Methods for improving the efficacy and growth of chimeric antigen receptor expressing cells

Abstract

To provide methods to improve the efficiency of T cell harvest and the ability to grow larger amounts of immune effector cells to generate clinical grade products.SOLUTION: The invention provides immune effector cells (e.g., T cells, NK cells) that can be engineered to express a chimeric antigen receptor (CAR), compositions and reaction mixtures comprising the same, and methods of treatment using the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Ser. No. 62 / 149,249, filed April 17, 2015, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on April 14, 2016, is named N2067-7094WO_SL.txt and is 720,042 bytes in size.

[0003] The present invention relates generally to immune effector cells (e.g., T cells, NK cells) engineered to express chimeric antigen receptors (CARs), compositions comprising same, and methods of making and using same. [Background technology]

[0004] Adoptive cell transfer (ACT) therapy using autologous T cells, particularly chimeric antigen receptor (CAR)-transduced T cells, has shown promise in clinical trials for several hematological cancers. Despite these encouraging results, many patients undergoing evaluation for enrollment ultimately fail to proceed due, at least in part, to insufficient yields of clinically available products. This limitation in clinically available products may result from multiple factors, including the inability to harvest adequate and sufficient lymphocytes from patients, limitations on harvested cells, and limited ex vivo cell expansion. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a need to improve the efficiency of T cell harvesting and the ability to expand larger numbers of immune effector cells to generate clinical-grade products. [Means for solving the problem]

[0006] The present disclosure relates, at least in part, to methods for improving the efficacy and / or expansion (e.g., ex vivo expansion) of immune effector cells (e.g., T cells, NK cells) for use in cell therapy. In some embodiments, applicants have discovered significant advantages of obtaining, e.g., harvesting, immune cells from a subject, e.g., a cancer patient, as early as possible after diagnosis (e.g., before treatment or early during the course of treatment). Some embodiments described herein provide criteria for evaluating and / or optimizing the suitability of immune cells for use in cell therapy, e.g., chimeric antigen receptor (CAR) therapy. Without wishing to be bound by theory, immune cells harvested as early as possible after diagnosis or treatment with a selected chemotherapy are believed to contain less differentiated immune effector cell populations (e.g., naive T cells and early memory T cells). Less differentiated immune effector cell populations are believed to be highly proliferative, self-renewing, and / or viable, and therefore have greater proliferative potential and / or greater anti-tumor efficacy.

[0007] Thus, in some embodiments, the methods disclosed herein advantageously improve the suitability (e.g., suitability for use in cell therapy) and efficacy of T cells by obtaining the cells from a subject early after cancer diagnosis, e.g., prior to chemotherapy or prior to certain / multiple chemotherapy cycles. In some embodiments, the methods disclosed herein utilize cells obtained from a patient prior to a chemotherapy cycle involving a T cell depleting agent (e.g., obtained prior to two, three, four, or five chemotherapy cycles), or prior to a late intensification or consolidation cycle. In other embodiments, the methods optimize the proliferative capacity and / or anti-tumor effect of immune cells by, for example, including patient selection based on malignancy type (e.g., leukemia versus lymphoma) or disease severity (e.g., standard risk, high risk, or very high risk). In still other embodiments, the methods optimize the proliferative capacity and / or anti-tumor effect of immune cells by, for example, including selection of cell populations with high absolute T cell numbers or a high proportion of poorly differentiated T cells. Thus, the methods disclosed herein can be used to optimize one or more of the compositions and methods for cell therapy and / or methods for generating immune effector cell populations (e.g., CAR-expressing cells).

[0008] Accordingly, in one aspect, the invention features a method for assessing the suitability of an immune cell (e.g., an immune effector cell or population thereof) for use in cell therapy, e.g., CAR therapy (e.g., cancer therapy). The method includes: (i) The timing of obtaining (e.g., harvesting) immune cells from a subject (e.g., a cancer patient); (ii) timing of treatment, e.g., chemotherapy, for immune cell acquisition (e.g., harvesting); (iii) treatment, e.g., type of chemotherapy; (iv) primary malignant tumor; (v) immune cell (e.g., T cell) parameters, e.g., one or more of T cell number, T cell phenotype, or T cell function obtaining values ​​for one, two, three, four, or more (all) of (i) through (v), or a combination of (i) through (v); The value of any of (i)-(v) indicates the suitability of the immune cells for use in cell therapy, e.g., CAR therapy. In one embodiment, the value of any of (i)-(v) indicates one or more of the ability of the immune cells to expand (e.g., ex vivo expansion), the efficacy of the immune cells for therapy, or the yield of the immune cells.

[0009] In another aspect, the invention features a method of treating a subject with cancer or providing anti-tumor immunity to the subject. The method includes administering to the subject immune cells (e.g., immune effector cells or a population thereof) capable of expressing a CAR molecule ("CAR-expressing cells" or "CAR therapy") in combination with a therapy, e.g., chemotherapy. In certain embodiments, the immune cells (e.g., immune effector cells or a population thereof) are (i) The timing of obtaining (e.g., harvesting) immune cells from a subject (e.g., a cancer patient); (ii) timing of treatment, e.g., chemotherapy, for immune cell acquisition (e.g., harvesting); (iii) treatment, e.g., type of chemotherapy; (iv) primary malignant tumor; (v) immune cell (e.g., T cell) parameters, e.g., one or more of T cell number, T cell phenotype, or T cell function or obtained (e.g., obtained or harvested) by optimizing one, two, three, four, or more (all) of (i) through (v), or a combination of (i) through (v).

[0010] In one embodiment, the immune cells are obtained before the introduction of the CAR molecule. In one embodiment, the obtained immune cells are expanded before the introduction of the CAR molecule. In other embodiments, the immune cells are obtained after the introduction of the CAR molecule. In one embodiment, the obtained immune cells are expanded after the introduction of the CAR molecule. The immune cells can be expanded and / or activated according to any of the methods described herein.

[0011] In some embodiments, the immune cells exhibit increased one or more of the expansion (e.g., ex vivo expansion) of the immune cell population, the efficacy of the immune cell population for therapy, or the yield of the immune cell population when any of (i)-(v) is optimized.

[0012] In some embodiments, the method includes introducing into the obtained immune cells (e.g., cell population) a nucleic acid encoding a CAR, e.g., a CAR molecule described herein, e.g., a CD19 CAR described herein (e.g., CTL019).

[0013] In yet another aspect, the invention features a method of enriching for or generating an immune cell population (e.g., an immune effector cell population) suitable for use in CAR therapy (e.g., cells expressing a CAR molecule). The method comprises: (i) The timing of obtaining (e.g., harvesting) immune cells from a subject (e.g., a cancer patient); (ii) timing of treatment, e.g., chemotherapy, for immune cell acquisition (e.g., harvesting); (iii) treatment, e.g., type of chemotherapy; (iv) primary malignant tumor; (v) immune cell (e.g., T cell) parameters, e.g., one or more of T cell number, T cell phenotype, or T cell function The method includes obtaining (e.g., harvesting) the immune cell population according to one, two, three, four, or more (all) of (i) through (v), or a combination of (i) through (v).

[0014] In one embodiment, the immune cell population is obtained before the introduction of the CAR molecule. In one embodiment, the obtained immune cells are expanded before the introduction of the CAR molecule. In other embodiments, the immune cells are obtained after the introduction of the CAR molecule. In one embodiment, the obtained immune cells are expanded after the introduction of the CAR molecule. The immune cells can be expanded and / or activated according to any of the methods described herein.

[0015] In some embodiments, the immune cell population exhibits an increase in one or more of the expansion (e.g., ex vivo expansion) of the immune cell population, the efficacy of the immune cell population for a therapy, or the yield of the immune cell population when any of (i)-(v) is optimized.

[0016] In some embodiments, the method further includes introducing into the immune cells (e.g., the cell population) a nucleic acid encoding a CAR, e.g., a CAR molecule described herein, e.g., a CD19 CAR described herein (e.g., CTL019).

[0017] In another aspect, the invention features an immune cell preparation or reaction mixture comprising an immune effector cell population (e.g., comprising a CAR molecule or a nucleic acid encoding a CAR molecule), e.g., produced by a method described herein.

[0018] Further features or embodiments of any of the foregoing methods, preparations, and reaction mixtures include one or more of the following:

[0019] subject In one embodiment, the subject, for example, the subject that immune cell is obtained from and / or the subject that receives treatment is human, for example, cancer patient.In certain embodiments, the subject is 18 years old or younger (for example, 18 years old, 17 years old, 16 years old, 15 years old, 14 years old, 13 years old, 12 years old, 11 years old, 10 years old, 9 years old, 8 years old, 7 years old, 6 years old, 5 years old, 4 years old, 3 years old, 2 years old, 1 year old or younger (for example, 12 months old, 6 months old, 3 months old or younger)).In one embodiment, the subject is a pediatric cancer patient.

[0020] In other embodiments, the subject is an adult, e.g., the subject is over 18 years of age (e.g., over 18, over 19, over 20, over 25, over 30, over 35, over 40, over 45, over 50, over 55, over 60, over 65, over 70, over 75, over 80, or older). In one embodiment, the subject is an adult cancer patient.

[0021] In certain embodiments, the subject has a disease associated with expression of a tumor-associated antigen or a cancer-associated antigen, e.g., a disease described herein. In one embodiment, the subject has cancer, e.g., a cancer described herein.

[0022] In one embodiment, the subject has a cancer selected from a hematological cancer, a solid tumor, or a metastatic lesion thereof. Exemplary cancers include B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell promyelocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or These include, but are not limited to, large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma (HL), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenstrom's macroglobulinemia. In one embodiment, the cancer is ALL. In another embodiment, the cancer is CLL.

[0023] In some embodiments, the subject has leukemia, for example, ALL. In some embodiments, the subject has leukemia, for example, ALL, and is a pediatric patient, for example, 18 years old or younger (e.g., 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or younger (e.g., 12 months, 6 months, 3 months, or younger)).

[0024] In some embodiments, the subject has ALL, for example, is classified as having standard risk, high risk, or very high risk ALL. In some embodiments, the subject has ALL, for example, is classified as having standard risk, high risk, or very high risk ALL, and is a pediatric subject, for example, 18 years old or younger. In one embodiment, the subject is classified as having standard risk ALL. In some embodiments, the subject is classified as having standard risk ALL and is a pediatric subject, for example, 18 years old or younger. In another embodiment, the subject is not classified as having high risk or very high risk ALL. In some embodiments, the subject is not classified as having high risk or very high risk ALL and is a pediatric subject, for example, 18 years old or younger.

[0025] In some embodiments, the subject does not have lymphoma, e.g., the subject does not have non-Hodgkin's lymphoma (NHL). In some embodiments, the subject is a pediatric subject, e.g., 18 years of age or younger and does not have NHL.

[0026] In other embodiments, the subject has lymphoma, e.g., NHL. In some embodiments, the subject is a pediatric subject, e.g., 18 years of age or younger, and has NHL.

[0027] In some embodiments, the subject does not have a recurrent cancer. In some embodiments, the subject is a pediatric subject, e.g., 18 years of age or younger, and does not have a recurrent cancer. In other embodiments, the subject has a recurrent cancer. In some embodiments, the subject is a pediatric subject, e.g., 18 years of age or younger, and has a recurrent cancer.

[0028] In one embodiment, the immune cells (e.g., immune effector cell population) are obtained, e.g., obtained, from a subject with a hematological cancer, e.g., a leukemia, e.g., CLL, ALL, or a lymphoma, e.g., MCL, NHL, or HL.

[0029] Timing of harvest / chemotherapy administration In certain embodiments, early timing of immune cell acquisition, e.g., harvesting (e.g., after diagnosis), indicates increased suitability of the immune cells for use in cell therapy, e.g., CAR therapy. In some embodiments, early timing of immune cell harvesting increases one or more of the following capabilities: immune cell expansion (e.g., ex vivo expansion), immune cell suitability for therapy, or immune cell production yield.

[0030] In one embodiment, harvesting immune cells before administering a therapy, e.g., chemotherapy, to a subject results in an increased suitability of the immune cells for use in cell therapy, e.g., CAR therapy. In other embodiments, harvesting immune cells early in the course of a therapy, e.g., chemotherapy, to a subject (e.g., before the subject has undergone two, three, four, or five chemotherapy cycles) results in an increased suitability of the immune cells for use in cell therapy, e.g., CAR therapy.

[0031] In some embodiments of the method disclosed herein, immune cell collection occurs before administering therapy, for example, chemotherapy, to the subject.In some embodiments, collection occurs at an early stage during the course of therapy, for example, chemotherapy, to the subject.In some embodiments, immune cell collection occurs before the subject undergoes two, three, four, or five chemotherapy cycles.In some embodiments, immune cell collection occurs after the subject undergoes one chemotherapy cycle, but before the subject undergoes more than one chemotherapy cycle (for example, more than one, more than two, more than three, more than four, or more than five cycles).

[0032] In certain embodiments, the chemotherapy or chemotherapy cycle comprises one or more of induction, consolidation, interim maintenance, late intensification, or maintenance cycles. In one embodiment, the immune effector cells are harvested from the subject before the subject undergoes a consolidation chemotherapy cycle or an induction chemotherapy cycle. In some embodiments, the cells are harvested before the subject undergoes a consolidation chemotherapy cycle. In some embodiments, the subject is classified as having high-risk or very high-risk cancer (e.g., ALL), and the cells are harvested before the subject undergoes a consolidation cycle. In another embodiment, the immune effector cells are harvested from the subject before the subject undergoes a late intensification cycle. In some embodiments, the subject is classified as having standard-risk cancer (e.g., ALL), and the cells are harvested before the subject undergoes a late intensification cycle. In some embodiments, the chemotherapy cycle is selected from Table 8.

[0033] In some embodiments, the chemotherapy comprises a drug selected from Table 8. In some embodiments, the chemotherapy comprises the drugs and dosing regimens set forth in Table 8. In some embodiments, the chemotherapy may include vincristine, dexamethasone, PEG-L-asparaginase, daunorubicin, 6-mercaptopurine, cyclophosphamide, cytarabine, methotrexate, doxorubicin, 6-thioguanine, and / or prednisone.

[0034] In some embodiments, the immune effector cells are harvested from the subject before the subject receives cyclophosphamide and / or cytarabine.

[0035] Immune effector cell parameters In some embodiments, the harvested immune effector cells comprise a greater number of less differentiated T cells, e.g., a greater number of one or more of naive T cells, stem central memory T cells, and / or central memory T cells, e.g., compared to a reference value (e.g., a sample from the subject at a later time point or after exposure to additional rounds of chemotherapy). In some embodiments, the harvested immune effector cells comprise at least 20% naive T cells, at least 2% stem central memory T cells, and / or at least 4% central memory T cells.

[0036] In other embodiments, an increase in absolute T cell count (ATC) indicates increased suitability of the immune cells for use in cell therapy, e.g., CAR therapy. In one embodiment, the harvested immune effector cells comprise an absolute T cell count of at least 400 per microliter, an absolute naive T cell count of at least 200 per microliter, an absolute stem central memory T cell count of at least 20 per microliter, and / or an absolute central memory T cell count of at least 40 per microliter.

[0037] In some embodiments, the immune effector cell population is selected based on expression of one or more markers, e.g., CCR7, CD62L, CD45RO, and CD95, e.g., the immune effector cell (e.g., T cell) population is CCR7+ and CD62L+.

[0038] In some embodiments, naive T cells are identified based on an expression pattern of CCR7+, CD62L+, CD45RO-, CD95-, stem central memory T cells are identified based on an expression pattern of CCR7+, CD62L+, CD45RO-, CD95+, and central memory T cells are identified based on an expression pattern of CCR7+, CD62L+, CD45RO+, CD95+.

[0039] CAR molecule In accordance with the methods, preparations, and reaction mixtures described herein, immune effector cells obtained, for example, by the methods described herein, can be engineered to contain CAR molecules (also referred to herein as "CARs") that target one or more cancer-associated antigens. In some embodiments, the cancer-associated antigen (tumor antigen) is selected from one or more of the following: CD19; CD123; CD22; CD30; CD171; CS-1 (CD2 subset 1, also referred to as CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor family member B-cell maturation (BCMA); Tn antigen [(Tn Ag) or (GalNAcα-Ser / Thr)]; prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (Tes tisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP);Insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta type 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high-molecular-weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor β data; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); protein G-linked receptor class C group 5, member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); protein G-linked receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation-mutant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer-testis antigen-1 (MAD-CT-1); melanoma cancer-testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53);p53 mutant; prostein; survival; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin-8), melanoma antigen 1 recognized by T cells (MelanA or MART1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; inhibitor of apoptosis in melanoma (ML-IAP); ERG [transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene]; N-acetylglucosaminyl-transferase V (NA17); association box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc chicken myeloblastoma viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS or sibling of regulator of imprinted sites); squamous cell carcinoma antigen 3 recognized by T cells (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal eccentric 1 (RU1); renal eccentric 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mutated) hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment receptor for IgA (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like molecule-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican 3 (GPC3); Fc receptor-like 5 (FCRL5);and immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0040] In one embodiment, the cancer-associated antigen targeted by a CAR molecule, e.g., a CD19 CAR described herein (e.g., CTL019), is CD19. In one embodiment, the CD19 CAR comprises the amino acids set forth in Table 4 or has the nucleotide sequence set forth in Table 4.

[0041] In one embodiment, the cancer-associated antigen targeted by a CAR molecule, e.g., an anti-BCMA CAR described herein, is BCMA. In one embodiment, the anti-BCMA CAR comprises the amino acid sequence shown in Table 11 or has the nucleotide sequence shown in Table 11.

[0042] In some embodiments, the cancer-associated antigen targeted by a CAR molecule, e.g., an anti-EGFRvIII CAR described herein, is EGFRvIII, e.g., as described in US2014 / 0322275A1, which is incorporated herein by reference. In some embodiments, the anti-EGFRvIII CAR comprises the amino acid sequence set forth in US2014 / 0322275A1, which is incorporated herein by reference, or has the nucleotide sequence set forth therein.

[0043] In some embodiments, the cancer-associated antigen targeted by a CAR molecule, e.g., an anti-CD123 CAR described herein, is CD123, e.g., as described in US2014 / 0322212A1 or US2016 / 0068601A1, both of which are incorporated herein by reference. In some embodiments, the anti-CD123 CAR comprises the amino acid sequence set forth in US2014 / 0322212A1 or US2016 / 0068601A1, both of which are incorporated herein by reference, or has the nucleotide sequence set forth therein.

[0044] In some embodiments, the cancer-associated antigen targeted by a CAR molecule, e.g., an anti-mesothelin CAR described herein, is mesothelin, e.g., as described in WO2015 / 090230, which is incorporated herein by reference. In some embodiments, the anti-mesothelin CAR comprises the amino acid sequence set forth in WO2015 / 090230, which is incorporated herein by reference, or has the nucleotide sequence set forth therein.

[0045] In some embodiments, the cancer-associated antigen targeted by the CAR molecule is mesothelin, e.g., an anti-CLL1 CAR described herein, e.g., in US2016 / 0051651A1, which is incorporated herein by reference. In some embodiments, the anti-CLL1 CAR comprises the amino acid sequence set forth in US2016 / 0051651A1, which is incorporated herein by reference, or has the nucleotide sequence set forth therein.

[0046] In some embodiments, the cancer-associated antigen targeted by the CAR molecule is mesothelin, e.g., an anti-CD33 CAR described herein, e.g., in US2016 / 0096892A1, which is incorporated herein by reference. In some embodiments, the anti-CD33 CAR comprises the amino acid sequence set forth in US2016 / 0096892A1, which is incorporated herein by reference, or has the nucleotide sequence set forth therein. In some embodiments, the antigen-binding domain of the CAR molecule comprises an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, single-domain antibody (SDAB), VH or VL domain, or camelid VHH domain.

[0047] In some embodiments, the transmembrane domain of the CAR molecule is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD 11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile ), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0048] In certain embodiments, the transmembrane domain of the CAR molecule comprises the amino acid sequence of a CD8 transmembrane domain having at least one, two, or three modifications but not more than 20, more than 10, or more than 5 modifications of the amino acid sequence of SEQ ID NO: 6, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 6. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO: 6.

[0049] In another embodiment, the nucleic acid sequence encoding the CD8 transmembrane domain comprises the sequence of SEQ ID NO: 17 or a sequence having 95-99% identity thereto.

[0050] In certain embodiments, the antigen-binding domain is connected to the transmembrane domain by a hinge region. In one embodiment, the hinge region comprises the amino acid sequence of a CD8 hinge, e.g., SEQ ID NO: 2, or the amino acid sequence of an IgG4 hinge, e.g., SEQ ID NO: 36, or a sequence having 95 to 99% identity to SEQ ID NO: 2 or 36. In other embodiments, the nucleic acid sequence encoding the hinge region comprises the sequence of SEQ ID NO: 13 or SEQ ID NO: 37, or a sequence having 95 to 99% identity to SEQ ID NO: 13 or 37, which correspond to a CD8 hinge or an IgG4 hinge, respectively.

[0051] In other embodiments, the CAR comprises an intracellular signaling domain, for example, a primary signaling domain and / or a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain.

[0052] In certain embodiments, the primary signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc epsilon R1b), CD79a, CD79b, Fc gamma RIIa, DAP10, and DAP12.

[0053] In one embodiment, the primary signaling domain of the CAR molecule comprises a functional signaling domain of CD3 zeta. The CD3 zeta primary signaling domain can comprise an amino acid sequence having at least one, two, or three modifications but not more than 20, more than 10, or more than 5 modifications of the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, the primary signaling domain comprises the sequence of SEQ ID NO:9 or SEQ ID NO:10. In other embodiments, the nucleic acid sequence encoding the primary signaling domain comprises the sequence of SEQ ID NO:20 or SEQ ID NO:21, or a sequence with 95-99% identity thereto.

[0054] In some embodiments, the intracellular signaling domain of the CAR molecule comprises a costimulatory signaling domain. For example, the intracellular signaling domain can comprise a primary signaling domain and a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD1 1b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, or NKG2D.

[0055] In certain embodiments, the costimulatory signaling domain of the CAR molecule comprises an amino acid sequence having at least one, two, or three modifications but not more than 20, more than 10, or more than 5 modifications of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 16, or a sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 16. In one embodiment, the costimulatory signaling domain comprises the sequence of SEQ ID NO: 7 or SEQ ID NO: 16. In other embodiments, the nucleic acid sequence encoding the costimulatory signaling domain comprises the sequence of SEQ ID NO: 18 or SEQ ID NO: 15, or a sequence with 95-99% identity thereto.

[0056] In other embodiments, the intracellular domain of the CAR molecule comprises the sequence of SEQ ID NO:9 or SEQ ID NO:10 and the sequence of SEQ ID NO:7 or SEQ ID NO:16, wherein the amino acid sequences comprising the intracellular signaling domain are expressed in the same frame as a single polypeptide chain.

[0057] In certain embodiments, the nucleic acid sequence encoding the intracellular signaling domain comprises the sequence of SEQ ID NO: 18 or SEQ ID NO: 15, or a sequence having 95-99% identity thereto, and the sequence of SEQ ID NO: 20 or SEQ ID NO: 21, or a sequence having 95-99% identity thereto.

[0058] In some embodiments, the CAR further comprises a leader sequence. In one embodiment, the leader sequence comprises the sequence of SEQ ID NO:1.

[0059] In certain embodiments, the antigen binding domain of the CAR molecule comprises 10 -4 M~10 -8 It has a binding affinity KD of M.

[0060] In one embodiment, the antigen binding domain of the CAR molecule is an antigen binding domain described herein, for example, an antigen binding domain described herein for the targets described above.

[0061] In some embodiments, the CAR comprises a CD19 CAR, e.g., a CD19 CAR described herein. In some embodiments, the CD19 CAR comprises an antigen-binding domain described herein, e.g., in Table 1 or 4.

[0062] Treatment methods / combination therapy According to the methods of treating a disorder (e.g., cancer) described herein and the methods of providing anti-tumor immunity described herein, in some embodiments, the method comprises administering to a subject a CAR molecule or an immune effector cell population produced by a method described herein. In some embodiments, the immune effector cell population is engineered to express a CAR molecule, e.g., a CAR described herein, e.g., a CD19 CAR described herein. In some embodiments, the method further comprises administering a therapy, e.g., a chemotherapy described herein, to the subject.

[0063] Also provided herein are compositions comprising immune effector cells (e.g., immune effector cell populations) comprising a CAR molecule (e.g., a CAR molecule described herein) for use in treating a subject having a disease associated with expression of a tumor antigen, e.g., a disorder described herein.

[0064] In some embodiments, the chemotherapy (e.g., chemotherapy cycle) and CAR therapy are administered concurrently. In other embodiments, the chemotherapy (e.g., chemotherapy cycle) and CAR therapy are administered sequentially, e.g., one after the other. In some embodiments, there is less than a week of overlap between the administration of the chemotherapy (e.g., chemotherapy cycle) and CAR therapy (e.g., less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day). In some embodiments, there is at least a week of overlap between the administration of the chemotherapy (e.g., chemotherapy cycle) and CAR therapy (e.g., at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more).

[0065] In some embodiments, CAR therapy is administered before chemotherapy, e.g., before a chemotherapy cycle. In some embodiments, CAR therapy is administered before the first chemotherapy cycle, the second chemotherapy cycle, the third chemotherapy cycle, the fourth chemotherapy cycle, or the fifth chemotherapy cycle. In some embodiments, CAR therapy is administered before a remission induction chemotherapy cycle, a consolidation chemotherapy cycle, an intermediate maintenance chemotherapy cycle, a late intensification chemotherapy cycle, or a maintenance chemotherapy cycle. In some embodiments, CAR therapy is administered before chemotherapy, e.g., a chemotherapy cycle comprising a drug described herein, e.g., a drug described in Table 8. In some embodiments, administration of CAR therapy begins before administration of chemotherapy, e.g., before a chemotherapy cycle, and then administration of CAR therapy and chemotherapy is continued in parallel for a period of time.

[0066] In some embodiments, CAR therapy is administered after chemotherapy, e.g., after a chemotherapy cycle. In some embodiments, CAR therapy is administered after the first chemotherapy cycle, the second chemotherapy cycle, the third chemotherapy cycle, the fourth chemotherapy cycle, or the fifth chemotherapy cycle. In some embodiments, CAR therapy is administered after a remission induction chemotherapy cycle, a consolidation chemotherapy cycle, an intermediate maintenance chemotherapy cycle, a late intensification chemotherapy cycle, or a maintenance chemotherapy cycle. In some embodiments, CAR therapy is administered after chemotherapy, e.g., a chemotherapy cycle comprising a drug described herein, e.g., a drug described in Table 8. In some embodiments, administration of CAR therapy begins after administration of chemotherapy, e.g., after a chemotherapy cycle, and then administration of CAR therapy and chemotherapy is continued in parallel for a period of time.

[0067] In one embodiment, the cancer is a hematological cancer, such as, for example, ALL or CLL. In one embodiment, the cancer is, for example, a hematological cancer described herein, for example, a leukemia (e.g., ALL or CLL) or a lymphoma (e.g., MCL, HL, or NHL).

