Methods for improving efficacy and expansion of chimeric antigen receptor-expressing cells

Abstract

To provide a method for enriching immune-effector cells suitable for use in a CART cell therapy.SOLUTION: The method comprises: (a) obtaining immune effector cell populations from a cancer patient in need of a CART cell therapy; (b) selecting an immune effector cell population comprising a plurality of markers selected from the group consisting of CCR7, CD62L, CD45RO and CD95; (c) generating a regulatory T cell-depleted immune effector cell population; (d) removing immune effector cells expressing a tumor antigen expressed in the cancer patient to form regulatory T cell-depleted and tumor antigen-depleted immune effector cell population; and (e) selecting a regulatory T cell-depleted and tumor antigen-depleted immune effector cell population expressing a plurality of markers selected from the group consisting of CD3, CD28, CD4, CD8, CD45RA and CD45RO thereby forming an enriched immune effector cell population suitable for a CART cell therapy.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims priority to U.S. Ser. No. 62 / 149,249, filed Apr. 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 those transduced with chimeric antigen receptors (CARs), has shown promise in clinical trials for several hematological cancers. Despite these encouraging results, many patients who are evaluated for enrollment ultimately fail to proceed, at least in part, due to insufficient yields of clinically available products. These limitations in clinically available products may result from multiple factors, including the inability to harvest adequate and sufficient lymphocytes from patients, limitations of harvested cells, and limited ex vivo cell expansion. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, 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 a clinical grade product. [Means for solving the problem]

[0006] The present disclosure relates, at least in part, to methods for improving the efficacy and / or proliferation (e.g., ex vivo proliferation) 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 therapy or early during the course of therapy). 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, it is believed that immune cells harvested as early as possible after diagnosis or treatment with a selected chemotherapy include a less differentiated immune effector cell population (e.g., naive T cells and early memory T cells). It is believed that the less differentiated immune effector cell population is highly proliferative, self-renewing, and / or viable, and therefore has a higher proliferation capacity and / or a higher anti-tumor effect.

[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 cells from a subject at an early time point after cancer diagnosis, for example, before chemotherapy or before a certain / several chemotherapy cycles. In some embodiments, the methods disclosed herein utilize cells obtained from a patient before a chemotherapy cycle that includes a T cell depleting drug (e.g., obtained before 2, 3, 4, or 5 chemotherapy cycles), or cells obtained before a late intensification or consolidation cycle. In other embodiments, the methods optimize the proliferation capacity and / or anti-tumor effect of immune cells, for example, by including patient selection based on type of malignancy (e.g., lymphoma versus leukemia) or disease severity (e.g., standard risk, high risk, or very high risk). In yet other embodiments, the methods optimize the proliferation capacity and / or anti-tumor effect of immune cells, for example, by including cell selection, for example, selection of a cell population with a high absolute number of T cells 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] Thus, 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) For example, the timing of treatment, e.g., chemotherapy, relative to 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 numbers, T cell phenotype, or T cell function. 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 following: the ability of the immune cells to expand (e.g., expand ex vivo), the efficacy of the immune cells for a 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 populations 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 populations thereof) are (i) The timing of obtaining (e.g., harvesting) immune cells from a subject (e.g., a cancer patient); (ii) For example, the timing of treatment, e.g., chemotherapy, relative to 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 numbers, T cell phenotype, or T cell function. or a combination of (i)-(v) may be obtained (e.g., obtained or harvested) by optimizing one, two, three, four, or more (all) of (i)-(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 a therapy, or the yield of the immune cell population when any of (i)-(v) are 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 includes: (i) The timing of obtaining (e.g., harvesting) immune cells from a subject (e.g., a cancer patient); (ii) For example, the timing of treatment, e.g., chemotherapy, relative to 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 numbers, 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 the above, or a combination of (i)-(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 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 a therapy, or the yield of the immune cell population when any of (i)-(v) are optimized.

[0016] In some embodiments, the method further includes introducing into the immune cell (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 that includes 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 from which immune cells are obtained and / or the subject from which treatment is received, is a human, for example, a 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, 6 months, 3 months 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 or cancer-associated antigen, such as a disease described herein. In one embodiment, the subject has cancer, such as a cancer described herein.

[0022] In one embodiment, the subject has a cancer selected from hematological cancer, solid tumors, or metastatic lesions thereof. Exemplary cancers include 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, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell type or pulmonary leukemia. The cancers 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 [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)].

[0024] In an embodiment, the subject has ALL, for example, classified as having standard risk, high risk, or very high risk ALL. In an embodiment, the subject has ALL, for example, 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 a 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 having 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 immune cells for use in cell therapy, e.g., CAR therapy. In another embodiment, harvesting immune cells early in the course of a therapy, e.g., chemotherapy, to a subject (e.g., before the subject has undergone 2, 3, 4, or 5 chemotherapy cycles) results in an increased suitability of 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 a treatment, such as chemotherapy, to the subject.In some embodiments, collection occurs at an early stage during the course of treatment, such as 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 (e.g., 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, intermediate 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 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 includes a drug selected from Table 8. In some embodiments, the chemotherapy includes a drug and dosing regimen 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 a 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 an 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 the expression pattern of CCR7+, CD62L+, CD45RO-, CD95-, stem central memory T cells are identified based on the expression pattern of CCR7+, CD62L+, CD45RO-, CD95+, and central memory T cells are identified based on the expression pattern of CCR7+, CD62L+, CD45RO+, CD95+.

[0039] CAR molecule In accordance with the methods, preparations, and reaction mixtures described herein, for example, immune effector cells obtained by the methods described herein can be engineered to contain a CAR molecule (also referred to herein as "CAR") that targets 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 (also referred to as CD2 subset 1, 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 fetal 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 beta 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; prosteine; 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); synaptic 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 recognized by T cells 3 (SART3); synapsis box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchoring 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 (mut 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 acid sequence shown in Table 4 or has the nucleotide sequence shown 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 an embodiment, the cancer associated antigen targeted by the CAR molecule, e.g., the 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 an embodiment, the cancer associated antigen targeted by the CAR molecule, e.g., the 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 or has the nucleotide sequence set forth in US2014 / 0322212A1 or US2016 / 0068601A1, both of which are incorporated herein by reference.

[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 as 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 as 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, an antibody fragment, scFv, Fv, Fab, (Fab')2, a single domain antibody (SDAB), a VH or VL domain, or a 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 an amino acid sequence of a CD8 hinge, e.g., SEQ ID NO: 2, or an amino acid sequence of an IgG4 hinge, e.g., SEQ ID NO: 36, or a sequence having 95-99% identity to SEQ ID NO: 2 or 36. In other embodiments, the nucleic acid sequence encoding the hinge region comprises a sequence of SEQ ID NO: 13 or SEQ ID NO: 37, or a sequence having 95-99% identity to SEQ ID NO: 13 or 37, corresponding to a CD8 hinge or an IgG4 hinge, respectively.

[0051] In other embodiments, the CAR comprises an intracellular signaling domain, such as 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 may 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 having 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 having 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 having 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 having 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, e.g., 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] Methods of Treatment / 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 generated 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 to the subject a therapy, e.g., a chemotherapy described herein.

[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 the CAR therapy are administered in parallel. In other embodiments, the chemotherapy (e.g., chemotherapy cycle) and the CAR therapy are administered sequentially, e.g., one after the other. In some embodiments, the administration of the chemotherapy (e.g., chemotherapy cycle) and the CAR therapy overlap by less than one week (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 one day). In some embodiments, the administration of the chemotherapy (e.g., chemotherapy cycle) and the CAR therapy overlap by at least one week (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 continues 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 continues 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 lymphoma (e.g., MCL, HL, or NHL).

[0068] In one embodiment, the disease associated with tumor antigen, for example, the tumor antigen described herein, for example, CD19, is selected from a proliferative disease, for example, 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 the 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 hematological 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 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 additional hematological cancers or conditions, including, but not limited to, "preleukemia" (a diverse collection of hematological conditions, such as those united by ineffective production (or dysplasia) of bone marrow blood cells). In certain embodiments, diseases associated with expression of the tumor antigens described herein include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases that express the tumor antigens described herein, and combinations thereof.

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

[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, malignant melanoma of the skin or eye, 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 esophagus, 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 said cancers, and metastatic lesions of said cancers.

[0071] In certain embodiments of any of the foregoing 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-B1, MAGE-C1, MAGE-D2, MAGE-D ...1, MAGE-D2, 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 breakpoints, 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 polypeptides 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 which the population is administered. In one embodiment, the cell population is allogeneic to the subject to which 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 a period of 8 days or less, e.g., 7 days, 6 days, 5 days, 4 days, or 3 days. In one embodiment, the cells, e.g., 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 4 ~10 6 In one embodiment, a subject is administered 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 10 / kg of the subject's body weight), 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 / kg of the subject's body weight or more). 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, in total about 10 immune effector cells per kg of the subject's body weight, in total, over the course of at least three administrations of the immune effector cell population. 6 For example, a subject may receive an initial dose of 1×10 immune effector cells. 5 immune effector cells, 3 × 10 for the second round 5 administration of immune effector cells, and the third dose of 6 × 10 5 administrations of immune effector cells, e.g., each administration is 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 the 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 of 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. In 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 anti-CD25 antibody or its fragment or CD25 binding ligand, e.g., IL-2. In one embodiment, anti-CD25 antibody or its fragment or CD25 binding ligand is conjugated to a substrate, e.g., a bead, or otherwise coated on a substrate, e.g., a bead. In one embodiment, anti-CD25 antibody or its fragment is conjugated to a substrate as 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 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.

