein are used to treat liquid tumors, including leukemias, including acute leukemias (e.g., ALL, AML and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemias (e.g., CLL, SLL, CML, HCL), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.

[0297] In certain embodiments, the CAR T cell compositions produced using the methods contemplated herein are used to treat B-cell malignancies, including but not limited to multiple myeloma (MM), non-Hodgkin lymphoma (NHL), and chronic lymphocytic leukemia (CLL).

[0298] Multiple myeloma is a B cell malignancy of mature plasma cell morphology, characterized in that the monoclonal tumorigenic transformation of these types of cells. These plasma cells propagate in the bone marrow (BM) and can invade adjacent bone, sometimes invading the blood. Variant forms of multiple myeloma include significant multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma and extramedullary plasmacytoma (see, for example, Braunwald et al. (eds), Harrison's Principles of Internal Medicine, 15th edition (McGraw-Hill 2001)).

[0299] Non-Hodgkin's lymphoma encompasses a large class of lymphocyte (white blood cell) cancers. Non-Hodgkin's lymphoma can occur at any age and is usually characterized by larger-than-normal lymph nodes, fever, and weight loss. There are many different types of non-Hodgkin's lymphoma. For example, non-Hodgkin's lymphoma can be divided into aggressive (rapid growth) and indolent (slow growth) types. Although non-Hodgkin's lymphoma can be derived from B cells and T cells, as used herein, the terms "non-Hodgkin's lymphoma" and "B cell non-Hodgkin's lymphoma" are used interchangeably. B cell non-Hodgkin's lymphoma (NHL) includes Burkitt's lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, and mantle cell lymphoma. Lymphomas that develop after a bone marrow or stem cell transplant are usually B-cell non-Hodgkin lymphomas.

[0300] Chronic lymphocytic leukemia (CLL) is an indolent (slow-growing) cancer that causes a slow increase in immature white blood cells called B lymphocytes, or B cells. The cancer cells spread through the blood and bone marrow and can also affect lymph nodes or other organs, such as the liver and spleen. CLL eventually leads to bone marrow failure. Sometimes, in the later stages of the disease, the disease is called small lymphocytic lymphoma.

[0301] In certain embodiments, a composition comprising a therapeutically effective amount of anti-BCMA CAR T cells is administered to a subject to treat multiple myeloma or lymphoma.

[0302] In a specific embodiment, a composition comprising a therapeutically effective amount of anti-BCMA CAR T cells is administered to a subject to treat relapsed / refractory multiple myeloma. "Relapse" refers to the reappearance or recurrence of signs and symptoms of cancer diagnosed after a period of improvement or remission. "Refractory" refers to the resistance or non-response of cancer to therapy with a specific therapeutic agent. Cancer can be refractory at the beginning of treatment (i.e., unresponsive to initial exposure to the therapeutic agent) or refractory due to resistance to the therapeutic agent during the first treatment period or during subsequent treatment periods.

[0303] In certain embodiments, the compositions contemplated herein are administered to a subject with relapsed / refractory multiple myeloma that has not been successfully treated with one, two, three, or more treatments including at least one proteasome inhibitor and / or immunomodulatory drug (IMiD). In one embodiment, the subject's multiple myeloma is refractory to three treatment regimens including at least one proteasome inhibitor and an IMiD. In one embodiment, the subject's multiple myeloma is double refractory to one or more treatment regimens.

[0304] Illustrative examples of proteasome inhibitors for a subject's multiple myeloma that is refractory include, but are not limited to, bortezomib and carfilzomib.

[0305] Illustrative examples of IMiDs that are refractory to a subject's multiple myeloma include, but are not limited to, thalidomide, lenalidomide, and pomalidomide.

[0306] Illustrative examples of other treatments for refractory multiple myeloma include, but are not limited to, dexamethasone, and antibody-based therapies selected from the group consisting of elotuzumab, daratumumab, MOR03087, isatuximab, bevacizumab, cetuximab, siltuximab, tocilizumab, elsilimomab, azintrol, and seleximab. el), rituximab, tositumomab, milatuzumab, lucatumumab, dacetuzumab, figitumumab, dalotuzumab, AVE1642, tabalumab, pembrolizumab, pidilizumab, and nivolumab.

[0307] In one embodiment, the subject's multiple myeloma is refractory to daratumumab treatment.

[0308] In certain embodiments, the subject's multiple myeloma is refractory to treatment with an IMiD, a proteasome inhibitor, and dexamethasone.

[0309] The methods contemplated herein may also include treating the subject with relapsed / refractory multiple myeloma with an autologous hematopoietic stem cell transplant prior to administering the anti-BCMA CAR T cell composition.

[0310] The method contemplated herein may also include performing lymphocyte clearance on the subject before administering the anti-BCMA CAR T cell composition contemplated herein, such as lymphocyte clearance chemotherapy 1-4 days (e.g., 1, 2, 3 or 4 days) before administration. In a specific embodiment, lymphocyte clearance includes administering one or more of melphalan, cytoxan, cyclophosphamide and fludarabine. In one embodiment, prior to administering the anti-BCMA CAR T cell composition contemplated herein, lymphocyte clearance is performed on the subject with 300 mg / m2 of cyclophosphamide and 30 mg / m2 of fludarabine.

[0311] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0312] Although the foregoing invention has been described in considerable detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art, based on the teachings of the present invention, that variations and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided for illustration only and are not intended to be limiting. Those skilled in the art will readily recognize a variety of non-critical parameters that may be changed or modified to produce substantially similar results.