[0068] In one embodiment, the disease associated with a tumor antigen, for example, a tumor antigen described herein, for example, CD19, is selected from a proliferative disease, for example, a cancer or malignant tumor, or a precancerous condition, for example, myelodysplasia, myelodysplastic syndrome, or preleukemia, or is a non-cancer-related indication associated with the expression of a tumor antigen described herein. In one embodiment, the disease is a cancer described herein, for example, a cancer described herein that is associated with a target described herein. In one embodiment, the blood cancer is leukemia. In one embodiment, the cancer is one or more acute leukemias, including but not limited to B-ALL, T-ALL, ALL; one or more chronic leukemias, including but not limited to chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL); B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MA and / or additional hematological cancers or conditions, including, but not limited to, LT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and / or "preleukemias" (a diverse collection of hematological conditions united by, e.g., ineffective production (or dysplasia) of bone marrow blood cells). In certain embodiments, diseases associated with expression of a tumor antigen described herein include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative disorders, and combinations thereof, that express a tumor antigen described herein.

[0069] In some embodiments, the disease associated with tumor antigen expression is selected from the group consisting of a proliferative disease, a precancerous condition, a cancer, and a non-cancer related indication associated with tumor antigen expression.

[0070] In another embodiment, the disease associated with the tumor antigens described herein is a solid tumor. In some embodiments, the cancer is colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, cancer of the endocrine system, thyroid cancer ... The cancer is selected from cancer of the bladder, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, combinations of the foregoing cancers, and metastatic lesions of the foregoing cancers.

[0071] In certain embodiments of any of the aforementioned methods or uses, the tumor antigen associated with the disease is CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1 (CLECL1), CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, FAP, IGF-I receptor, CAIX, LMP2, gp100, bcr-a bl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, legumain, HPVThe proteins are selected from one or more of E6, E7, intestinal carboxylelastase, hsp70-2 mutant, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.

[0072] In one embodiment, the cell population is autologous to the subject to whom the population is administered. In one embodiment, the cell population is allogeneic to the subject to whom the population is administered. In one embodiment, the subject is a human.

[0073] In one embodiment, a population of immune effector cells transduced with a nucleic acid encoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein, is expanded, e.g., by a method described herein. In one embodiment, the cells are expanded for 8 days or less, e.g., 7 days, 6 days, 5 days, 4 days, or 3 days. In one embodiment, the cells, e.g., the CD19 CAR cells described herein, are expanded in culture for 5 days, and the resulting cells are more potent than the same cells expanded in culture for 9 days under the same culture conditions, e.g., as described herein.

[0074] In one embodiment, the subject is administered 10 mg / kg of the subject's body weight. 4 ~10 6 In one embodiment, a subject receives an initial dose of the immune effector cell population (e.g., 10 per kg of the subject's body weight). 4 ~10 6 immune effector cells, e.g., 10 per kg of subject body weight 4 ~10 5 a first administration of immune effector cells, a plurality of which comprises a nucleic acid encoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein, and further receive one or more subsequent administrations of a population of immune effector cells (e.g., 10 per kg of the subject's body weight or more than 10 per kg of the subject's body weight). 4 ~10 6immune effector cells, e.g., 10 per kg of subject body weight 4 ~10 5 In one embodiment, the subject receives a total of about 10 immune effector cells per kg of the subject's body weight over the course of at least three administrations of the immune effector cell population, wherein the immune effector cells comprise a nucleic acid encoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein. In one embodiment, the one or more subsequent administrations are administered less than 15 days, e.g., less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, or less than 2 days after the previous administration. In one embodiment, the subject receives a total of about 10 immune effector cells per kg of the subject's body weight over the course of at least three administrations of the immune effector cell population. 6 immune effector cells, e.g., a subject receives an initial dose of 1 x 10 5 immune effector cells, 3 × 10 for the second round 5 administration of immune effector cells, and the third 6 × 10 5 administrations of immune effector cells, for example, each administration being administered less than 4 days, less than 3 days, less than 2 days after the previous administration.

[0075] In certain embodiments, the method or use is performed in combination with an agent that increases the effectiveness of immune effector cells, such as an agent described herein.

[0076] Further embodiments of the methods, preparations, and reaction mixtures According to the methods of treatment and / or methods of making preparations and reaction mixtures described herein, in some embodiments, the method further comprises removing regulatory T cells, e.g., CD25+ T cells, from the immune cell population, e.g., thereby obtaining a population of regulatory T cell-depleted cells, e.g., CD25+-depleted cells, suitable for expression of a CAR.

[0077] In one embodiment, the population of regulatory T cell depleted cells contains less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% CD25+ cells.

[0078] In one embodiment, the immune cell population comprises cells from a subject with cancer, e.g., a subject with a CD25-expressing cancer, e.g., chronic lymphocytic leukemia (CLL). In one embodiment, the regulatory T cell-depleted cell population contains less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% CD25+ cells and less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% tumor cells.

[0079] In one embodiment, the population of immune cells is autologous to the subject to whom the cells are administered therapeutically, hi one embodiment, the population of immune effector cells is allogeneic to the subject to whom the cells are administered therapeutically.

[0080] In one embodiment, regulatory T cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody or a fragment thereof, or a CD25-binding ligand, e.g., IL-2. In one embodiment, the anti-CD25 antibody or a fragment thereof, or the CD25-binding ligand, is conjugated to a substrate, e.g., a bead, or is otherwise coated on a substrate, e.g., a bead. In one embodiment, the anti-CD25 antibody or a fragment thereof is conjugated to a substrate described herein.

[0081] In one embodiment, regulatory T cells, e.g., CD25+ T cells, are removed from the population using CD25 depletion reagent from Militenyi™. In one embodiment, the ratio of cells to CD25 depletion reagent is 20 uL to 1e7 cells, or 15 uL to 1e7 cells, or 10 uL to 1e7 cells, or 5 uL to 1e7 cells, or 2.5 uL to 1e7 cells, or 1.25 uL to 1e7 cells.

[0082] In one embodiment, the population of regulatory T cell-depleted cells, e.g., CD25+ depleted cells, is suitable for expression of a CAR described herein, e.g., a CD19 CAR described herein. In one embodiment, the regulatory T cell-depleted cell population contains less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of leukemia cells, e.g., CLL cells, ALL cells, or lymphoma cells, e.g., MCL cells, NHL cells, or HL cells. In one embodiment, the immune effector cell population is obtained from a subject with CLL, and the population of regulatory T cell-depleted cells, e.g., CD25+ depleted cells, contains less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of leukemia cells, e.g., CLL cells, and is suitable for expression of a CD19 CAR described herein. In one embodiment, the regulatory T cell depleted cell population contains less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% CD25+ cells and less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% tumor cells, e.g., CD25-expressing tumor cells, e.g., CLL cells. In one embodiment, the regulatory T cell depleted cell population contains less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% CD25+ cells and less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% tumor cells, e.g., CD25-expressing tumor cells, e.g., CLL cells.

[0083] In some embodiments, the immune effector cells are harvested from the subject's blood, e.g., peripheral blood.

[0084] In one embodiment, immune effector cell population is T cell that is isolated from peripheral blood lymphocyte.In some embodiments, T cell population is obtained by lysing red blood cells and / or depleting monocytes.In some embodiments, T cell population is isolated from peripheral lymphocyte, for example, by using the method described herein.

[0085] In one embodiment, the immune effector cell population can be obtained from a blood sample from a subject, for example, by apheresis. In one embodiment, the cells collected by apheresis are washed to remove the plasma fraction, and optionally, the cells are placed in an appropriate buffer or medium for subsequent processing steps. In one embodiment, the cells are washed with a buffer, for example, phosphate-buffered saline (PBS). In some embodiments, the cells are washed in a washing solution lacking one or more divalent cations, such as calcium and magnesium. In one embodiment, the cells are washed in a buffer that is substantially free of divalent cations.

[0086] In some embodiments, immune effector cells are harvested from blood using density centrifugation.

[0087] In one embodiment, the method further comprises removing cells expressing a tumor antigen, e.g., a tumor antigen not containing CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14, or CD11b, from the population, thereby obtaining a population of regulatory T cell-depleted cells, e.g., CD25+ depleted cells, and tumor antigen-depleted cells suitable for expressing a CAR, e.g., a CAR described herein. In one embodiment, tumor antigen-expressing cells are removed simultaneously with regulatory T cells, e.g., CD25+ cells. For example, an anti-CD25 antibody or a fragment thereof and an anti-tumor antigen antibody or a fragment thereof can be attached to the same substrate, e.g., beads, and used to remove cells, or an anti-CD25 antibody or a fragment thereof, or an anti-tumor antigen antibody or a fragment thereof can be attached to separate beads, and a mixture thereof can be used to remove cells. In other embodiments, the removal of regulatory T cells, e.g., CD25+ cells, and the removal of tumor antigen-expressing cells are sequential, e.g., can occur in either order.

[0088] In one embodiment, the method further comprises removing cells expressing a checkpoint inhibitor, e.g., a checkpoint inhibitor described herein, e.g., one or more of PD1+ cells, LAG3+ cells, and TIM3+ cells, from the population, thereby obtaining a population of regulatory T cell-depleted cells, e.g., CD25+ depleted cells, and checkpoint inhibitor-depleted cells, e.g., PD1+, LAG3+, and / or TIM3+ depleted cells. In one embodiment, the checkpoint inhibitor-expressing cells are removed simultaneously with the regulatory T cells, e.g., CD25+ cells. For example, an anti-CD25 antibody or fragment thereof and an anti-checkpoint inhibitor antibody or fragment thereof can be attached to the same beads, which can be used to remove the cells, or an anti-CD25 antibody or fragment thereof and an anti-checkpoint inhibitor antibody or fragment thereof can be attached to separate beads, a mixture of which can be used to remove the cells. In other embodiments, the removal of regulatory T cells, e.g., CD25+ cells, and the removal of checkpoint inhibitor-expressing cells are sequential, e.g., can occur in either order.

[0089] In one embodiment, the cell population to be depleted is neither regulatory T cells nor tumor cells, but cells that would otherwise negatively affect the proliferation and / or function of CART cells, e.g., cells expressing CD14, CD11b, CD33, CD15, or potentially other markers expressed by immunosuppressive cells. In one embodiment, it is envisioned that such cells are depleted in parallel with, or after, the depletion of regulatory T cells and / or tumor cells, or in a different sequence.

[0090] In one embodiment, the method further comprises removing cells expressing CD14 from the population, thereby obtaining a population of regulatory T cell-depleted cells, e.g., CD25+ depleted cells, and CD14+ depleted cells. In one embodiment, CD14+ cells are removed simultaneously with regulatory T cells, e.g., CD25+ cells. For example, an anti-CD25 antibody or a fragment thereof and an anti-CD14 antibody or a fragment thereof can be attached to the same beads, which can be used to remove cells, or an anti-CD25 antibody or a fragment thereof, or an anti-CD14 antibody or a fragment thereof can be attached to separate beads, and a mixture thereof can be used to remove cells. In other embodiments, the removal of regulatory T cells, e.g., CD25+ cells, and the removal of CD14+ cells can be sequential, e.g., can occur in any order.

[0091] In one embodiment, the population of immune effector cells provided is selected based on expression of one or more markers, e.g., CD3, CD28, CD4, CD8, CD27, CD127, CD45RA, and CD45RO, e.g., the population of immune effector cells (e.g., T cells) provided is CD3+ and / or CD28+.

[0092] In one embodiment, the method further comprises obtaining a population of immune effector cells, e.g., T cells, enriched for expression of one or more markers, e.g., CD3, CD28, CD4, CD8, CD27, CD127, CD45RA, and CD45RO. In an embodiment, the immune effector cell population is enriched for CD3+ cells and / or CD28+ cells. For example, the isolated T cells are obtained by incubation with anti-CD3 / anti-CD28 conjugated beads. In one embodiment, the method further comprises selecting cells expressing one or more markers, e.g., CD3, CD28, CD4, CD8, CD45RA, and CD45RO, from a population of regulatory T cell-depleted cells, e.g., CD25+-depleted cells.

[0093] In one embodiment, the method further comprises activating the population of regulatory T cell depleted cells, eg, CD25+ depleted cells, eg, by a method described herein.

[0094] In some embodiments, the method further includes transducing cells from the immune effector cell population with a vector comprising a nucleic acid encoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein. In some embodiments, the method further includes expanding the immune effector cell population, e.g., engineered to express a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein, e.g., by a method described herein. In some embodiments, the cell population is expanded in the presence of a cytokine, e.g., IL-15 and / or IL-7 (e.g., IL-15 and IL-7).

[0095] In one embodiment, the method further comprises transducing cells from a regulatory T cell-depleted cell population, e.g., a CD25+ depleted cell population, with a vector comprising a nucleic acid encoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein. In one embodiment, the vector is selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector. In one embodiment, the cells from a regulatory T cell-depleted cell population, e.g., a CD25+ depleted cell population, have been transduced with the vector once within one day after the immune effector cell population has been obtained, e.g., obtained from a blood sample from a subject, e.g., by apheresis.

[0096] In one embodiment, the method further comprises generating a population of RNA-engineered cells that transiently express an exogenous RNA from a regulatory T cell-depleted cell population, e.g., a CD25+-depleted cell population. The method comprises introducing in vitro transcribed or synthetic RNA into cells from the population, wherein the RNA comprises a nucleic acid encoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein.

[0097] In one embodiment, cells transduced with a nucleic acid encoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein, are expanded, e.g., by a method described herein. In one embodiment, the cells are expanded in culture for a period of several hours (e.g., about 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 18 hours, 21 hours) to about 14 days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days). In one embodiment, the cells are expanded for a period of 4 to 9 days. In one embodiment, the cells are expanded for a period of 8 days or less, e.g., 7 days, 6 days, 5 days, 4 days, or 3 days. In one embodiment, cells, e.g., CD19 CAR cells described herein, are grown in culture for 5 days, and the resulting cells are more potent than the same cells grown in culture for 9 days under the same culture conditions. Potency can be defined, for example, by various T cell functions, such as proliferation, target cell killing, cytokine production, activation, migration, or a combination thereof. In one embodiment, cells grown for 5 days, e.g., CD19 CAR cells described herein, exhibit at least a 1-fold, 2-fold, 3-fold, or 4-fold increase in cell doubling upon antigen stimulation, compared to the same cells grown in culture for 9 days under the same culture conditions. In one embodiment, cells, e.g., cells expressing a CD19 CAR described herein, are grown in culture for 5 days, and the resulting cells exhibit higher pro-inflammatory cytokine production, e.g., IFN-γ and / or GM-CSF levels, compared to the same cells grown in culture for 9 days under the same culture conditions. In one embodiment, cells expanded for 5 days, e.g., CD19 CAR cells described herein, exhibit at least a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more increase in pro-inflammatory cytokine production, e.g., INF-γ and / or GM-CSF levels in pg / ml, compared to the same cells expanded in culture for 9 days under the same culture conditions.

[0098] In one embodiment, the cells are expanded by culturing the cells in the presence of an agent that stimulates a CD3 / TCR complex-associated signal, e.g., as described herein, and a ligand that stimulates a costimulatory molecule on the surface of the cells. In one embodiment, the agent is a bead conjugated with an anti-CD3 antibody or fragment thereof, and / or an anti-CD28 antibody or fragment thereof.

[0099] In one embodiment, the cells are grown in an appropriate medium (e.g., a medium described herein), which may contain one or more factors for proliferation and / or survival, as appropriate, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21, TGFβ, and TNF-α, or any other additives for cell growth.

[0100] In one embodiment, the cells are grown in an appropriate medium (e.g., a medium described herein) containing one or more interleukins, which results in at least a 200-fold (e.g., 200-fold, 250-fold, 300-fold, 350-fold) increase in cells over a 14-day growth period, as measured by a method described herein, such as, for example, flow cytometry. In one embodiment, the cells are grown in the presence of IL-15 and / or IL-7 (e.g., IL-15 and IL-7).

[0101] In one embodiment, the cells are cryopreserved after a suitable expansion period. In one embodiment, the cells are cryopreserved according to the methods described herein. In one embodiment, the expanded cells are cryopreserved in an appropriate medium, for example, an injectable medium, such as those described herein by way of example.

[0102] In one embodiment, the method further comprises contacting the immune effector cell population with a nucleic acid encoding a telomerase subunit, e.g., hTERT. In certain embodiments, the nucleic acid is DNA or RNA.

[0103] In one embodiment, the method further comprises removing regulatory T cells, e.g., CD25+ T cells, from the population prior to expansion, thereby obtaining a population of regulatory T cell-depleted cells, e.g., CD25+ depleted cells, to be expanded. In one embodiment, the regulatory T cells, e.g., CD25+ cells, are removed by a method described herein.

[0104] In one embodiment, the method further comprises removing regulatory T cells, e.g., CD14+ cells, from the population prior to expansion, thereby obtaining a population of CD14+-depleted cells to be expanded. In one embodiment, regulatory T cells, e.g., CD14+ cells, are removed by a method described herein.

[0105] In one embodiment, the method further comprises contacting the immune effector cell population with a nucleic acid encoding a telomerase subunit, e.g., hTERT. In certain embodiments, the nucleic acid is DNA or RNA.

[0106] In some embodiments, the method includes contacting a population of immune effector cells with a nucleic acid encoding a CAR and a nucleic acid encoding a telomerase subunit, e.g., hTERT, under conditions that allow expression of the CAR and telomerase.

[0107] In some embodiments, the nucleic acid encoding the telomerase subunit is RNA. In other embodiments, the nucleic acid encoding the telomerase subunit is DNA. In some embodiments, the nucleic acid encoding the telomerase subunit comprises a promoter capable of driving expression of the telomerase subunit.

[0108] In some embodiments, the method of production includes contacting a population of immune effector cells with a nucleic acid encoding a CAR and an RNA encoding a telomerase subunit, e.g., hTERT, under conditions that allow expression of the CAR and telomerase.

[0109] In one embodiment, the nucleic acid encoding the CAR and the RNA encoding the telomerase subunit are part of the same nucleic acid molecule. In one embodiment, the nucleic acid encoding the CAR and the RNA encoding the telomerase subunit are part of separate nucleic acid molecules.

[0110] In some embodiments, the method comprises contacting an immune effector cell population with a nucleic acid encoding a CAR and an RNA encoding a telomerase subunit substantially simultaneously. In some embodiments, the method comprises contacting an immune effector cell population with a nucleic acid encoding a CAR before contacting the immune effector cell population with the RNA encoding a telomerase subunit. In some embodiments, the method comprises contacting an immune effector cell population with a nucleic acid encoding a CAR after contacting the immune effector cell population with the RNA encoding a telomerase subunit.

[0111] In some embodiments, the RNA encoding the telomerase subunit is mRNA. In some embodiments, the RNA encoding the telomerase subunit comprises a poly(A) tail. In some embodiments, the RNA encoding the telomerase subunit comprises a 5' cap structure.

[0112] In some embodiments, the method comprises transfecting immune effector cells with RNA encoding a telomerase subunit. In some embodiments, the method comprises transducing immune effector cells with RNA encoding a telomerase subunit. In some embodiments, the method comprises electroporating immune effector cells with RNA encoding a telomerase subunit under conditions that allow expression of the CAR and telomerase.

[0113] In some embodiments, the method includes providing a population of immune effector cells (e.g., T cells or NK cells) that express a CAR and / or include a nucleic acid encoding a CAR, and contacting the immune effector cell population with a nucleic acid encoding a telomerase subunit, e.g., hTERT, under conditions that allow expression of hTERT.

[0114] In some embodiments, the method includes providing a population of immune effector cells (e.g., T cells or NK cells) that express a nucleic acid encoding a telomerase subunit, e.g., hTERT, and contacting the immune effector cell population with a nucleic acid encoding a CAR under conditions that allow expression of the CAR.

[0115] Immune effector cell preparations In some embodiments, an immune effector cell preparation (eg, a reaction mixture, or an immune effector cell population) described herein is produced by a method described herein.

[0116] In some embodiments, the immune effector cell preparation (e.g., reaction mixture) comprises: (i) an immune effector cell population comprising at least 20% naive T cells, at least 2% stem central memory T cells, and / or at least 4% central memory T cells; and / or (ii) an immune effector cell population comprising an absolute number of at least 400 T cells per microliter, an absolute number of at least 200 naive T cells per microliter, an absolute number of at least 20 stem central memory T cells per microliter, and / or an absolute number of at least 40 central memory T cells per microliter; is selected from.

[0117] In some embodiments, the immune effector cell population is selected based on expression of one or more markers, e.g., CCR7, CD62L, CD45RO, and CD95, e.g., the immune effector cell (e.g., T cell) population is CCR7+ and CD62L+.

[0118] In some embodiments, naive T cells are identified based on an expression pattern of CCR7+, CD62L+, CD45RO-, CD95-, stem central memory T cells are identified based on an expression pattern of CCR7+, CD62L+, CD45RO-, CD95+, and central memory T cells are identified based on an expression pattern of CCR7+, CD62L+, CD45RO+, CD95+.

[0119] In some embodiments, an immune effector cell preparation described herein comprises a nucleic acid encoding a CAR, e.g., a CAR described herein.

[0120] In some embodiments, the immune effector cell preparations described herein include a nucleic acid encoding an exogenous telomerase subunit, e.g., hTERT. In certain embodiments, the nucleic acid encoding the exogenous telomerase subunit is RNA, e.g., mRNA.

[0121] In some embodiments, the immune effector cell preparation described herein comprises a CAR, for example, a CAR described herein, and an exogenous telomerase subunit, for example, hTERT. In some embodiments, the cells do not contain DNA encoding the exogenous telomerase subunit. For example, the cells may be contacted with mRNA encoding the exogenous telomerase subunit.

[0122] In one embodiment, the immune effector cell preparation is a regulatory T cell-depleted cell population containing less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% CD25+ cells. In one embodiment, the immune effector cell preparation is a regulatory T cell-depleted cell population containing less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% CD25+ cells and less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% CD25-expressing tumor cells, e.g., CLL cells. In one embodiment, the immune effector cell preparation contains less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% CD25+ cells and less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% tumor cells, e.g., CD25-expressing tumor cells, e.g., CLL cells. In one embodiment, the immune effector cell preparation contains less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% CD25+ cells and less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% tumor cells, e.g., CD25-expressing tumor cells, e.g., CLL cells.

[0123] In one embodiment, the immune effector cell preparation is a regulatory T cell-depleted cell population containing less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% CD25+ cells and less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% checkpoint inhibitor-expressing cells, e.g., PD1+ cells, LAG3+ cells, or TIM3+ cells.

[0124] In one embodiment, the immune effector cell preparation is a regulatory T cell depleted cell population that contains less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% CD25+ cells and less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% CD14+ cells.

[0125] In some embodiments, an immune effector cell preparation described herein comprises an autologous immune effector cell population, e.g., a plurality of which have been transfected or transduced with a vector comprising a nucleic acid molecule encoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein, wherein the immune effector cell preparation contains less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% CD25+ cells and less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% tumor cells, e.g., CLL cells. In one embodiment, the immune effector cell preparation contains less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% CD25+ cells and less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% tumor cells, e.g., CD25-expressing tumor cells, e.g., CLL cells. In one embodiment, the immune effector cell preparation contains less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% CD25+ cells and less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% tumor cells, e.g., CD25-expressing tumor cells, e.g., CLL cells.

[0126] In one embodiment, the reaction mixture may further comprise an agent that activates and / or expands cells in the population, e.g., an agent that stimulates a CD3 / TCR complex-associated signal and / or a ligand that stimulates a costimulatory molecule on the surface of the cells, e.g., as described herein. In one embodiment, the agent is a bead conjugated to an anti-CD3 antibody or fragment thereof, and / or an anti-CD28 antibody or fragment thereof.

[0127] In some embodiments, the reaction mixture described herein comprises a regulatory T cell-depleted cell population containing less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% CD25+ cells. In one embodiment, the reaction mixture comprises a regulatory T cell-depleted cell population containing less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% CD25+ cells and less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% CD25-expressing tumor cells, e.g., CLL cells. In one embodiment, the cell population contains less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% CD25+ cells and less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% tumor cells, e.g., CD25-expressing tumor cells, e.g., CLL cells. In one embodiment, the cell population contains less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% CD25+ cells and less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% tumor cells, e.g., CD25-expressing tumor cells, e.g., CLL cells.

[0128] In one embodiment, the reaction mixture comprises a regulatory T cell-depleted cell population containing less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% CD25+ cells and less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% checkpoint inhibitor-expressing cells, e.g., PD1+ cells, LAG3+ cells, or TIM3+ cells. The reaction mixture may further comprise a buffer or other reagent, e.g., a PBS-containing solution.

[0129] In one embodiment, the reaction mixture comprises a regulatory T cell-depleted cell population containing less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% CD25+ cells and less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% CD14+ cells. The reaction mixture may further comprise a buffer or other reagent, such as a PBS-containing solution.

[0130] In one embodiment, the reaction mixture further comprises one or more factors for proliferation and / or survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21, TGFβ, and TNF-α, or any other additive for cell growth. In one embodiment, the reaction mixture further comprises IL-15 and / or IL-7.

[0131] In one embodiment, a plurality of cells in the population in the reaction mixture comprises a nucleic acid molecule, e.g., a nucleic acid molecule described herein, that comprises a CAR-encoding sequence, e.g., a CD19 CAR-encoding sequence, e.g., as described herein.

[0132] In one embodiment, the plurality of cells in the population in the reaction mixture comprises a vector comprising a nucleic acid sequence encoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein. In one embodiment, the vector is a vector described herein, e.g., a vector selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

[0133] In one embodiment, the reaction mixture further comprises a cryoprotectant or stabilizer, such as monosaccharides, oligosaccharides, polysaccharides, and polyols (e.g., trehalose, mannitol, sorbitol, lactose, sucrose, glucose, and dextran), salts, crown ethers, etc. In one embodiment, the cryoprotectant is dextran.

[0134] In some embodiments, the reaction mixture comprises an immune effector cell population, wherein a plurality of cells within the population in the reaction mixture comprises a nucleic acid molecule, e.g., a nucleic acid molecule described herein, comprising a CAR-encoding sequence, e.g., a CD19 CAR-encoding sequence, e.g., as described herein, and IL-7 and / or IL-15.