[0082] In one embodiment, the population of regulatory T cell depleted cells, e.g., CD25+ depleted cells, is suitable for expression of the CAR described herein, e.g., the 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%, 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%, less than 1% of leukemia cells, e.g., CLL cells, and is suitable for expression of the 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 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 a suitable buffer or medium for subsequent processing steps. In one embodiment, the cells are washed with a buffer, for example, phosphate-buffered saline (PBS). In an embodiment, the cells are washed in a washing solution that lacks 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 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 as described herein. In one embodiment, the tumor antigen-expressing cells are removed simultaneously with the regulatory T cells, e.g., CD25+ cells. For example, the anti-CD25 antibody or fragment thereof and the anti-tumor antigen antibody or fragment thereof can be attached to the same substrate, e.g., beads, which can be used to remove the cells, or the anti-CD25 antibody or fragment thereof, or the anti-tumor antigen antibody or fragment thereof can be attached to separate beads, and a mixture of these can be used to remove the cells. In other embodiments, the removal of the regulatory T cells, e.g., CD25+ cells, and the removal of the 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., one or more of the checkpoint inhibitors described herein, e.g., 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, the anti-CD25 antibody or fragment thereof and the anti-checkpoint inhibitor antibody or fragment thereof can be attached to the same bead, which can be used to remove the cells, or the anti-CD25 antibody or fragment thereof and the anti-checkpoint inhibitor antibody or fragment thereof can be attached to separate beads, which mixture can be used to remove the cells. In other embodiments, the removal of the regulatory T cells, e.g., CD25+ cells, and the removal of the checkpoint inhibitor-expressing cells are sequential, e.g., can occur in either order.

[0089] In one embodiment, the cell population to be removed is neither regulatory T cells nor tumor cells, but cells that would otherwise negatively affect the proliferation and / or function of CART cells, such as cells expressing CD14, CD11b, CD33, CD15, or other markers potentially expressed by immunosuppressive cells. In one embodiment, it is envisioned that such cells are removed in parallel with the regulatory T cells and / or tumor cells, or after said depletion, 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, anti-CD25 antibody or fragment thereof and anti-CD14 antibody or fragment thereof can be attached to the same bead, which can be used to remove cells, or anti-CD25 antibody or fragment thereof, or anti-CD14 antibody or fragment thereof can be attached to separate beads, which mixture 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., 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 incubating 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, e.g., CD25+ depleted cells, e.g., by a method described herein.

[0094] In some embodiments, the method further comprises 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 comprises expanding the immune effector cell population engineered to express, e.g., 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 a cell 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 cell from a regulatory T cell depleted cell population, e.g., a CD25+ depleted cell population, is transduced with a vector once within one day after the immune effector cell population is obtained, e.g., 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, where 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 4-9 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 as 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, e.g., 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 as 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 CD19 CAR as 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, for example, by culturing the cells in the presence of an agent that stimulates a CD3 / TCR complex-associated signal 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 the growth of cells.

[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, by 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 growth period. In one embodiment, the cells are cryopreserved according to the methods described herein. In one embodiment, the expanded cells are cryopreserved in a suitable 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 an embodiment, 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, the 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 an embodiment, the method comprises contacting the immune effector cell population with a nucleic acid encoding a CAR and an RNA encoding a telomerase subunit substantially simultaneously. In an embodiment, the method comprises contacting the immune effector cell population with a nucleic acid encoding a CAR before contacting the immune effector cell population with an RNA encoding a telomerase subunit. In an embodiment, the method comprises contacting the immune effector cell population with a nucleic acid encoding a CAR after contacting the immune effector cell population with an RNA encoding a telomerase subunit.

[0111] In some embodiments, the RNA encoding the telomerase subunit is an 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 an embodiment, the method comprises transfecting an immune effector cell with an RNA encoding a telomerase subunit. In an embodiment, the method comprises transducing an immune effector cell with an RNA encoding a telomerase subunit. In an embodiment, the method comprises electroporating an immune effector cell with an 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 contain 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 for 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 T cells per microliter of at least 400, an absolute number of naive T cells per microliter of at least 200, an absolute number of stem central memory T cells per microliter of at least 20, and / or an absolute number of central memory T cells per microliter of at least 40; 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 the expression pattern of CCR7+, CD62L+, CD45RO-, CD95-, stem central memory T cells are identified based on the expression pattern of CCR7+, CD62L+, CD45RO-, CD95+, and central memory T cells are identified based on the 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 preparation described herein comprises a nucleic acid encoding an exogenous telomerase subunit, e.g., hTERT. In some 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, e.g., a CAR described herein, and an exogenous telomerase subunit, e.g., hTERT. In some embodiments, the cell does not comprise DNA encoding an exogenous telomerase subunit. For example, the cell may be contacted with an mRNA encoding an exogenous telomerase subunit.

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

[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, and 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%, 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 immune effector cell preparation 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.