[0313] Example

[0314] Example 1

[0315] Improved manufacturing process

[0316] Cells are harvested from multiple myeloma donors by leukocyte separation, and PBMC is separated using density gradients on Cell Saver Elite. PBMC is washed and then resuspended in T cell growth medium (TCGM) with 250IU IU / mL IL-2. Cell count, viability and PBMC FACS analysis are performed before and after washing. Washed PBMCs are stored at low temperatures until activated or freshly used. At day 0, PBMCs are activated and stimulated by culturing TCGM with 250IU / mL IL-2, 1 μM ZSTK474 (CAS NO.475110-96-4), 50ng / mL anti-CD3 antibodies and 50ng / mL anti-CD28 antibodies for culture and cultured for about 18 hours to 24 hours. PBMC cultures are transduced with lentivirus encoding anti-BCMA CAR (e.g., SEQ ID NO: 1, SEQ ID NO: 2) for about 18 hours to about 24 hours. Then PBMC culture is cultivated in the TCGM containing 250IU / mL IL-2 and 1 μM ZSTK474 and carries out T cell expansion, continues 4 days, 6 days or 9 days (being respectively 5 days, 7 days, 10 days manufacturing process).In each day of one day or more day expansion, cell aliquot is optionally taken and cell is counted, viability is measured, cryopreserved and characterizes PBMC using FACS analysis.The cell of expansion is recovered and washed, and cryopreserved at a temperature of at least -80 DEG C in the freezing box of controlled rate, and is stored in the gas phase of liquid nitrogen storage tank.

[0317] Example 2

[0318] Improved manufacturing process modulates T cell phenotype

[0319] Five multiple myeloma donor PBMC cell batches were used to manufacture anti-BMCA CAR T cells using the 7-day or 10-day manufacturing process described in Example 1 in the presence or absence of the PI3K inhibitor ZSTK474. At the end of the T cell expansion culture, the cells were stained with anti-human antibodies against CD3, CD62L, CCR7, and CD45RA and analyzed by flow cytometry. Each dot plot was gated on live CD3+ lymphocytes. Compared to anti-BCMA CAR T cell DPs manufactured for 10 days in the presence of ZSTK474 or in the absence of a PI3K inhibitor, the anti-BCMA CAR T cell drug product (DP) manufactured for 7 days in the presence of ZSTK474 had increased expression of markers for a stronger T cell phenotype. Figure 1 .

[0320] Example 3

[0321] Improved manufacturing process regulates T cell differentiation

[0322] Five batches of multiple myeloma donor PBMC cells were used to manufacture anti-BMCA CAR T cells in the presence of the PI3K inhibitor ZSTK474 using either a 7-day or 10-day manufacturing process as described in Example 1. At the end of T cell expansion culture, cells were stained with metal-labeled anti-human antibodies against CCR7, CD25, CD28, CD122, ICOS, CD45RO, CD57, and TIM3 and analyzed by CyTOF. Each dot plot was gated on live CD3+ lymphocytes. Compared to anti-BCMA CAR T cell DPs manufactured for 10 days in the presence of ZSTK474, anti-BCMA CAR T cell DPs manufactured for 7 days in the presence of ZSTK474 showed increased expression of markers of the poorly differentiated T cell phenotype, while expression of markers of the well-differentiated T cell phenotype was decreased. Figure 2 .

[0323] Example 4

[0324] Improved manufacturing process to enrich CD27 + T cells

[0325] Five batches of multiple myeloma donor PBMC cells were used to manufacture anti-BMCA CAR T cells in the presence or absence of the PI3K inhibitor ZSTK474 using the 7-day or 10-day manufacturing process described in Example 1. At the end of the T cell expansion culture, the cells were stained with metal-labeled anti-human antibodies against CD4, CD8, and CD27 and analyzed by CyTOF. VISNE images show CD27 expression in different cell populations. The gated population represents CD27. + Compared with anti-BCMA CAR T cell DPs produced for 10 days in the presence of ZSTK474 or in the absence of PI3K inhibitors, anti-BCMA CAR T cell DPs produced for 7 days in the presence of ZSTK474 had unexpected and significant CD27 + 、LEF1 + and / or TCF1 + Enrichment of T cells increased. Figure 3 .

[0326] Example 5

[0327] Enriched CD27 + T cell populations have a potent T cell phenotype

[0328] Five batches of multiple myeloma donor PBMC cells were used to manufacture anti-BMCA CAR T cells in the presence of the PI3K inhibitor ZSTK474 using the 7-day or 10-day manufacturing process described in Example 1. At the end of the T cell expansion culture, the cells were incubated with antibodies against CCR7, CD25, CD28, HLA-DR, and TIM3 ( Figure 4A ) and CD45RO, CD57, CD70, CD244 and PD-1 ( Figure 4B ) were stained with metal-labeled anti-human antibodies and analyzed by CyTOF. VISNE diagrams show the marker expression in different cell populations. The gated group represents CD27 + T cell enrichment. Compared to anti-BCMA CART cell DP produced in the presence of ZSTK474 for 10 days, anti-BCMA CAR T cell DP produced in the presence of ZSTK474 for 7 days showed increased expression of markers of poorly differentiated T cell phenotypes and decreased expression of markers of well-differentiated T cell phenotypes. Figure 4A-4B .

[0329] Example 6

[0330] Improved manufacturing process to regulate CD27 + T cell activation characteristics

[0331] Five batches of multiple myeloma donor PBMC cells were used to manufacture anti-BMCA CAR T cells in the presence of the PI3K inhibitor ZSTK474 using either the 7-day or 10-day manufacturing process described in Example 1. At the end of the T cell expansion culture, the cells were stained with metal-labeled anti-human antibodies against CD27, CD28, ICOS, HLA-DR, CD25, and TIM3 and analyzed by CyTOF. CD27 identified by VISNE was analyzed. + T cell phenotype of enriched cells to obtain CD4+ T cells ( Figure 5 , top) and CD8 + T cells ( Figure 5 , bottom). Anti-BCMA CAR T cell DPs manufactured in the presence of ZSTK474 for 10 days had reduced activation properties and increased exhaustion properties compared to anti-BCMA CAR T cell DPs manufactured in the presence of ZSTK474 for 7 days.