[0135] In one embodiment, the plurality of cells in the population in the reaction mixture comprises a vector comprising a nucleic acid sequence encoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein. In one embodiment, the vector is a vector described herein, e.g., a vector selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

[0136] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, and suitable methods and materials are described below.All publications, patent applications, patents, and other references described herein are incorporated by reference in their entirety.In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. [Brief explanation of the drawings]

[0137] [Figure 1A] Figures 1A–1D show the differential effects of γc cytokines and IL-18 on CAR-T cell accumulation. Figure 1A is a schematic diagram of the C4-27z CAR vector. Figure 1B is a graph showing the total accumulation of CAR-T cells in response to various cytokine exposures. T cells were transduced and exposed to various exogenous cytokines at a final concentration of 10 ng / mL starting the following day (day 0). CAR-T cell numbers were calculated based on T cell counts and the percentage of CAR expression. Curves are representative of six donors. *P<0.05, ***P<0.001. NC, no cytokine. Figure 1C is a histogram showing T cell proliferation in response to various cytokines. Seven days after lentiviral transduction, T cells in the NC group were labeled with CFSE (2.5 μM) and then exposed to various cytokines. After 7 days, T cells were analyzed for CFSE dilution by flow cytometry. Figure ID is a graph showing T cell viability 15 days after lentiviral transduction. T cells from various cytokine groups were stained with Annexin V and 7-AAD and then analyzed for the percentage of viable cells (negative for both Annexin V and 7-AAD). *P<0.05, **P<0.01 compared to the IL-2 group (n=6). [Figure 1B]Figures 1A–1D show the differential effects of γc cytokines and IL-18 on CAR-T cell accumulation. Figure 1A is a schematic diagram of the C4-27z CAR vector. Figure 1B is a graph showing the total accumulation of CAR-T cells in response to various cytokine exposures. T cells were transduced and exposed to various exogenous cytokines at a final concentration of 10 ng / mL starting the following day (day 0). CAR-T cell numbers were calculated based on T cell counts and the percentage of CAR expression. Curves are representative of six donors. *P<0.05, ***P<0.001. NC, no cytokine. Figure 1C is a histogram showing T cell proliferation in response to various cytokines. Seven days after lentiviral transduction, T cells in the NC group were labeled with CFSE (2.5 μM) and then exposed to various cytokines. After 7 days, T cells were analyzed for CFSE dilution by flow cytometry. Figure ID is a graph showing T cell viability 15 days after lentiviral transduction. T cells from various cytokine groups were stained with Annexin V and 7-AAD and then analyzed for the percentage of viable cells (negative for both Annexin V and 7-AAD). *P<0.05, **P<0.01 compared to the IL-2 group (n=6). [Figure 1C]Figures 1A–1D show the differential effects of γc cytokines and IL-18 on CAR-T cell accumulation. Figure 1A is a schematic diagram of the C4-27z CAR vector. Figure 1B is a graph showing the total accumulation of CAR-T cells in response to various cytokine exposures. T cells were transduced and exposed to various exogenous cytokines at a final concentration of 10 ng / mL starting the following day (day 0). CAR-T cell numbers were calculated based on T cell counts and the percentage of CAR expression. Curves are representative of six donors. *P<0.05, ***P<0.001. NC, no cytokine. Figure 1C is a histogram showing T cell proliferation in response to various cytokines. Seven days after lentiviral transduction, T cells in the NC group were labeled with CFSE (2.5 μM) and then exposed to various cytokines. After 7 days, T cells were analyzed for CFSE dilution by flow cytometry. Figure ID is a graph showing T cell viability 15 days after lentiviral transduction. T cells from various cytokine groups were stained with Annexin V and 7-AAD and then analyzed for the percentage of viable cells (negative for both Annexin V and 7-AAD). *P<0.05, **P<0.01 compared to the IL-2 group (n=6). [Figure 1D]Figures 1A–1D show the differential effects of γc cytokines and IL-18 on CAR-T cell accumulation. Figure 1A is a schematic diagram of the C4-27z CAR vector. Figure 1B is a graph showing the total accumulation of CAR-T cells in response to various cytokine exposures. T cells were transduced and exposed to various exogenous cytokines at a final concentration of 10 ng / mL starting the following day (day 0). CAR-T cell numbers were calculated based on T cell counts and the percentage of CAR expression. Curves are representative of six donors. *P<0.05, ***P<0.001. NC, no cytokine. Figure 1C is a histogram showing T cell proliferation in response to various cytokines. Seven days after lentiviral transduction, T cells in the NC group were labeled with CFSE (2.5 μM) and then exposed to various cytokines. After 7 days, T cells were analyzed for CFSE dilution by flow cytometry. Figure ID is a graph showing T cell viability 15 days after lentiviral transduction. T cells from various cytokine groups were stained with Annexin V and 7-AAD and then analyzed for the percentage of viable cells (negative for both Annexin V and 7-AAD). *P<0.05, **P<0.01 compared to the IL-2 group (n=6). [Figure 2A]Figures 2A-2F show memory T cell subsets of CAR-T cells. Figure 2A shows CD95 expression in the CD45RA+CD62L+ subpopulation of T cells before transduction and 15 days after transduction. Figures 2B and 2C are graphs showing the increase in the proportion of memory stem T cells (Tscm) in CD4+ (Figure 2B) and CD8+ T cells (Figure 2C) after lentiviral transduction. Tscm is defined as the CD45RA+CD62L+CD95+CCR7+ T cell subset. Figure 2D is a graph showing the correlation between the amount of naive T (Tn, defined as the CD45RA+CD62L+CD95- subpopulation) in T cells before transduction and the proportion of Tscm in CAR-T cells after transduction (n = 6). The left bar represents the percentage of Tn in CD4+ and CD8+ T cells before transduction, and the right bar represents the percentage of Tscm in CD4+ and CD8+ CAR-T cells. *P<0.05, **P<0.01. Figure 2E is a graph showing the self-renewal and differentiation of various subsets of CAR-T cells. FACS-sorted CAR+ Tscm, Tcm, Tem, and Tempra cells were cultured and exposed to IL-2 (10 ng / mL) for 3 days, then phenotypically analyzed based on CD45RA and CD62L expression (n=3). Figure 2F is a histogram plot showing the proliferation of various subsets of CAR-T cells in response to IL-2. FACS-sorted CAR+ Tscm, Tcm, Tem, and Tempra cells were labeled with CFSE (2.5 μM) and then cultured and exposed to IL-2 (10 ng / mL) for 3 days. After 3 days, T cells were analyzed for CFSE dilution. [Figure 2B]Figures 2A-2F show memory T cell subsets of CAR-T cells. Figure 2A shows CD95 expression in the CD45RA+CD62L+ subpopulation of T cells before transduction and 15 days after transduction. Figures 2B and 2C are graphs showing the increase in the proportion of memory stem T cells (Tscm) in CD4+ (Figure 2B) and CD8+ T cells (Figure 2C) after lentiviral transduction. Tscm is defined as the CD45RA+CD62L+CD95+CCR7+ T cell subset. Figure 2D is a graph showing the correlation between the amount of naive T (Tn, defined as the CD45RA+CD62L+CD95- subpopulation) in T cells before transduction and the proportion of Tscm in CAR-T cells after transduction (n = 6). The left bar represents the percentage of Tn in CD4+ and CD8+ T cells before transduction, and the right bar represents the percentage of Tscm in CD4+ and CD8+ CAR-T cells. *P<0.05, **P<0.01. Figure 2E is a graph showing the self-renewal and differentiation of various subsets of CAR-T cells. FACS-sorted CAR+ Tscm, Tcm, Tem, and Tempra cells were cultured and exposed to IL-2 (10 ng / mL) for 3 days, then phenotypically analyzed based on CD45RA and CD62L expression (n=3). Figure 2F is a histogram plot showing the proliferation of various subsets of CAR-T cells in response to IL-2. FACS-sorted CAR+ Tscm, Tcm, Tem, and Tempra cells were labeled with CFSE (2.5 μM) and then cultured and exposed to IL-2 (10 ng / mL) for 3 days. After 3 days, T cells were analyzed for CFSE dilution. [Figure 2C]Figures 2A-2F show memory T cell subsets of CAR-T cells. Figure 2A shows CD95 expression in the CD45RA+CD62L+ subpopulation of T cells before transduction and 15 days after transduction. Figures 2B and 2C are graphs showing the increase in the proportion of memory stem T cells (Tscm) in CD4+ (Figure 2B) and CD8+ T cells (Figure 2C) after lentiviral transduction. Tscm is defined as the CD45RA+CD62L+CD95+CCR7+ T cell subset. Figure 2D is a graph showing the correlation between the amount of naive T (Tn, defined as the CD45RA+CD62L+CD95- subpopulation) in T cells before transduction and the proportion of Tscm in CAR-T cells after transduction (n = 6). The left bar represents the percentage of Tn in CD4+ and CD8+ T cells before transduction, and the right bar represents the percentage of Tscm in CD4+ and CD8+ CAR-T cells. *P<0.05, **P<0.01. Figure 2E is a graph showing the self-renewal and differentiation of various subsets of CAR-T cells. FACS-sorted CAR+ Tscm, Tcm, Tem, and Tempra cells were cultured and exposed to IL-2 (10 ng / mL) for 3 days, then phenotypically analyzed based on CD45RA and CD62L expression (n=3). Figure 2F is a histogram plot showing the proliferation of various subsets of CAR-T cells in response to IL-2. FACS-sorted CAR+ Tscm, Tcm, Tem, and Tempra cells were labeled with CFSE (2.5 μM) and then cultured and exposed to IL-2 (10 ng / mL) for 3 days. After 3 days, T cells were analyzed for CFSE dilution. [Figure 2D]Figures 2A-2F show memory T cell subsets of CAR-T cells. Figure 2A shows CD95 expression in the CD45RA+CD62L+ subpopulation of T cells before transduction and 15 days after transduction. Figures 2B and 2C are graphs showing the increase in the proportion of memory stem T cells (Tscm) in CD4+ (Figure 2B) and CD8+ T cells (Figure 2C) after lentiviral transduction. Tscm is defined as the CD45RA+CD62L+CD95+CCR7+ T cell subset. Figure 2D is a graph showing the correlation between the amount of naive T (Tn, defined as the CD45RA+CD62L+CD95- subpopulation) in T cells before transduction and the proportion of Tscm in CAR-T cells after transduction (n = 6). The left bar represents the percentage of Tn in CD4+ and CD8+ T cells before transduction, and the right bar represents the percentage of Tscm in CD4+ and CD8+ CAR-T cells. *P<0.05, **P<0.01. Figure 2E is a graph showing the self-renewal and differentiation of various subsets of CAR-T cells. FACS-sorted CAR+ Tscm, Tcm, Tem, and Tempra cells were cultured and exposed to IL-2 (10 ng / mL) for 3 days, then phenotypically analyzed based on CD45RA and CD62L expression (n=3). Figure 2F is a histogram plot showing the proliferation of various subsets of CAR-T cells in response to IL-2. FACS-sorted CAR+ Tscm, Tcm, Tem, and Tempra cells were labeled with CFSE (2.5 μM) and then cultured and exposed to IL-2 (10 ng / mL) for 3 days. After 3 days, T cells were analyzed for CFSE dilution. [Figure 2E]Figures 2A-2F show memory T cell subsets of CAR-T cells. Figure 2A shows CD95 expression in the CD45RA+CD62L+ subpopulation of T cells before transduction and 15 days after transduction. Figures 2B and 2C are graphs showing the increase in the proportion of memory stem T cells (Tscm) in CD4+ (Figure 2B) and CD8+ T cells (Figure 2C) after lentiviral transduction. Tscm is defined as the CD45RA+CD62L+CD95+CCR7+ T cell subset. Figure 2D is a graph showing the correlation between the amount of naive T (Tn, defined as the CD45RA+CD62L+CD95- subpopulation) in T cells before transduction and the proportion of Tscm in CAR-T cells after transduction (n = 6). The left bar represents the percentage of Tn in CD4+ and CD8+ T cells before transduction, and the right bar represents the percentage of Tscm in CD4+ and CD8+ CAR-T cells. *P<0.05, **P<0.01. Figure 2E is a graph showing the self-renewal and differentiation of various subsets of CAR-T cells. FACS-sorted CAR+ Tscm, Tcm, Tem, and Tempra cells were cultured and exposed to IL-2 (10 ng / mL) for 3 days, then phenotypically analyzed based on CD45RA and CD62L expression (n=3). Figure 2F is a histogram plot showing the proliferation of various subsets of CAR-T cells in response to IL-2. FACS-sorted CAR+ Tscm, Tcm, Tem, and Tempra cells were labeled with CFSE (2.5 μM) and then cultured and exposed to IL-2 (10 ng / mL) for 3 days. After 3 days, T cells were analyzed for CFSE dilution. [Figure 2F]Figures 2A-2F show memory T cell subsets of CAR-T cells. Figure 2A shows CD95 expression in the CD45RA+CD62L+ subpopulation of T cells before transduction and 15 days after transduction. Figures 2B and 2C are graphs showing the increase in the proportion of memory stem T cells (Tscm) in CD4+ (Figure 2B) and CD8+ T cells (Figure 2C) after lentiviral transduction. Tscm is defined as the CD45RA+CD62L+CD95+CCR7+ T cell subset. Figure 2D is a graph showing the correlation between the amount of naive T (Tn, defined as the CD45RA+CD62L+CD95- subpopulation) in T cells before transduction and the proportion of Tscm in CAR-T cells after transduction (n = 6). The left bar represents the percentage of Tn in CD4+ and CD8+ T cells before transduction, and the right bar represents the percentage of Tscm in CD4+ and CD8+ CAR-T cells. *P<0.05, **P<0.01. Figure 2E is a graph showing the self-renewal and differentiation of various subsets of CAR-T cells. FACS-sorted CAR+ Tscm, Tcm, Tem, and Tempra cells were cultured and exposed to IL-2 (10 ng / mL) for 3 days, then phenotypically analyzed based on CD45RA and CD62L expression (n=3). Figure 2F is a histogram plot showing the proliferation of various subsets of CAR-T cells in response to IL-2. FACS-sorted CAR+ Tscm, Tcm, Tem, and Tempra cells were labeled with CFSE (2.5 μM) and then cultured and exposed to IL-2 (10 ng / mL) for 3 days. After 3 days, T cells were analyzed for CFSE dilution. [Figure 3A] Figures 3A-3B show the correlation between CD45RA expression and CFSE intensity. Figure 3A shows that CD45RA expression is inversely correlated with CFSE intensity. Figure 3B shows that for all cytokine groups (IL-2, IL-7, IL-15, IL-18, and IL-21), CD45RA+ T cells showed much lower CFSE levels than CD45RA weakly positive and negative T cells. This indicates that CD45RA+ T cells had stronger proliferative activity than CD45RA- T cells. [Figure 3B]Figures 3A-3B show the correlation between CD45RA expression and CFSE intensity. Figure 3A shows that CD45RA expression is inversely correlated with CFSE intensity. Figure 3B shows that for all cytokine groups (IL-2, IL-7, IL-15, IL-18, and IL-21), CD45RA+ T cells showed much lower CFSE levels than CD45RA weakly positive and negative T cells. This indicates that CD45RA+ T cells had stronger proliferative activity than CD45RA- T cells. [Figure 4] Figure 4 shows the phenotype of CAR-T cells resulting from exposure to various cytokines. Figure 4 is a series of graphs showing quantification by FACS of CD45RA, CD62L, CCR7, CD27, CD28, and IL7Rα expression on the surface of CAR-T cells in the indicated cytokine groups. Histograms represent the mean ± SEM of expression levels from six independent donors. *P<0.05, **P<0.01 compared to the IL-2 group. [Figure 5A] Figures 5A-5D show functional analysis of CAR-T cells exposed to various cytokines. Figures 5A, 5B, and 5C are quantitative plots showing the percentage of cytokine-producing CAR-T cells in various cytokine groups (n = 6) for the production of IFNγ (Figure 5A), TNF-α (Figure 5B), and IL-2 (Figure 5C). Lentiviral-transduced T cells were exposed to the indicated cytokines for 14 days and then co-cultured with SKOV3 cells for 5 hours before harvesting for flow cytometry analysis. Figure 5D is a graph showing the antigen-specific cytotoxic activity of CAR-T cells. After 14 days of exposure to the indicated cytokines, CAR-T cells were evaluated for cytolytic potential using a luciferase-based assay after 18 hours of co-culture with SKOV3 cells at the indicated E / T ratios. Untransduced T cells (UNT) served as a negative effector control. Data shown are the mean ± SEM of six independent cytolytic assays. [Figure 5B]Figures 5A-5D show functional analysis of CAR-T cells exposed to various cytokines. Figures 5A, 5B, and 5C are quantitative plots showing the percentage of cytokine-producing CAR-T cells in various cytokine groups (n = 6) for the production of IFNγ (Figure 5A), TNF-α (Figure 5B), and IL-2 (Figure 5C). Lentiviral-transduced T cells were exposed to the indicated cytokines for 14 days and then co-cultured with SKOV3 cells for 5 hours before harvesting for flow cytometry analysis. Figure 5D is a graph showing the antigen-specific cytotoxic activity of CAR-T cells. After 14 days of exposure to the indicated cytokines, CAR-T cells were evaluated for cytolytic potential using a luciferase-based assay after 18 hours of co-culture with SKOV3 cells at the indicated E / T ratios. Untransduced T cells (UNT) served as a negative effector control. Data shown are the mean ± SEM of six independent cytolytic assays. [Figure 5C] Figures 5A-5D show functional analysis of CAR-T cells exposed to various cytokines. Figures 5A, 5B, and 5C are quantitative plots showing the percentage of cytokine-producing CAR-T cells in various cytokine groups (n = 6) for the production of IFNγ (Figure 5A), TNF-α (Figure 5B), and IL-2 (Figure 5C). Lentiviral-transduced T cells were exposed to the indicated cytokines for 14 days and then co-cultured with SKOV3 cells for 5 hours before harvesting for flow cytometry analysis. Figure 5D is a graph showing the antigen-specific cytotoxic activity of CAR-T cells. After 14 days of exposure to the indicated cytokines, CAR-T cells were evaluated for cytolytic potential using a luciferase-based assay after 18 hours of co-culture with SKOV3 cells at the indicated E / T ratios. Untransduced T cells (UNT) served as a negative effector control. Data shown are the mean ± SEM of six independent cytolytic assays. [Figure 5D]Figures 5A-5D show functional analysis of CAR-T cells exposed to various cytokines. Figures 5A, 5B, and 5C are quantitative plots showing the percentage of cytokine-producing CAR-T cells in various cytokine groups (n = 6) for the production of IFNγ (Figure 5A), TNF-α (Figure 5B), and IL-2 (Figure 5C). Lentiviral-transduced T cells were exposed to the indicated cytokines for 14 days and then co-cultured with SKOV3 cells for 5 hours before harvesting for flow cytometry analysis. Figure 5D is a graph showing the antigen-specific cytotoxic activity of CAR-T cells. After 14 days of exposure to the indicated cytokines, CAR-T cells were evaluated for cytolytic potential using a luciferase-based assay after 18 hours of co-culture with SKOV3 cells at the indicated E / T ratios. Untransduced T cells (UNT) served as a negative effector control. Data shown are the mean ± SEM of six independent cytolytic assays. [Figure 6A] Figures 6A-6C show the phenotype and function of the CAR-T cells described above in Figure 5. Figures 6A and 6B show that CD62L+ CAR-T cells (Tscm and Tcm) exhibited lower cytokine production activity (Figures 6A and 6B) and weaker cytolytic ability (Figure 6C) when compared to CD62L- CAR-T cells (Tem and Temra). [Figure 6B] Figures 6A-6C show the phenotype and function of the CAR-T cells described above in Figure 5. Figures 6A and 6B show that CD62L+ CAR-T cells (Tscm and Tcm) exhibited lower cytokine production activity (Figures 6A and 6B) and weaker cytolytic ability (Figure 6C) when compared to CD62L- CAR-T cells (Tem and Temra). [Figure 6C] Figures 6A-6C show the phenotype and function of the CAR-T cells described above in Figure 5. Figures 6A and 6B show that CD62L+ CAR-T cells (Tscm and Tcm) exhibited lower cytokine production activity (Figures 6A and 6B) and weaker cytolytic ability (Figure 6C) when compared to CD62L- CAR-T cells (Tem and Temra). [Figure 7A]Figures 7A-7B show the proliferation and phenotype of CAR-T cells exposed to antigen challenge. Figure 7A presents two graphs showing the overall accumulation and viability of CAR-T cells previously exposed to the indicated cytokines upon antigen challenge. T cells exposed to the indicated cytokines were harvested on day 15 and then cocultured with SKOV3 at a 5:1 E / T ratio for 7 days. CAR-T cell proliferation was calculated, and T cell viability was assessed on day 17. Figure 7B presents two graphs showing the distribution of memory T subsets of CD4+ and CD8+ CAR-T cells in the various cytokine groups. NS, no statistical difference. [Figure 7B] Figures 7A-7B show the proliferation and phenotype of CAR-T cells exposed to antigen challenge. Figure 7A presents two graphs showing the overall accumulation and viability of CAR-T cells previously exposed to the indicated cytokines upon antigen challenge. T cells exposed to the indicated cytokines were harvested on day 15 and then cocultured with SKOV3 at a 5:1 E / T ratio for 7 days. CAR-T cell proliferation was calculated, and T cell viability was assessed on day 17. Figure 7B presents two graphs showing the distribution of memory T subsets of CD4+ and CD8+ CAR-T cells in the various cytokine groups. NS, no statistical difference. [Figure 8A] Figures 8A-8C show the antitumor activity of various cytokine-exposed CAR-T cells. Figure 8A shows tumor growth curves for mice treated with various cytokine-exposed C4-27z CAR-T cells, anti-CD19-27z CAR-T cells, and untransduced T cells. Data are expressed as mean ± SEM. Arrows indicate the time of T cell infusion. Figure 8B shows quantification of circulating human CD4+ and CD8+ T cell numbers in the peripheral blood of mice 15 days after the first dose of CAR-T cell infusion. Figure 8C shows quantification of CAR expression on circulating human CD4+ and CD8+ T cells in mouse blood. [Figure 8B]Figures 8A-8C show the antitumor activity of various cytokine-exposed CAR-T cells. Figure 8A shows tumor growth curves for mice treated with various cytokine-exposed C4-27z CAR-T cells, anti-CD19-27z CAR-T cells, and untransduced T cells. Data are expressed as mean ± SEM. Arrows indicate the time of T cell infusion. Figure 8B shows quantification of circulating human CD4+ and CD8+ T cell numbers in the peripheral blood of mice 15 days after the first dose of CAR-T cell infusion. Figure 8C shows quantification of CAR expression on circulating human CD4+ and CD8+ T cells in mouse blood. [Figure 8C] Figures 8A-8C show the antitumor activity of various cytokine-exposed CAR-T cells. Figure 8A shows tumor growth curves for mice treated with various cytokine-exposed C4-27z CAR-T cells, anti-CD19-27z CAR-T cells, and untransduced T cells. Data are expressed as mean ± SEM. Arrows indicate the time of T cell infusion. Figure 8B shows quantification of circulating human CD4+ and CD8+ T cell numbers in the peripheral blood of mice 15 days after the first dose of CAR-T cell infusion. Figure 8C shows quantification of CAR expression on circulating human CD4+ and CD8+ T cells in mouse blood. [Figure 9] Figure 9 is a series of FACS plots (top) showing CD3 and CD19 populations, and histograms (bottom) showing CD14 expression in cells from apheresis, anti-CD3 / CD28-selected cells, CD25-depleted cells, and CD25-enriched cells. [Figure 10A] Figures 10A, 10B, and 10C show a comparison of the proliferation potential of CD3 / CD28-selected cells and CD25-depleted cells. Figure 10A is a graph showing total cell counts for the indicated days of culture. Figure 10B is a graph showing quantified population doublings for each indicated day of culture. Figure 10C shows the percentage of viable cells for the indicated days of culture. [Figure 10B]Figures 10A, 10B, and 10C show a comparison of the proliferation potential of CD3 / CD28-selected cells and CD25-depleted cells. Figure 10A is a graph showing total cell counts for the indicated days of culture. Figure 10B is a graph showing quantified population doublings for each indicated day of culture. Figure 10C shows the percentage of viable cells for the indicated days of culture. [Figure 10C] Figures 10A, 10B, and 10C show a comparison of the proliferation potential of CD3 / CD28-selected cells and CD25-depleted cells. Figure 10A is a graph showing total cell counts for the indicated days of culture. Figure 10B is a graph showing quantified population doublings for each indicated day of culture. Figure 10C shows the percentage of viable cells for the indicated days of culture. [Figure 11] FIG. 11 is a series of FACs plots showing the distribution of CD3 and CD19 in unmanipulated and CD25-depleted PBMCs after culture with the indicated cytokine supplements (IL-7, IL-15, or IL-7 and IL-15). [Figure 12] Figure 12 is a set of graphs showing the proliferation profile in population doublings (left hand graph) and mean size (fL) (right hand graph) of PBMCs stimulated with anti-CD3 and CD28 beads and either left unmanipulated (UTD) or transduced with CD19 CAR (CD19.BBz), de-beaded, and then harvested on day 5 and D9. [Figure 13] Figure 13 is a set of graphs depicting cytotoxicity as percent lysis of CD19-expressing K562 cells stimulated with anti-CD3 and CD28 beads and left unmanipulated (UTD) or transduced with CD19 CAR (CD19.BBz), beads removed, and then treated with PBMCs collected on day 5 and D9. [Figure 14]Figure 14 is a set of graphs depicting the proliferation of PBMCs stimulated with anti-CD3 and CD28 beads (3x28 beads), wild-type K562 cells, CD19-expressing K562 cells, ALL cells (Nalm6), or CLL cells (PI14). PBMCs were left unmanipulated (UTD) or transduced with a CD19 CAR (CART19), beads were removed, and then harvested on day 5 and D9. [Figure 15] FIG. 15 is a schematic diagram of an exemplary fabrication scheme. [Figure 16] FIG. 16 is a schematic diagram of an exemplary fabrication scheme. [Figure 17] Figure 17 is a set of graphs depicting the levels of cell proliferation of two different production batches of donor cells transfected with CTL019 CAR, CHP959-115, and CHP959-121 and grown over a period of 0 to 9 days. [Figure 18] FIG. 18 is a set of graphs showing pro-inflammatory cytokine production, IFN-γ, GM-CSF, TNF-α, and IL-4, of two different production batches of donor cells transfected with either the CTL019 CAR, i.e., CHP959-115, or the ss1-mesoCAR, i.e., CHP959-121, and expanded over a period of 0 to 9 days after apheresis. [Figure 19] Figure 19 is a set of graphs depicting the production levels of IFN-γ, TNF-α, IL-6, IL-8, IL-2, IL-1β, GM-CSF, and IL-4 in donor cells transfected with CTL019 CAR and grown for 5-9 days, stimulated with anti-CAR19 idiotype antibody beads or control beads. No cytokines or low cytokine levels (<200 pg / ml) were detected with control beads. [Figure 20]Figure 20 is a graph showing total lysate-based cell killing using luciferase assay of Nalm6 (ALL) cells left unmanipulated (UTD) or transduced with CD19 CAR (CART19), beads removed, and then PBMCs collected on day 5 and D9. Various ratios of PBMCs to Nalm6 cells (effector (E): target (T)) were cultured. As shown, CART19 cells collected on day 5 have better killing ability. [Figure 21] Figure 21 is a graph depicting the long-term in vivo killing capacity of PBMCs that were left unmanipulated (UTD) or transduced with CD19 CAR (CART19), beads were removed, and then collected on day 5 and D9. PBMCs were transferred into non-obese diabetic / severe combined immunodeficiency mice inoculated with Nalm6 cells. [Figure 22A] Figure 22A is a bar graph showing the percentage of peripheral blood samples from ALL and NHL patients that passed the proliferation test. Figure 22B is a bar graph showing the percentage of peripheral blood samples from SR-ALL and HR / VHR ALL patients that passed the proliferation test. Figure 22C is a bar graph showing absolute lymphocyte counts from peripheral blood at various collection times from all patients who expanded more than (passed) or less than (failed) five-fold at the in vitro proliferation threshold. Figure 22D is a bar graph showing absolute T cell counts from peripheral blood at various collection times from all patients who passed or failed the five-fold at the in vitro proliferation threshold. Figure 22E is a bar graph showing absolute lymphocyte counts (in cells per microliter) from peripheral blood at various collection times from all patients with leukemia or lymphoma. Figure 22F is a bar graph showing absolute T cell counts (in cells per microliter) from peripheral blood at various collection times from leukemia or lymphoma patients. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analysis can be found in Table 12. [Figure 22B]Figure 22A is a bar graph showing the percentage of peripheral blood samples from ALL and NHL patients that passed the proliferation test. Figure 22B is a bar graph showing the percentage of peripheral blood samples from SR-ALL and HR / VHR ALL patients that passed the proliferation test. Figure 22C is a bar graph showing absolute lymphocyte counts from peripheral blood at various collection times from all patients who expanded more than (passed) or less than (failed) five-fold at the in vitro proliferation threshold. Figure 22D is a bar graph showing absolute T cell counts from peripheral blood at various collection times from all patients who passed or failed the five-fold at the in vitro proliferation threshold. Figure 22E is a bar graph showing absolute lymphocyte counts (in cells per microliter) from peripheral blood at various collection times from all patients with leukemia or lymphoma. Figure 22F is a bar graph showing absolute T cell counts (in cells per microliter) from peripheral blood at various collection times from leukemia or lymphoma patients. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analysis can be found in Table 12. [Figure 22C] Figure 22A is a bar graph showing the percentage of peripheral blood samples from ALL and NHL patients that passed the proliferation test. Figure 22B is a bar graph showing the percentage of peripheral blood samples from SR-ALL and HR / VHR ALL patients that passed the proliferation test. Figure 22C is a bar graph showing absolute lymphocyte counts from peripheral blood at various collection times from all patients who expanded more than (passed) or less than (failed) five-fold at the in vitro proliferation threshold. Figure 22D is a bar graph showing absolute T cell counts from peripheral blood at various collection times from all patients who passed or failed the five-fold at the in vitro proliferation threshold. Figure 22E is a bar graph showing absolute lymphocyte counts (in cells per microliter) from peripheral blood at various collection times from all patients with leukemia or lymphoma. Figure 22F is a bar graph showing absolute T cell counts (in cells per microliter) from peripheral blood at various collection times from leukemia or lymphoma patients. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analysis can be found in Table 12. [Figure 22D] Figure 22A is a bar graph showing the percentage of peripheral blood samples from ALL and NHL patients that passed the proliferation test. Figure 22B is a bar graph showing the percentage of peripheral blood samples from SR-ALL and HR / VHR ALL patients that passed the proliferation test. Figure 22C is a bar graph showing absolute lymphocyte counts from peripheral blood at various collection times from all patients who expanded more than (passed) or less than (failed) five-fold at the in vitro proliferation threshold. Figure 22D is a bar graph showing absolute T cell counts from peripheral blood at various collection times from all patients who passed or failed the five-fold at the in vitro proliferation threshold. Figure 22E is a bar graph showing absolute lymphocyte counts (in cells per microliter) from peripheral blood at various collection times from all patients with leukemia or lymphoma. Figure 22F is a bar graph showing absolute T cell counts (in cells per microliter) from peripheral blood at various collection times from leukemia or lymphoma patients. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analysis can be found in Table 12. [Figure 22E] Figure 22A is a bar graph showing the percentage of peripheral blood samples from ALL and NHL patients that passed the proliferation test. Figure 22B is a bar graph showing the percentage of peripheral blood samples from SR-ALL and HR / VHR ALL patients that passed the proliferation test. Figure 22C is a bar graph showing absolute lymphocyte counts from peripheral blood at various collection times from all patients who expanded more than (passed) or less than (failed) five-fold at the in vitro proliferation threshold. Figure 22D is a bar graph showing absolute T cell counts from peripheral blood at various collection times from all patients who passed or failed the five-fold at the in vitro proliferation threshold. Figure 22E is a bar graph showing absolute lymphocyte counts (in cells per microliter) from peripheral blood at various collection times from all patients with leukemia or lymphoma. Figure 22F is a bar graph showing absolute T cell counts (in cells per microliter) from peripheral blood at various collection times from leukemia or lymphoma patients. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analysis can be found in Table 12. [Figure 22F] Figure 22A is a bar graph showing the percentage of peripheral blood samples from ALL and NHL patients that passed the proliferation test. Figure 22B is a bar graph showing the percentage of peripheral blood samples from SR-ALL and HR / VHR ALL patients that passed the proliferation test. Figure 22C is a bar graph showing absolute lymphocyte counts from peripheral blood at various collection times from all patients who expanded more than (passed) or less than (failed) five-fold at the in vitro proliferation threshold. Figure 22D is a bar graph showing absolute T cell counts from peripheral blood at various collection times from all patients who passed or failed the five-fold at the in vitro proliferation threshold. Figure 22E is a bar graph showing absolute lymphocyte counts (in cells per microliter) from peripheral blood at various collection times from all patients with leukemia or lymphoma. Figure 22F is a bar graph showing absolute T cell counts (in cells per microliter) from peripheral blood at various collection times from leukemia or lymphoma patients. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analysis can be found in Table 12. [Figure 23A] Figures 23A-23O are bar graphs showing the memory phenotype of T cells collected from the peripheral blood of patients undergoing chemotherapy. Absolute cell counts are shown from samples that passed and failed a >5-fold proliferation threshold [column 1, Figures 23A-23E], ALL or NHL patients [column 2, Figures 23F-23J], and SR- and HR / VHR-ALL patients [column 3, Figures 23K-23O]. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analyses can be found in Table 13, and a summary table can be found in Table 9. [Figure 23B]Figures 23A-23O are bar graphs showing the memory phenotype of T cells collected from the peripheral blood of patients undergoing chemotherapy. Absolute cell counts are shown from samples that passed and failed a >5-fold proliferation threshold [column 1, Figures 23A-23E], ALL or NHL patients [column 2, Figures 23F-23J], and SR- and HR / VHR-ALL patients [column 3, Figures 23K-23O]. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analyses can be found in Table 13, and a summary table can be found in Table 9. [Figure 23C] Figures 23A-23O are bar graphs showing the memory phenotype of T cells collected from the peripheral blood of patients undergoing chemotherapy. Absolute cell counts are shown from samples that passed and failed a >5-fold proliferation threshold [column 1, Figures 23A-23E], ALL or NHL patients [column 2, Figures 23F-23J], and SR- and HR / VHR-ALL patients [column 3, Figures 23K-23O]. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analyses can be found in Table 13, and a summary table can be found in Table 9. [Figure 23D] Figures 23A-23O are bar graphs showing the memory phenotype of T cells collected from the peripheral blood of patients undergoing chemotherapy. Absolute cell counts are shown from samples that passed and failed a >5-fold proliferation threshold [column 1, Figures 23A-23E], ALL or NHL patients [column 2, Figures 23F-23J], and SR- and HR / VHR-ALL patients [column 3, Figures 23K-23O]. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analyses can be found in Table 13, and a summary table can be found in Table 9. [Figure 23E]Figures 23A-23O are bar graphs showing the memory phenotype of T cells collected from the peripheral blood of patients undergoing chemotherapy. Absolute cell counts are shown from samples that passed and failed a >5-fold proliferation threshold [column 1, Figures 23A-23E], ALL or NHL patients [column 2, Figures 23F-23J], and SR- and HR / VHR-ALL patients [column 3, Figures 23K-23O]. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analyses can be found in Table 13, and a summary table can be found in Table 9. [Figure 23F] Figures 23A-23O are bar graphs showing the memory phenotype of T cells collected from the peripheral blood of patients undergoing chemotherapy. Absolute cell counts are shown from samples that passed and failed a >5-fold proliferation threshold [column 1, Figures 23A-23E], ALL or NHL patients [column 2, Figures 23F-23J], and SR- and HR / VHR-ALL patients [column 3, Figures 23K-23O]. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analyses can be found in Table 13, and a summary table can be found in Table 9. [Figure 23G] Figures 23A-23O are bar graphs showing the memory phenotype of T cells collected from the peripheral blood of patients undergoing chemotherapy. Absolute cell counts are shown from samples that passed and failed a >5-fold proliferation threshold [column 1, Figures 23A-23E], ALL or NHL patients [column 2, Figures 23F-23J], and SR- and HR / VHR-ALL patients [column 3, Figures 23K-23O]. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analyses can be found in Table 13, and a summary table can be found in Table 9. [Figure 23H]Figures 23A-23O are bar graphs showing the memory phenotype of T cells collected from the peripheral blood of patients undergoing chemotherapy. Absolute cell counts are shown from samples that passed and failed a >5-fold proliferation threshold [column 1, Figures 23A-23E], ALL or NHL patients [column 2, Figures 23F-23J], and SR- and HR / VHR-ALL patients [column 3, Figures 23K-23O]. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analyses can be found in Table 13, and a summary table can be found in Table 9. [Figure 23I] Figures 23A-23O are bar graphs showing the memory phenotype of T cells collected from the peripheral blood of patients undergoing chemotherapy. Absolute cell counts are shown from samples that passed and failed a >5-fold proliferation threshold [column 1, Figures 23A-23E], ALL or NHL patients [column 2, Figures 23F-23J], and SR- and HR / VHR-ALL patients [column 3, Figures 23K-23O]. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analyses can be found in Table 13, and a summary table can be found in Table 9. [Figure 23J] Figures 23A-23O are bar graphs showing the memory phenotype of T cells collected from the peripheral blood of patients undergoing chemotherapy. Absolute cell counts are shown from samples that passed and failed a >5-fold proliferation threshold [column 1, Figures 23A-23E], ALL or NHL patients [column 2, Figures 23F-23J], and SR- and HR / VHR-ALL patients [column 3, Figures 23K-23O]. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analyses can be found in Table 13, and a summary table can be found in Table 9. [Figure 23K]Figures 23A-23O are bar graphs showing the memory phenotype of T cells collected from the peripheral blood of patients undergoing chemotherapy. Absolute cell counts are shown from samples that passed and failed a >5-fold proliferation threshold [column 1, Figures 23A-23E], ALL or NHL patients [column 2, Figures 23F-23J], and SR- and HR / VHR-ALL patients [column 3, Figures 23K-23O]. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analyses can be found in Table 13, and a summary table can be found in Table 9. [Figure 23L] Figures 23A-23O are bar graphs showing the memory phenotype of T cells collected from the peripheral blood of patients undergoing chemotherapy. Absolute cell counts are shown from samples that passed and failed a >5-fold proliferation threshold [column 1, Figures 23A-23E], ALL or NHL patients [column 2, Figures 23F-23J], and SR- and HR / VHR-ALL patients [column 3, Figures 23K-23O]. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analyses can be found in Table 13, and a summary table can be found in Table 9. [Figure 23M] Figures 23A-23O are bar graphs showing the memory phenotype of T cells collected from the peripheral blood of patients undergoing chemotherapy. Absolute cell counts are shown from samples that passed and failed a >5-fold proliferation threshold [column 1, Figures 23A-23E], ALL or NHL patients [column 2, Figures 23F-23J], and SR- and HR / VHR-ALL patients [column 3, Figures 23K-23O]. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analyses can be found in Table 13, and a summary table can be found in Table 9. [Figure 23N]Figures 23A-23O are bar graphs showing the memory phenotype of T cells collected from the peripheral blood of patients undergoing chemotherapy. Absolute cell counts are shown from samples that passed and failed a >5-fold proliferation threshold [column 1, Figures 23A-23E], ALL or NHL patients [column 2, Figures 23F-23J], and SR- and HR / VHR-ALL patients [column 3, Figures 23K-23O]. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analyses can be found in Table 13, and a summary table can be found in Table 9. [Figure 23O] Figures 23A-23O are bar graphs showing the memory phenotype of T cells collected from the peripheral blood of patients undergoing chemotherapy. Absolute cell counts are shown from samples that passed and failed a >5-fold proliferation threshold [column 1, Figures 23A-23E], ALL or NHL patients [column 2, Figures 23F-23J], and SR- and HR / VHR-ALL patients [column 3, Figures 23K-23O]. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analyses can be found in Table 13, and a summary table can be found in Table 9. [Figure 24] Figure 24 is a bar graph showing absolute T cell counts (in cells per microliter) from peripheral blood at various collection times from all patients with SR- or HR / VHR-ALL. Significant differences are indicated by an "*" above each column, representing P<0.05. Statistical analysis can be found in Table 12. [Figure 25A]Figures 25A and 25B are bar graphs showing the effects of IL-7 and IL-15 on T cell phenotype and proliferation. Harvested samples were split into two stimulation cultures, with or without IL-7 and IL-15, as described in Example 4. Figure 25A is a bar graph showing the percent change in absolute memory phenotype cell numbers in leukemia and lymphoma patients after culture with cytokines. Significant differences are indicated by an "*" above each column, representing P<0.05. Figure 25B is a bar graph showing the percent of test proliferation-passing samples evaluated after culture with or without cytokines. "Total samples" represents all samples grown without cytokines, some of which were harvested before the split culture protocol. Statistical analysis can be found in Table 14. [Figure 25B] Figures 25A and 25B are bar graphs showing the effects of IL-7 and IL-15 on T cell phenotype and proliferation. Harvested samples were split into two stimulation cultures, with or without IL-7 and IL-15, as described in Example 4. Figure 25A is a bar graph showing the percent change in absolute memory phenotype cell numbers in leukemia and lymphoma patients after culture with cytokines. Significant differences are indicated by an "*" above each column, representing P<0.05. Figure 25B is a bar graph showing the percent of test proliferation-passing samples evaluated after culture with or without cytokines. "Total samples" represents all samples grown without cytokines, some of which were harvested before the split culture protocol. Statistical analysis can be found in Table 14. [Figure 26] Figure 26 is a set of pie charts showing the post-growth phenotypes of test growth passing and failing samples. Phenotypes were assessed at the end of test growth. DETAILED DESCRIPTION OF THE INVENTION