[0126] In one embodiment, the reaction mixture may further comprise an agent that activates and / or expands the 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 with 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 that contains less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells. In one embodiment, the reaction mixture comprises a regulatory T cell depleted cell population that contains less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells and less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 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%, 1% CD25+ cells and less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 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 that contains less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells and less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 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 within the population in the reaction mixture comprises a nucleic acid molecule, e.g., a nucleic acid molecule described herein, that comprises a CAR coding sequence, e.g., a CD19 CAR coding 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, where a plurality of cells within the population in the reaction mixture comprises a nucleic acid molecule, e.g., a nucleic acid molecule described herein, that comprises a CAR coding sequence, e.g., a CD19 CAR coding 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 this invention, but 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 description 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 next day (day 0). CAR-T cell numbers were calculated based on T cell numbers and percentages of CAR expression. Curves are representative of six donors. *P<0.05, ***P<0.001. NC, no cytokines. Figure 1C is a histogram showing the proliferation of T cells 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 the viability of T cells 15 days after lentiviral transduction. T cells from various cytokine groups are stained with Annexin V and 7-AAD and then analyzed for the percentage of live cells (both Annexin V and 7-AAD negative). *P<0.05, **P<0.01 compared to 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 next day (day 0). CAR-T cell numbers were calculated based on T cell numbers and percentages of CAR expression. Curves are representative of six donors. *P<0.05, ***P<0.001. NC, no cytokines. Figure 1C is a histogram showing the proliferation of T cells 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 the viability of T cells 15 days after lentiviral transduction. T cells from various cytokine groups are stained with Annexin V and 7-AAD and then analyzed for the percentage of live cells (both Annexin V and 7-AAD negative). *P<0.05, **P<0.01 compared to 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 next day (day 0). CAR-T cell numbers were calculated based on T cell numbers and percentages of CAR expression. Curves are representative of six donors. *P<0.05, ***P<0.001. NC, no cytokines. Figure 1C is a histogram showing the proliferation of T cells 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 the viability of T cells 15 days after lentiviral transduction. T cells from various cytokine groups are stained with Annexin V and 7-AAD and then analyzed for the percentage of live cells (both Annexin V and 7-AAD negative). *P<0.05, **P<0.01 compared to 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 next day (day 0). CAR-T cell numbers were calculated based on T cell numbers and percentages of CAR expression. Curves are representative of six donors. *P<0.05, ***P<0.001. NC, no cytokines. Figure 1C is a histogram showing the proliferation of T cells 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 the viability of T cells 15 days after lentiviral transduction. T cells from various cytokine groups are stained with Annexin V and 7-AAD and then analyzed for the percentage of live cells (both Annexin V and 7-AAD negative). *P<0.05, **P<0.01 compared to 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 CD45RA+CD62L+ subpopulation of T cells before transduction and 15 days after transduction of CAR-T cells. Figures 2B and 2C are graphs showing the increase in memory stem T cell (Tscm) percentage in CD4+ (Figure 2B) and CD8+ T cells (Figure 2C) after lentiviral transduction. Tscm is defined as CD45RA+CD62L+CD95+CCR7+ T cell subset. Figure 2D is a graph showing the correlation between the amount of naive T (Tn, defined as CD45RA+CD62L+CD95- subpopulation) in T cells before transduction and the percentage 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 Temra cells were cultured and exposed to IL-2 (10 ng / mL) for 3 days, and then phenotype was 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 Temra cells were labeled with CFSE (2.5 μM), 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 CD45RA+CD62L+ subpopulation of T cells before transduction and 15 days after transduction of CAR-T cells. Figures 2B and 2C are graphs showing the increase in memory stem T cell (Tscm) percentage 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 percentage 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 Temra cells were cultured and exposed to IL-2 (10 ng / mL) for 3 days, and then phenotype was 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 Temra cells were labeled with CFSE (2.5 μM), 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 CD45RA+CD62L+ subpopulation of T cells before transduction and 15 days after transduction of CAR-T cells. Figures 2B and 2C are graphs showing the increase in memory stem T cell (Tscm) percentage 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 percentage 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 Temra cells were cultured and exposed to IL-2 (10 ng / mL) for 3 days, and then phenotype was 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 Temra cells were labeled with CFSE (2.5 μM), 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 CD45RA+CD62L+ subpopulation of T cells before transduction and 15 days after transduction of CAR-T cells. Figures 2B and 2C are graphs showing the increase in memory stem T cell (Tscm) percentage in CD4+ (Figure 2B) and CD8+ T cells (Figure 2C) after lentiviral transduction. Tscm is defined as CD45RA+CD62L+CD95+CCR7+ T cell subset. Figure 2D is a graph showing the correlation between the amount of naive T (Tn, defined as CD45RA+CD62L+CD95- subpopulation) in T cells before transduction and the percentage 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 Temra cells were cultured and exposed to IL-2 (10 ng / mL) for 3 days, and then phenotype was 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 Temra cells were labeled with CFSE (2.5 μM), 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 CD45RA+CD62L+ subpopulation of T cells before transduction and 15 days after transduction of CAR-T cells. Figures 2B and 2C are graphs showing the increase in memory stem T cell (Tscm) percentage in CD4+ (Figure 2B) and CD8+ T cells (Figure 2C) after lentiviral transduction. Tscm is defined as CD45RA+CD62L+CD95+CCR7+ T cell subset. Figure 2D is a graph showing the correlation between the amount of naive T (Tn, defined as CD45RA+CD62L+CD95- subpopulation) in T cells before transduction and the percentage 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 Temra cells were cultured and exposed to IL-2 (10 ng / mL) for 3 days, and then phenotype was 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 Temra cells were labeled with CFSE (2.5 μM), 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 CD45RA+CD62L+ subpopulation of T cells before transduction and 15 days after transduction of CAR-T cells. Figures 2B and 2C are graphs showing the increase in memory stem T cell (Tscm) percentage in CD4+ (Figure 2B) and CD8+ T cells (Figure 2C) after lentiviral transduction. Tscm is defined as CD45RA+CD62L+CD95+CCR7+ T cell subset. Figure 2D is a graph showing the correlation between the amount of naive T (Tn, defined as CD45RA+CD62L+CD95- subpopulation) in T cells before transduction and the percentage 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 Temra cells were cultured and exposed to IL-2 (10 ng / mL) for 3 days, and then phenotype was 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 Temra cells were labeled with CFSE (2.5 μM), 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 proliferation 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 proliferation 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 vs. 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 production of IFNγ (Figure 5A), TNF-α (Figure 5B), and IL-2 (Figure 5C). Lentiviral-transduced T cells are exposed to the indicated cytokines for 14 days and then co-cultured with SKOV3 cells for 5 h before harvesting for flow cytometry analysis. Figure 5D is a graph showing antigen-specific cytotoxic activity of CAR-T cells. After 14 days of the indicated cytokine exposure, CAR-T cells were assessed for cytolytic potential using a luciferase-based assay after 18 h co-culture with SKOV3 at the indicated E / T ratios. Untransduced T cells (UNT) served as negative effector controls. 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 production of IFNγ (Figure 5A), TNF-α (Figure 5B), and IL-2 (Figure 5C). Lentiviral-transduced T cells are exposed to the indicated cytokines for 14 days and then co-cultured with SKOV3 cells for 5 h before harvesting for flow cytometry analysis. Figure 5D is a graph showing antigen-specific cytotoxic activity of CAR-T cells. After 14 days of the indicated cytokine exposure, CAR-T cells were assessed for cytolytic potential using a luciferase-based assay after 18 h co-culture with SKOV3 at the indicated E / T ratios. Untransduced T cells (UNT) served as negative effector controls. 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 production of IFNγ (Figure 5A), TNF-α (Figure 5B), and IL-2 (Figure 5C). Lentiviral-transduced T cells are exposed to the indicated cytokines for 14 days and then co-cultured with SKOV3 cells for 5 h before harvesting for flow cytometry analysis. Figure 5D is a graph showing antigen-specific cytotoxic activity of CAR-T cells. After 14 days of the indicated cytokine exposure, CAR-T cells were assessed for cytolytic potential using a luciferase-based assay after 18 h co-culture with SKOV3 at the indicated E / T ratios. Untransduced T cells (UNT) served as negative effector controls. 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 production of IFNγ (Figure 5A), TNF-α (Figure 5B), and IL-2 (Figure 5C). Lentiviral-transduced T cells are exposed to the indicated cytokines for 14 days and then co-cultured with SKOV3 cells for 5 h before harvesting for flow cytometry analysis. Figure 5D is a graph showing antigen-specific cytotoxic activity of CAR-T cells. After 14 days of the indicated cytokine exposure, CAR-T cells were assessed for cytolytic potential using a luciferase-based assay after 18 h co-culture with SKOV3 at the indicated E / T ratios. Untransduced T cells (UNT) served as negative effector controls. Data shown are the mean ± SEM of six independent cytolytic assays. [Figure 6A] Figures 6A-6C show the phenotype and function of CAR-T cells as 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 weak 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 CAR-T cells as 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 weak 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 CAR-T cells as 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 weak 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 represents two graphs showing the total accumulation and viability of CAR-T previously exposed to the indicated cytokines upon antigen challenge. T cells exposed to the indicated cytokines are harvested on day 15 and then co-cultured with SKOV3 at an E / T ratio of 5:1 for 7 days. CAR-T cell proliferation is calculated and T cell viability is assessed on day 17. Figure 7B is 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 represents two graphs showing the total accumulation and viability of CAR-T previously exposed to the indicated cytokines upon antigen challenge. T cells exposed to the indicated cytokines are harvested on day 15 and then co-cultured with SKOV3 at an E / T ratio of 5:1 for 7 days. CAR-T cell proliferation is calculated and T cell viability is assessed on day 17. Figure 7B is 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 anti-tumor activity of various CAR-T cells previously exposed to cytokines. Figure 8A shows the tumor growth curves of mice treated with various cytokine-exposed C4-27z CAR-T cells, anti-CD19-27z CAR-T cells, and non-transduced T cells. Data are expressed as mean ± SEM. Arrows indicate the time of T cell infusion. Figure 8B shows the quantification of circulating human CD4+ and CD8+ T cell numbers in mouse peripheral blood 15 days after the first dose of CAR-T cell infusion. Figure 8C shows the quantification of CAR expression in circulating human CD4+ and CD8+ T cells in mouse blood. [Figure 8B]Figures 8A-8C show the anti-tumor activity of various CAR-T cells previously exposed to cytokines. Figure 8A shows the tumor growth curves of mice treated with various cytokine-exposed C4-27z CAR-T cells, anti-CD19-27z CAR-T cells, and non-transduced T cells. Data are expressed as mean ± SEM. Arrows indicate the time of T cell infusion. Figure 8B shows the quantification of circulating human CD4+ and CD8+ T cell numbers in mouse peripheral blood 15 days after the first dose of CAR-T cell infusion. Figure 8C shows the quantification of CAR expression in circulating human CD4+ and CD8+ T cells in mouse blood. [Figure 8C] Figures 8A-8C show the anti-tumor activity of various CAR-T cells previously exposed to cytokines. Figure 8A shows the tumor growth curves of mice treated with various cytokine-exposed C4-27z CAR-T cells, anti-CD19-27z CAR-T cells, and non-transduced T cells. Data are expressed as mean ± SEM. Arrows indicate the time of T cell infusion. Figure 8B shows the quantification of circulating human CD4+ and CD8+ T cell numbers in mouse peripheral blood 15 days after the first dose of CAR-T cell infusion. Figure 8C shows the quantification of CAR expression in circulating human CD4+ and CD8+ T cells in mouse blood. [Figure 9] FIG. 9 is a series of FACS plots showing CD3 and CD19 populations (top) and histograms showing CD14 expression of cells from apheresis, anti-CD3 / CD28 selected cells, CD25-depleted cells, and CD25-enriched cells (bottom). [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 the indicated days 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 the indicated days 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 the indicated days 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 following culture with the indicated cytokine supplements (IL-7, IL-15, or IL-7 and IL-15). [Figure 12] FIG. 12 is a set of graphs showing the proliferation profile in population doublings (left hand graph) and average 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), the beads removed (de-beaded), and then harvested on day 5 and D9. [Figure 13] FIG. 13 is a set of graphs depicting cytotoxicity as percent lysis of CD19-expressing K562 cells stimulated with anti-CD3 and CD28 beads and either left unmanipulated (UTD) or transduced with CD19 CAR (CD19.BBz), the beads removed, and then treated with PBMCs harvested 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 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 0-9 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 CTL019 CAR, i.e., CHP959-115, or 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 cell killing based on total lysate using luciferase assay of Nalm6 (ALL) cells left unmanipulated (UTD) or transduced with CD19 CAR (CART19), beads were removed, and then PBMCs were harvested on day 5 and D9. Various ratios of PBMCs to Nalm6 cells (effector (E): target (T)) were cultured. As shown, CART19 cells harvested 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), the beads were removed, and then harvested 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 test proliferation. Figure 22B is a bar graph showing the percentage of peripheral blood samples from SR-ALL and HR / VHR ALL patients that passed the test proliferation. Figure 22C is a bar graph showing the absolute lymphocyte counts from peripheral blood at various collection times from all patients who expanded more than 5 times (passed) or less than 5 times (failed) at the in vitro proliferation threshold. Figure 22D is a bar graph showing the absolute T cell counts from peripheral blood at various collection times from all patients who passed or failed the in vitro proliferation threshold 5 times. Figure 22E is a bar graph showing the 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 the absolute T cell counts (in cells per microliter) from peripheral blood at various collection times from leukemia or lymphoma patients. Significant differences are indicated with 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 test proliferation. Figure 22B is a bar graph showing the percentage of peripheral blood samples from SR-ALL and HR / VHR ALL patients that passed the test proliferation. Figure 22C is a bar graph showing the absolute lymphocyte counts from peripheral blood at various collection times from all patients who expanded more than 5 times (passed) or less than 5 times (failed) at the in vitro proliferation threshold. Figure 22D is a bar graph showing the absolute T cell counts from peripheral blood at various collection times from all patients who passed or failed the in vitro proliferation threshold 5 times. Figure 22E is a bar graph showing the 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 the absolute T cell counts (in cells per microliter) from peripheral blood at various collection times from leukemia or lymphoma patients. Significant differences are indicated with 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 test proliferation. Figure 22B is a bar graph showing the percentage of peripheral blood samples from SR-ALL and HR / VHR ALL patients that passed the test proliferation. Figure 22C is a bar graph showing the absolute lymphocyte counts from peripheral blood at various collection times from all patients who expanded more than 5 times (passed) or less than 5 times (failed) at the in vitro proliferation threshold. Figure 22D is a bar graph showing the absolute T cell counts from peripheral blood at various collection times from all patients who passed or failed the in vitro proliferation threshold 5 times. Figure 22E is a bar graph showing the 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 the absolute T cell counts (in cells per microliter) from peripheral blood at various collection times from leukemia or lymphoma patients. Significant differences are indicated with 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 test proliferation. Figure 22B is a bar graph showing the percentage of peripheral blood samples from SR-ALL and HR / VHR ALL patients that passed the test proliferation. Figure 22C is a bar graph showing the absolute lymphocyte counts from peripheral blood at various collection times from all patients who expanded more than 5 times (passed) or less than 5 times (failed) at the in vitro proliferation threshold. Figure 22D is a bar graph showing the absolute T cell counts from peripheral blood at various collection times from all patients who passed or failed the in vitro proliferation threshold 5 times. Figure 22E is a bar graph showing the 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 the absolute T cell counts (in cells per microliter) from peripheral blood at various collection times from leukemia or lymphoma patients. Significant differences are indicated with 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 test proliferation. Figure 22B is a bar graph showing the percentage of peripheral blood samples from SR-ALL and HR / VHR ALL patients that passed the test proliferation. Figure 22C is a bar graph showing the absolute lymphocyte counts from peripheral blood at various collection times from all patients who expanded more than 5 times (passed) or less than 5 times (failed) at the in vitro proliferation threshold. Figure 22D is a bar graph showing the absolute T cell counts from peripheral blood at various collection times from all patients who passed or failed the in vitro proliferation threshold 5 times. Figure 22E is a bar graph showing the 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 the absolute T cell counts (in cells per microliter) from peripheral blood at various collection times from leukemia or lymphoma patients. Significant differences are indicated with 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 test proliferation. Figure 22B is a bar graph showing the percentage of peripheral blood samples from SR-ALL and HR / VHR ALL patients that passed the test proliferation. Figure 22C is a bar graph showing the absolute lymphocyte counts from peripheral blood at various collection times from all patients who expanded more than 5 times (passed) or less than 5 times (failed) at the in vitro proliferation threshold. Figure 22D is a bar graph showing the absolute T cell counts from peripheral blood at various collection times from all patients who passed or failed the in vitro proliferation threshold 5 times. Figure 22E is a bar graph showing the 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 the absolute T cell counts (in cells per microliter) from peripheral blood at various collection times from leukemia or lymphoma patients. Significant differences are indicated with 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 taken from 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 with an "*" above each column, representing P<0.05. Statistical analysis 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 taken from 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 with an "*" above each column, representing P<0.05. Statistical analysis 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 taken from 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 with an "*" above each column, representing P<0.05. Statistical analysis 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 taken from 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 with an "*" above each column, representing P<0.05. Statistical analysis 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 taken from 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 with an "*" above each column, representing P<0.05. Statistical analysis 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 taken from 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 with an "*" above each column, representing P<0.05. Statistical analysis 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 taken from 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 with an "*" above each column, representing P<0.05. Statistical analysis 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 taken from 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 with an "*" above each column, representing P<0.05. Statistical analysis 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 taken from 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 with an "*" above each column, representing P<0.05. Statistical analysis 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 taken from 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 with an "*" above each column, representing P<0.05. Statistical analysis 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 taken from 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 with an "*" above each column, representing P<0.05. Statistical analysis 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 taken from 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 with an "*" above each column, representing P<0.05. Statistical analysis 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 taken from 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 with an "*" above each column, representing P<0.05. Statistical analysis 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 taken from 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 with an "*" above each column, representing P<0.05. Statistical analysis 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 taken from 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 with an "*" above each column, representing P<0.05. Statistical analysis 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 with 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 effect of IL-7 and IL-15 on T cell phenotype and proliferation. The 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 culturing with cytokines. Significant differences are indicated with an "*" above each column, representing P<0.05. Figure 25B is a bar graph showing the percent of test proliferation passing samples evaluated after culturing 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 effect of IL-7 and IL-15 on T cell phenotype and proliferation. The 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 culturing with cytokines. Significant differences are indicated with an "*" above each column, representing P<0.05. Figure 25B is a bar graph showing the percent of test proliferation passing samples evaluated after culturing 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] 26 is a set of pie charts showing the post-growth phenotypes of samples that passed and failed test growth. Phenotypes were assessed at the end of test growth. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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, 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 poorly 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 counts) obtained from the subject.