[0332] Example 7

[0333] Improved manufacturing process modulates T cell gene expression

[0334] Anti-BCMA CAR T cells were produced as described in Example 1 using multiple myeloma PBMC batches in the absence of the PI3K inhibitor ZSTK474 for 7 days (n=1) or 10 days (n=13) or in the presence of the PI3K inhibitor for 7 days (n=10) or 10 days (n=6). Approximately 100 ng of total RNA was extracted from the anti-BCMA CAR T cell DP and mixed with the ImmunoV2 probe kit from Nanostring and analyzed for transcriptional properties. Figure 6 A heat map of the top 50 differentially expressed genes between manufacturing conditions is shown in Figure 1. Compared to DP manufactured for 10 days, DP of anti-BCMA CAR T cells manufactured for 7 days generally showed increased expression of T cell memory phenotype genes and genes associated with T cell activation and proliferation, and decreased expression of genes associated with cell death.

[0335] Example 8

[0336] D AUDI Anti-BCMA CAR T cells in tumor mouse models

[0337] A Daudi tumor mouse model was established to compare the efficacy of drug products manufactured using a 7-day and 10-day process. Healthy donor PBMCs were activated and stimulated, transduced with a lentiviral vector encoding an anti-BCMA CAR, and expanded for 7 or 10 days in the presence of IL-2 and a PI3K inhibitor (see Example 1). Ten days before adoptive cell therapy, NSG mice were intravenously injected with 2×10 6 Firefly luciferase-labeled Daudi tumor cells were injected into mice with 2.5, 5, or 10 × 10 6 Anti-BCMACAR + Anti-BCMA CAR T cells manufactured using the 7-day process showed better efficacy compared to cells manufactured at 10 days, which was achieved at lower CAR + This is evidenced by the ability to control tumor growth at higher doses. Figure 7 .

[0338] Example 9

[0339] Anti-BCMA CAR T cell phenotype

[0340] Fifteen multiple myeloma donor PBMC cell batches were used to manufacture anti-BMCA CAR T cells using the 7-day or 10-day manufacturing process described in Example 1 in the presence of the PI3K inhibitor ZSTK474. At the end of the T cell expansion culture, the cells were stained with a panel of ~36 T cell phenotyping metal-labeled anti-human antibodies and analyzed using CyTOF. Phenotyping antibodies enable the distinction of the following T cell phenotypes: naive T cells (T naive), central memory T cells (TCM), effector memory T cells (EM), effector T cells (TEff), and stem cell memory T cells (TSCM). The naive T cell quadrant (CCR7 + CD45RO - ) to identify T stem cell memory subsets. The data presented show that according to CD27 + The % of enriched cells relative to T cell subsets analyzed for each DP batch. + CD4 + T cells are positively correlated with TCM-like phenotype, while CD27 + CD8 + T cells were positively correlated with TSCM-like phenotype. Figure 8 .

[0341] Example 10

[0342] CD8 + Anti-BCMA CAR T cell phenotype

[0343] Analysis of CD8 produced in Example 9 using FlowSOM + T cell data. FlowSOM identified 20 different T cell clusters. Three major categories of T cells were identified based on cluster 4 (enriched in memory T cell markers such as CD27, CD25, CD127, TCF1, LEF1, CD28, CCR7) and cluster 5 (enriched in effector T cell markers such as granzyme A, granzyme B, perforin, T-Bet, EOMES). Analysis of %CD27 + CD8 + Anti-BCMA CAR T cells, methods of manufacturing, and clinical responses in subjects treated with anti-BCMA CAR T cells. The 7-day manufacturing process generally resulted in anti-BCMA CAR T cells with increased expression of T cell memory markers and increased CD27 compared to the 10-day manufacturing process. + Enriched cell population. Figure 9 .

[0344] Example 11

[0345] Anti-BCMA CAR T cell gene expression analysis

[0346] Twelve multiple myeloma donor PBMC cell batches were used to manufacture anti-BMCA CAR T cells using the 7-day (n=8) or 10-day (n=4) manufacturing process described in Example 1 in the presence of the PI3K inhibitor ZSTK474. Approximately 100 ng of total RNA was extracted from the anti-BCMA CAR T cell DP and mixed with the ImmunoV2 probe kit from Nanostring. The data were QC'd in NSolver software (Nanostring) and differential gene expression analysis was performed. A heat map of the top 25 differentially expressed genes between the 7-day and 10-day manufacturing processes was generated (p value 0.05). Analysis of %CD27 + Anti-BCMA CAR T cells, manufacturing methods, and clinical responses in subjects treated with anti-BCMA CAR T cells. The 7-day manufacturing process generally resulted in anti-BCMA CAR T cells with increased expression of T cell memory markers and increased CD27 compared to the 10-day manufacturing process. + Enriched cell population. Figure 10 .

[0347] Example 12

[0348] Anti-BCMA CAR T cell gene expression analysis

[0349] Five batches of multiple myeloma donor PBMC cells were each divided into two groups, one for manufacturing anti-BMCA CAR T cells using a 7-day manufacturing process and the other for manufacturing anti-BMCA CAR T cells using a 10-day manufacturing process. CAR T cells were manufactured in the presence of the PI3K inhibitor ZSTK4 as described in Example 1.

[0350] Approximately 100 ng of total RNA was extracted from anti-BCMA CAR T cell DP and mixed with the ImmunoV2 probe kit from Nanostring. The data were QC'd using NSolver software (Nanostring) and differential gene expression analysis was performed.