[0138] The present invention is based, at least in part, on the discovery that the suitability of immune effector cells (e.g., T cells or NK cells) for use in cell therapy (e.g., CAR therapy, e.g., as described herein, by way of example) is correlated with a number of factors, including the timing of chemotherapy relative to obtaining the cells from a subject, the type of chemotherapy administered to the subject from whom the cells are obtained, the primary malignancy of the subject (from whom the cells are obtained), the severity of the malignancy (e.g., standard risk, high risk, or very high risk ALL), the proportion of less differentiated T cells (e.g., naive T cells, stem central memory T cells, or central memory T cells), the proportion of terminal effector T cells, and the number of T cells (e.g., absolute T cell count) obtained from the subject.

[0139] As described in detail in the Examples, improved T cell proliferation (e.g., which may indicate suitability for use in cell therapy) was observed by using cells obtained from a patient at an early time point during the patient's chemotherapy (e.g., before administration of a chemotherapy cycle that depletes T cells with high proliferative potential). Without wishing to be bound by theory, it is believed that obtaining cells from a patient at an early time point during the patient's chemotherapy provides, for example, a cell population with less deteriorated proliferative potential. Improved T cell proliferation, for example, was observed by using cells obtained from a patient before a later intensive or consolidation chemotherapy cycle. In some embodiments, the severity of the disease affects cell deterioration, e.g., cell proliferative potential. In some embodiments, cells obtained from patients with less severe disease (e.g., standard-risk ALL) proliferated better than cells obtained from patients with more severe disease (e.g., high-risk or very high-risk ALL). In some embodiments, patients with more severe disease (e.g., high-risk or very high-risk ALL) may require cell acquisition earlier during chemotherapy (e.g., before the second or consolidation cycle) compared to patients with less severe disease (e.g., standard-risk ALL). In some embodiments, cells capable of proliferation may be acquired later during chemotherapy (e.g., before the fourth or late intensification cycle) from patients with less severe disease (e.g., standard-risk ALL) compared to patients with more severe disease. In other embodiments, cells acquired from patients with leukemia (e.g., ALL, e.g., pediatric ALL) had higher proliferative capacity than cells acquired from patients with lymphoma (e.g., NHL, e.g., pediatric NHL). Furthermore, acquired cell populations with higher absolute T cell numbers also tended to proliferate better than those with lower absolute T cell numbers. Acquired cell populations that contained a greater proportion of less differentiated immune effector cells (e.g., less differentiated T cells), which tend to have higher proliferative and viable capacity, similarly exhibited higher proliferative capacity.

[0140] Without wishing to be bound by theory, it is believed that drugs used in some chemotherapy cycles, such as cyclophosphamide and cytarabine, significantly deplete highly proliferative T cells (e.g., low-differentiation T cells), and this effect is more pronounced in patients with more severe disease.Longer exposure to chemotherapy drugs or higher doses can further deplete these T cell populations.In addition, it is believed that a larger number of low-differentiation T cell populations leads to higher T cell proliferation capacity, persistence, and effectiveness as anti-cancer T cell therapy.

[0141] Thus, in some embodiments, the methods advantageously improve the suitability and efficacy of T cells (suitability for use in cell therapy) by obtaining cells from a subject early after cancer diagnosis, e.g., before chemotherapy or before certain / multiple chemotherapy cycles. In some embodiments, the methods utilize cells obtained from a patient before a chemotherapy cycle involving a T cell depleting agent, before two, three, four, or five chemotherapy cycles, or before a late intensification or consolidation cycle. In other embodiments, the methods optimize the proliferative capacity and / or anti-tumor effect of immune cells by, for example, including patient selection based on malignancy type (e.g., leukemia vs. lymphoma) or disease severity (e.g., standard risk, high risk, or very high risk). In still other embodiments, the methods optimize the proliferative capacity and / or anti-tumor effect of immune cells by, for example, including selection of cell populations with high absolute T cell numbers or a high proportion of poorly differentiated T cells. Additional embodiments are described in further detail herein.

[0142] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0143] The terms "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the item. By way of example, "one element" means one or more than one element.

[0144] The term "about" when referring to a measurable value, e.g., an amount, a time period, etc., is meant to include a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the stated value, as such variations are appropriate for performing the disclosed methods.

[0145] As used herein, the term "obtain" or "obtaining" refers to obtaining a physical entity (e.g., a sample, a cell or cell population, a polypeptide, a nucleic acid, or a sequence) or a value, e.g., a numerical value, by "directly obtaining" or "indirectly obtaining" that physical entity or value. In one embodiment, obtaining refers to obtaining or harvesting a cell or cell population (e.g., an immune effector cell or population described herein). "Directly obtaining" means performing a process (e.g., performing a synthetic or analytical or purification method) to obtain the physical entity or value. "Indirectly obtaining" refers to receiving the physical entity or value from another population or source (e.g., a third-party laboratory that directly obtained the physical entity or value). Directly obtaining a physical entity includes performing a process that involves a physical change of a physical substance, e.g., a starting material. Exemplary transformations include creating a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more separate entities into a mixture, and performing a chemical reaction that involves breaking or forming covalent or non-covalent bonds. Obtaining a value directly includes performing a process that involves a physical change of a sample or another substance, for example, performing an analytical process (often referred to herein as "physical analysis") that involves a physical change of a substance, e.g., a sample, analyte, or reagent, an analytical method, for example, performing a method that involves one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or a fragment or other derivative thereof, with another substance, e.g., a buffer, solvent, or reactant; or altering the structure of an analyte, or a fragment or other derivative thereof, for example, by breaking or forming a covalent or non-covalent bond between a first atom and a second atom of the analyte; or altering the structure of a reagent, or a fragment or other derivative thereof, for example, by breaking or forming a covalent or non-covalent bond between a first atom and a second atom of the reagent.

[0146] "Bioequivalent" refers to the amount of an agent other than a reference compound (e.g., RAD001) required to produce an effect equivalent to that produced by a reference dose or reference amount of the reference compound (e.g., RAD001). In some embodiments, the effect is the level of mTOR inhibition, e.g., as measured by P70 S6 kinase inhibition, e.g., as assessed in an in vivo or in vitro assay, e.g., an assay described herein, e.g., the Boulay assay, or by measuring phosphorylated S6 levels by Western blot. In some embodiments, the effect is a change in the ratio of PD-1-positive T cells / PD-1-negative T cells, as measured by cell sorting. In some embodiments, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of P70 S6 kinase inhibition as a reference dose or reference amount of the reference compound. In some embodiments, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of change in the ratio of PD-1-positive T cells / PD-1-negative T cells as a reference dose or reference amount of the reference compound.

[0147] The term "chimeric antigen receptor" or alternatively "CAR" refers to a set of polypeptides, typically two polypeptides in the simplest embodiment, that provide specificity for a target cell, typically a cancer cell, and provide intracellular signal generation in an immune effector cell. In some embodiments, a CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule, as defined below. In some aspects, the set of polypeptides are contiguous with each other, e.g., on the same polypeptide chain, e.g., comprising a chimeric fusion protein. In some embodiments, the set of polypeptides are not contiguous with each other, e.g., on different polypeptide chains. In some embodiments, the set of polypeptides includes a dimerization switch that allows the polypeptides to bind to each other when a dimerization molecule is present, e.g., allows the antigen-binding domain to bind to the intracellular signaling domain. In one aspect, the stimulatory molecule is a zeta chain associated with a T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, as defined below. In one embodiment, the costimulatory molecule is selected from a costimulatory molecule described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule.In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and one functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and one functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR may comprise a leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, where the leader sequence is optionally cleaved from the antigen-binding domain (e.g., scFv) upon cellular processing and localization of the CAR to the cell membrane.

[0148] A CAR comprising an antigen-binding domain (e.g., scFv or TCR) that targets a specific tumor antigen X, such as those described herein, is also referred to as an XCAR. For example, a CAR comprising an antigen-binding domain that targets CD19 is referred to as a CD19CAR.

[0149] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within the cell to control cellular activity through a given signaling pathway, either by generating second messengers or by functioning as an effector by responding to such messengers.

[0150] The term "antibody," as used herein, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. An antibody can also be a tetramer of an immunoglobulin particle.

[0151] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) with an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFvs), Fd fragments consisting of a VH domain and a CH1 domain, linear antibodies, single-domain antibodies such as sdAbs (either VL or VH), camelid VHH domains, multispecific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, isolated CDRs, or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides, such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, describing fibronectin polypeptide minibodies).

[0152] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are contiguously linked, for example, via a synthetic linker, e.g., a short flexible polypeptide linker, and can be expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, an scFv may have the VL and VH variable regions in either order; for example, with respect to the N- and C-termini of the polypeptide, an scFv may comprise VL-linker-VH or VH-linker-VL.

[0153] The portion of the CAR comprising an antibody or antibody fragment thereof can exist in various forms in which the antigen-binding domain is expressed as part of a continuous polypeptide chain, including, for example, single-domain antibody fragments (sdAbs), single-chain antibodies (scFvs), and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen-binding domain of the CAR comprises an antibody fragment. In a further embodiment, the CAR comprises an antibody fragment comprising an scFv. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the "Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (the "Chothia" numbering scheme), or a combination thereof.

[0154] As used herein, the term "binding domain" or "antibody molecule" refers to a protein, e.g., an immunoglobulin chain or fragment thereof, that comprises at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In certain embodiments, an antibody molecule is a multispecific antibody molecule, e.g., it comprises multiple immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In certain embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0155] The term "complementarity determining region" or "CDR" as used herein refers to the amino acid sequence in the variable region of an antibody that confers antigen specificity and binding affinity.For example, generally, each heavy chain variable region has three CDRs (for example, HCDR1, HCDR2, and HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3).The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme), or a combination thereof. In accordance with the Kabat numbering scheme, in some embodiments, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In accordance with the Chothia numbering scheme, in some embodiments, the CDR amino acids of the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both.For example, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of a VH, e.g., a mammalian VH, e.g., a human VH, and correspond to amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of a VL, e.g., a mammalian VL, e.g., a human VL.

[0156] The portion of the CAR of the present invention comprising an antibody or antibody fragment thereof can exist in various forms, in which the antigen-binding domain is expressed as part of a continuous polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen-binding domain of the CAR composition of the present invention comprises an antibody fragment. In a further embodiment, the CAR comprises an antibody fragment comprising an scFv.

[0157] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in the natural configuration of an antibody molecule, which usually determines the class to which the antibody belongs.

[0158] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in the naturally occurring configuration of an antibody molecule. Kappa (κ) light chain and lambda (λ) light chain refer to the two major antibody light chain isotypes.

[0159] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed in a bacteriophage or yeast expression system. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence was obtained using recombinant DNA or amino acid sequence technology available and well known in the art.

[0160] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may include antibody production or activation of specific immunologically compatible cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as the term is used herein. Furthermore, those skilled in the art will understand that an antigen is not necessarily encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences can be arranged in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It will be readily apparent that an antigen can be generated, synthesized, or derived from a biological sample, or can be a macromolecule other than a polypeptide. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells, or fluids having other biological components.

[0161] The term "autologous" refers to any material that originates from the same individual, which material is later reintroduced into that individual.

[0162] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some embodiments, allogeneic material derived from individuals of the same species is sufficiently genetically distinct to be able to interact antigenically.

[0163] The term "xenogenic" refers to any material derived from an animal of a different species.

[0164] The term "cancer" refers to a disease characterized by the uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Various examples of cancer are described herein, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "cancer" are used interchangeably herein, for example, both terms encompass solid tumors and liquid tumors, such as diffuse tumors or circulating tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant and malignant cancers and tumors.

[0165] The term "derived from," as used herein, indicates a relationship between a first molecule and a second molecule. This term generally refers to the structural similarity between the first and second molecules and does not imply or imply any limitation regarding the process or source by which the first molecule is derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure so that it has the required function, i.e., the ability to generate a signal under appropriate conditions. This does not imply or imply any limitation to a particular process for generating the intracellular signaling domain; for example, it does not mean that one must start with the CD3 zeta sequence and delete or mutate unnecessary sequences to achieve the intracellular signaling domain to provide this intracellular signaling domain.

[0166] The phrase "disease associated with expression of a tumor antigen" as used herein includes, but is not limited to, a disease associated with expression of a tumor antigen described herein or a condition associated with cells expressing a tumor antigen described herein, including, for example, a proliferative disease such as a cancer or malignant tumor, or a precancerous condition, e.g., myelodysplasia, myelodysplastic syndrome, or preleukemia, or a non-cancer-related indication associated with cells expressing a tumor antigen described herein. In one embodiment, the cancer associated with expression of a tumor antigen described herein is a hematological cancer. In one embodiment, the cancer associated with expression of a tumor antigen described herein is a solid cancer. Additional diseases associated with expression of a tumor antigen described herein include, but are not limited to, atypical and / or non-classical cancers, malignant tumors, precancerous conditions, or proliferative diseases associated with expression of a tumor antigen described herein. Non-cancer-related indications associated with expression of a tumor antigen described herein include, but are not limited to, for example, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, the tumor antigen-expressing cells express, or have expressed at any time, mRNA encoding a tumor antigen. In certain embodiments, the tumor antigen-expressing cells produce a tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal or reduced levels. In certain embodiments, the tumor antigen-expressing cells produced a detectable level of the tumor antigen protein at one time, and then did not substantially produce any detectable tumor antigen protein thereafter.

[0167] As used herein, the term "CD19" refers to the Cluster of Differentiation 19 protein, an antigenic determinant detectable on leukemia precursor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found under UniProt / Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 can be found under accession number NM_001178098. As used herein, "CD19" includes proteins containing mutations of full-length wild-type CD19, such as point mutations, fragments, insertions, deletions, and splice variants. CD19 is expressed in most B-lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Other cells that express CD19 are provided below in the definition of "diseases associated with CD19 expression." It is also an early marker of B cell precursors. See, for example, Nicholson et al. Mol. Immun. 34(16-17):1157-1165(1997). In one embodiment, the antigen-binding portion of the CART recognizes and binds to an antigen within the extracellular domain of the CD19 protein. In one embodiment, the CD19 protein is expressed on cancer cells.