[0139] As described in detail in the Examples, 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 proliferation capacity) have been observed to improve T cell proliferation (e.g., may indicate suitability for use in cell therapy). 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, for example, provides a cell population with less deterioration in proliferation capacity. Using cells obtained from a patient before a late intensive or consolidation chemotherapy cycle, for example, has been observed to improve T cell proliferation. In some embodiments, the severity of the disease affects cell deterioration, for example, cell proliferation capacity. 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 consolidation 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 proliferation 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 proliferation and survival capacity, also exhibited higher proliferation 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 result in further depletion of 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 efficacy as anti-cancer T cell therapy.

[0141] Thus, in some embodiments, the method advantageously improves the suitability (suitability for use in cell therapy) and efficacy of T cells by obtaining cells from a subject at an early time point after cancer diagnosis, for example, before chemotherapy or before a certain / several chemotherapy cycles. In some embodiments, the method utilizes cells obtained from a patient before a chemotherapy cycle that includes a T cell depleting drug, before two, three, four, or five chemotherapy cycles, or before a late intensification or consolidation cycle. In other embodiments, the method optimizes the proliferation capacity and / or anti-tumor effect of immune cells, for example, by including patient selection based on type of malignancy (e.g., lymphoma versus leukemia) or disease severity (e.g., standard risk, high risk, or very high risk). In yet other embodiments, the method optimizes the proliferation capacity and / or anti-tumor effect of immune cells, for example, by including cell selection, for example, selection of a cell population with a high absolute number of T cells or a high proportion of poorly differentiated T cells. Further embodiments are described in more 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, "an 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 indicated value, as such variations are appropriate for performing the disclosed methods.

[0145] The term "obtain" or "obtaining" as used herein 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 involving a physical change of a sample or another substance, e.g., performing an analytical process (often referred to herein as "physical analysis") involving a physical change of a substance, e.g., a sample, analyte, or reagent, an analytical method, e.g., performing a method that includes 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, e.g., 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, e.g., 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 amount of a reference compound (e.g., RAD001). In an embodiment, the effect is the level of mTOR inhibition, e.g., measured by P70 S6 kinase inhibition, e.g., measured by an in vivo or in vitro assay, e.g., an assay described herein, e.g., Boulay assay, or measuring phosphorylated S6 levels by Western blot. In an embodiment, the effect is a change in the ratio of PD-1 positive T cells / PD-1 negative T cells, measured by cell sorting. In an embodiment, 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 amount of a reference compound. In an embodiment, 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 amount of a 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 the cell with specificity for a target cell, typically a cancer cell, and provide intracellular signal generation in an immune effector cell. In some embodiments, the 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., in 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., in different polypeptide chains. In some embodiments, the set of polypeptides comprises a dimerization switch that allows the polypeptides to bind to each other when a dimerization molecule is present, e.g., the antigen-binding domain to bind to the intracellular signaling domain. In one aspect, the stimulatory molecule is a zeta chain associated with the 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 the costimulatory molecules 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 cleaved from the antigen-binding domain (e.g., scFv) upon cellular processing and localization of the CAR to the cell membrane, as appropriate.

[0148] A CAR that includes 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 that includes 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 a 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, multichain or single chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. An antibody can 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 Fab, Fab', F(ab') 2 , 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 bivalent fragments 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 variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, where the light chain variable region and the heavy chain variable region are linked contiguously, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and can be expressed as a single-chain polypeptide, where 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, e.g., with respect to the N-terminus and C-terminus of the polypeptide, an scFv may comprise VL-linker-VH or VH-linker-VL.

[0153] The portion of the CAR that comprises 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 that comprises an scFv. The exact 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 an embodiment, the antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, a first immunoglobulin variable domain sequence of the plurality having binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality having binding specificity for a second epitope. In an embodiment, the 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 having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having 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 (e.g., 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 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 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 that comprises 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 that comprises 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 natural 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 by 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 that specifies the antibody, where the DNA or amino acid sequence has been 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 that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response thus 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 is 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 are 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 is readily apparent that an antigen may be generated, synthesized, or derived from a biological sample, or may 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 derived from the same individual that 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 to 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 antigenically interactive.

[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 uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers 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, and the like. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid tumors and liquid tumors, e.g., 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 molecule and the second molecule, and does not indicate or include any limitation on the process or origin from 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 indicate or include any limitation to a particular process of 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 in order to provide this intracellular signaling domain.

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

[0167] As used herein, the term "CD19" refers to Cluster of Differentiation 19 protein, which is an antigenic determinant detectable on leukemia progenitor cells. Human and murine 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 that contain 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 expressing CD19 are provided in the definition of "diseases associated with CD19 expression" below. 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 CART recognizes and binds to an antigen within the extracellular domain of CD19 protein. In one embodiment, CD19 protein is expressed on cancer cells.