[0351] Aliquots of anti-BCMA CAR T cell DP total RNA were also used for RNA sequencing (RNA-Seq). Cells were thawed / washed / counted and tested for viability (>70% viability was required). TRIAZOL was used to extract the total RNA from 2-3×10 6 Total RNA from each cell was collected. For total RNA, phenol / chloroform extraction and the Qiagen miRNA-easy kit were used to harvest RNA. RNA was isolated using a poly A bead capture strategy. RNA quality / quantity was determined using a Tapestation 2200 (RIN value > 7 was required). Sequencing libraries were prepared using an Illumina TruSeq RNA analyzer. Libraries were quality checked using a Tapestation 2200 (DNA kit) and sequenced using a NextSeq 550 instrument. Data were analyzed using QC / alignment methods.

[0352] Table 1 shows the top 11 up-regulated genes and the top 9 down-regulated genes by fold change (FC) relative to the 7-day manufacturing process.

[0353]

[0354] Example 13

[0355] Anti-BCMA CAR T cell gene expression analysis

[0356] Five batches of multiple myeloma donor PBMC cells were each divided into two groups, one for manufacturing anti-BMCA CAR T cells using a 7-day manufacturing process and the other for manufacturing anti-BMCA CAR T cells using a 10-day manufacturing process. CAR T cells were manufactured in the presence of the PI3K inhibitor ZSTK4 as described in Example 1.

[0357] RNA sequencing (RNA-Seq) was performed using an aliquot of total RNA from anti-BCMA CAR T cell DPs. Cells were thawed / washed / counted and tested for viability (>70% viability was required). TRIAZOL was used to extract the total RNA from 2-3 × 10 6 Total RNA from each cell was collected. For total RNA, phenol / chloroform extraction and the Qiagen miRNA-easy kit were used to harvest RNA. rRNA was depleted using RiboErase. RNA quality / quantity was determined using a Tapestation 2200 (RIN value > 7 was required). RNA quality / quantity was determined using a Tapestation 2200 (RIN value > 7 was required). Sequencing libraries were prepared using Illumina TruSeq RNA. Libraries were quality checked using a Tapestation 2200 (DNA kit) and sequenced using a NextSeq 550 instrument. Data were analyzed using QC / alignment methods.

[0358] Relative to the 7-day manufacturing process, CCL1, NR4A2, ATF3, CCL5, and WNT5B were among the top 25 upregulated genes, and NKD2 and NQO1 were among the top 10 downregulated genes by fold change (FC).

[0359] Example 14

[0360] Anti-BCMA CAR T cell therapy

[0361] Harvest PBMC from multiple myeloma patients, wash and be resuspended in T cell growth medium (TCGM) with 250IU IU / mL IL-2.Carry out cell counting, viability and PBMC flow cytometric analysis before and after washing.Washed PBMC is cryopreserved until activation or fresh use.At the 0th day, by culturing PBMC in TCGM with 250IU / mL IL-2, 50ng / mL anti-CD3 antibody and 50ng / mL anti-CD28 antibody to activate and stimulate T cells and cultivate about 18 hours-24 hours.With coding anti-BCMA CAR (for example, SEQ ID NO:1, SEQ ID NO:2) lentiviral transduction PBMC culture about 18 hours to about 24 hours.Then PBMC culture is cultivated in TCGM containing 250IU / mL IL-2 and carries out T cell expansion, continues 9 days (10 days manufacturing process). The expanded cells were recovered, washed and cryopreserved at a temperature of at least -80°C in a controlled rate freezer and subsequently stored in the vapor phase of a liquid nitrogen storage tank.

[0362] The frozen cells were then thawed / washed / counted and tested for viability (>70% viability was required). The cells were then used for CyTOF experiments or frozen, when cell pellets were preserved in TRIzol, for subsequent RNA extraction and gene expression analysis.

[0363] Experiment 1. Cells were stained with metal-labeled anti-human antibodies against T cell markers and analyzed using a Fluidigm CyTOF Helios mass spectrometer. Protein marker expression was gated based on a single marker compared to a negative population established in a reference sample spiked into each sample prior to antibody staining. Cells were classified as memory cell types using a combination of markers, and positive marker expression was gated by shadowing. Memory populations of CD4 and CD8 T cells were gated using the following marker combinations, respectively: T 原初 (CCR7+CD45RO-CD95-), T SCM (CCR7+CD45RO-CD95+), T CM (CCR7+CD45RO+CD95+), T EM (CCR7-CD45RO+CD95+), T EF (CCR7-CD45RO-CD95+). The following marker combinations were used to gate the major immune populations: CD4 T cells (CD3+CD4+CD8-CD14-CD19-CD56-), CD8 T cells (CD3+CD4-CD8+CD14-CD19-CD56-), NK cells (CD3-CD19-CD14-CD56+), NKT cells (CD3+CD56+CD19-CD14-), B cells (CD3-CD19+CD14-CD56-) and monocytes (CD3-CD19-CD14+CD56-). The differential abundance of cell proportions was inferred using a quasi-binomial generalized linear model adjusted for sex. The Wilcoxon rank sum test was used to infer the differences in the proportions of each marker in each cell type. The CAR T cell composition was compared between patients with a response duration of better than 18 months (persistent responders) compared to all patients with a response duration of less than 18 months (non-persistent responders). Figure 11A and Figure 11B .

[0364] Experiment 2. Cells were stained with metal-labeled anti-human antibodies for T cell markers (including LEF-1) and analyzed by CyTOF. CyTOF data were quality checked and analyzed to elicit the expression of each marker of CD4 and CD8 immune cell populations. The Wilcoxon rank sum test was used to infer the proportional differences of each marker in each cell type. Persistent responders were compared with non-persistent responders and males were compared with females, and differential expression analysis was used to analyze the gene level counts from drug product samples. Figure 12A .