[0168] The phrase "diseases associated with CD19 expression" includes, but is not limited to, diseases associated with the expression of CD19 (e.g., wild-type or mutant CD19) or conditions associated with cells that express or have expressed CD19 (e.g., wild-type or mutant CD19) at any time, including, for example, proliferative diseases such as cancer or malignant tumors, or precancerous conditions, such as myelodysplasia, myelodysplastic syndrome, or preleukemia, or non-cancer-related indications associated with cells expressing CD19. For avoidance of doubt, diseases associated with CD19 expression may include conditions associated with cells that do not currently express CD19, for example, because they once expressed CD19, but whose expression has been downregulated, for example, by treatment with a CD19-targeting molecule, e.g., a CD19 CAR. In one embodiment, the cancer associated with CD19 expression is a blood cancer. In one embodiment, the blood cancer is leukemia or lymphoma. In one aspect, cancers associated with CD19 expression include, by way of example only, cancers and malignancies including, but not limited to, one or more acute leukemias, including but not limited to, for example, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL); one or more chronic leukemias, including but not limited to, for example, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL). Additional cancers or hematologic conditions associated with CD19 expression include, but are not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemia," a diverse collection of hematologic conditions united by ineffective production (or dysplasia) of bone marrow blood cells.Additional diseases associated with CD19 expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with CD19 expression. Non-cancer-related indications associated with CD19 expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, CD19-expressing cells express CD19 mRNA or have expressed it at any time. In some embodiments, CD19-expressing cells produce CD19 protein (e.g., wild-type or mutant), and the CD19 protein may be present at normal or reduced levels. In some embodiments, CD19-expressing cells produced detectable levels of CD19 protein at one time, and then substantially no detectable CD19 protein was produced thereafter.

[0169] The term "conservative sequence modification" refers to an amino acid modification that does not substantially affect or change the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or antibody fragment of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CARs described herein can be replaced with other amino acid residues from the same side chain family, and the altered CARs can be tested using the functional assays described herein.

[0170] The term "stimulation" refers to a primary response elicited by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex or a CAR) to its cognate ligand (or tumor antigen in the case of a CAR), thereby mediating a signaling event, such as, but not limited to, signaling through the TCR / CD3 complex or signaling through the signaling domain of an appropriate NK receptor or CAR. Stimulation can mediate changes in the expression of certain molecules.

[0171] The term "stimulatory molecule" refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that provides cytoplasmic signaling sequences that regulate immune cell activation in a stimulatory manner for at least some aspects of immune cell signaling pathways. In one aspect, the signal is a primary signal initiated, for example, by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, resulting in mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. Primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that act in a stimulatory manner can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. Examples of cytoplasmic signaling sequences containing ITAMs that are particularly useful in the present invention include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In certain CARs of the invention, the intracellular signaling domain of any one or more CARs of the invention comprises an intracellular signaling sequence, e.g., the primary signaling sequence of CD3 zeta. In certain CARs of the invention, the primary signaling sequence of CD3 zeta is the sequence provided as SEQ ID NO: 9, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc. In certain CARs of the invention, the primary signaling sequence of CD3 zeta is the sequence provided as SEQ ID NO: 10, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.

[0172] The term "antigen-presenting cell" or "APC" refers to immune system cells, such as accessory cells (e.g., B cells, dendritic cells, etc.), that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0173] The term "intracellular signaling domain," as used herein, refers to the intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes immune effector function in a CAR-containing cell, e.g., a CART cell. For example, examples of immune effector function in a CART cell include cytolytic activity and helper activity, including cytokine secretion. In some embodiments, the intracellular signaling domain is a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits the effector function signal. The term intracellular signaling domain is therefore meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0174] In some embodiments, the intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary or antigen-dependent stimulation. In some embodiments, the intracellular signaling domain may comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain may comprise the cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain may comprise the cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0175] The primary intracellular signaling domain may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, CD278 ("ICOS"), FcεRI, CD66d, CD32, DAP10, and DAP12.

[0176] The terms "zeta" or alternatively "zeta chain," "CD3 zeta," or "TCR zeta" are identified as the protein provided under GenBank Accession No. BAG36664.1, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and a "zeta stimulatory domain" or alternatively "CD3 zeta stimulatory domain" or "TCR zeta stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit an early signal necessary for T cell activity. In one embodiment, the cytoplasmic domain of zeta comprises residues 52 to 164 of GenBank Accession No. BAG36664.1, or equivalent residues from a non-human species, e.g., its functional ortholog, mouse, rodent, monkey, ape, etc. In one embodiment, a "zeta stimulatory domain" or "CD3 zeta stimulatory domain" is the sequence provided as SEQ ID NO:9. In one embodiment, the "zeta stimulatory domain" or "CD3 zeta stimulatory domain" is the sequence provided as SEQ ID NO:10.

[0177] The term "costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7- H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD 49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT These include, but are not limited to, ligands that specifically bind to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83. The costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule.The intracellular signaling domain can include the entire intracellular portion or the entire native intracellular signaling domain of the molecule from which it is derived or a functional fragment thereof.

[0178] The term "4-1BB" refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and a "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc. In one embodiment, the "4-1BB costimulatory domain" is the sequence provided as SEQ ID NO: 7, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.

[0179] "Immune effector cells," as the term is used herein, refer to cells that are involved in an immune response, e.g., that are involved in promoting an immune effector response. Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloblast-derived phagocytes.

[0180] "Immune effector function or immune effector response," as used herein, refers to a function or response, e.g., a function or response of an immune effector cell, that enhances or promotes the immune attack of a target cell. For example, an immune effector function or response refers to a property of a T cell or NK cell that promotes the killing of a target cell or the inhibition of the growth or proliferation of a target cell. In the case of a T cell, primary stimulation and costimulation are examples of immune effector functions or responses.

[0181] The term "encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, e.g., a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers or macromolecules in biological processes that have either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence and have the biological properties conferred thereby. Thus, a gene, cDNA, or RNA encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of the gene or cDNA.

[0182] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" can also include introns, to the extent that the nucleotide sequence encoding the protein may contain introns in some forms.

[0183] The term "endogenous" refers to any material that originates from or is produced within an organism, cell, tissue, or system.

[0184] The term "exogenous" refers to any material that is introduced into or produced outside of an organism, cell, tissue, or system.

[0185] The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0186] The term "transfer vector" refers to a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes autonomously replicating plasmids or viruses. This term should also be considered to include non-plasmid or non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0187] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0188] The term "lentivirus" refers to a genus of the retrovirus family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and they can deliver significant amounts of genetic information into the DNA of host cells, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0189] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, including, inter alia, self-inactivating lentiviral vectors, such as those provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, but are not limited to, the LENTIVECTOR® gene delivery technology from Oxford BioMedica and the LENTIMAX™ vector system from Lentigen. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.

[0190] The term "homologous" or "identity" refers to the sequence identity of subunits between two polymer molecules, e.g., between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. Two molecules are homologous or identical at a subunit position if that position is occupied by the same monomer subunit in both molecules, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half the positions in two sequences (e.g., 5 positions in a polymer 10 subunits in length) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

[0191] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications allow for further refinement and optimization of antibody or antibody fragment performance. Generally, a humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or most of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0192] "Fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, in which the entire molecule is of human origin or consists of an amino acid sequence identical to a human form of an antibody or immunoglobulin.

[0193] The term "isolated" means changed or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide is "isolated" if it is partially or completely separated from the coexisting materials of its natural state. An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as a host cell.

[0194] In the context of the present invention, the following abbreviations are used for commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0195] The term "operably linked" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other, and, for example, when necessary to link two protein coding regions, can be in the same reading frame.

[0196] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, intratumoral, or infusion techniques.

[0197] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), as well as polymers thereof, in either single- or double-stranded form. The term "nucleic acid" includes a gene, cDNA, or mRNA. In one embodiment, a nucleic acid molecule is synthetic (e.g., chemically synthesized) or recombinant. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues [Batzer et al., Nucleic Acid Res. 19:5081 (1991), Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985), and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)].

[0198] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, but there is no limit regarding the maximum number of amino acids that may comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, as well as longer chains, of which there are numerous varieties and commonly referred to in the art as proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others. A polypeptide includes naturally occurring polypeptides, recombinant polypeptides, or combinations thereof.

[0199] The term "promoter" refers to a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

[0200] The term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be the core promoter sequence, and in other cases, this sequence may also include an enhancer sequence and other control elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0201] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0202] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0203] The term "tissue-specific" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes that gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0204] The terms "cancer-associated antigen" or "tumor antigen" refer interchangeably to a molecule (typically a protein, carbohydrate, or lipid) that is preferentially expressed, either in whole or as fragments (e.g., MHC / peptide), on the surface of cancer cells compared to normal cells and is useful for preferentially targeting pharmacological agents to cancer cells. In some embodiments, a cancer-associated antigen is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more, compared to normal cells. In some embodiments, a cancer-associated antigen is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule that contains a deletion, addition, or mutation compared to the molecule expressed in normal cells. In some embodiments, a cancer-associated antigen will be expressed only on the cell surface of cancer cells, either in whole or as fragments (e.g., MHC / peptide), and will not be synthesized or expressed on the surface of normal cells. In some embodiments, the CARs of the present invention include CARs that include an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presented peptide. Typically, peptides derived from endogenous proteins fill the pockets of major histocompatibility complex (MHC) class I molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In the case of cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy.TCR-like antibodies that target peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21) :1601-1608; Dao et al., Sci Transl Med 2013 5(176) :176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified by screening libraries such as human scFv phage display libraries.

[0205] The term "flexible polypeptide linker" or "linker" as used in the context of scFvs refers to a peptide linker composed of amino acids such as glycine and / or serine residues, used alone or in combination to link the heavy chain variable region and the light chain variable region together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n (SEQ ID NO:22), where n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5, and n=6, n=7, n=8, n=9, and n=10. In one embodiment, flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 (SEQ ID NO:27) or (Gly4Ser)3 (SEQ ID NO:28). In another embodiment, the linker comprises multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO:29). In one embodiment, the linker is GSTSGSGKPGSGEGSTKG (SEQ ID NO: 104). Also included within the scope of the present invention are linkers described in WO2012 / 138475, which is incorporated herein by reference.

[0206] As used herein, a 5' cap (also referred to as an RNA cap, RNA 7-methylguanosine cap, or RNA m7G cap) is a modified guanine nucleotide added to the "front" or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of a terminal group attached to the first transcribed nucleotide. Its presence is important for ribosome recognition and protection from RNases. Capping is coupled to transcription and occurs simultaneously with transcription in a reciprocal manner. Shortly after transcription initiation, the 5' end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzyme complex catalyzes the chemical reactions required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping moiety can be modified to modulate functions such as mRNA stability or translation efficiency.

[0207] As used herein, "in vitro transcribed RNA" refers to RNA, preferably mRNA, synthesized in vitro. Generally, in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector contains a template used to generate the in vitro transcribed RNA.

[0208] As used herein, "poly(A)" refers to a series of adenosines attached to mRNA by polyadenylation. In a preferred embodiment of a construct for transient expression, the poly(A) is 50 to 5,000 (SEQ ID NO: 30), preferably greater than 64, more preferably greater than 100, and most preferably 300 or 400. The poly(A) sequence may be chemically or enzymatically modified to regulate mRNA function, such as localization, stability, or translation efficiency.

[0209] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety, or modified variants thereof, to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at their 3' ends. The 3' poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to a pre-mRNA through the action of a polyadenylation polymerase enzyme. In higher eukaryotes, the poly(A) tail is added to transcripts containing a specific sequence, the polyadenylation signal. The poly(A) tail and its associated proteins help protect the mRNA from exonuclease degradation. Polyadenylation is also important for transcription termination, mRNA transport from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after DNA-to-RNA transcription, but can also occur later in the cytoplasm. After transcription is terminated, the mRNA strand is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

[0210] As used herein, "transient" refers to expression of a non-integrated transgene for a period of hours, days, or weeks, where the period of expression is shorter than the period of expression of the gene when integrated into the genome or contained within a stable plasmid replicon in the host cell.

[0211] Apheresis is the process of removing whole blood from an individual, separating it into selected components, and returning the remainder to the circulation. Generally, there are two methods for separating blood components: centrifugal and non-centrifugal. Leukapheresis involves the active selection and removal of a patient's white blood cells.

[0212] As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or alleviation of the progression, severity, and / or duration of a proliferative disorder, or the alleviation of one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder, resulting from the administration of one or more therapies (e.g., one or more therapeutic agents, such as a CAR of the invention). In specific embodiments, the terms "treat," "treatment," and "treating" refer to the alleviation of at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which is not necessarily discernible by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to the inhibition of progression of a proliferative disorder, either physical, e.g., by stabilization of a discernible symptom, physiological, e.g., by stabilization of a physical parameter, or both. In other embodiments, the terms "treat," "treatment," and "treating" refer to the reduction or stabilization of tumor size or cancer cell count. Treatment need not be 100%, and in some embodiments, a reduction or delay of at least one symptom of a disease or disorder by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% is sufficient to be considered within the scope of these terms.

[0213] As used herein, "refractory" refers to a disease, e.g., cancer, that does not respond to treatment. In some embodiments, a refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, a refractory cancer may become refractory during treatment.

[0214] The term "recurrence" as used herein refers to the reappearance of a disease (e.g., cancer) after an initial response period (e.g., complete response or partial response). The initial response period may be accompanied by a decrease in cancer cell levels below a certain threshold, e.g., below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Recurrence may be accompanied by an increase in cancer cell levels above a certain threshold, e.g., above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Recurrence can be identified using, for example, the Cheson criteria described in Cheson et al., J Clin Oncol 17:1244 (1999) and Cheson et al., "Revised Response Criteria for Malignant Lymphoma", J Clin Oncol 25:579-586 (2007), both of which are incorporated herein by reference in their entireties. For example, in the context of B-ALL, after a complete response, reappearance can involve the reappearance of blasts, for example, in the blood, bone marrow (greater than 5%), or any non-bone marrow site. A complete response, in this context, can involve less than 5% BM blasts. More generally, in some embodiments, a response (e.g., a complete response or a partial response) can involve the absence of detectable MRD (minimal residual disease). In some embodiments, the initial response period lasts for at least 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days; at least 1 week, 2 weeks, 3 weeks, or 4 weeks; at least 1 month, 2 months, 3 months, 4 months, 6 months, 8 months, 10 months, or 12 months; or at least 1 year, 2 years, 3 years, 4 years, or 5 years.

[0215] The term "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and complexes of molecules that can receive a signal and transmit the signal across the cell membrane.

[0216] The term "subject" is intended to include organisms in which an immune response is elicited (eg, mammals, humans).

[0217] The term "substantially purified" cells refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which the cells are normally associated in their natural state. In some cases, a population of substantially purified cells refers to a homogenous cell population. In other cases, the term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells have been cultured in vitro. In other aspects, the cells have not been cultured in vitro.

[0218] In the context of the present invention, a "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen that is common to a particular hyperproliferative disorder. In certain embodiments, the tumor antigen is derived from a cancer, including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma, such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc.

[0219] The terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary cell and its progeny.

[0220] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to its cognate binding partner (e.g., stimulatory and / or costimulatory molecule present on a T cell) protein present in a sample, but which does not substantially recognize or bind to other molecules in the sample.

[0221] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes those with 95%, 96%, 97%, 98%, or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This is also true with respect to the breadth of the range.

[0222] Treatment method In one aspect, the present disclosure provides methods for treating a disease associated with expression of a tumor antigen described herein.

[0223] In one aspect, the present disclosure provides a method of treating cancer (e.g., hematological cancers such as ALL and CLL) by providing to a subject in need thereof immune effector cells (e.g., T cells, NK cells) engineered to express a CAR, e.g., a CAR described herein. In one embodiment, the cancer being treated is a B-cell malignancy. In one embodiment, the cancer being treated is ALL (acute lymphoblastic leukemia), CLL (chronic lymphocytic leukemia), DLBCL (diffuse large B-cell lymphoma), MCL (mantle cell lymphoma), or MM (multiple myeloma).

[0224] In one aspect, the present disclosure provides a method of treating cancer (e.g., a hematological cancer such as ALL and CLL) by providing to a subject in need thereof immune effector cells (e.g., T cells, NK cells) engineered to express a CAR, e.g., a CAR described herein, e.g., a CD19 CAR, wherein the cancer cells express CD19. In one embodiment, the cancer being treated is a B-cell malignancy. In one embodiment, the cancer being treated is ALL (acute lymphoblastic leukemia), CLL (chronic lymphocytic leukemia), DLBCL (diffuse large B-cell lymphoma), MCL (mantle cell lymphoma), Hodgkin's lymphoma, or MM (multiple myeloma).

[0225] The present disclosure includes a type of cell therapy in which immune effector cells (e.g., T cells, NK cells) are genetically modified (e.g., by transduction with a lentiviral vector) to express a CAR, and the CAR-expressing cells are infused into a recipient in need thereof. The infused cells can kill tumor cells in the recipient. Unlike antibody therapy, CAR-modified immune effector cells (e.g., T cells, NK cells) are capable of replication in vivo, resulting in long-term persistence that can lead to sustained tumor control. CAR-expressing cells (e.g., T cells or NK cells) generated using lentiviral vectors will have stable CAR expression. In various embodiments, the immune effector cells (e.g., T cells, NK cells) or their progeny administered to a patient persist in the patient for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, or 5 years after administration of the T cells to the patient.

[0226] The present invention also includes a type of cell therapy in which immune effector cells (e.g., T cells, NK cells) are modified, for example, by in vitro transcribed RNA, to transiently express a CAR, and the CAR-expressing cells are infused into a recipient in need thereof. CAR-expressing cells (e.g., T cells, NK cells) generated by transduction of CAR RNA (e.g., by transfection or electroporation) transiently express the RNA CAR for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after transduction. The infused cells can kill tumor cells in the recipient. Thus, in various embodiments, the immune effector cells (e.g., T cells, NK cells) administered to a patient exist for less than one month, for example, less than three weeks, less than two weeks, or less than one week, after the T cells are administered to the patient.

[0227] In one embodiment, the present disclosure provides a method of treating cancer (e.g., hematological cancers such as ALL and CLL) by providing to a subject in need thereof immune effector cells (e.g., T cells, NK cells) engineered to express a CAR that specifically targets or binds to a tumor antigen (or cancer-associated antigen) described herein. In yet another embodiment, the method of treatment includes altering the production of CAR-expressing cells, e.g., to enrich for naive T cells as described herein.

[0228] In one embodiment, immune effector cells (e.g., T cells, NK cells) are engineered to express a CD19 CAR to treat a subject with cancer (e.g., a hematological cancer such as ALL and CLL), wherein the cancer cells express CD19. In one embodiment, the cancer being treated is ALL or CLL. The CD19 CAR molecule expressed in the immune effector cells can include any anti-CD19 antigen-binding domain in the art (e.g., those provided in Tables 1 or 4) in combination with any of the CAR domains described herein to generate a complete CAR construct. For example, the complete CAR construct is a CAR listed in Table 4. Table 4 provides exemplary complete CD19 CAR constructs generated using various CAR domains (e.g., transmembrane domains and intracellular signaling domains) described herein and the anti-CD19 antigen-binding domain listed in Tables 1 or 4. Amino acid sequences are designated (aa) and nucleic acid sequences are designated (nt).

[0229] In one aspect, the present disclosure provides a method for treating cancer, e.g., cancer associated with CD19 expression, using CAR-expressing cell (e.g., T cell, NK cell) therapy. Exemplary cancers include, but are not limited to, one or more acute leukemias, e.g., B-ALL, T-ALL, ALL, e.g., one or more chronic leukemias, e.g., chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), but are not limited to.

[0230] Additional cancers or hematological conditions that can be treated using the methods described herein include, but are not limited to, B-cell promyelocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemia," a diverse collection of hematological conditions united by ineffective production (or dysplasia) of bone marrow blood cells.

[0231] The aforementioned blood conditions may be associated with the expression of CD19. Furthermore, diseases associated with the expression of CD19 include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative disorders associated with the expression of CD19.

[0232] In one embodiment, the present disclosure provides a method for treating CLL.

[0233] In another embodiment, the present disclosure provides a method for treating ALL.

[0234] In another embodiment, the present disclosure provides a method for treating B-cell ALL.

[0235] In one aspect, the present disclosure provides a method of treating a subject with cancer (e.g., a hematological cancer such as ALL and CLL) with a CAR-expressing cell (e.g., a T cell, an NK cell) (e.g., a CD19 CAR-expressing cell (e.g., a T cell, an NK cell) described herein, such as, for example, CTL019). In an embodiment, the present disclosure provides a method of treating a subject with a CAR-expressing cell (e.g., a T cell, an NK cell) in combination with another therapeutic agent, e.g., another therapeutic agent described herein (e.g., another CAR, e.g., another CAR described herein, an inhibitory CAR, e.g., an inhibitory CAR described herein; chemotherapy; a kinase inhibitor (e.g., a kinase inhibitor described herein, e.g., an mTOR inhibitor, a BTK inhibitor), a checkpoint inhibitor, e.g., a checkpoint inhibitor described herein, a standard of care therapy, etc.). The combination can be, for example, with any agent described herein.

[0236] In some embodiments, the treatment of cancer in a subject comprises administering a CAR therapy described herein in combination with one or more chemotherapy cycles. In some embodiments, exemplary chemotherapy cycles include induction, consolidation, intermediate maintenance, late intensification, and maintenance.

[0237] In some embodiments, the first chemotherapy cycle is remission induction, where the goal is to eliminate as many cancerous cells as possible. In some embodiments, in a subject with leukemia (e.g., ALL), the remission induction cycle includes treatment with one or more drugs, such as vincristine, dexamethasone, and / or polyethylene glycol (PEG)-L-asparaginase. In an embodiment, in a subject with leukemia (e.g., ALL, e.g., standard-risk ALL), the remission induction cycle includes treatment with vincristine, dexamethasone, and PEG-L-asparaginase, for example, at 6 mg / m each. 2 , 6 mg / m 2 , and 2500 U / m 2In certain embodiments, in a subject with leukemia (e.g., ALL, e.g., high-risk ALL), a remission induction cycle includes treatment with vincristine, dexamethasone, PEG-L-asparaginase, and daunorubicin, e.g., at a dose of 6 mg / m each. 2 , 6 mg / m 2 , and 2500 U / m 2 , and 100 mg / m 2 This includes treatments using doses of

[0238] In some embodiments, the additional chemotherapy cycle is consolidation, where the goal is to destroy any remaining cancerous cells. In some embodiments, a consolidation cycle is administered to a subject after remission is achieved following an induction cycle. In some embodiments, in a subject with leukemia (e.g., ALL), the consolidation cycle includes treatment with one or more drugs, e.g., vincristine, 6-mercaptopurine, cyclophosphamide, cytarabine, and / or PEG-L-asparaginase. In an embodiment, in a subject with leukemia (e.g., ALL, e.g., standard-risk ALL), the consolidation cycle includes treatment with vincristine and 6-mercaptopurine, e.g., at 1.5 mg / m each. 2 , and 75 mg / m 2 In certain embodiments, in a subject with leukemia (e.g., ALL, e.g., high-risk ALL), a consolidation cycle includes treatment with vincristine, 6-mercaptopurine, cyclophosphamide, cytarabine, and PEG-L-asparaginase, e.g., at a dose of 1.5 mg / m each. 2 , 60 mg / m 2 , 1g / m 2 , 75 mg / m 2 , and 2500 mg / m 2 This includes treatments using doses of

[0239] In some embodiments, the additional chemotherapy cycles include maintenance cycles, where the goal is to reduce the long-term risk of cancer recurrence, or intermediate maintenance cycles, where the goal is to provide a break from intensive treatment. In some embodiments, the intermediate maintenance cycles are performed after induction, and in some cases, after consolidation. In some embodiments, the intermediate maintenance cycles are performed before the late intensification cycles. In some embodiments, the intermediate maintenance cycles are performed after the late intensification cycles. In some embodiments, the maintenance cycles are the last chemotherapy cycles, e.g., after induction, consolidation, intermediate maintenance, and / or late intensification. In some embodiments, in subjects with leukemia (e.g., ALL, e.g., standard-risk ALL), the intermediate maintenance cycles include vincristine and methotrexate, e.g., at 7.5 mg / m 2 each. 2 , and 500 mg / m 2 In some embodiments, in a subject with leukemia (e.g., ALL, e.g., high-risk ALL), an interim maintenance cycle includes treatment with vincristine, methotrexate, and 6-mercaptopurine, e.g., at a dose of 7.5 mg / m each. 2 , 20 mg / m 2 , and 25 mg / m 2 In some embodiments, in a subject with leukemia (e.g., ALL, e.g., standard-risk ALL), maintenance cycles include treatment with vincristine, dexamethasone, 6-mercaptopurine, and methotrexate, e.g., at a dose of 1.5 mg / m each. 2 , 6 mg / m 2 , 75 mg / m 2 , and 20 mg / m 2 In some embodiments, in a subject with leukemia (e.g., ALL, e.g., high-risk ALL), maintenance cycles include treatment with vincristine, prednisone, 6-mercaptopurine, and methotrexate, e.g., at a dose of 1.5 mg / m each. 2 , 40 mg / m 2 , 75 mg / m 2 , and 20 mg / m2 This includes treatments using doses of

[0240] In some embodiments, the additional chemotherapy cycles include a late intensification cycle, where the goal is to eliminate any remaining cancerous cells. In some embodiments, the late intensification cycle is performed after the consolidation cycle and, for example, after an interim maintenance cycle. In some embodiments, in a subject with leukemia (e.g., ALL, e.g., standard-risk ALL), the late intensification cycle includes vincristine, dexamethasone, doxorubicin, PEG-L-asparaginase, cyclophosphamide, cytarabine, and 6-thioguanine, for example, at 4.5 mg / m each. 2 , 10 mg / m 2 , 75 mg / m 2 , 2500U / m 2 , 1g / m 2 , 60 mg / m 2 , and 60 mg / m 2 In some embodiments, in a subject with leukemia (e.g., ALL, e.g., high-risk ALL), a post-consolidation cycle includes treatment with vincristine, dexamethasone, doxorubicin, PEG-L-asparaginase, cyclophosphamide, cytarabine, and 6-thioguanine, e.g., at a dose of 7.5 mg / m each. 2 , 10 mg / m 2 , 75 mg / m 2 , 5000U / m 2 , 1g / m 2 , 60 mg / m 2 , and 60 mg / m 2 This includes treatments using doses of

[0241] In some embodiments, subjects are treated with chemotherapy regimens of varying intensity based on risk measures, e.g., to maximize survival while minimizing toxicity. In some embodiments, for example, subjects with ALL (e.g., childhood ALL) are classified into standard-risk or high-risk groups based on white blood cell count and age, e.g., the National Cancer Institute (NCI) risk group classification. See, e.g., Smith et al. J. Clin. Oncol. 14.1(1996):18-24. For example, a subject is classified into the standard-risk group if the white blood cell count is less than 50,000 cells per microliter and the subject is between 1 and 10 years old. For example, a subject is classified into the high-risk group if the white blood cell count is more than 50,000 cells per microliter and / or the subject is 10 years old or older. In some embodiments, for example, subjects with ALL (e.g., childhood ALL) are classified into a very high-risk group, as described in Moeriche et al. Blood 111.9 (2008): 4477-89 and Pui et al. Blood 92.2 (1998): 411-5. For example, ALL subjects with one or more of the following characteristics are classified as very high-risk ALL: subjects who are infants; subjects with harmful chromosomal abnormalities (e.g., t(9;22), MLL gene rearrangement, and hypodiploidy (chromosome number < 44); subjects with a slow initial response to initial therapy; subjects with high minimal residual disease (MRD) levels at the end of the remission induction cycle or in subsequent cycles; subjects with morphologically persistent disease after the remission induction cycle.