[0168] The phrase "disease associated with CD19 expression" includes, but is not limited to, disease associated with CD19 (e.g., wild-type or mutant CD19) expression or conditions associated with cells that express or have expressed CD19 (e.g., wild-type or mutant CD19) at any time, including, for example, cancer or malignant tumor, or proliferative disease such as precancerous condition, e.g., myelodysplasia, myelodysplastic syndrome, or preleukemia, or non-cancer-related indications associated with cells expressing CD19. For avoidance of doubt, disease 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 is downregulated, for example, by treatment with a molecule that targets CD19, e.g., CD19 CAR. In one embodiment, the cancer associated with CD19 expression is a blood cancer. In one embodiment, the blood cancer is a leukemia or lymphoma. In one aspect, cancers associated with expression of CD19 include, by way of example only, cancers and malignancies including, but not limited to, one or more acute leukemias, including, but not limited to, 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, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL). Additional cancers or hematological conditions associated with expression of CD19 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 hematological conditions united by ineffective production (or dysplasia) of bone marrow blood cells.Further diseases associated with the expression of CD19 expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with the expression of CD19. Non-cancer-related indications associated with the expression of CD19 include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergy and asthma), and transplantation. In some embodiments, CD19-expressing cells express or have expressed CD19 mRNA at any time. In some embodiments, CD19-expressing cells produce CD19 protein (e.g., wild-type or mutant), and CD19 protein may be present at normal or reduced levels. In some embodiments, CD19-expressing cells produce detectable levels of CD19 protein at one time, and then do not produce substantially detectable CD19 protein thereafter.

[0169] The term "conservative sequence modification" refers to an amino acid modification that does not substantially affect or change the binding characteristics of the 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 are 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), as well as aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CARs described herein may 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) with 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 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 a cytoplasmic signaling sequence that controls the activation of immune cells in a stimulatory manner for at least some aspects of the immune cell signaling pathway. In one aspect, the signal is a primary signal, e.g., initiated by binding of the TCR / CD3 complex with a peptide-loaded MHC molecule, resulting in mediation of T cell responses, including but not limited to proliferation, activation, differentiation, and the like. Primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that act in a stimulatory manner may contain signaling motifs known as immune receptor 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., a 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 of 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 secretion of cytokines. In some embodiments, the intracellular signaling domain is the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Although 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 that is 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 T cell receptor, and the costimulatory intracellular signaling domain may comprise the cytoplasmic sequence from 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 term "zeta" or alternatively "zeta chain", "CD3 zeta", or "TCR zeta" is specified as the protein provided under GenBan 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, the "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. A 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 aspect, 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 immune responses, e.g., cells that are involved in promoting immune effector responses. 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 this term is used herein refers to a function or response, such as an immune effector cell function or response, that enhances or promotes the immune attack of target cells. For example, immune effector function or response refers to the property of T cells or NK cells that promotes the killing of target cells or the inhibition of the growth or proliferation of target cells. For T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0181] The term "encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as 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 thus have the biological properties conferred thereon. Thus, a gene, cDNA, or RNA codes for a protein when transcription and translation of the mRNA corresponding to that gene results in the protein in a cell or other biological system. Both the coding strand, which is the nucleotide sequence that is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of a gene or cDNA, can be referred to as encoding a protein or other product of that 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 is derived from or produced within an organism, cell, tissue, or system.

[0184] The term "exogenous" refers to any material that is introduced into, or produced outside, 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 isolated nucleic acid and can be used to deliver isolated nucleic acid to the inside of 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 acid 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 that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and 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 a 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 a significant amount of genetic information to the DNA of host cells, making them one of the most efficient methods of 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, in particular, self-inactivating lentiviral vectors as 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, for example, LENTIVECTOR® gene delivery technology from Oxford BioMedica, LENTIMAX™ vector system from Lentigen, and the like. 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. If a subunit position in both of the two molecules is occupied by the same monomer subunit, e.g., if a position in each of the 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, e.g., 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, and if 90% of the positions (e.g., 9 out of 10) are matching 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 further refinement and optimization of antibody or antibody fragment performance. Generally, a humanized antibody or antibody fragment thereof will contain substantially all of at least one, 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. A humanized antibody or antibody fragment may also contain 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 a 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, for example, 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 link between a control sequence and a heterologous nucleic acid sequence that results in the 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 when it affects the transcription or expression of the coding sequence. The operably linked DNA sequences may be contiguous with each other, e.g., in the same reading frame when necessary to join the coding regions of two proteins.

[0196] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, intratumor, 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-stranded or double-stranded form. The term "nucleic acid" includes genes, cDNA, or mRNA. In one embodiment, the nucleic acid molecule is synthetic (e.g., chemically synthesized) or recombinant. Unless specifically limited, the term encompasses nucleic acids that contain 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 as to the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, for example, and longer chains, of which there are numerous varieties and which are commonly referred to in the art as proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural polypeptide, a recombinant polypeptide, or a combination 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 a core promoter sequence, and in other cases, this sequence may also include enhancer sequences 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 the 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 molecules (typically proteins, carbohydrates, or lipids) that are preferentially expressed on the surface of cancer cells, either in whole or as fragments (e.g., MHC / peptides), as compared to normal cells, and are useful for preferentially targeting pharmacological agents to cancer cells. In some embodiments, the cancer-associated antigen is a cell surface molecule that is overexpressed in cancer cells as compared to normal cells, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more, as compared to normal cells. In some embodiments, the cancer-associated antigen is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule that contains deletions, additions, or mutations as compared to molecules expressed in normal cells. In some embodiments, the cancer-associated antigen will be expressed only on the cell surface of cancer cells, either in whole or as fragments (e.g., MHC / peptides), 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 the T cell receptor (TCR) 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 targeting 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 a library, such as a human scFv phage display library.

[0205] The term "flexible polypeptide linker" or "linker" as used in the context of scFv refers to a peptide linker consisting of amino acids such as glycine and / or serine residues used alone or in combination to link the heavy and light chain variable regions 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 the 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, an RNA 7-methylguanosine cap, or an 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 linked to the first transcribed nucleotide. Its presence is important for recognition by ribosomes 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 synthesis 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. In general, in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector contains the template used to generate the in vitro transcribed RNA.

[0208] As used herein, "poly(A)" is a series of adenosines attached to an mRNA by polyadenylation. In a preferred embodiment of a construct for transient expression, the polyA is 50-5000 (SEQ ID NO: 30), preferably greater than 64, more preferably greater than 100, and most preferably 300 or 400, and the poly(A) sequence may be chemically or enzymatically modified to modulate mRNA function such as localization, stability, or efficiency of translation.

[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 the 3' end. The 3' poly(A) tail is a long sequence of adenine nucleotides (often hundreds) added to a pre-mRNA through the action of polyadenylation polymerase enzymes. In higher eukaryotes, the poly(A) tail is added to transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the proteins bound to it help protect the mRNA from exonuclease degradation. Polyadenylation is also important for transcription termination, transport of mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but can additionally 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 of the host cell.

[0211] Apheresis is the process of removing whole blood from an individual, separating it into select components, and returning the remainder to the circulation. Generally, there are two methods for separating blood components: centrifugal and non-centrifugal. Leukapheresis results in 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 present 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 does not have to be 100%, 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 involve a decrease in cancer cell levels below a certain threshold, e.g., below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Reappearance may involve an increase in cancer cell levels above a certain threshold, e.g., above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Recurrence may be identified using the Cheson criteria, for example, as 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 entirety. For example, in the context of B-ALL, after a complete response, reappearance may involve, for example, the reappearance of blasts in blood, bone marrow (greater than 5%), or any non-bone marrow site. A complete response, in this context, may involve less than 5% BM blasts. More generally, in an embodiment, a response (e.g., a complete response or a partial response) may involve the absence of detectable MRD (minimal residual disease). In an embodiment, the initial response period lasts for at least 1, 2, 3, 4, 5, or 6 days, at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, or 4 months, at least 1, 2, 3, or 4 months, at least 6, 8, 10, or 12 months, or at least 1, 2, 3, or 4 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 a 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 are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[0218] In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with 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 considered as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc. as well as individual numerical values ​​within that range, for example, 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, including 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 disclosure provides methods for treating a disease associated with expression of a tumor antigen described herein.

[0223] In one aspect, the disclosure provides a method of treating cancer (e.g., hematological cancers such as ALL and CLL) by providing to a subject in need thereof an immune effector cell (e.g., T cell, NK cell) 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 disclosure provides a method of treating cancer (e.g., hematological cancers such as ALL and CLL) by providing a subject in need thereof with 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, where the cancer cells express CD19. In one embodiment, the cancer to be treated is a B-cell malignancy. In one embodiment, the cancer to be 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 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) can replicate in vivo, providing 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 remain 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 kind of cell therapy, in which immune effector cells (e.g., T cells, NK cells) are modified to transiently express a CAR, for example, by in vitro transcribed RNA, and the CAR-expressing cells are infused into a recipient in need thereof. The CAR-expressing cells (e.g., T cells, NK cells) generated by transduction (e.g., by transfection or electroporation) of the CAR RNA transiently express the RNA CAR for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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 are present for less than one month, for example, less than three weeks, less than two weeks, less than one week after the T cells are administered to the patient.

[0227] In one embodiment, the disclosure provides a method of treating cancer (e.g., hematological cancers such as ALL and CLL) by providing a subject in need thereof with 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) as 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 CD19 CARs to treat subjects with cancer (e.g., hematological cancers such as ALL and CLL), where the cancer cells express CD19. In one embodiment, the cancer to be 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 Table 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 an exemplary complete CD19 CAR construct generated using various CAR domains (e.g., transmembrane and intracellular signaling domains) described herein and the anti-CD19 antigen binding domain listed in Table 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, for example, 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, for example, including but not limited to, B-ALL, T-ALL, ALL, one or more chronic leukemias, for example, including but not limited to, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL).

[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 that are united by ineffective production (or dysplasia) of bone marrow blood cells, etc.

[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 diseases associated with the expression of CD19.