[0365] For total RNA, phenol / chloroform extraction and Qiagen miRNA-easy kit were used to harvest RNA and rRNA was depleted using the Kapa RNA HyperPrep kit with RiboErase. RNA quality / quantity was determined using a Tapestation 2200 (RNA integrity index or RIN>7 was required). Sequencing libraries were prepared using the Illumina TruSeq RNA library preparation kit. Library quality and quantity were determined using a Tapestation 2200 (DNA kit) and sequenced using an Illumina NextSeq 550 instrument. Sequencing data were analyzed. Spearman rank correlation was used to determine the correlation between LEF1 gene expression and serum BCMA (sBCMA) levels. Figure 12B .

[0366] Example 15

[0367] Anti-BCMA CAR T cell therapy

[0368] PBMC from multiple myeloma patients are harvested, washed and resuspended in T cell growth medium (TCGM) with 250IU IU / mL IL-2. Cell count, viability and PBMC flow cytometry analysis are performed before and after washing. The washed PBMCs are cryopreserved until activated or freshly used. At day 0, PBMCs are activated and stimulated by culturing them in TCGM with 250IU / mL IL-2, 50ng / mL anti-CD3 antibodies, 50ng / mL anti-CD28 antibodies and cultured for about 18 hours to 24 hours in the presence of 1 μM ZSTK474 (PI3K inhibitor, CAS NO.475110-96-4). PBMC cultures are transduced for about 18 hours to about 24 hours with a lentiviral transduction encoding anti-BCMA CAR (e.g., SEQ ID NO: 1, SEQ ID NO: 2). The PBMC cultures were then cultured in TCGM containing 250 IU / mL of IL-2 and 1 μM ZSTK474 for T cell expansion for 9 days (10-day manufacturing process). The expanded cells were recovered, washed, and cryopreserved at a temperature of at least -80°C in a controlled rate freezer and subsequently stored in the vapor phase of a liquid nitrogen storage tank.

[0369] The cryopreserved samples were thawed and stained with metal-labeled anti-human antibodies for T cell markers including CD3, CD27, CCR7, and CD57. The labeled cells were analyzed using a Fluidigm CyTOF Helios mass spectrometer. Manual analysis of CyTOF phenotyping was performed using the FlowJo software package. Based on the negative population established in the reference sample spiked into each subject sample before antibody staining, the expression of protein markers was gated on the basis of a single marker. The expression of CCR7 ( Figure 13 , upper left), LEF1( Figure 13 , upper middle panel) and CD57 ( Figure 13 , upper right panel). This demonstrates that the PI3-K inhibitor-based manufacturing process enriches for early-stage memory, less differentiated cells.

[0370] The y-axis shows the expression of CCR7 ( Figure 13 , lower left), LEF-1( Figure 13 , lower middle panel) and CD57 ( Figure 13, lower right figure) of the percentage of CD3+ viable cells. The x-axis shows the maximum vector copy number (VCN) on CD3+ cells extracted from whole blood at different time points after infusion as determined by PCR. These figures show a positive correlation between the maximum expansion of anti-BCMA CAR+ cells after infusion and the percentage of CD3+ DP cells expressing LEF-1, and a negative correlation with the percentage of CD3+ DP expressing CD57. This indicates that CCR7 and LEF-2 enrichment in DP leads to a more robust expansion of anti-BCMA CAR in vivo.

[0371] The percentage of live CD3+ cells expressing CD57 (senescence marker), LEF-1, CCR7, and CD27 (memory cells) is shown as a cluster heat map. Figure 14 .Red indicates that for the marker, the proportion of cells in the sample is relatively higher than that in other samples. Blue indicates that for the marker, the proportion of cells in the sample is relatively lower than that in other samples. The data were grouped using average linkage hierarchical clustering, and as determined by the cluster tree diagram, the first three clusters were associated with the patient's clinical response at 6 months (whether the disease progressed). Only patients with available follow-up data were included in this analysis to clinically evaluate the response at 6 months. Unsupervised clustering showed that the high CD57 expression, low LEF-1 / CCR7 / CD27 expression group was associated with the progressors at 6 months (4 / 6 progressed), while the group with high LEF-1 / CCR7 / CD27 expression and low CD57 expression was mainly non-progressors (1 / 7 progressed). The intermediate group had 1 / 5 progressors. This demonstrates the correlation between memory markers and fluorescent markers in the drug product and sustained clinical responses.