[0242] In certain embodiments, the standard of care for CLL includes, but is not limited to, the exemplary treatments described herein, such as those described in Table 5, and combinations thereof.

[0243] [Table 1]

[0244] In some embodiments, standard care for CLL includes (1) radiation therapy, (2) chemotherapy, (3) surgery (e.g., removal of the spleen), (4) targeted therapy, (5) stem cell transplant, and combinations thereof. In some embodiments, standard care includes external beam radiation therapy. In some embodiments, standard care includes internal radiation therapy (e.g., radioactive material enclosed in needles, wires, or catheters that are placed directly into or near the cancer, for example).

[0245] In certain embodiments, the standard of care for ALL includes, but is not limited to, the exemplary treatments described herein, such as those described in Table 6, and combinations thereof.

[0246] [Table 2]

[0247] In one embodiment, the standard of care for ALL includes: (1) chemotherapy, (2) radiation therapy, (3) stem cell transplant, (4) biotherapy, (5) targeted therapy, and combinations thereof.

[0248] In certain embodiments, standard of care includes, but is not limited to, fludarabine and cyclophosphamide (FC); fludarabine and rituximab (FR); fludarabine, cyclophosphamide, and rituximab (FCR); cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP); and combinations thereof. Common chemotherapy drugs that may be used include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), and cytarabine liposome injection (D epoCyt®), dacarbazine [DTIC-Dome®], dactinomycin (actinomycin D, Cosmegan), daunorubicin hydrochloride [Cerubidine®], daunorubicin citrate liposomal injection [DaunoXome®], dexamethasone, docetaxel [Taxotere®], doxorubicin hydrochloride [Adriamycin®, Rubex®], etoposide tetracycline (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®),Irinotecan [Camptosar®], L-asparaginase [ELSPAR®], leucovorin calcium, melphalan [Alkeran®], 6-mercaptopurine [Purinethol®], methotrexate [Folex®], mitoxantrone [Novantrone®], mylotarg, paclitaxel [Taxol®], phoenix (yttrium-90 / MX-DTPA), pentostatin, carmustine These include, but are not limited to, polifeprosan 20 with steroid implant [Gliadel®], tamoxifen citrate [Nolvadex®], teniposide [Vumon®], 6-thioguanine, thiotepa, tirapazamine [Tirazone®], injectable topotecan hydrochloride [Hycamptin®], vinblastine [Velban®], vincristine [Oncovin®], vinorelbine [Navelbine®], and combinations thereof.

[0249] In certain embodiments, chemotherapy is administered using a combination of folate antagonists (also referred to herein as antifolates), pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors: methotrexate [Rheumatrex®, Trexall®], 5-fluorouracil [Adrucil®, Efudex®, Fluoroplex®], floxuridine [FUDF®], cytarabine [Cytosar-U®, Tarabine PFS], 6-mercaptopurine [Puri-Nethol®], 6-thioguanine [Thioguanine Tabloid®), fludarabine phosphate [Fludara®], pentostatin [Nipent®], pemetrexed [Alimta®], raltitrexed [Tomudex®], cladribine [Leustatin®], clofarabine [Clofarex®, Clolar®], cytarabine liposome (Cytarabine liposomal) [liposomal Ara-C, also known as DepoCyt™], decitabine [Dacogen™]; hydroxyurea [Hydrea™, Droxia™, and Mylocel™]; mercaptopurine [Puri-Nethol™], pralatrexate [Folotyn™]; capecitabine [Xeloda™], nelarabine [Arranon™], azacitidine [Vidaza™], and gemcitabine [Gemzar™]. Preferred antimetabolites include, for example, 5-fluorouracil (Adrucil®, Efudex®, Fluoroplex®), floxuridine (FUDF®), capecitabine (Xeloda®), pemetrexed (Alimta®), raltitrexed (Tomudex®), and gemcitabine (Gemzar®), and combinations thereof. In one embodiment, the purine analog is fludarabine.

[0250] In some embodiments, the chemotherapy includes any of the following: nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas and triazenes, uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard, etc.) mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine [Mustargen®], cyclophosphamide [Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune®], ifosfamide [Mitoxana®], melphalan [Alkeran®], chlorambucil [Leukeran®], pipobroman [Amedel®] ), Vercyte®)], triethylenemelamine [Hemel®, Hexalen®, Hexastat®], triethylenethiophosphoramine, temozolomide [Temodar®], thiotepa [Thioplex®], busulfan [Busilvex®, Myleran®], carmustine [BiCNU®], lomustine [CeeNU®], streptozocin [Zanosar®], and dacarbazine [DTIC-Dome®], and combinations thereof.Further exemplary alkylating agents include, without limitation, oxaliplatin (Eloxatin®), temozolomide (Temodar® and Temodal®), dactinomycin (actinomycin-D, also known as Cosmegen®), melphalan (L-PAM, L-sarcolysin, and phenylalanine mustard, also known as Alkeran®), altretamine (hexamethylmelamine (HMM), also known as Hexalen®), ], carmustine [BiCNU®], bendamustine [Treanda®], busulfan [Busulfex® and Myleran®], carboplatin [Paraplatin®], lomustine [also known as CCNU, CeeNU®], cisplatin [also known as CDDP, Platinol®, and Platinol®-AQ], chlorambucil [Leukeran®], cyclophosphamide [Cytoxan®], and Neosar®), dacarbazine [DTIC, DIC, and imidazole carboxamide, also known as DTIC-Dome®], altretamine [hexamethylmelamine (HMM), also known as Hexalen®], ifosfamide [Ifex®], prednumustine, procarbazine [Matulane®], mechlorethamine [nitrogen mustard, mustine, and mechlorethamine hydrochloride, Musta®], In one embodiment, the alkylating agent is bendamustine HCl, and combinations thereof.In certain embodiments, the alkylating agent is cyclophosphamide.

[0251] In certain embodiments, the chemotherapeutic agent is a kinase inhibitor, for example, erlotinib hydrochloride (Tarceva®), linifanib (N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N'-(2-fluoro-5-methylphenyl)urea], also known as ABT 869 and available from Genentech), sunitinib malate (Sutent®), bosutinib (4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methylphenyl)urea], also known as SKI-606 and described in U.S. Pat. No. 6,780,996). and tyrosine kinase inhibitors, including, but not limited to, ibrutinib (Piperazin-1-yl)propoxy]quinoline-3-carbonitrile), dasatinib (Sprycel®), pazopanib (Votrient®), sorafenib (Nexavar®), zactima (ZD6474), and imatinib or imatinib mesylate (Gilvec® and Gleevec®). In one embodiment, the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765), GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, and LFM-A13.In one embodiment, the kinase inhibitor is aloisine A, flavopiridol or HMR-1275, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone, crizotinib (PF-02341066, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one, hydrochloride (P276-00), 1-methyl-5-[[2- [5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine (RAF265), indisulam (E7070), roscovitine (CYC202), palbociclib (PD0332991), dinaciclib (SCH727965), N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide (BMS 387032), 4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoic acid (MLN8054), 5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine (AG-024322), 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid N-(piperidin-4-yl)amide (AT7519), 4-[2-methyl-1-(1-methylethyl) In one embodiment, the kinase inhibitor is a CDK4 inhibitor selected from CGP052088, 4-amino-3-(p-fluorophenylamino)-pyrazolo[3,4-d]pyrimidine (CGP57380), cercosporamide, ETC-1780445-2, and 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine.

[0252] In certain embodiments, targeted therapies include, but are not limited to, anti-CD20 antibodies or functional fragments thereof, such as rituximab (Riuxan® and MabThera®), tositumomab (Bexxar®), and ofatumumab (Arzerra®), and combinations thereof. In one embodiment, targeted therapies include, but are not limited to, anti-CD52 antibodies or functional fragments thereof, such as alemtuzumab (Campath®).

[0253] In certain embodiments, the biotherapy comprises immunotherapy. Exemplary anthracyclines include, without limitation, doxorubicin (Adriamycin® and Rubex®), bleomycin (lenoxane®), daunorubicin (daunorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®), daunorubicin liposome (daunorubicin citrate liposome, DaunoXome®), mitoxantrone (DHAD, Novantrone®), epirubicin (Ellence™), idarubicin (Idamycin®, Idamycin PFS®), mitomycin C (Mutamycin®), geldanamycin, herbimycin, rabidomycin, desacetylrabidomycin, and combinations thereof.

[0254] In some embodiments, the stem cell transplantation comprises autogeneic stem cell transplantation. In some embodiments, the stem cell transplantation comprises allogeneic stem cell transplantation. In some embodiments, the stem cell transplantation comprises allogeneic bone marrow transplantation. In some embodiments, the stem cell transplantation comprises hematopoietic stem cell transplantation (HSCT). In some embodiments, the hematopoietic stem cells are derived from various tissues, including, but not limited to, bone marrow, peripheral blood, umbilical cord blood, and combinations thereof.

[0255] In one aspect, the present disclosure provides a method for treating a disease associated with CD19 expression. In one aspect, the present invention provides a method for treating a disease in which a portion of the tumor is negative for CD19 and a portion of the tumor is positive for CD19. For example, the provided method is useful for treating a subject who has been treated for a disease associated with increased expression of CD19, wherein the subject who has been treated for elevated CD19 levels exhibits a disease associated with elevated CD19 levels.

[0256] In one embodiment, the methods provided include a vector comprising a CD19 CAR operably linked to a promoter for expression in mammalian cells (e.g., T cells or NK cells). In one embodiment, the methods provided include recombinant cells (e.g., T cells or NK cells) expressing a CD19 CAR for use in treating CD19-expressing tumors, wherein the recombinant T cells expressing a CD19 CAR are referred to as CD19 CAR-expressing cells. In one embodiment, the CD19 CAR-expressing cells (e.g., T cells, NK cells) administered according to the methods provided can contact tumor cells with at least one CD19 CAR expressed on the surface of the CD19 CAR-expressing cells, such that the CAR-expressing cells target the tumor cells and tumor growth is inhibited.

[0257] In one aspect, the disclosure features a method of inhibiting the growth of a tumor cell that expresses CD19, the method including contacting the tumor cell with a CD19 CAR-expressing cell (e.g., a T cell, an NK cell) described herein, such that the CAR-expressing cell is activated in response to an antigen and targets the cancer cell, and tumor growth is inhibited.

[0258] In one aspect, the present disclosure includes a type of cell therapy in which T cells are genetically modified to express a CAR, and the CAR-expressing cells (e.g., T cells, NK cells) are infused into a recipient in need thereof. The infused cells can kill tumor cells in the recipient. Unlike antibody therapy, CAR-modified cells (e.g., T cells or NK cells) are capable of replication in vivo, providing long-term persistence that can lead to sustained tumor control. In various aspects, the cells administered to a patient or their progeny persist in the patient for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, or 5 years after the cells are administered to the patient.

[0259] The present disclosure also includes a type of cell therapy in which cells (e.g., T cells, NK cells) are modified, e.g., by in vitro transcribed RNA, to transiently express a chimeric antigen receptor (CAR), and the CAR-expressing cells (e.g., T cells, NK cells) are infused into a recipient in need thereof. The infused cells can kill tumor cells in the recipient. Thus, in various embodiments, the cells administered to a patient are present for less than one month, e.g., less than three weeks, less than two weeks, or less than one week, after the cells (e.g., T cells, NK cells) are administered to the patient.

[0260] Without wishing to be bound by any particular theory, the anti-tumor immune response induced by CAR-modified cells (e.g., T cells, NK cells) can be an active or passive immune response, or alternatively, can result from a direct versus indirect immune response. In one embodiment, CAR-transduced T cells respond to CD19-expressing human cancer cells by exhibiting specific pro-inflammatory cytokine secretion and potent cytolytic activity, resisting soluble CD19 inhibition, mediating bystander killing, and mediating the regression of established human tumors. For example, antigen-free tumor cells within heterogeneous regions of CD19-expressing tumors may be susceptible to indirect destruction by CD19-redirected T cells that previously reacted against adjacent antigen-positive cancer cells.

[0261] In one embodiment, the fully human CAR-modified cells (e.g., T cells, NK cells) described herein can be a type of vaccine for ex vivo immunization and / or in vivo therapy in a mammal. In one embodiment, the mammal is a human.

[0262] For ex vivo immunization, at least one of the following occurs prior to administering the cells to a subject: i) expansion of the cells, ii) introduction of a nucleic acid encoding a CAR into the cells, or iii) cryopreservation of the cells.

[0263] Ex vivo procedures are known in the art and are described in more detail below. Briefly, cells are isolated from a subject (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to obtain therapeutic benefits. The mammalian recipient can be a human, and the CAR-modified cells can be autologous to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic to the recipient.

[0264] blood cancer Hematological cancer conditions are types of cancer that affect the blood, bone marrow, and lymphatic system, such as leukemia and malignant lymphoproliferative conditions.

[0265] Leukemia can be classified as acute leukemia and chronic leukemia. Acute leukemia can be further classified as acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). Chronic leukemia includes chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). Other related conditions include myelodysplastic syndromes (MDS, formerly known as "preleukemias"), a diverse collection of blood conditions united by ineffective production (or dysplasia) of bone marrow blood cells and the risk of transformation to AML.

[0266] The present disclosure provides compositions and methods for treating cancer. In one embodiment, the cancer is a blood cancer, including but not limited to, leukemia or lymphoma. In one embodiment, the CAR-expressing cells (e.g., T cells, NK cells) of the present invention can be used to treat, for example, acute leukemia, including but not limited to, B-ALL, T-ALL, ALL, one or more chronic leukemias, including but not limited to, CML, CLL, for example, B-cell promyelocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, It can be used to treat cancers and malignancies such as mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and additional blood cancers or conditions including, but not limited to, "preleukemia," a diverse collection of blood conditions united by, for example, ineffective production (or dysplasia) of bone marrow blood cells.

[0267] The present disclosure also provides a method for inhibiting or reducing the proliferation of a CD19-expressing cell population, comprising contacting a cell population comprising CD19-expressing cells with a CD19 CAR-expressing cell (e.g., T cell, NK cell) described herein that binds to the CD19-expressing cells. In a specific aspect, the present disclosure provides a method for inhibiting or reducing the proliferation of a CD19-expressing cancer cell population, comprising contacting the CD19-expressing cancer cell population with a CD19 CAR-expressing cell (e.g., T cell, NK cell) described herein that binds to the CD19-expressing cells. In one aspect, the present disclosure provides a method for inhibiting or reducing the proliferation of a CD19-expressing cancer cell population, comprising contacting the CD19-expressing cancer cell population with a CD19 CAR-expressing cell (e.g., T cell, NK cell) described herein that binds to the CD19-expressing cells. In certain embodiments, the anti-CD19 CAR-expressing cells (e.g., T cells, NK cells) reduce the quantity, number, amount, or proportion of cells and / or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in a subject with, or an animal model of, myeloid leukemia or another cancer associated with CD19-expressing cells compared to a negative control. In one embodiment, the subject is a human.

[0268] The present disclosure also provides a method for preventing, treating, and / or managing a disease associated with CD19-expressing cells (e.g., a hematological cancer or a variant cancer that expresses CD19), comprising administering to a subject in need thereof a CAR-expressing cell (e.g., a T cell, an NK cell) described herein that binds to the CD19-expressing cell. In one embodiment, the subject is a human. Non-limiting examples of disorders associated with CD19-expressing cells include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma), and cancer (hematological cancer or a variant cancer that expresses CD19).

[0269] The present disclosure also provides a method for preventing, treating, and / or managing a disease associated with CD19-expressing cells, comprising administering to a subject in need thereof a CD19 CAR-expressing cell (e.g., a T cell, an NK cell) described herein that binds to a CD19-expressing cell. In one embodiment, the subject is a human.

[0270] The present disclosure provides a method for preventing the recurrence of a cancer associated with CD19-expressing cells (e.g., a hematological cancer such as ALL and CLL), comprising administering to a subject in need thereof a CD19 CAR-expressing cell (e.g., T cell, NK cell) described herein that binds to the CD19-expressing cell. In one embodiment, the method comprises administering to a subject in need thereof an effective amount of a CD19 CAR-expressing cell (e.g., T cell, NK cell) described herein that binds to the CD19-expressing cell in combination with an effective amount of another therapeutic agent.

[0271] Combination Therapy It is understood that any cancer therapy described above and herein may be administered in combination with one or more additional therapeutic agents to treat and / or reduce the symptoms of cancer described herein. Pharmaceutical compositions may be administered simultaneously with, before, or after one or more other additional therapeutic agents or therapeutic agents. In certain embodiments, the CAR-expressing cells described herein can be used in combination with other known drugs and therapeutic agents. Administered "in combination," as used herein, means that two (or more) different therapies are delivered to a subject during the course of suffering associated with the subject's disorder, e.g., two or more therapies are delivered after the subject is diagnosed with a disorder and before the disorder is cured or eliminated, or before treatment is terminated for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so there is an overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In either case, in some embodiments, the treatments are more effective due to the combined administration. For example, the second treatment may be more effective, for example, when the second treatment is administered without the first treatment, or when a similar situation occurs with the first treatment, a smaller amount of the second treatment may be administered to achieve the same effect, or the second treatment may reduce symptoms to a greater extent. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed when one treatment is delivered in the absence of the other. The effects of the two treatments may be partially additive, entirely additive, or greater than additive. The delivery may be such that the effect of the first treatment delivered remains detectable when the second treatment is delivered.

[0272] The CAR-expressing cells described herein and at least one additional therapeutic agent may be administered simultaneously in the same or separate compositions, or may be administered sequentially. For sequential administration, the CAR-expressing cells described herein may be administered first and the additional agent may be administered second, or the order of administration may be reversed.

[0273] In further embodiments, the CAR-expressing cells described herein can be used in treatment regimens in combination with surgery, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies or other immunoadsorbents such as CAMPATH, anti-CD3 antibodies or other antibody therapeutics, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation peptide vaccines such as those described in Izumoto et al. 2008 J NEUROSURG 108:963-971.

[0274] In one embodiment, the CAR-expressing cells described herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., bendamustine, cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzumab, gemtuzumab, rituximab, tositumomab), antimetabolites (e.g., levothyroxine ... acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine)], mTOR inhibitors, TNFR glucocorticoid-inducible TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclacinomycin A, gliotoxin, or bortezomib), immunomodulatory agents such as thalidomide or thalidomide derivatives (e.g., lenalidomide), and combinations thereof.

[0275] Common chemotherapeutic agents that may be considered for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentaoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), and cyclosporine (Cyclosporin®). trademarks)], chlorambucil [Leukeran®], cisplatin [Platinol®], cladribine [Leustatin®], cyclophosphamide [Cytoxan® or Neosar®], cytarabine, cytosine arabinoside [Cytosar-U®], cytarabine liposome injection [DepoCyt®], dacarbazine [DTIC-Dome®], dactinomycin (actinomycin D, Cosmegan), daunorubicin hydrochloride [Cer ubidine (registered trademark)], daunorubicin citrate liposomal injection [DaunoXome (registered trademark)], dexamethasone, docetaxel [Taxotere (registered trademark)], doxorubicin hydrochloride [Adriamycin (registered trademark), Rubex (registered trademark)], etoposide [Vepesid (registered trademark)], fludarabine phosphate [Fludara (registered trademark)], 5-fluorouracil [Adrucil (registered trademark), Efudex (registered trademark)], flutamide [Eulexin (registered trademark)], tezacitibine, gemcitabine (Dif fluorodeoxycytidine), hydroxyurea [Hydrea®], idarubicin [Idamycin®], ifosfamide [IFEX®], irinotecan [Camptosar®], L-asparaginase [ELSPAR®], leucovorin calcium, melphalan [Alkeran®], 6-mercaptopurine [Purinethol®], methotrexate [Folex®], mitoxantrone [Novantrone®], Mylotarg,Paclitaxel [Taxol®], Phoenix (Yttrium 90 / MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant [Gliadel®], tamoxifen citrate [Nolvadex®], teniposide [Vumon®], 6-thioguanine, thiotepa, tirapazamine [Tirazone®], injectable topotecan hydrochloride [Hycamptin®], vinblastine [Velban®], vincristine [Oncovin®], and vinorelbine [Navelbine®].

[0276] Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas and triazenes, uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard, etc.). mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine [Mustargen®], cyclophosphamide [Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune®], ifosfamide [Mitoxana®], melphalan [Alkeran®], chlorambucil [Leukeran®], pipobroman [Am edel®, Vercyte®), triethylenemelamine [Hemel®, Hexalen®, Hexastat®], triethylenethiophosphoramine, temozolomide [Temodar®], thiotepa [Thioplex®], busulfan [Busilvex®, Myleran®], carmustine [BiCNU®], lomustine [CeeNU®], streptozocin [Zanosar®], and dacarbazine [DTIC-Dome®], and combinations thereof.Further exemplary alkylating agents include, without limitation, oxaliplatin (Eloxatin®), temozolomide (Temodar® and Temodal®), dactinomycin (actinomycin-D, also known as Cosmegen®), melphalan (L-PAM, L-sarcolysin, and phenylalanine mustard, also known as Alkeran®), altretamine (hexamethylmelamine (HMM), also known as Hexalen®), ], carmustine [BiCNU®], bendamustine [Treanda®], busulfan [Busulfex® and Myleran®], carboplatin [Paraplatin®], lomustine [also known as CCNU, CeeNU®], cisplatin [also known as CDDP, Platinol®, and Platinol®-AQ], chlorambucil [Leukeran®], cyclophosphamide [Cytoxan®], and Neosar®), dacarbazine [DTIC, DIC, and imidazole carboxamide, also known as DTIC-Dome®], altretamine [hexamethylmelamine (HMM), also known as Hexalen®], ifosfamide [Ifex®], prednumustine, procarbazine [Matulane®], mechlorethamine [nitrogen mustard, mustine, and mechlorethamine hydrochloride, Musta®], rgen®], streptozocin [Zanosar®], thiotepa [thiophosphoamide, TESPA, and TSPA, also known as Thioplex®], cyclophosphamide [Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®], bendamustine HCl [Treanda®], and combinations thereof.

[0277] Exemplary mTOR inhibitors include, without limitation, RAD001, temsirolimus, ridaforolimus [formerly known as deferolimus, (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0] 4,9 ]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03 / 064383], everolimus [Afinitor® or RAD001], rapamycin [AY22989, Sirolimus®], simapimod (CAS 164301-51-3), emsirolimus, (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055), 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4), and N 2 -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine- (SEQ ID NO: 356), inner salt (SF1126, CAS 936487-67-1), XL765, and combinations thereof.

[0278] Exemplary immunomodulatory agents include, without limitation, afutuzumab (available from Roche®), pegfilgrastim (Neulasta®), lenalidomide (CC-5013, Revlimid®), thalidomide (Thalomid®), actimid (CC4047), IRX-2 (a mixture of human cytokines including interleukin-1, interleukin-2, and interferon-gamma, CAS 951209-71-5, available from IRX Therapeutics), and combinations thereof.

[0279] Exemplary anthracyclines include, without limitation, doxorubicin [Adriamycin® and Rubex®], bleomycin [lenoxane®], daunorubicin [daunorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®], daunorubicin liposome [daunorubicin citrate liposome, DaunoXome®], mitoxantrone [DHAD, Novantrone®], epirubicin [Ellence™], idarubicin [Idamycin®, Idamycin PFS®], mitomycin C [Mutamycin®], geldanamycin, herbimycin, rabidomycin, desacetylrabidomycin, and combinations thereof.

[0280] Exemplary vinca alkaloids include, without limitation, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®), vindesine (Eldisine®), vinblastine (also known as vinblastine sulfate, vincaleukoblastine, and VLB, Alkaban-AQ® and Velban®), vinorelbine (Navelbine®), and combinations thereof.

[0281] Exemplary proteosome inhibitors include, without limitation, bortezomib [Velcade®], carfilzomib [PX-171-007, (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutane amide)-pentanamide], marizomib (NPI-0052), ixazomib citrate (MLN-9708), delanzomib (CEP-18770), O-methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912), and combinations thereof.

[0282] Exemplary GITR agonists include, without limitation, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as those described in U.S. Pat. No. 6,111,090, European Patent No. 090505B1, U.S. Pat. No. 8,586,023, PCT Publication Nos. WO 2010 / 003118 and 2011 / 090754, or those described in, for example, U.S. Pat. No. 7,025,962, European Patent No. 1947183B1, U.S. Pat. No. 7,812,135, U.S. Pat. No. 8,388,967, U.S. Pat. No. 8,591,886, European Patent No. EP 1866 339, PCT Publication No. WO2011 / 028683, PCT Publication No. WO2013 / 039954, PCT Publication No. WO2005 / 007190, PCT Publication No. WO2007 / 133822, PCT Publication No. WO2005 / 055808, PCT Publication No. WO99 / 40196, PCT Publication No. WO2001 / 03720, PCT Publication No. WO99 / 20758, PCT Publication No. WO2006 / 083289, PCT Publication No. WO2005 / 115451, U.S. Pat. No. 7,618,632, and PCT Publication No. WO2011 / 051726.

[0283] In some embodiments, a CAR-expressing cell described herein, e.g., a CD19 CAR-expressing cell (e.g., T cell, NK cell), such as, e.g., CTL019, is administered to a subject, e.g., a subject identified as a partial responder or non-responder, in combination with an mTOR inhibitor, such as RAD001, which is a target of the rapamycin signaling pathway, e.g., an mTOR inhibitor described herein. In some embodiments, the mTOR inhibitor is administered before the CAR-expressing cell. For example, in some embodiments, the mTOR inhibitor may be administered before apheresis of the cells. In some embodiments, the subject has cancer (e.g., a hematological cancer such as ALL and CLL). In some embodiments, the subject has ALL. In some embodiments, the subject has CLL.

[0284] In some embodiments, a CAR-expressing cell described herein, e.g., a CD19 CAR-expressing cell (e.g., T cell, NK cell), such as CTL019, is administered in combination with a GITR agonist, e.g., a GITR agonist described herein, to a subject, e.g., a subject identified as a partial responder or non-responder. In some embodiments, the GITR agonist is administered before the CAR-expressing cell. For example, in some embodiments, the GITR agonist may be administered before apheresis of the cells. In some embodiments, the subject has cancer (e.g., a hematological cancer such as ALL and CLL). In some embodiments, the subject has ALL. In some embodiments, the subject has CLL.

[0285] Kinase inhibitors In one embodiment, the CAR-expressing cells described herein can be used in a treatment regimen in combination with a kinase inhibitor, e.g., a CDK4 inhibitor, a BTK inhibitor, an MNK inhibitor, an mTOR inhibitor, an ITK inhibitor, etc. In one embodiment, the subject is a complete responder, and the subject is administered a treatment regimen comprising administration of a CAR-expressing cell described herein in combination with a kinase inhibitor, e.g., a kinase inhibitor described herein, e.g., at a dose or dosing schedule described herein. In one embodiment, the subject is a partial responder or non-responder, and the subject is administered a treatment regimen comprising administration of a CAR-expressing cell described herein in combination with a kinase inhibitor, e.g., a kinase inhibitor described herein, e.g., at a dose or dosing schedule described herein.