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

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

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

[0235] In one aspect, the disclosure provides a method of treating a subject with cancer (e.g., hematological cancers such as ALL and CLL) with a CAR-expressing cell (e.g., T cell, NK cell) (e.g., a CD19 CAR-expressing cell (e.g., T cell, NK cell) described herein, such as, for example, CTL019). In an embodiment, the disclosure provides a method of treating a subject with a CAR-expressing cell (e.g., T cell, 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 as described herein in combination with one or more chemotherapy cycles. In some embodiments, exemplary chemotherapy cycles include induction, consolidation, intermediate maintenance, late consolidation, 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 subjects 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 some embodiments, in subjects 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 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 doses 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 further chemotherapy cycle is consolidation, where the goal is to destroy any remaining cancerous cells. In some embodiments, a consolidation cycle is given to a subject after remission is achieved following an induction cycle. In some embodiments, in a subject with leukemia (e.g., ALL), a 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), a consolidation cycle includes treatment with vincristine and 6-mercaptopurine, e.g., 1.5 mg / m2 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 doses 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 further chemotherapy cycles include a maintenance cycle, where the goal is to reduce the long-term risk of cancer recurrence, or an intermediate maintenance cycle, where the goal is to provide a break from intensive treatment. In some embodiments, the intermediate maintenance cycle is performed after induction and, in some cases, after consolidation. In some embodiments, the intermediate maintenance cycle is performed before the late intensification cycle. In some embodiments, the intermediate maintenance cycle is performed after the late intensification cycle. In some embodiments, the maintenance cycle is the last chemotherapy cycle, 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 cycle comprises vincristine and methotrexate, e.g., at 7.5 mg / m, respectively. 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, respectively. 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 doses 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 doses 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 further 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 intermediate maintenance cycle. In some embodiments, in subjects 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 later intensification cycle includes treatment with vincristine, dexamethasone, doxorubicin, PEG-L-asparaginase, cyclophosphamide, cytarabine, and 6-thioguanine, e.g., at doses 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 measurements, e.g., to maximize survival while minimizing toxicity. In some embodiments, subjects with, e.g., ALL (e.g., pediatric ALL) are classified into standard risk or high risk groups, e.g., based on white blood cell count and age, e.g., based on 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 a 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 a high risk group if the white blood cell count is 50,000 cells per microliter or more and / or the subject is 10 years old or older. In some embodiments, for example, subjects with ALL (e.g., pediatric ALL) are classified into very high-risk groups, 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 adverse chromosomal abnormalities (e.g., t(9;22), MLL gene rearrangement, and hypodiploidy (chromosome number<44); subjects with slow initial response to first therapy; subjects with high minimal residual disease (MRD) levels at the end of remission induction cycle or subsequent cycles; subjects with morphologically persistent disease after remission induction cycle.

[0242] In certain embodiments, the standard of care for CLL includes, but is not limited to, the exemplary therapies 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 beam 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 therapies 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 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®), 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, polypheneprosan 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 includes 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 PFS), 5-fluorouracil (Adrucil®, Efudex®, Fluoroplex®), 6-thioguanine (Thioguanine PFS), 5-fluorouracil (Adrucil®, Efudex®, Fluoroplex®), 5-fluorouracil (FuCi®, FuCi®, FuCi®), 5-fluorouracil (F ... Tabloid®), fludarabine phosphate [Fludara®], pentostatin [Nipent®], pemetrexed [Alimta®], raltitrexed [Tomudex®], cladribine [Leustatin®], clofarabine [Clofarex®, Clolar®], cytarabine liposomal (Cytarabine liposomal) [liposomal Ara-C, also known as DepoCyt™], decitabine [Dacogen™]; hydroxyurea [Hydrea™, Droxia™, and Mylocel™]; mercaptopurine [Puri-Nethol™], pralatrexate [Folotyn™]; antibody therapeutics, including but not limited to 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 certain embodiments, the purine analog is fludarabine.

[0250] In certain embodiments, chemotherapy includes any of the following: nitrogen mustards, ethylenimine 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 (also known as Actinomycin-D, Cosmegen®), melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®), altretamine (also known as hexamethylmelamine (HMM), 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 [also known as DTIC, DIC, and imidazole carboxamide, 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. In certain embodiments, the alkylating agent is bendamustine.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 (also known as ABT 869 and available from Genentech, N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N'-(2-fluoro-5-methylphenyl)urea), sunitinib malate (Sutent®), bosutinib (also known as SKI-606 and described in U.S. Pat. No. 6,780,996, 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methylphenyl)urea]- tyrosine kinase inhibitors, including but not limited to, ibrutinib (1-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) -1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine (AZD5438), and XL281 (BMS908662). In one embodiment, the kinase inhibitor is a 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.

[0252] In certain embodiments, targeted therapies include, but are not limited to, anti-CD20 antibodies or functional fragments thereof, such as, for example, 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, for example, alemtuzumab (Campath®).

[0253] In some embodiments, the biological therapy includes immunotherapy. Exemplary anthracyclines include, but are not limited to, 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 transplant comprises an autogeneic stem cell transplant. In some embodiments, the stem cell transplant comprises an allogeneic stem cell transplant. In some embodiments, the stem cell transplant comprises an allogeneic bone marrow transplant. In some embodiments, the stem cell transplant comprises a hematopoietic stem cell transplant (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 tumor is negative for CD19 and a portion of 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, where the subject who has been treated for increased CD19 levels exhibits a disease associated with increased CD19 levels.

[0256] In one embodiment, the methods provided include a vector comprising a CD19 CAR operably linked to a promoter for expression in a mammalian cell (e.g., a T cell or an NK cell). In one embodiment, the methods provided include a recombinant cell (e.g., a T cell or an NK cell) expressing a CD19 CAR for use in treating a CD19-expressing tumor, where the recombinant T cell expressing a CD19 CAR is referred to as a CD19 CAR-expressing cell. In one embodiment, the CD19 CAR-expressing cell (e.g., a T cell, an NK cell) administered according to the methods provided can contact a tumor cell with at least one CD19 CAR expressed on the surface of the CD19 CAR-expressing cell, such that the CAR-expressing cell targets the tumor cell 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, comprising contacting the tumor cell with a CD19 CAR-expressing cell (e.g., T cell, 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 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) can replicate in vivo, providing long-term persistence that can lead to sustained tumor control. In various aspects, the cells administered to the 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 the patient are present for less than one month, e.g., less than three weeks, less than two weeks, 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) may be an active or passive immune response, or alternatively, may result from a direct versus indirect immune response. In one embodiment, CAR-transduced T cells respond to human cancer cells expressing CD19 by exhibiting specific pro-inflammatory cytokine secretion and potent cytolytic activity, resisting soluble CD19 inhibition, mediating bystander killing, and mediating 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., human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR as 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, such as leukemia and malignant lymphoproliferative conditions, that affect the blood, bone marrow, and lymphatic system.

[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 "preleukemia"), a diverse collection of blood conditions that are 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, such as B-cell promyelocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small 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 "preleukemia," a diverse collection of blood conditions united by ineffective production (or dysplasia) of bone marrow blood cells, etc.

[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 cells, NK cells) described herein that binds to the CD19-expressing cells. In a specific embodiment, the present disclosure provides a method for inhibiting or reducing the proliferation of a cancer cell population expressing CD19, comprising contacting a CD19-expressing cancer cell population with a CD19 CAR-expressing cell (e.g., T cells, NK cells) described herein that binds to the CD19-expressing cells. In one embodiment, the present disclosure provides a method for inhibiting or reducing the proliferation of a cancer cell population expressing CD19, comprising contacting a CD19-expressing cancer cell population with a CD19 CAR-expressing cell (e.g., T cells, NK cells) 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 percentage 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 having myeloid leukemia or another cancer associated with CD19-expressing cells or in an animal model thereof, 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., hematological cancer or atypical cancers expressing CD19), comprising administering to a subject in need thereof a CAR-expressing cell (e.g., T cell, NK cell) as described herein that binds to CD19-expressing cells. In one embodiment, the subject is a human. Non-limiting examples of disorders associated with CD19-expressing cells include autoimmune disorders (e.g., lupus), inflammatory disorders (e.g., allergy and asthma), and cancers (e.g., hematological cancer or atypical cancers expressing 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) as 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 recurrence of cancer associated with CD19-expressing cells (e.g., hematological cancers such as ALL and CLL), comprising administering to a subject in need thereof a CD19 CAR-expressing cell (e.g., T cell, NK cell) as 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) as 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 as described herein. The pharmaceutical composition may be administered simultaneously with, prior to, or after one or more other additional therapeutic agents or 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, for example, two or more therapies are delivered after the subject is diagnosed with a disorder and before the disorder is cured or eliminated or before the 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 may be 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 is more effective, for example, when the second treatment is administered without the first treatment, or when a similar situation occurs with the first treatment, the second treatment is less effective or the degree to which the second treatment reduces symptoms is greater.In some embodiments, the delivery is such that the reduction in symptoms, or other parameters related to the disorder, is greater than that which would be observed if one treatment is delivered in the absence of the other.The effect of the two treatments may be partially additive, totally additive, or greater than additive.The delivery may be such that the effect of the first treatment delivered is still detectable when the second one 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 aspects, the CAR-expressing cells described herein can be used in combination treatment regimens 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 chemotherapeutic agents. 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, decarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzumab, gemtuzumab, rituximab, tositumomab), antimetabolites (e.g., leiomyomab ... 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), immunomodulators 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 rifampicin (Ricin®). 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 (difluprit), 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, polipheprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

[0276] Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes, uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard, and the like). 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 (also known as Actinomycin-D, Cosmegen®), melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®), altretamine (also known as hexamethylmelamine (HMM), 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 [also known as DTIC, DIC, and imidazole carboxamide, 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. WO03 / 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-aspartyl L-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-[(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 the GITR fusion proteins 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. Patent No. 7,618,632, and PCT Publication No. WO2011 / 051726.