[0372] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to encompass all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Therefore, the claims are not limited by this disclosure. Sequence Listing <110> Bluebird Bio, Inc. Kevin Friedman Eric Scott Alonzo <120> Production of anti-BCMA CAR T cells <130> BLUE-118.PC <150> US 62 / 830,004 <151> 2019-04-05 <150> US 62 / 944,485 <151> 2019 / 12 / 6 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 493 <212> PRT <213> Artificial sequence <220> <223> Laboratory-made - synthetic anti-BMCA CAR <400> 1 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu 20 25 30 Ala Met Ser Leu Gly Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu 35 40 45 Ser Val Thr Ile Leu Gly Ser His Leu Ile His Trp Tyr Gln Gln Lys 50 55 60 Pro Gly Gln Pro Pro Thr Leu Leu Ile Gln Leu Ala Ser Asn Val Gln 65 70 75 80 Thr Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe 85 90 95 Thr Leu Thr Ile Asp Pro Val Glu Glu Asp Asp Val Ala Val Tyr Tyr 100 105 110 Cys Leu Gln Ser Arg Thr Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly 130 135 140 Glu Gly Ser Thr Lys Gly Gln Ile Gln Leu Val Gln Ser Gly Pro Glu 145 150 155 160 Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly 165 170 175 Tyr Thr Phe Thr Asp Tyr Ser Ile Asn Trp Val Lys Arg Ala Pro Gly 180 185 190 Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro 195 200 205 Ala Tyr Ala Tyr Asp Phe Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr 210 215 220 Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Tyr Glu Asp 225 230 235 240 Thr Ala Thr Tyr Phe Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr 245 250 255 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala Thr Thr 260 265 270 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 275 280 285 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 290 295 300 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 305 310 315 320 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 325 330 335 Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 340 345 350 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 355 360 365 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 370 375 380 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 385 390 395 400 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 405 410 415 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 420 425 430 Light Aspen Pro Glu Gly Leu Tire Asn Glu Leu Gln Light Asp Light Met 435 440 445 White Glue White Tyre Gly Ile Gly Met Light Gly Gly Arg Arg Arg Gly 450 455 460 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 465 470 475 480 Thr Tyr Asp Ala Leu His Met Gln Free Mp3 Download Arg 485 490 <210> 2 <211> 1485 <212> DNA <213> The snowstorm <220> <223> Railroad Railroad - Remote BMCA CAR <400> 2 atggcactcc ccgtcaccgc ccttctcttg cccctcgccc tgctgctgca tgctgccagg cccgacattg tgctcactca gtcacctccc agcctggcca tgagcctggg aaaaaagggcc 120 accatctcct gtagagccag tgagtccgtc acaatcttgg ggagccatct tattcactgg tatcagcaga agccgggca gcctccaacc cttcttattc agctcgcgtc aaacgtccag 240 acgggtgtac ctgccagatt ttctggtagc gggtcccgca ctgattttac actgaccata gatccagtgg aagaagacga tgtggccgtg tattattgtc tgcagagcag aacgattcct 360 cgcacatttg gtgggggtac taagctggag attaagggaa gcacgtccgg ctcagggaag 420 ccgggctccg gcgagggaag cacgaagggg caaattcagc tggtccagag cggacctgag 480 ctgaaaaaac ccggcgagac tgttaagatc agttgtaaag catctggcta taccttcacc 540 gactacagca taaattgggt gaaacgggcc cctggaaagg gcctcaaatg gatgggttgg 600 atcaataccg aaactaggga gcctgcttat gcatatgact tccgcgggag attcgccttt 660 tcactcgaga catctgcctc tactgcttac ctccaaataa acaacctcaa gtatgaagat 720 acagccactt acttttgcgc cctcgactat agttacgcca tggactactg gggacaggga 780 acctccgtta ccgtcagttc cgcggccgca accacaacac ctgctccaag gccccccaca 840 cccgctccaa ctatagccag ccaaccattg agcctcagac ctgaagcttg caggcccgca 900 gcaggaggcg ccgtccatac gcgaggcctg gacttcgcgt gtgatattta tatttgggcc 960 cctttggccg gaacatgtgg ggtgttgctt ctctcccttg tgatcactct gtattgtaag 1020 cgcgggagaa agaagctcct gtacatcttc aagcagcctt ttatgcgacc tgtgcaaacc 1080 actcaggaag aagatgggtg ttcatgccgc ttccccgagg aggaagaagg agggtgtgaa 1140 ctgagggtga aattttctag aagcgccgat gctcccgcat atcagcaggg tcagaatcag 1200 ctctacaatg aattgaatct cggcaggcga gaagtacg atgttctgga caagacgg 1260 ggcagggatc ccgagatggg gggaaagccc cggagaaaaa atcctcagga ggggttgtac 1320 aatgagctgc agaaggacaa gatggctgaa gcctatagcg agatcggaat gaaaggcgaa 1380 agacgcagag gcaaggggca tgacggtctg taccagggtc tctctacagc caccaaggac 1440 acttatgatg cgttgcatat gcaagccttg ccaccccgct aatga 1485

Claims

1. A method for producing anti-BCMACAR T cells, comprising: (a) activating a T cell population and stimulating the proliferation of the T cell population; (b) transducing the T cells with a lentiviral vector encoding an anti-BCMACAR comprising the amino acid sequence set forth in SEQ ID NO: 1; (c) culturing the transduced T cells to proliferate for a period of 5 to 7 days; wherein steps (a)-(c) are performed in the presence of a PI3K inhibitor, wherein the PI3K inhibitor is ZSTK474, and wherein the gene expression of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A and CCL5 or (ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of CCL1, NR4A2, ATF3, CCL5 and WNT5B is at least 1.5-fold or at least two-fold higher in the T cells cultured in step (c) than in the T cells correspondingly transduced and cultured for proliferation for a period of 10 days in step (b).

2. A method for producing anti-BCMACAR T cells, comprising: (a) activating a T cell population and stimulating the proliferation of the T cell population; (b) transducing the T cells with a lentiviral vector encoding an anti-BCMACAR comprising the amino acid sequence set forth in SEQ ID NO: 1; (c) culturing the transduced T cells to proliferate for a period of 5 to 7 days; wherein steps (a)-(c) are performed in the presence of a PI3K inhibitor, wherein the PI3K inhibitor is ZSTK474, and wherein the gene expression of (i) 1, 2, 3, 4, 5, 6, 7, 8, 9 or all of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2 and WDR62 or (ii) NKD2 and NQO1 in the T cells cultured in step (c) is at least 1.5-fold or at least two-fold lower than in the T cells correspondingly transduced and cultured for proliferation for a period of 10 days in step (b).

3. A method for producing anti-BCMACAR T cells, comprising: (a) activating a T cell population and stimulating the proliferation of the T cell population; (b) transducing the T cells with a lentiviral vector encoding an anti-BCMACAR comprising the amino acid sequence set forth in SEQ ID NO: 1; (c) culturing the transduced T cells to proliferate for a period of 5 to 7 days; wherein steps (a)-(c) are performed in the presence of a PI3K inhibitor, wherein the PI3K inhibitor is ZSTK474, and wherein in the T cells cultured in step (c), compared to the T cells correspondingly transduced and cultured for proliferation in step (b), the expression of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A and CCL5, or (ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of the genes of CCL1, NR4A2, ATF3, CCL5 and WNT5B is at least 1.5-fold or at least two-fold higher, and the expression of 1, 2, 3, 4, 5, 6, 7, 8, 9 or all of the genes of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2 and WDR62, or (ii) NKD2 and NQO1 is at least 1.5-fold or at least two-fold lower.