[0286] In some embodiments, the kinase inhibitor is a CDK4 inhibitor, such as a CDK4 inhibitor described herein, for example, a CDK4 / 6 inhibitor, such as 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one, hydrochloride (also referred to as palbociclib or PD0332991). In one embodiment, the kinase inhibitor is a BTK inhibitor, such as a BTK inhibitor described herein, for example, ibrutinib. In one embodiment, the kinase inhibitor is an mTOR inhibitor, such as an mTOR inhibitor described herein, for example, rapamycin, a rapamycin analog, OSI-027, or the like. The mTOR inhibitor can be, for example, an mTORC1 inhibitor and / or an mTORC2 inhibitor, such as an mTORC1 inhibitor and / or an mTORC2 inhibitor described herein. In one embodiment, the kinase inhibitor is an MNK inhibitor, such as an MNK inhibitor described herein, such as 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine. The MNK inhibitor can be, for example, an MNK1a, MNK1b, MNK2a, and / or MNK2b inhibitor.

[0287] In one embodiment, the kinase inhibitor is aloisine A, flavopiridol or HMR-1275, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone, crizotinib (PF-02341066, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one, hydrochloride (P276-00), 1-methyl-5-[[2- [5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine (RAF265), indisulam (E7070), roscovitine (CYC202), palbociclib (PD0332991), dinaciclib (SCH727965), N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide (BMS 387032), 4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoic acid (MLN8054), 5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine (AG-024322), 4-(2, 6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid N-(piperidin-4-yl)amide (AT7519), 4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine (AZD5438), and XL281 (BMS908662).

[0288] In one embodiment, the kinase inhibitor is a CDK4 inhibitor, e.g., palbociclib (PD0332991), and palbociclib is administered at a dose of about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g., 75 mg, 100 mg, or 125 mg) daily for a period of time, e.g., daily for 14 to 21 days of a 28-day cycle, daily for 7 to 12 days of a 21-day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more cycles of palbociclib are administered.

[0289] In one embodiment, the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765), GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, and LFM-A13.

[0290] In one embodiment, the kinase inhibitor is a BTK inhibitor, e.g., ibrutinib (PCI-32765), and ibrutinib is administered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g., 250 mg, 420 mg, or 560 mg) daily for a period of time, e.g., daily for a 21-day cycle or daily for a 28-day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more cycles of ibrutinib are administered.

[0291] In one embodiment, the kinase inhibitor is (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0], also known as temsirolimus, ridaforolimus, AP23573, and MK8669. 4,9 ]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, everolimus (RAD001), rapamycin (AY22989), simapimod, (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055), 2-mmino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502), N 2 -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine- (SEQ ID NO: 356), inner salt (SF1126), and XL765.

[0292] In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., rapamycin, and the rapamycin is administered at a dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg) daily for a period of time, e.g., daily for a 21-day cycle or daily for a 28-day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more cycles of rapamycin are administered. In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., everolimus, and the everolimus is administered at a dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (e.g., 10 mg) daily for a period of time, e.g., daily for a 28-day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more cycles of everolimus are administered.

[0293] In one embodiment, the kinase inhibitor is an MNK inhibitor selected from CGP052088, 4-amino-3-(p-fluorophenylamino)-pyrazolo[3,4-d]pyrimidine (CGP57380), cercosporamide, ETC-1780445-2, and 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine.

[0294] Combination with low-dose mTOR inhibitors The methods described herein can use low, immune-enhancing doses of mTOR inhibitors, such as allosteric mTOR inhibitors, including rapalogs such as RAD001. Administration of low, immune-enhancing doses of mTOR inhibitors (e.g., doses that are insufficient to completely suppress the immune system but sufficient to improve immune function) can optimize the performance of immune effector cells, such as T cells or CAR-expressing cells, in a subject. Methods, dosages, treatment regimens, and suitable pharmaceutical compositions for measuring mTOR inhibition are described in U.S. Patent Application No. 2015 / 0140036, filed November 13, 2014, and incorporated herein by reference.

[0295] In certain embodiments, administration of a low immune enhancing dose of an mTOR inhibitor may be achieved by: i) a decrease in the number of PD-1-positive immune effector cells, ii) an increase in the number of PD-1-negative immune effector cells; iii) an increase in the ratio of PD-1 negative immune effector cells / PD-1 positive immune effector cells; iv) an increase in the number of naive T cells; v) For example, the following markers on memory T cells, e.g., memory T cell precursors: CD62L high , CD127 high , CD27 + and increased expression of one or more of BCL2; vi) a decrease in the expression of KLRG1, e.g., in memory T cells, e.g., memory T cell precursors, or vii) memory T cell precursors, e.g., those with the following characteristics: CD62L high Increased CD127 high Increased CD27 + an increase in the number of cells having any one or more combinations of an increase in KLRG1, a decrease in KLRG1, and an increase in BCL2; wherein any of the foregoing, e.g., i), ii), iii), iv), v), vi), or vii), occurs, e.g., at least transiently, e.g., compared to an untreated subject.

[0296] In another embodiment, administration of a low, immune-enhancing dose of an mTOR inhibitor results in increased or prolonged proliferation of CAR-expressing cells, e.g., in culture or in a subject, compared to, e.g., untreated CAR-expressing cells or an untreated subject. In some embodiments, the increased proliferation is accompanied by an increase in the number of CAR-expressing cells. In another embodiment, administration of a low, immune-enhancing dose of an mTOR inhibitor results in increased cancer cell killing by CAR-expressing cells, e.g., in culture or in a subject, compared to, e.g., untreated CAR-expressing cells or an untreated subject. In some embodiments, increased cancer cell killing is associated with a decrease in tumor volume.

[0297] In one embodiment, cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered in combination with a low, immune-enhancing dose of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001, or a catalytic mTOR inhibitor. For example, administration of the low, immune-enhancing dose of the mTOR inhibitor can be initiated before administration of the CAR-expressing cells described herein, can be completed before administration of the CAR-expressing cells described herein, can be initiated simultaneously with administration of the CAR-expressing cells described herein, can overlap with administration of the CAR-expressing cells described herein, or can continue after administration of the CAR-expressing cells described herein.

[0298] Alternatively or additionally, administration of a low, immune-enhancing dose of an mTOR inhibitor can be optimized to engineer immune effector cells to express a CAR molecule described herein. In such embodiments, administration of a low, immune-enhancing dose of an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or a catalytic inhibitor, is initiated or completed before harvesting immune effector cells, e.g., T cells or NK cells, from a subject to be engineered to express a CAR molecule described herein.

[0299] In another embodiment, e.g., immune effector cells, e.g., T cells or NK cells, to be engineered to express a CAR molecule described herein after removal from a subject, or CAR-expressing immune effector cells, e.g., T cells or NK cells, before administration to a subject, can be cultured in the presence of a low, immune-enhancing dose of an mTOR inhibitor.

[0300] In some embodiments, administering a low, immune-enhancing dose of an mTOR inhibitor to a subject includes administering, for example, once per week, 0.1-20 mg, 0.5-10 mg, 2.5-7.5 mg, 3-6 mg, or about 5 mg of RAD001, or a biologically equivalent dose thereof, e.g., in an immediate-release dosage form. In some embodiments, administering a low, immune-enhancing dose of an mTOR inhibitor to a subject includes administering, for example, once per week, 0.3-60 mg, 1.5-30 mg, 7.5-22.5 mg, 9-18 mg, or about 15 mg of RAD001, or a biologically equivalent dose thereof, e.g., in a sustained-release dosage form.

[0301] In certain embodiments, the dose of the mTOR inhibitor is at least 5% but not more than 90%, at least 10% but not more than 90%, at least 15% but not more than 90%, at least 20% but not more than 90%, at least 30% but not more than 90%, at least 40% but not more than 90%, at least 50% but not more than 90%, at least 60% but not more than 90%, at least 70% but not more than 90%, at least 5% but not more than 80%, at least 10% but not more than 80%, at least 10% but not more than 100%, at least 100% 5% but not more than 80%, at least 20% but not more than 80%, at least 30% but not more than 80%, at least 40% but not more than 80%, at least 50% but not more than 80%, at least 60% but not more than 80%, at least 5% but not more than 70%, at least 10% but not more than 70%, at least 15% but not more than 70%, at least 20% but not more than 70%, at least 30% but not more than 70%, at least 40% but not more than 70%, at least 50% but 70% not exceeding, at least 5% but not exceeding 60%, at least 10% but not exceeding 60%, at least 15% but not exceeding 60%, at least 20% but not exceeding 60%, at least 30% but not exceeding 60%, at least 40% but not exceeding 60%, at least 5% but not exceeding 50%, at least 10% but not exceeding 50%, at least 15% but not exceeding 50%, at least 20% but not exceeding 50%, at least 30% but not exceeding 50%, at least 40% but not exceeding 50%, or at least 5% but not more than 40%, at least 10% but not more than 40%, at least 15% but not more than 40%, at least 20% but not more than 40%, at least 30% but not more than 40%, at least 35% but not more than 40%, at least 5% but not more than 30%, at least 10% but not more than 30%, at least 15% but not more than 30%, at least 20% but not more than 30%, or at least 25% but not more than 30% mTOR inhibition.

[0302] The degree of mTOR inhibition can be expressed as or correspond to the degree of P70 S6 kinase inhibition, for example, the degree of mTOR inhibition can be determined by the level of reduction in P70 S6 kinase activity, for example, by a reduction in the phosphorylation of a P70 S6 kinase substrate. The level of mTOR inhibition can be assessed by various methods, such as measuring P70 S6 kinase activity by Boulay assay, measuring the level of phosphorylated S6 by Western blot, or assessing changes in the ratio of PD1-negative to PD1-positive immune effector cells, as described in U.S. Patent Application No. 2015 / 01240036, which is incorporated herein by reference, or as described in U.S. Patent No. 7,727,950, which is incorporated herein by reference.

[0303] As used herein, the term "mTOR inhibitor" refers to a compound or ligand that inhibits mTOR kinase in cells, or its pharmaceutically acceptable salt. In some embodiments, the mTOR inhibitor is an allosteric inhibitor. Allosteric mTOR inhibitors include the naturally occurring tricyclic compound rapamycin (sirolimus), compounds that have structural and functional similarity with rapamycin, such as rapamycin derivatives, rapamycin analogs (also called rapalogs), and rapamycin-related compounds, including other macrolide compounds that inhibit mTOR activity. In some embodiments, the mTOR inhibitor is a catalytic inhibitor.

[0304] Rapamycin is a known macrolide antibiotic produced by Streptomyces hygroscopicus having the structure shown in Formula A.

[0305] [ka]

[0306] See, e.g., McAlpine, JB, et al., J. Antibiotics (1991) 44: 688; Schreiber, SL, et al., J. Am. Chem. Soc. (1991) 113: 7433; U.S. Patent No. 3,929,992. Various numbering schemes have been proposed for rapamycin. To avoid confusion, when naming specific rapamycin analogs herein, the names are given in reference to rapamycin using the numbering scheme of Formula A.

[0307] Rapamycin analogs useful in the present invention include, for example, O-substituted analogs in which the hydroxyl group on the cyclohexyl ring of rapamycin is substituted with OR, where R is hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl, such as RAD001, also known as everolimus, described in U.S. Pat. No. 5,665,772 and WO 94 / 09010, the contents of which are incorporated by reference. Other suitable rapamycin analogs include those substituted at the 26- or 28-position. The rapamycin analog may be an epimer of the above-mentioned analogs, for example, an epimer of an analog substituted at position 40, 28, or 26, as described in US Pat. No. 6,015,815, WO 95 / 14023, and WO 99 / 15530, among others, the contents of which are incorporated by reference, and optionally further hydrogenated, for example, ABT578, also known as zotarolimus or a rapamycin analog, e.g., AP23573, also known as ridaforolimus, as described in US Pat. No. 7,091,213, WO 98 / 02441, and WO 01 / 14387, the contents of which are incorporated by reference.

[0308] Examples of rapamycin analogs from U.S. Pat. No. 5,665,772 that are suitable for use in the present invention include 40-O-benzyl-rapamycin, 40-O-(4'-hydroxymethyl)benzyl-rapamycin, 40-O-[4'-(1,2-dihydroxyethyl)]benzyl-rapamycin, 40-O-allyl-rapamycin, 40-O-[3'-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-prop-2'-en-1'-yl]-rapamycin, (2'E,4'S)-40-O-(4',5'-dihydroxypenta-2'-en-1'-yl) ...4'-(1,2-dihydroxyethyl)]benzyl-rapamycin, 40-O-[4'-(1,2-dihydroxyethyl)]benzyl-rapamycin, 40-O-allyl-rapamycin, 40-O-[4'-(1,2-dihydroxyethyl)]benzyl-rapamycin, 40-O-[4'-(1,2-dihydroxyethyl)]benzyl 40-O-(2-hydroxy)ethoxycarbonylmethyl-rapamycin, 40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin, 40-O-(6-hydroxy)hexyl-rapamycin, 40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-[(3S)-2,2-dimethyldioxolan-3-yl]methyl-rapamycin, 40-O-[(2S)-2,3-dihydroxyprop-1-yl]-rapamycin, 40-O-( 2-acetoxy)ethyl-rapamycin, 40-O-(2-nicotinoyloxy)ethyl-rapamycin, 40-O-[2-(N-morpholino)acetoxy]ethyl-rapamycin, 40-O-(2-N-imidazolylacetoxy)ethyl-rapamycin, 40-O-[2-(N-methyl-N'-piperazinyl)acetoxy]ethyl-rapamycin, 39-O-desmethyl-39,40-O,O-ethylene-rapamycin, (26R)-26-dihydro-40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(2-aminoethyl)-rapamycin rapamycin, 40-O-(2-acetaminoethyl)-rapamycin, 40-O-(2-nicotinamidoethyl)-rapamycin, 40-O-(2-(N-methyl-imidazo-2'-ylcarbethoxamido)ethyl)-rapamycin, 40-O-(2-ethoxycarbonylaminoethyl)-rapamycin, 40-O-(2-tolylsulfonamidoethyl)-rapamycin, and 40-O-[2-(4',5'-dicarbethoxy-1',2',3'-triazol-1'-yl)-ethyl]-rapamycin.

[0309] Other rapamycin analogs useful in the present invention are analogs in which the hydroxyl group on the cyclohexyl ring of rapamycin and / or the hydroxy group at position 28 are replaced with a hydroxyester group, e.g., rapamycin analogs such as temsirolimus are known, e.g., those found in U.S. Patent No. 44,768.

[0310] Other rapamycin analogs useful in the present invention include those in which the methoxy group at position 16 is replaced with another substituent, preferably alkynyloxy (optionally hydroxy-substituted), benzyl, orthomethoxybenzyl, or cyclobenzyl, and / or those in which the methoxy group at position 39 is deleted along with the 39 carbon, resulting in the cyclohexyl ring of rapamycin becoming a cyclopentyl ring lacking the methoxy group at position 39, as described, for example, in WO 95 / 16691 and WO 96 / 41807, the contents of which are incorporated by reference. Analogs can be further modified so that the hydroxy at position 40 of rapamycin is alkylated and / or the 32-carbonyl is reduced.

[0311] Rapamycin analogues from WO 95 / 16691 include 16-demethoxy-16-(pent-2-ynyl)oxy-rapamycin, 16-demethoxy-16-(but-2-ynyl)oxy-rapamycin, 16-demethoxy-16-(propargyl)oxy-rapamycin, 16-demethoxy-16-(4-hydroxy-but-2-ynyl)oxy-rapamycin, 16-demethoxy-16-benzyloxy-40-O-(2-hydroxyethyl)-rapamycin, 16-demethoxy-16-benzyloxy-rapamycin, 16-demethoxy-16-ortho-methoxybenzyl-rapamycin, 16-demethoxy-40-O-(2-methoxyethyl)-16-pent-2-ynyl)oxy-rapamycin, 39-demethoxy-40-desoxy-39-formyl-42 42-nor-rapamycin, 39-demethoxy-40-desoxy-39-hydroxymethyl-42-nor-rapamycin, 39-demethoxy-40-desoxy-39-carboxy-42-nor-rapamycin, 39-demethoxy-40-desoxy-39-(4-methyl-piperazin-1-yl)carbonyl-42-nor-rapamycin, 39-demethoxy-40-desoxy-39-(morpholin-4-yl)carbonyl-42-nor-rapamycin, 39-demethoxy-40-desoxy-39-[N-methyl,N-(2-pyridin-2-yl-ethyl)]carbamoyl-42-nor-rapamycin, and 39-demethoxy-40-desoxy-39-(p-toluenesulfonylhydrazonomethyl)-42-nor-rapamycin.

[0312] Rapamycin analogs from WO 96 / 41807 include, but are not limited to, 32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-40-O-(2-hydroxy-ethyl)-rapamycin, 16-O-pent-2-ynyl-32-(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin, 32(S)-dihydro-40-O-(2-methoxy)ethyl-rapamycin, and 32(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin.

[0313] Another suitable rapamycin analog is umirolimus, which is described in US 2005 / 0101624, the contents of which are incorporated by reference.

[0314] RAD001 is otherwise known as everolimus (Afinitor®) and has the chemical name (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-{(1R)-2-[(1S,3R,4R)-4-(2-hydroxybenzoate)} (3-ethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-aza-tricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentaone.

[0315] Further examples of allosteric mTOR inhibitors include sirolimus (rapamycin, AY-22989), 40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin (also known as temsirolimus or CCI-779), and ridaforolimus (AP-23573 / MK-8669). Other examples of allosteric mTor inhibitors include zotarolimus (ABT578) and umirolimus.

[0316] Alternatively or additionally, catalytic ATP-competitive mTOR inhibitors directly target the mTOR kinase domain and have been found to target both mTORC1 and mTORC2. They are also more effective inhibitors of mTORC1 than allosteric mTOR inhibitors such as rapamycin because they modulate rapamycin-resistant mTORC1-mediated outcomes, such as 4EBP1-T37 / 46 phosphorylation and cap-dependent translation.

[0317] Catalytic inhibitors include: BEZ235 or 2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrile, or the monotosylate salt form, the synthesis of BEZ235 is described in WO2006 / 122806; CCG168 (otherwise known as AZD-8055, Chresta, CM, et al., Cancer Res, 2010, 70(1), 288-298), which has the chemical name {5-[2,4-bis-((S)-3-methyl-morpholin-4-yl)-pyrido[2,3-d]pyrimidin-7-yl]-2-methoxy-phenyl}-methanol; 3-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-methylbenzamide (WO09104019); 3-(2-aminobenzo[d]oxazoline) N-(3-(N-(3-((3,5-dimethoxyphenyl)amino)quinoxalin-2-yl)sulfamoyl)phenyl)-3-methoxy-4-methylbenzamide (WO07044729 and WO12006552); PKI-587 (Venkatesan, AM, J. Med.Chem., 2010, 53, 2636-2645), which has the chemical name 1-[4-[4-(dimethylamino)piperidine-1-carbonyl]phenyl]-3-[4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl]urea; GSK-2126458 (ACS Med. Chem. Lett., 2010, 1, 39-43), which has the chemical name 2,4-difluoro-N-{2-methoxy-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide; 5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine (WO10114484); (E)-N-(8-(6-amino-5-(trifluoromethyl)pyridin-3-yl)-1-(6-(2-cyanopropan-2-yl)pyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-ylidene)cyanamide (WO12007926).

[0318] Further examples of catalytic mTOR inhibitors include 8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one (WO2006 / 122806) and Ku-0063794 (Garcia-Martinez JM, et al., Biochem J., 2009, 421(1), 29-42. Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR). WYE-354 is another example of a catalytic mTor inhibitor (Yu K, et al. (2009). Biochemical, Cellular, and In vivo Activity of Novel ATP-Competitive and Selective Inhibitors of the Mammalian Target of Rapamycin. Cancer Res. 69(15): 6232-6240).

[0319] mTOR inhibitors useful according to the present invention also include prodrugs, derivatives, pharmaceutically acceptable salts, or analogs thereof of any of the foregoing.

[0320] The mTOR inhibitors such as RAD001 may be formulated for delivery according to methods well established in the art based on the specific dosages described herein. Specifically, U.S. Patent No. 6,004,973 (incorporated herein by reference) provides examples of formulations that can be used with the mTOR inhibitors described herein.

[0321] Further combination therapies may include anti-allergens, anti-emetics, analgesics, adjunctive therapies.

[0322] Some patients may experience allergic reactions to the therapeutic agents described herein and / or other anti-cancer agents during or after administration, and therefore, anti-allergy agents may be administered to minimize the risk of an allergic reaction. Suitable anti-allergy agents include corticosteroids such as dexamethasone (e.g., Decadron®), beclomethasone (e.g., Beclovent®), hydrocortisone (also known as cortisone, hydrocortisone sodium succinate, and hydrocortisone sodium phosphate, sold under the trade names Ala-Cort®, hydrocortisone phosphate, Solu-Cortef®, Hydrocort Acetate®, and Lanacort®), prednisolone (sold under the trade names Delta-Cortel®, Orapred®, Pediapred®, and Prelone®), prednisone (sold under the trade names Deltasone®, Liquid Red®, Meticorten®, and Orasone®], methylprednisolone (also known as 6-methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, sold under the trade names Duralone®, Medralone®, Medrol®, M-Prednisol®, and Solu-Medrol®), antihistamines such as diphenhydramine [e.g., Benadryl®], hydroxyzine, and cyproheptadine, and bronchodilators such as the beta-adrenergic receptor agonists albuterol [e.g., Proventil®], and terbutaline [Brethine®].

[0323] Some patients may experience nausea during and after administration of the therapeutic agents described herein and / or other anticancer agents, and therefore antiemetic drugs are used to prevent nausea (upper stomach) and vomiting. Suitable antiemetic drugs include aprepitant (Emend®), ondansetron (Zofran®), granisetron HCl (Kytril®), lorazepam (Ativan®), dexamethasone (Decadron®), prochlorperazine (Compazine®), casopitant (Rezonic® and Zunrisa®), and combinations thereof.

[0324] Medications to alleviate pain experienced during treatment are often prescribed to make patients more comfortable. Common non-prescription painkillers, such as Tylenol®, are often used. However, opioid analgesics, such as hydrocodone / paracetamol or hydrocodone / acetaminophen (e.g., Vicodin®), morphine (e.g., Astramorph® or Avinza®), oxycodone (e.g., OxyContin® or Percocet®), oxymorphone hydrochloride (Opana®), and fentanyl (e.g., Duragesic®), are also useful for moderate or severe pain.

[0325] In an effort to protect normal cells from treatment toxicity and limit organ toxicity, cytoprotective agents (such as neuroprotective agents, free radical scavengers, cardioprotective agents, anthracycline extravasation neutralizers, and nutrients) may be used as adjunctive therapy. Suitable cytoprotective agents include amifostine (Ethyol®), glutamine, dimesna (Tavocept®), mesna (Mesnex®), dexrazoxane (Zinecard® or Totect®), xaliproden (Xaprila®), and leucovorin (also known as leucovorin calcium, citrovorum factor, and folinic acid).

[0326] The structures of active compounds identified by code number, generic name or trade name can be obtained from the current edition of the standard compendium "The Merck Index" or from databases such as Patents International (e.g. IMS World Publications).

[0327] The compounds described above that can be used in combination with the compounds of the present invention can be prepared and administered as described in the art, such as in the documents listed above.

[0328] In one embodiment, the present invention provides a pharmaceutical composition comprising at least one compound of the present invention (e.g., a compound of the present invention) or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier suitable for administration to a human or animal subject, either alone or with other anti-cancer agents.

[0329] In one embodiment, the present invention provides a method of treating a human or animal subject suffering from a cell proliferative disorder, such as cancer. The present invention provides a method of treating a human or animal subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a compound of the present invention (e.g., a compound of the present invention) or a pharmaceutically acceptable salt thereof, either alone or in combination with other anti-cancer agents.

[0330] Specifically, the compositions will either be formulated together as a combination therapy or will be administered separately.

[0331] In combination therapy, the compounds of the present invention and other anti-cancer agents may be administered either simultaneously, in parallel, or sequentially without any specific time limit, and such administration provides therapeutically effective levels of the two compounds in the patient's body.

[0332] In a preferred embodiment, the compound of the present invention and other anticancer drugs are generally administered sequentially in any order by injection or oral administration.Dosage regimens can vary depending on the stage of disease, the patient's physical compatibility, the safety profile and tolerability of each drug, and other criteria known to the physician and medical practitioner who administers the combination drug.The compound of the present invention and other anticancer drugs can be administered within minutes, hours, days, or weeks of each other, depending on the specific cycle used in treatment.In addition, this cycle can include administering one drug more frequently than the other drug during the treatment cycle, and at different doses each time the drug is administered.

[0333] In another aspect of the present invention, kits are provided that include one or more compounds of the present invention and a combination partner disclosed herein. Exemplary kits include (a) a compound of the present invention or a pharmaceutically acceptable salt thereof, and (b) at least one combination partner, such as those described above, where such kits may include a package insert or other label containing instructions for administration.

[0334] The compounds of the present invention may also be used to advantage in combination with known therapeutic processes, such as the administration of hormones or, in particular, radiation. The compounds of the present invention may be used, in particular, as radiosensitizers for the treatment of tumors that are poorly sensitive to radiation therapy.

[0335] Generation and method of immune effector cells Provided herein are methods of making immune effector cells (e.g., T cells, NK cells) that can be engineered to carry a CAR, e.g., a CAR described herein, as well as reaction mixtures and compositions comprising such cells.

[0336] In one aspect, the present disclosure features immune effector cells (e.g., T cells, NK cells) engineered to express a CAR, where the engineered immune effector cells exhibit anti-tumor properties. A preferred antigen is a cancer-associated antigen (i.e., tumor antigen) described herein. In one aspect, the cell is transformed with a CAR, and the CAR is expressed on the cell surface. In some embodiments, the cell (e.g., T cells, NK cells) is transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell may stably express the CAR. In another embodiment, the cell (e.g., T cells, NK cells) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding the CAR. In some such embodiments, the cell may transiently express the CAR.

[0337] Additionally, the present invention provides CART compositions and their use in medicaments or methods for treating, among other diseases, cancer or any malignancy or autoimmune disease involving cells or tissues that express the tumor antigens described herein.

[0338] In one aspect, the CARs of the present invention can be used to eradicate normal cells that express the tumor antigens described herein, and are therefore applicable for use as a conditioning therapy for cells prior to cell transplantation.

[0339] Source of immune effector cells In some embodiments, prior to expansion and genetic or other modification, a source of cells, e.g., T cells or natural killer (NK) cells, e.g., a T cell population or an NK cell population, can be obtained, e.g., obtained, from a subject. The term "subject" is intended to include an organism (e.g., a mammal) in which an immune response is elicited. Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.

[0340] In some embodiments, the cell population, e.g., the harvested cell population, comprises T cells or T cell populations, e.g., at various stages of differentiation. Differentiation stages of T cells, from least differentiated to most differentiated, include naive T cells, stem central memory T cells, central memory T cells, effector memory T cells, and terminal effector T cells. After antigen exposure, naive T cells proliferate and differentiate into memory T cells, e.g., stem central memory T cells and central memory T cells, which then differentiate into effector memory T cells. Upon receiving the appropriate T cell receptor, costimulatory signals, and inflammatory signals, memory T cells further differentiate into terminal effector T cells. See, e.g., Restifo. Blood. 124.4 (2014): 476-77 and Joshi et al. J. Immunol. 180.3 (2008): 1309-15.