[0283] In an embodiment, a CAR-expressing cell described herein, e.g., a CD19 CAR-expressing cell (e.g., T cell, NK cell), 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, e.g., RAD001, a target of the rapamycin signaling pathway, e.g., an mTOR inhibitor described herein. In an embodiment, the mTOR inhibitor is administered prior to the CAR-expressing cell. For example, in an embodiment, the mTOR inhibitor may be administered prior to apheresis of the cells. In an embodiment, the subject has cancer (e.g., a hematological cancer such as ALL and CLL). In an embodiment, the subject has ALL. In an embodiment, the subject has CLL.

[0284] In an embodiment, a CAR-expressing cell described herein, e.g., a CD19 CAR-expressing cell (e.g., T cell, NK cell), e.g., CTL019, is administered to a subject, e.g., a subject identified as a partial responder or non-responder, in combination with a GITR agonist, e.g., a GITR agonist described herein. In an embodiment, the GITR agonist is administered before the CAR-expressing cell. For example, in an embodiment, the GITR agonist can be administered before apheresis of the cells. In an embodiment, the subject has cancer (e.g., hematological cancer such as ALL and CLL). In an embodiment, the subject has ALL. In an embodiment, 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, such as 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 the administration of a CAR-expressing cell described herein in combination with a kinase inhibitor, such as 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 the administration of a CAR-expressing cell described herein in combination with a kinase inhibitor, such as a kinase inhibitor described herein, e.g., at a dose or dosing schedule described herein.

[0286] In an embodiment, the kinase inhibitor is a CDK4 inhibitor, such as a CDK4 inhibitor described herein, such as 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 an embodiment, the kinase inhibitor is a BTK inhibitor, such as a BTK inhibitor described herein, such as ibrutinib. In an embodiment, the kinase inhibitor is an mTOR inhibitor, such as an mTOR inhibitor described herein, such as rapamycin, rapamycin analogs, OSI-027, etc. 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 14-21 days of a 28 day cycle, daily for 7-12 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-aspartyl-L-serine- (SEQ ID NO: 356), inner salt (SF1126), and an mTOR inhibitor selected from 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, including rapalogs such as RAD001, and allosteric mTOR inhibitors. 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 subjects. 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 one embodiment, administration of a low immune enhancing dose of an mTOR inhibitor comprises the following: 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 to PD-1 positive immune effector cells; iv) 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 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 Increase in CD27 + an increase in the number of cells having any one or more combinations of an increase in IL-1, 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 untreated subjects. 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 untreated subjects. In some embodiments, the 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 an mTOR inhibitor may be initiated prior to administration of a CAR-expressing cell described herein, may be completed prior to administration of a CAR-expressing cell described herein, may be initiated simultaneously with administration of a CAR-expressing cell described herein, may overlap with administration of a CAR-expressing cell described herein, or may be continued after administration of a CAR-expressing cell 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 as described herein. In such an embodiment, administration of a low immune enhancing dose of an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or a catalytic inhibitor, is started or completed before harvesting immune effector cells, e.g., T cells or NK cells, from a subject that are to be engineered to express a CAR molecule as 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% or at least 150%. 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% at least 5% but not more than 60%, at least 10% but not more than 60%, at least 15% but not more than 60%, at least 20% but not more than 60%, at least 30% but not more than 60%, at least 40% but not more than 60%, at least 5% but not more than 50%, at least 10% but not more than 50%, at least 15% but not more than 50%, at least 20% but not more than 50%, at least 30% but not more than 50%, at least 40% but not more than 50%, In some embodiments, the method is associated with or provides 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 conveyed 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 the reduction in 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 evaluating the change 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 pharma- ceutically acceptable salt. In some embodiments, the mTOR inhibitor is an allosteric inhibitor. Allosteric mTOR inhibitors include the natural tricyclic compound rapamycin (sirolimus), rapamycin-related compounds, which are compounds that have structural and functional similarity with rapamycin, such as rapamycin derivatives, rapamycin analogs (also called rapalogs), and 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, for example, 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 name is given in terms of rapamycin using the numbering scheme of formula A.

[0307] Rapamycin analogs useful in the present invention include those in which the hydroxyl group on the cyclohexyl ring of rapamycin is OR 1 wherein R 1 O-substituted analogs in which R is hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl, e.g., RAD001, also known as everolimus, described in US 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 positions. The rapamycin analog may be an epimer of the above-mentioned analogs, for example, an epimer of an analog substituted at the 40, 28 or 26 positions, and optionally hydrogenated, as described in US 6,015,815, WO 95 / 14023, and WO 99 / 15530, the contents of which are incorporated by reference, for example, ABT578, also known as zotarolimus or a rapamycin analog, for example, AP23573, also known as ridaforolimus, as described in US 7,091,213, WO 98 / 02441, and WO 01 / 14387, the contents of which are incorporated by reference.

[0308] Examples of rapamycin analogues from US 5,665,772 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'-dihydroxypent-2'-ene-1'-yl)-rapamycin, 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'-dicarboethoxy-1',2',3'-triazol-1'-yl)-ethyl]-rapamycin.

[0309] Other rapamycin analogs useful in the present invention are those 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 found in U.S. RE 44,768, e.g., temsirolimus, are known.

[0310] Other rapamycin analogs useful in the present invention include those in which the methoxy group at position 16 is replaced by other substituents, preferably alkynyloxy (optionally hydroxy substituted), benzyl, orthomethoxybenzyl, or cyclobenzyl, and / or the methoxy group at position 39 is missing 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 may 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-hydroxy {1-(2-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 called 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-related 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]oxazo 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, pharma- ceutically acceptable salts, or analogs thereof of any of the foregoing.

[0320] mTOR inhibitors such as RAD001 may be formulated for delivery based on the specific dosages described herein and methods well established in the art. In particular, 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 have an allergic reaction to the therapeutic agents described herein and / or other anti-cancer agents during or after administration, and therefore, to minimize the risk of an allergic reaction, an anti-allergy agent may be administered. 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, and 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 reduce the pain experienced during treatment are often prescribed to make patients more comfortable. Common non-prescription painkillers, such as Tylenol®, are often used. However, opioid painkillers, 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, cytoprotectants (such as neuroprotectants, free radical scavengers, cardioprotectants, anthracycline extravasation neutralizers, and nutrients) may be used as adjunctive therapy.Suitable cytoprotectants 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 overview book "The Merck Index" or from databases such as Patents International (e.g. IMS World Publications).

[0327] The above-mentioned compounds 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 pharma- ceutically acceptable salt thereof, together with a pharma- ceutically 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 disease, 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 pharma- ceutically 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 therapeutic agent or will be administered separately.

[0331] In combination therapy, the compounds of the present invention and other anti-cancer drugs can be administered either simultaneously, in parallel, or sequentially without any specific time restriction, such administration providing 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 may vary according to the stage of disease, the physical compatibility of the patient, the safety profile of each drug, and the tolerability of each drug, as well as other criteria known to the doctor and medical practitioner who administers the combination drug.The compound of the present invention and other anticancer drugs may be administered within minutes, hours, days, or weeks of each other, depending on the specific cycle used in treatment.In addition, the cycle may 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, a kit is provided that includes one or more compounds of the present invention and combination partners disclosed herein.A representative kit includes (a) a compound of the present invention or a pharma- ceutically acceptable salt thereof, (b) at least one combination partner, such as those described above, and such a kit may include a package insert or other label that includes instructions for administration.

[0334] The compounds of the 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 invention may be used, in particular, as radiosensitizers for the treatment of tumors that are poorly sensitive to radiation therapy.

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

[0336] In one aspect, the disclosure features immune effector cells (e.g., T cells, NK cells) engineered to express a CAR, which exhibits anti-tumor properties. A preferred antigen is a cancer-associated antigen (i.e., tumor antigen) as 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 expressing the tumor antigens described herein.

[0338] In one aspect, the CARs of the present invention can be used to eradicate normal cells expressing 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 differentiation stages. Differentiation stages of T cells include, from least differentiated to most differentiated, 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 appropriate T cell receptors, 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-, and CD95-. Stem central memory T cells (T SCM ) are characterized by the expression pattern of the following cell surface markers: CCR7+, CD62L+, CD45RO-, and 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 expression pattern of cell surface markers: 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 white blood cells, red blood cells, and platelets. In one aspect, cells collected by apheresis may be washed to remove the plasma fraction and, if appropriate, place the cells in a suitable buffer or medium for subsequent processing steps. In one embodiment, the cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the washing solution may be calcium-free and magnesium-free, or may be free of many, but not all, divalent cations.

[0343] An initial activation step in the absence of calcium may result in the expansion of activation. As will be readily understood by those skilled in the art, the washing step may 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 a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline with or without buffer. Alternatively, the undesired components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0344] It is understood that the methods 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, the selection of 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 herein by way of example. 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%, less than 1% CD25+ cells.

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

[0348] In one embodiment, regulatory T cells, e.g., CD25+ T cells, are removed from the population using 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, more 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 about 6×10 9 In other embodiments, the population of immune effector cells to be depleted includes about 1×10 9 ~1×10 10In one embodiment, the resulting regulatory T cell depleted cell population comprises 2×10 CD25+ T cells, 1×10 CD25+ T cells, or any integer value therebetween. 9 10 or less regulatory T cells, e.g., CD25+ cells (e.g., 1×10 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 with a depletion tube set, e.g., tube 162-01, etc. In one embodiment, the CliniMAC system is run in a depletion setting, e.g., DEPLETION2.1.