4. A method for producing anti-BCMACAR T cells, comprising: (a) activating a T cell population and stimulating the proliferation of the T cell population; (b) transducing the T cells with a lentiviral vector encoding an anti-BCMACAR comprising the amino acid sequence set forth in SEQ ID NO: 1; (c) culturing the transduced T cells to proliferate for a period of 5 to 7 days; wherein steps (a)-(c) are performed in the presence of a PI3K inhibitor, wherein the PI3K inhibitor is ZSTK474, and wherein the proliferating cells are CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + .

5. The method of any one of claims 1 to 3, wherein the anti-BCMACAR T cells comprise at least 10% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

6. The method of any one of claims 1 to 3, wherein the anti-BCMACAR T cells comprise at least 15% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

7. The method of any one of claims 1 to 3, wherein the anti-BCMACAR T cells comprise at least 20% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

8. The method of any one of claims 1 to 3, wherein the anti-BCMACAR T cells comprise at least 25% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

9. The method of any one of claims 1 to 3, wherein the anti-BCMACAR T cells comprise at least 30% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

10. The method of any one of claims 1 to 3, wherein the CD27 + LEF1 + and / or CCR7 + and / or TCF1 + .

11. The method of any one of claims 1 to 3, wherein the CD27 + LEF1 + and / or CCR7 + and TCF1 + .

12. The method of any one of claims 1 to 3, wherein the CD27 + Anti-BCMA CAR T cells contain CD4 + Anti-BCMACAR T cells.

13. The method of any one of claims 1 to 3, wherein the CD27 + Anti-BCMA CAR T cells contain CD8 + Anti-BCMACAR T cells.

14. The method of any one of claims 1 to 3, wherein the CD27 + Anti-BCMA CAR T cells contain CD4 + and CD8 + Anti-BCMACAR T cells.

15. The method of any one of claims 1 to 4, wherein the T cells are autologous.

16. The method of any one of claims 1 to 4, wherein the method further comprises isolating peripheral blood mononuclear cells (PBMCs) as a source of the T cells.

17. The method of claim 16, wherein the PBMCs are isolated from a subject suffering from multiple myeloma or lymphoma.

18. The method of claim 17, wherein the subject has relapsed / refractory multiple myeloma.

19. The method of any one of claims 1 to 4, wherein the method further comprises cryopreserving the PBMCs prior to step (a).

20. The method of any one of claims 1 to 4, wherein the T cells are cryopreserved after step (c).

21. The method of any one of claims 1 to 4, wherein the T cells are activated and stimulated to proliferate for 18 to 24 hours.

22. The method of any one of claims 1 to 4, wherein activation of the T cells comprises contacting the T cells with an anti-CD3 antibody or antigen-binding fragment thereof.

23. The method of claim 22, wherein the anti-CD3 antibody or antigen-binding fragment thereof is soluble.

24. The method of claim 23, wherein the anti-CD3 antibody or antigen-binding fragment thereof is bound to a surface.

25. The method of claim 24, wherein the surface is a bead, optionally a paramagnetic bead.

26. The method of any one of claims 1 to 4, wherein stimulation of the T cells comprises contacting the T cells with an anti-CD28 antibody or antigen-binding fragment thereof.

27. The method of claim 26, wherein the anti-CD28 antibody or antigen-binding fragment thereof is soluble.

28. The method of claim 26, wherein the anti-CD28 antibody or antigen-binding fragment thereof is bound to a surface.

29. The method of claim 28, wherein the surface is a bead, optionally a paramagnetic bead, optionally the paramagnetic bead is bound to the anti-CD3 antibody or antigen-binding fragment thereof.

30. The method of any one of claims 1 to 4, wherein the cells are transduced with an HIV-1 derived lentiviral vector.

31. The method of any one of claims 1 to 4, wherein the anti-BCMACAR is encoded by the polynucleotide sequence set forth in SEQ ID NO:

2.

32. A method for increasing CD4 + TCM-like anti-BCMACAR T cells and CD8 + A method for TSCM-like anti-BCMACAR T cells, comprising contacting anti-BCMACAR T cells with a PI3K inhibitor ex vivo for 5 to 7 days, wherein the PI3K inhibitor is ZSTK474, and wherein CD4 T cells are increased in the anti-BCMACAR T cells compared to the anti-BCMACAR T cells contacted with the PI3K inhibitor ex vivo for 10 days. + TCM-like anti-BCMACAR T cells and CD8 + The number of TSCM-like anti-BCMACAR T cells was at least two-fold higher.

33. The method of claim 32, wherein the anti-BCMACAR T cells comprise at least 10% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

34. The method of claim 32, wherein the anti-BCMACAR T cells comprise at least 15% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

35. The method of claim 32, wherein the anti-BCMACAR T cells comprise at least 20% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

36. The method of claim 32, wherein the anti-BCMACAR T cells comprise at least 25% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

37. The method of claim 32, wherein the anti-BCMACAR T cells comprise at least 30% CD27 + and / or LEF1 + and / or CCR7 + and / or TCF1 + T cells.

38. The method of any one of claims 32 to 37, wherein the T cells are autologous.

39. The method of any one of claims 32 to 37, wherein the method further comprises isolating peripheral blood mononuclear cells (PBMCs) as a source of the T cells.

40. The method of claim 39, wherein the PBMCs are isolated from a subject with multiple myeloma or lymphoma.

41. The method of claim 40, wherein the subject has relapsed / refractory multiple myeloma.

42. The method of any one of claims 32 to 37, wherein the anti-BCMACAR T cells comprise an HIV-1 derived lentiviral vector.