[0341] Naive T cells (T N ) are characterized by the expression pattern of the following cell surface markers: CCR7+, CD62L+, CD45RO-, CD95-. SCM ) are characterized by the expression pattern of the following cell surface markers: CCR7+, CD62L+, CD45RO-, CD95+. Central memory T cells (T CM) are characterized by the expression pattern of the following cell surface markers: CCR7+, CD62L+, CD45RO+, CD95+. Effector memory T cells (T EM ) are characterized by the expression pattern of the following cell surface markers: CCR7-, CD62L-, CD45RO+, CD95+. Terminal effector memory T cells (T Eff ) are characterized by the following cell surface marker expression pattern: CCR7-, CD62L-, CD45RO-, CD95+. See, e.g., Gattinoni et al. Nat. Med. 17(2011):1290-7 and Flynn et al. Clin. Translat. Immunol. 3(2014):e20.

[0342] In certain aspects of the present disclosure, immune effector cells, e.g., T cells, can be obtained from a blood unit collected from a subject using any of a number of techniques known to those skilled in the art, such as Ficoll™ separation. In one preferred aspect, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one aspect, cells collected by apheresis can be washed to remove the plasma fraction and, if appropriate, placed in an appropriate buffer or medium for subsequent processing steps. In one embodiment, cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the wash solution can be calcium-free and magnesium-free, or can be free of many, but not all, divalent cations.

[0343] An initial activation step in the absence of calcium can result in expanded activation. As will be readily understood by those skilled in the art, the washing step can be accomplished by methods known to those skilled in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processing device, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in various biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0344] It is understood that the method of the present application can use culture medium conditions containing 5% or less, for example, 2% human AB serum, and known culture medium conditions and compositions, for example, those described in Smith et al., "Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement," Clinical & Translational Immunology (2015) 4, e31; doi:10.1038 / cti.2014.31, can be employed.

[0345] In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by PERCOLL™ gradient centrifugation or counterflow centrifugal elution.

[0346] The methods described herein include, for example, selecting a particular subpopulation of immune effector cells, e.g., T cells, that are regulatory T cell-depleted populations, CD25+ depleted cells, e.g., using negative selection techniques, e.g., as described by way of example herein. Preferably, the population of regulatory T cell-depleted cells contains less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% CD25+ cells.

[0347] In one embodiment, regulatory T cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody or a fragment thereof, or a CD25-binding ligand, e.g., IL-2. In one embodiment, the anti-CD25 antibody or a fragment thereof, or the CD25-binding ligand, is conjugated to a substrate, e.g., a bead, or is otherwise coated on a substrate, e.g., a bead. In one embodiment, the anti-CD25 antibody or a fragment thereof is conjugated to a substrate described herein.

[0348] In one embodiment, regulatory T cells, e.g., CD25+ T cells, are removed from the population using a CD25 depletion reagent from Miltenyi™. In one embodiment, the ratio of cells to CD25 depletion reagent is 20uL to 1e7 cells, or 15uL to 1e7 cells, or 10uL to 1e7 cells, or 5uL to 1e7 cells, or 2.5uL to 1e7 cells, or 1.25uL to 1e7 cells. In one embodiment, for example, for regulatory T cells, e.g., CD25+ depletion, greater than 500 million cells / ml are used. In further aspects, cell concentrations of 600 million, 700 million, 800 million, or 900 million cells / ml are used.

[0349] In one embodiment, the population of immune effector cells to be depleted includes approximately 6×10 9 In other embodiments, the population of immune effector cells to be depleted includes about 1 x 10 CD25+ T cells. 9 ~1×10 10In one embodiment, the resulting regulatory T cell depleted cell population comprises 2 x 10 CD25+ T cells, 1 x 10 CD25+ T cells, or any integer value therebetween. 9 or fewer regulatory T cells, e.g., CD25+ cells (e.g., 1 x 10 9 pieces, 5×10 8 pieces, 1×10 8 pieces, 5×10 7 pieces, 1×10 7 or fewer CD25+ cells).

[0350] In one embodiment, regulatory T cells, e.g., CD25+ cells, are removed from the population using the CliniMAC system in conjunction with a depletion tubing set, e.g., tubing 162-01, etc. In one embodiment, the CliniMAC system is operated in a depletion setting, e.g., DEPLETION2.1.

[0351] Without wishing to be bound by a particular theory, it is believed that reducing the levels of negative regulators of immune cells in a subject prior to apheresis or during production of the CAR-expressing cell product (e.g., reducing the levels of undesirable immune cells, e.g., T REG By reducing the number of T cells, the risk of recurrence in the subject is significantly reduced. REG Methods for depleting cells are known in the art. REG Methods for reducing cells include, but are not limited to, cyclophosphamide, anti-GITR antibodies (anti-GITR antibodies described herein), CD25 depletion, and combinations thereof.

[0352] In some embodiments, the production method includes generating a T cell prior to generating the CAR-expressing cell. REG For example, production methods can include reducing (e.g., depleting) the number of cells, e.g., T cells, NK cells, prior to production of the CAR-expressing cell (e.g., T cell, NK cell) product. REG This involves contacting the sample, eg, an apheresis sample, with an anti-GITR antibody and / or an anti-CD25 antibody (or a fragment thereof or a CD25-binding ligand) to deplete the cells.

[0353] In one embodiment, the subject is treated with T REG In one embodiment, the subject is pre-treated with one or more therapies that reduce the risk of relapse to CAR-expressing cell therapy. REG Methods for reducing cells include, but are not limited to, administering to a subject one or more of cyclophosphamide, anti-GITR antibodies, CD25 depletion, or a combination thereof. Administration of one or more of cyclophosphamide, anti-GITR antibodies, CD25 depletion, or a combination thereof can occur before, during, or after infusion of the CAR-expressing cell product.

[0354] In one embodiment, the subject is pre-treated with cyclophosphamide prior to harvesting cells for CAR-expressing cell product production, thereby reducing the subject's risk of relapse to CAR-expressing cell therapy. In one embodiment, the subject is pre-treated with an anti-GITR antibody prior to harvesting cells for CAR-expressing cell product production, thereby reducing the subject's risk of relapse to CAR-expressing cell therapy.

[0355] In one embodiment, the cell population to be depleted is neither regulatory T cells nor tumor cells, but cells that would otherwise negatively affect the proliferation and / or function of CART cells, e.g., cells expressing CD14, CD11b, CD33, CD15, or potentially other markers expressed by immunosuppressive cells. In one embodiment, it is envisioned that such cells are depleted in parallel with, or after, the depletion of regulatory T cells and / or tumor cells, or in a different sequence.

[0356] In an embodiment, the CAR-expressing cell (e.g., T cell, NK cell) production process includes the step of generating a CAR-expressing cell (e.g., T cell, NK cell) product (e.g., CTL019 product) prior to generating the CAR-expressing cell (e.g., T cell, NK cell) product. REGIn one embodiment, CD25 depletion is used to deplete T cells prior to generation of a CAR-expressing cell (e.g., T cell, NK cell) product (e.g., CTL019 product). REG Deplete the cells.

[0357] The method described herein can comprise more than one selection step, for example, more than one depletion step.The enrichment of T cells by negative selection can be achieved, for example, by combining antibodies against the surface markers specific to the cells that have undergone negative selection.One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, using a cocktail of monoclonal antibodies against the cell surface markers present on the cells that have undergone negative selection.For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail can comprise antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8.

[0358] The methods described herein may further include removing cells expressing tumor antigens, e.g., tumor antigens not containing CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14, or CD11b, from the population, thereby obtaining a population of regulatory T cell-depleted cells, e.g., CD25+ depleted cells, and tumor antigen-depleted cells suitable for expressing a CAR, e.g., a CAR described herein. In one embodiment, tumor antigen-expressing cells are removed simultaneously with regulatory T cells, e.g., CD25+ cells. For example, an anti-CD25 antibody or a fragment thereof and an anti-tumor antigen antibody or a fragment thereof can be attached to the same substrate, e.g., beads, and used to remove cells, or an anti-CD25 antibody or a fragment thereof, or an anti-tumor antigen antibody or a fragment thereof can be attached to separate beads, and a mixture thereof can be used to remove cells. In other embodiments, the removal of regulatory T cells, e.g., CD25+ cells, and the removal of tumor antigen-expressing cells are sequential, e.g., can occur in either order.

[0359] Cells that express a checkpoint inhibitor, e.g., a checkpoint inhibitor described herein, e.g., one or more of PD1+ cells, LAG3+ cells, and TIM3+ cells, are removed from the population, thereby producing, e.g., regulatory T cell-depleted cells, e.g., CD25+ depleted cells, and checkpoint inhibitor-depleted cells, e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM), as described herein. Also provided are methods for obtaining a population of antibodies expressing a checkpoint inhibitor, including those expressing a CD4+ antibody or fragment thereof, ... In other embodiments, the depletion of regulatory T cells, e.g., CD25+ cells, and the depletion of checkpoint inhibitor-expressing cells are sequential, e.g., can occur in either order.

[0360] The methods described herein may include a positive selection step. For example, T cells can be isolated by incubating with anti-CD3 / anti-CD28 (e.g., 3x28) conjugated beads, e.g., DYNABEADS® M-450 CD3 / CD28 T, for a period sufficient for positive selection of the desired T cells. In one embodiment, this period is approximately 30 minutes. In a further embodiment, this period ranges from 30 minutes to 36 hours or more, and all integer values ​​therebetween. In a further embodiment, this period is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. In yet another embodiment, this period is 10 to 24 hours, e.g., 24 hours. Longer incubation times may be used to isolate T cells in any situation where few T cells are present compared to other cell types, such as isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. Furthermore, using longer incubation times increases the efficiency of capture of CD8+ T cells. Thus, simply by shortening or lengthening the time that T cells are bound to the CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells (as further described herein), subpopulations of T cells can be preferentially selected for or against at the beginning of culture or at other times during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, subpopulations of T cells can be preferentially selected for or against at the beginning of culture or at other desired times.

[0361] In one embodiment, IFN- γT cell populations can be selected that express one or more of the following: TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other suitable molecules, e.g., other cytokines. Methods for screening for cell expression can be determined, for example, by the methods described in PCT Publication No. WO2013 / 126712.

[0362] To isolate a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the amount of beads and cells mixed together (e.g., increase the cell concentration) to ensure maximum contact between the cells and beads. For example, in one embodiment, a concentration of 10 billion cells / ml, 9 billion cells / ml, 8 billion cells / ml, 7 billion cells / ml, 6 billion cells / ml, or 5 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, a concentration of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml is used. In a further embodiment, a concentration of 125 million or 150 million cells / ml can be used.

[0363] Using a high concentration can result in increased cell yield, cell activation, and cell proliferation. Furthermore, using a high cell concentration allows for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells, or from samples containing a large number of tumor cells (e.g., leukemia blood, tumor tissue, etc.). Such cell populations may have therapeutic value and would be desirable to obtain. For example, using a high cell concentration allows for more efficient selection of CD8+ T cells, which typically have weak CD28 expression.

[0364] In related embodiments, it may be desirable to use lower cell concentrations. By significantly diluting the mixture of T cells and surfaces (e.g., particles such as beads), interactions between the particles and the cells are minimized. This selects for cells that express high amounts of the desired antigen that binds to these particles. For example, CD4+ T cells express higher levels of CD28 and are captured more efficiently than CD8+ T cells at dilute concentrations. In one embodiment, the concentration of cells used is 5×10 6 In other embodiments, the concentration used is about 1 x 10 5 / ml ~ 1 × 10 6 / ml, and any integer value therebetween.

[0365] In other embodiments, cells may be incubated on a rotator at various speeds for various lengths of time at either 2-10°C or room temperature.

[0366] In one embodiment, a plurality of immune effector cells in the population do not express diglycerol kinase (DGK), e.g., are DGK-deficient. In one embodiment, a plurality of immune effector cells in the population do not express Ikaros, e.g., are Ikaros-deficient. In one embodiment, a plurality of immune effector cells in the population do not express DGK and Ikaros, e.g., are both DGK-deficient and Ikaros-deficient.

[0367] Alternatively, T cells for stimulation may be frozen after a washing step. Without wishing to be bound by theory, the freezing step and subsequent thawing step remove granulocytes and some monocytes from the cell population, resulting in a more uniform product. After removing plasma and platelets through a washing step, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and are useful in this context, one method involves the use of PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A. The cells are then frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods can be used, as well as uncontrolled immediate freezing at -20°C or in liquid nitrogen.

[0368] In certain embodiments, cryopreserved cells are thawed as described herein, washed, and allowed to stand at room temperature for 1 hour before being activated using the methods of the invention.

[0369] The collection of a blood sample or apheresis product from a subject at a time before the expanded cells described herein may be needed is also contemplated in the context of the present invention. Thus, a source of cells for expansion can be collected at any time needed, and the desired cells, such as T cells, can be isolated and frozen for future use in immune effector cell therapy for any of a number of diseases or conditions that would benefit from immune effector cell therapy, such as those described herein. In one embodiment, a blood sample or apheresis is obtained from a generally healthy subject. In certain embodiments, a blood sample or apheresis is obtained from a generally healthy subject who is at risk of developing a disease but has not yet developed the disease, and the desired cells are isolated and frozen for future use. In certain embodiments, T cells can be expanded, frozen, and used at a later time. In certain embodiments, a sample is collected from a patient shortly after diagnosis of a specific disease described herein, but before any treatment. In further embodiments, the cells are isolated from a blood sample or apheresis from a subject prior to any of a number of relevant therapeutic modalities, including, but not limited to, treatment with agents such as natalizumab, efalizumab, antivirals, chemotherapy, irradiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation.

[0370] In a further embodiment of the present invention, T cells are obtained from a patient immediately after a treatment that leaves the subject with functional T cells. In this regard, it has been observed that after certain cancer treatments, particularly treatments with drugs that damage the immune system, the quality of the T cells obtained may be optimal or improved in terms of their ability to expand ex vivo shortly after treatment, when the patient would normally be recovering from the treatment. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a state favorable for enhanced engraftment and in vivo expansion. Therefore, it is contemplated within the context of the present invention to collect blood cells, including T cells, dendritic cells, or other hematopoietic cells, during this recovery phase. Furthermore, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create conditions in the subject that are favorable for the repopulation, recirculation, regeneration, and / or proliferation of specific cell types, particularly during a predetermined time frame after the treatment. Exemplary cell types include T cells, B cells, dendritic cells, and other immune system cells.

[0371] In one embodiment, immune effector cells expressing a CAR molecule, e.g., a CAR molecule described herein, are obtained from a subject who has received a low, immune-enhancing dose of an mTOR inhibitor. In an embodiment, the population of immune effector cells, e.g., T cells, to be engineered to express a CAR are harvested after a sufficient time from, or sufficient dosing with, the low, immune-enhancing dose of an mTOR inhibitor such that the level of PD1-negative immune effector cells, e.g., T cells, or the ratio of PD1-negative immune effector cells, e.g., T cells / PD1-positive immune effector cells, e.g., T cells, in or harvested from the subject is at least transiently increased.

[0372] In other embodiments, a population of immune effector cells, e.g., T cells, that have been or will be engineered to express a CAR can be treated ex vivo by contacting them with an amount of an mTOR inhibitor that increases the number of PD1 negative immune effector cells, e.g., T cells, or increases the ratio of PD1 negative immune effector cells, e.g., T cells / PD1 positive immune effector cells, e.g., T cells.

[0373] It is understood that the method of the present application can use culture medium conditions containing 5% or less, for example, 2% human AB serum, and known culture medium conditions and compositions, for example, those described in Smith et al., "Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement," Clinical & Translational Immunology (2015) 4, e31; doi:10.1038 / cti.2014.31, can be employed.

[0374] In one embodiment, the T cell population is diglycerol kinase (DGK) deficient.DGK-deficient cells include cells that do not express DGK RNA or protein, or that have reduced or inhibited DGK activity.DGK-deficient cells can be generated by genetic approaches, for example, by administering RNA interference agents, such as siRNA, shRNA, miRNA, to reduce or prevent DGK expression.Alternatively, DGK-deficient cells can be generated by treating with DGK inhibitors as described herein.

[0375] In one embodiment, the T cell population is Ikaros-deficient.Ikaros-deficient cells include the cells that do not express Ikaros RNA or protein, or that have reduced or inhibited Ikaros activity, and Ikaros-deficient cells can be generated by genetic approach, for example, by administering RNA interference agents, such as siRNA, shRNA, miRNA, to reduce or prevent Ikaros expression.Alternatively, Ikaros-deficient cells can be generated by treating with Ikaros inhibitors, such as lenalidomide.

[0376] In some embodiments, the T cell population is DGK-deficient and Ikaros-deficient, e.g., does not express DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity. Such DGK-deficient and Ikaros-deficient cells can be generated by any of the methods described herein.

[0377] In one embodiment, the NK cells are obtained from a subject. In another embodiment, the NK cells are an NK cell line, e.g., the NK-92 cell line (Conkwest).

[0378] Similar Car In the embodiments described herein, the immune effector cells can be allogeneic immune effector cells, such as T cells or NK cells. For example, the cells can be allogeneic T cells, such as allogeneic T cells that lack expression of a functional T cell receptor (TCR) and / or human leukocyte antigen (HLA), such as HLA class I and / or HLA class II.

[0379] T cells lacking a functional TCR can, for example, be engineered not to express any functional TCR on their surface, engineered not to express one or more subunits that comprise a functional TCR (e.g., not expressing (or exhibiting reduced expression of) TCR alpha, TCR beta, TCR gamma, TCR delta, TCR epsilon, and / or TCR zeta), or engineered to produce little to no functional TCR on their surface. Alternatively, T cells can express a substantially impaired TCR, for example, by expression of mutated or truncated forms of one or more of the subunits of the TCR. The term "substantially impaired TCR" means that the TCR does not elicit a harmful immune response in the host.

[0380] The T cells described herein can be engineered, for example, to not express functional HLA on their surface. For example, the T cells described herein can be engineered to downregulate the cell surface expression of HLA, for example, HLA class 1 and / or HLA class II. In some embodiments, downregulation of HLA can be achieved by reducing or eliminating the expression of beta-2 microglobulin (B2M).

[0381] In some embodiments, the T cells may lack a functional TCR and a functional HLA, for example, HLA class I and / or HLA class II.

[0382] Modified T cells lacking expression of a functional TCR and / or HLA can be obtained by any suitable means, including knockout or knockdown of one or more subunits of the TCR or HLA. For example, the T cells can include knockdown of the TCR and / or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN), or zinc finger endonuclease (ZFN).

[0383] In some embodiments, the allogeneic cells may be cells that do not express or express at low levels of inhibitory molecules, for example, by any method described herein. For example, the cells may be cells that do not express or express at low levels of inhibitory molecules that can reduce the ability of CAR-expressing cells to initiate immune effector responses. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. For example, inhibiting inhibitory molecules by inhibiting at the DNA, RNA, or protein level can optimize the performance of CAR-expressing cells. In some embodiments, an inhibitory nucleic acid may be used, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, e.g., as described herein, a clustered regularly interspaced sh...

Claims

1. 1. A method for assessing the suitability of an immune effector cell population for use in CAR therapy, the method comprising: (i) providing an immune effector cell population, the immune effector cell population being obtained from a pediatric subject with leukemia before the subject receives cyclophosphamide and / or cytarabine; and (ii) assessing the population for the presence of at least 20% naive T cells, at least 2% stem central memory T cells, and / or at least 4% central memory T cells; wherein the presence of at least 20% naive T cells, at least 2% stem central memory T cells, and / or at least 4% central memory T cells indicates suitability of the immune effector cell population for use in CAR therapy.

2. the presence of at least 20% naive T cells, at least 2% stem central memory T cells, and / or at least 4% central memory T cells; (i) the ability to expand immune effector cell populations; (ii) the efficacy of the immune effector cell population in a treatment regimen; or (iii) Yield of immune effector cell population The method of claim 1 , wherein the method further comprises one or more of:

3. 3. The method of claim 1 or 2, wherein the immune effector cell population being evaluated is derived from a subject who is a human cancer patient.

4. The method of claim 3, wherein the subject is 18 years of age or younger.

5. 5. The method of any of claims 1 to 4, wherein the leukemia is selected from one or more of B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell promyelocytic leukemia, or hairy cell leukemia.

6. The method of any of claims 1 to 5, wherein the subject is classified as having standard risk, high risk, or very high risk ALL.

7. The method of any of claims 1 to 6, wherein the subject does not have lymphoma.

8. The method of any of claims 1 to 7, wherein the subject does not have recurrent cancer.

9. The immune effector cell population being evaluated is (a) prior to administering chemotherapy to the subject; (b) before the subject has received 2, 3, 4, or 5 chemotherapy cycles; (c) after the subject has undergone one chemotherapy cycle, but before the subject has undergone more than one chemotherapy cycle; (d) before the subject undergoes a consolidation chemotherapy cycle or an induction chemotherapy cycle; (e) before the subject undergoes a consolidation chemotherapy cycle; or (f) before the subject undergoes a late potentiated cycle. The method according to any one of claims 1 to 8, which has been obtained by

10. 10. The method of claim 9, wherein the chemotherapy or chemotherapy cycle comprises one or more of induction therapy, consolidation therapy, intermediate maintenance therapy, late intensification therapy, or maintenance therapy cycles.

11. (a) the subject is classified as having high-risk or very high-risk leukemia and the cells are harvested before the subject undergoes a consolidation cycle; or (b) the subject is classified as having high-risk or very high-risk leukemia, and the cells are harvested before the subject undergoes a late intensification cycle. The method of claim 10.

12. 12. The method of any of claims 9-11, wherein the chemotherapy comprises one or more of vincristine, dexamethasone, PEG-L-asparaginase, daunorubicin, 6-mercaptopurine, cyclophosphamide, cytarabine, methotrexate, doxorubicin, 6-thioguanine, and / or prednisone.

13. (a) the one or more chemotherapy cycles or drugs (i) Vincristine, dexamethasone, PEG-L-asparaginase, daunorubicin; (ii) Vincristine, 6-mercaptopurine, cyclophosphamide, cytarabine, PEG-L-asparaginase; (iii) Vincristine, methotrexate, 6-mercaptopurine; (iv) Vincristine, dexamethasone, doxorubicin, PEG-L-asparaginase, cyclophosphamide, cytarabine, 6-thioguanine; (v) vincristine, methotrexate, and (vi) Vincristine, dexamethasone, prednisone, 6-mercaptopurine, methotrexate Selected from the group consisting of: (b) the chemotherapy comprises the following for the treatment of subjects classified as having standard risk cancer: (i) Vincristine 6 mg / m ２ , dexamethasone 6 mg / m ２ , PEG-L-asparaginase 2500 U / m ２ ; (ii) Vincristine 1.5 mg / m ２ , 6-mercaptopurine 75 mg / m ２ ; (iii) Vincristine 7.5 mg / m ２ , methotrexate 500 mg / m ２ ; (iv) vincristine 4.5 mg / m ２ , dexamethasone 10 mg / m ２ , doxorubicin 75 mg / m ２ , PEG-L-asparaginase 2500 U / m ２ , cyclophosphamide 1g / m ２ , cytarabine 60 mg / m ２ , 6-thioguanine 60 mg / m ２ ; (v) Vincristine 7.5 mg / m ２ , methotrexate 500 mg / m ２ and (vi) Vincristine 1.5 mg / m ２ , dexamethasone 6 mg / m ２ , 6-mercaptopurine 75 mg / m ２ , methotrexate 20 mg / m ２ or (c) the chemotherapy comprises one or more of the following for the treatment of subjects classified as having high risk cancer: (i) Vincristine 6 mg / m ２ , dexamethasone 6 mg / m ２ , PEG-L-asparaginase 2500 U / m ２ , doxorubicin 100 mg / m ２ ; (ii) Vincristine 1.5 mg / m ２ , 6-mercaptopurine 60 mg / m ２ , cyclophosphamide 1g / m ２ , cytarabine 75 mg / m ２ , PEG-L-asparaginase 2500 mg / m ２ ; (iii) Vincristine 7.5 mg / m ２ , methotrexate 20 mg / m ２ , 6-mercaptopurine 25 mg / m ２ ; (iv) vincristine 7.5 mg / m ２ , dexamethasone 10 mg / m ２ , doxorubicin 75 mg / m ２ , PEG-L-asparaginase 5000 U / m ２ , cyclophosphamide 1g / m ２ , cytarabine 60 mg / m ２ , 6-thioguanine 60 mg / m ２ ; (v) Vincristine 1.5 mg / m ２ , prednisone 40 mg / m ２ , 6-mercaptopurine 75 mg / m ２ , methotrexate 20 mg / m ２ The drug and dosing regimen is selected from the group consisting of: or (d) the chemotherapy comprises one or more of vincristine, dexamethasone, PEG-L-asparaginase, daunorubicin, 6-mercaptopurine, cyclophosphamide, cytarabine, methotrexate, doxorubicin, 6-thioguanine, and / or prednisone; The method according to any one of claims 9 to 12.

14. The method of any of claims 1 to 13, wherein at least 50% of central memory T cells in the obtained immune effector cell population identify the cells as suitable for use in CAR therapy.

15. 14. The method of any of claims 1-13, wherein less than 55% combined effector memory T cells and terminal effector T cells in the obtained immune effector cell population identifies the cells as suitable for use in CAR therapy.

16. 14. The method of any of claims 1-13, wherein an increase in absolute T cell counts (ATC) in the immune effector cell population compared to a reference value obtained from a sample from the subject at a later time point or after exposure to additional rounds of chemotherapy identifies the immune effector cell population as suitable for use in CAR therapy.

17. 14. The method of any of claims 1-13, wherein an absolute number of at least 400 T cells per microliter, an absolute number of at least 200 naive T cells per microliter, an absolute number of at least 20 stem central memory T cells per microliter, and / or an absolute number of at least 40 central memory T cells per microliter in the obtained immune effector cell population identifies the immune effector cell population as suitable for use in CAR therapy.

18. The method according to any of claims 1 to 17, wherein the obtained immune effector cell population is selected based on the expression of one or more markers selected from CCR7, CD62L, CD45RO and CD95.

19. 20. The method of claim 18, wherein the population of immune effector cells is CCR7+ and CD62L+.

20. The method according to any one of claims 1 to 19, wherein the naive T cells are identified based on an expression pattern of CCR7+, CD62L+, CD45RO-, CD95-, the stem central memory T cells are identified based on an expression pattern of CCR7+, CD62L+, CD45RO-, CD95+, and the central memory T cells are identified based on an expression pattern of CCR7+, CD62L+, CD45RO+, CD95+.

21. The method of any of claims 1 to 20, wherein the immune effector cell population being evaluated is depleted of regulatory T cells.

22. 22. The method of claim 21, wherein the regulatory T cells are CD25+ and the population comprises less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% CD25+ cells.

23. The method of any of claims 1 to 22, wherein the immune effector cell population being evaluated is tumor cell depleted.

24. 24. The method of claim 23, wherein the population comprises less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% CD25+ cells and less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% tumor cells.

25. 25. The method of any of claims 1 to 24, wherein the immune effector cell population to be evaluated is selected based on expression of one or more markers selected from CD3, CD28, CD4, CD8, CD45RA, and CD45RO.

26. 26. The method of claim 25, wherein the population of immune effector cells obtained is CD3+ and / or CD28+.

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