43. The method of any one of claims 32 to 37, wherein the anti-BCMACAR comprises the amino acid sequence set forth in SEQ ID NO:

1.

44. The method of any one of claims 32 to 37, wherein the anti-BCMACAR is encoded by the polynucleotide sequence set forth in SEQ ID NO:

2.

45. A method for increasing gene expression of each of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A, and CCL5, or (ii) CCL1, NR4A2, ATF3, CCL5, and WNT5B in anti-BCMACAR T cells, comprising contacting the anti-BCMACAR T cells with a PI3K inhibitor ex vivo for 5 to 7 days, wherein the PI3K inhibitor is ZSTK474, and wherein the anti-BCMACAR T cells are more efficacious than those in the anti-BCMACAR T cells contacted with the PI3K inhibitor ex vivo for 10 days. In T cells, gene expression of each of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A, and CCL5, or (ii) CCL1, NR4A2, ATF3, CCL5, and WNT5B is at least 1.5-fold higher.

46. ​​A method for reducing gene expression of each of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2 and WDR62, or (ii) NKD2 and NQO1 in anti-BCMACAR T cells, comprising contacting anti-BCMACAR T cells with a PI3K inhibitor ex vivo for 5 to 7 days, wherein the PI3K inhibitor is ZSTK474, and wherein gene expression of each of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2 and WDR62, or (ii) NKD2 and NQO1 in the anti-BCMACAR T cells is at least 1.5-fold lower than in anti-BCMACAR T cells contacted with the PI3K inhibitor ex vivo for 10 days.

47. A method for increasing gene expression of each of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A, and CCL5, or (ii) CCL1, NR4A2, ATF3, CCL5, and WNT5B, and decreasing gene expression of each of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2, and WDR62, or (ii) NKD2 and NQO1 in anti-BCMACAR T cells, comprising contacting the anti-BCMACAR T cells with a PI3K inhibitor ex vivo for 5 to 7 days, wherein the PI3K inhibitor is ZSTK474, and wherein the anti-BCMACAR T cells are more effectively expressed than those in the anti-BCMACAR T cells contacted with the PI3K inhibitor ex vivo for 10 days. In T cells, the gene expression of each of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A, and CCL5 or (ii) CCL1, NR4A2, ATF3, CCL5, and WNT5B is at least 1.5-fold higher and the gene expression of each of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2, and WDR62 or (ii) NKD2 and NQO1 is at least 1.5-fold lower.

48. Use of anti-BCMACAR T cells in the preparation of a medicament for treating multiple myeloma, wherein the anti-BCMACAR T cells are prepared by a method of contacting anti-BCMACAR T cells with a PI3K inhibitor ex vivo for 5 to 7 days, wherein the PI3K inhibitor is ZSTK474, and wherein in the anti-BCMACAR T cells and in the anti-BCMACAR T cells contacted with the PI3K inhibitor ex vivo for 10 days, the gene expression of each of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A and CCL5 or (ii) CCL1, NR4A2, ATF3, CCL5 and WNT5B is increased, and at least 1.5-fold higher indicates increased therapeutic efficacy.

49. Use of anti-BCMACAR T cells in the preparation of a medicament for treating multiple myeloma, wherein the anti-BCMACAR T cells are prepared by a method of contacting the anti-BCMACAR T cells with a PI3K inhibitor ex vivo for 5 to 7 days, wherein the PI3K inhibitor is ZSTK474, and wherein the gene expression of each of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR 22, ILDR2, ATAD3, NKD2 and WDR62 or (ii) NKD2 and NQO1 is reduced in the anti-BCMACAR T cells and in the anti-BCMACAR T cells contacted with the PI3K inhibitor ex vivo for 10 days, and at least 1.5-fold reduction indicates increased therapeutic efficacy.

50. Use of anti-BCMACAR T cells in the preparation of a medicament for treating multiple myeloma, wherein the anti-BCMACAR T cells are prepared by contacting the anti-BCMACAR T cells with a PI3K inhibitor ex vivo for 5 to 7 days, wherein the PI3K inhibitor is ZSTK474, and wherein the anti-BCMACAR T cells are contacted with the PI3K inhibitor ex vivo for 10 days. An increase of at least 1.5-fold in gene expression of each of (i) NR4A2, LY9, LIN7A, WNT5B, BCL6, EGR1, EGR2, ATF3, CCL1, IL-1A, and CCL5, or (ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of CCL1, NR4A2, ATF3, CCL5, and WNT5B, and a decrease of at least 1.5-fold in gene expression of each of (i) NQO1, CCNA1, IL17F, EMP1, SNHG19, PRR22, ILDR2, ATAD3, NKD2, and WDR62, or (ii) NKD2 and NQO1, as compared to T cells, indicates increased therapeutic efficacy.

51. The use of any one of claims 48 to 50, wherein the multiple myeloma is relapsed / refractory multiple myeloma.

52. The method of any one of claims 45 to 47 or the use of any one of claims 48 to 50, wherein the anti-BCMACAR T cells are from a subject with multiple myeloma.

53. The method or use of claim 52, wherein the anti-BCMACAR T cells are from a subject with relapsed / refractory multiple myeloma.

54. The method or use of claim 53, wherein the anti-BCMA CAR T cell comprises an HIV-1-derived lentiviral vector comprising a polynucleotide encoding the anti-BCMA CAR.

55. The method of any one of claims 45 to 47 or the use of any one of claims 48 to 50, wherein the anti-BCMACAR comprises the amino acid sequence set forth in SEQ ID NO:

1.

56. The method of any one of claims 45 to 47 or the use of any one of claims 48 to 50, wherein the anti-BCMACAR is encoded by the polynucleotide sequence set forth in SEQ ID NO:

2.

57. The method of any one of claims 45 to 47 or the use of any one of claims 48 to 50, wherein the anti-BCMACAR T cells are autologous.

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