[0351] Without wishing to be bound by a particular theory, it is contemplated that the level of a negative regulator of immune cells in a subject prior to apheresis or during production of the CAR-expressing cell product may be reduced (e.g., by reducing the level 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 method of production includes generating a T cell prior to generation of the CAR-expressing cell. REG For example, the production method can include reducing (e.g., depleting) the number of cells, e.g., prior to production of the CAR-expressing cell (e.g., T cell, NK cell) product. REG To deplete cells, the method includes contacting the sample, e.g., an apheresis sample, with an anti-GITR antibody and / or an anti-CD25 antibody (or a fragment thereof or a CD25-binding ligand).

[0353] In one embodiment, the subject is treated with T REG In one embodiment, the subject is pretreated with one or more therapies that reduce the risk of relapse to the 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 removed is neither regulatory T cells nor tumor cells, but cells that would otherwise negatively affect the proliferation and / or function of CART cells, such as cells expressing CD14, CD11b, CD33, CD15, or other markers potentially expressed by immunosuppressive cells. In one embodiment, it is envisioned that such cells are removed in parallel with the regulatory T cells and / or tumor cells, or after said depletion, or in a different sequence.

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

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

[0358] The methods described herein may further comprise 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, the tumor antigen-expressing cells are removed simultaneously with the regulatory T cells, e.g., CD25+ cells. For example, the anti-CD25 antibody or fragment thereof and the anti-tumor antigen antibody or fragment thereof can be attached to the same substrate, e.g., beads, which can be used to remove the cells, or the anti-CD25 antibody or fragment thereof, or the anti-tumor antigen antibody or fragment thereof can be attached to separate beads, and a mixture of these can be used to remove the cells. In other embodiments, the removal of the regulatory T cells, e.g., CD25+ cells, and the removal of the tumor antigen-expressing cells are sequential, e.g., can occur in either order.

[0359] 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, are removed from the population, thereby producing 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-1, CEACAM-2, CEACAM-3, CEACAM-4, CEACAM-5, CEACAM-6, CEACAM-7, CEACAM-8, CEACAM-9, CEACAM-10, CEACAM-11, CEACAM-12, CEACAM-13, CEACAM-14, CEACAM-15, CEACAM-16, CEACAM-17, CEACAM-18, CEACAM-19, CEACAM-20, CEACAM-21, CEACAM-22, CEACAM-23, CEACAM-24, CEACAM-25, CEACAM-26, CEACAM-27, CEACAM-28, CEACAM-29, CEACAM-30, CEACAM-31, CEACAM-32, CEACAM-33, CEACAM-34, CEACAM-35, CEACAM-36, CEACAM-37, CEACAM-38, CEACAM-39, CEACAM-41, CEACAM-42, CEACAM-43, CEACAM-44, CEACAM-45, CEACAM-56, CEACAM-19, CEACAM-25, CEACAM-37, CEACAM-45, CEACAM-19, CEACAM-26, CEACAM-38, CEACAM-45, CEACAM-19, CEACAM-27, CEACAM-39 Also provided are methods that include obtaining a population of AM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR (e.g., TGFR beta). In one embodiment, checkpoint inhibitor expressing cells are simultaneously removed with regulatory T cells, e.g., CD25+ cells. For example, anti-CD25 antibody or fragment thereof and anti-checkpoint inhibitor antibody or fragment thereof can be attached to the same bead, which can be used to remove cells, or anti-CD25 antibody or fragment thereof and anti-checkpoint inhibitor antibody or fragment thereof can be attached to separate beads, which mixture can be used to remove cells. 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 about 30 minutes. In further embodiments, this period ranges from 30 minutes to 36 hours or more, as well as all integer values ​​therebetween. In further embodiments, 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. In any situation where there are few T cells present compared to other cell types, such as isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals, longer incubation times may be used to isolate T cells. Furthermore, the use of 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 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 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 beads or other surfaces, subpopulations of T cells can be selected for or against at the beginning of culture or at other desired times.

[0361] In one embodiment, IFN- γA T cell population can be selected that expresses 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, such as 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 the 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 concentration of cells) to ensure maximum contact between 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 further embodiments, a concentration of 125 million or 150 million cells / ml can be used.

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

[0364] In related embodiments, it may be desirable to use lower cell concentrations. By greatly 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 antigens that bind 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 1×10 5 / ml~1×10 6 / ml, and any integer value therebetween.

[0365] In other embodiments, the cells may be incubated on a rotator at various speeds for various lengths of time at either 2-10° C. or at 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 removes granulocytes and to some extent monocytes in the cell population, resulting in a more homogenous product. After removing plasma and platelets by a washing step, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and 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, and 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] Also contemplated in the context of the present invention is the collection of blood samples or apheresis products from a subject at a time before the expanded cells described herein may be required. Thus, the source of cells for expansion can be collected at any time required, and the desired cells, e.g., 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, blood samples or apheresis are obtained from generally healthy subjects. In certain embodiments, blood samples or apheresis are obtained from generally healthy subjects who are at risk of developing disease, but have not yet developed disease, and the cells of interest 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, samples are collected from patients shortly after diagnosis of a particular disease described herein, but prior to 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 after treatment with drugs that damage the immune system, the quality of T cells obtained may be optimal or improved in terms of their ability to expand ex vivo at a time 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 favorable state for enhanced engraftment and in vivo expansion. Thus, it is contemplated in 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 expansion of certain cell types, particularly during a given 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 that 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 is harvested after a sufficient time from, or after sufficient dosing of, 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 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 methods 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 deficient in diglycerol kinase (DGK).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, for example, siRNA, shRNA, miRNA, to reduce or prevent DGK expression.Alternatively, DGK-deficient cells can be generated by treatment with DGK inhibitors as described herein.

[0375] In one embodiment, T cell population is Ikaros-deficient.Ikaros-deficient cell includes the cell that does not express Ikaros RNA or protein, or has reduced or inhibited Ikaros activity, and Ikaros-deficient cell can be generated by genetic approach, for example, by administering RNA interference agent, for example, siRNA, shRNA, miRNA, to reduce or prevent Ikaros expression.Alternatively, Ikaros-deficient cell can be generated by treatment with Ikaros inhibitor, for example, 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 cell can be an allogeneic immune effector cell, such as a T cell or a NK cell. For example, the cell can be an allogeneic T cell, such as an allogeneic T cell that lacks expression of a functional T cell receptor (TCR) and / or a human leukocyte antigen (HLA), such as HLA class I and / or HLA class II.

[0379] A T cell lacking a functional TCR may, for example, be engineered to not express any functional TCR on its surface, or to not express one or more subunits that comprise a functional TCR (e.g., not express (or exhibit reduced expression of) TCR alpha, TCR beta, TCR gamma, TCR delta, TCR epsilon, and / or TCR zeta), or to produce very little functional TCR on its surface. Alternatively, the T cell may express a substantially impaired TCR, for example, by expression of a mutated or truncated form 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 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 as well as a functional HLA, e.g., HLA class I and / or HLA class II.

[0382] Modified T cells lacking expression of functional TCR and / or HLA can be obtained by any suitable means, including knocking out or knocking down one or more subunits of TCR or HLA. For example, T cells can include knockdown of 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 can 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 can be cells that do not express or express at low levels of inhibitory molecules, for example, 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. Inhibition of inhibitory molecules, for example, by inhibition at 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...

Claims

1. 1. A method of enriching or generating immune effector cells (e.g., cell populations) suitable for use in CAR therapy, comprising: (i) the timing of obtaining immune effector cells from a subject (e.g., a hematological cancer patient); (ii) the timing of chemotherapy relative to said immune effector cell acquisition; (iii) type of chemotherapy; (iv) primary malignant tumors, (v) immune effector cell (e.g., T cell) parameters, e.g., one or more of T cell numbers, T cell phenotype, or T cell function. obtaining (e.g., obtaining or harvesting) said immune effector cells according to one, two, three, four, five or more (all) of, or a combination of two, three, four or five of (i)-(v); thereby enriching or generating said immune effector cells suitable for use in CAR therapy.

2. 1. A composition comprising immune effector cells (e.g., cell populations) expressing a CAR molecule ("CAR-expressing cells") for use in combination with chemotherapy to treat or provide anti-tumor immunity to a subject with a hematological cancer, wherein the immune effector cells are (i) the timing of obtaining immune effector cells from a subject (e.g., a cancer patient); (ii) the timing of chemotherapy relative to said immune effector cell acquisition; (iii) type of chemotherapy; (iv) primary malignant tumors, (v) immune effector cell (e.g., T cell) parameters, e.g., one or more of T cell numbers, T cell phenotype, or T cell function. or a combination of two, three, four, or five of (i)-(v) are obtained (e.g., obtained or harvested) from the subject; (Optionally) the immune effector cells are obtained from the subject prior to introduction of the CAR molecule.

3. 1. A method of treating a subject having a hematological cancer or providing anti-tumor immunity to said subject, comprising administering to said subject an effective amount of immune effector cells (e.g., a population of cells) expressing a CAR molecule ("CAR-expressing cells" or "CAR therapy") in combination with chemotherapy, said immune effector cells being (i) the timing of obtaining immune effector cells from a subject (e.g., a cancer patient); (ii) the timing of chemotherapy relative to said immune effector cell acquisition; (iii) type of chemotherapy; (iv) primary malignant tumors, (v) immune effector cell (e.g., T cell) parameters, e.g., one or more of T cell numbers, T cell phenotype, or T cell function. or a combination of two, three, four, or five of (i)-(v) are obtained (e.g., obtained or harvested) from the subject; (Optionally) the immune effector cells are obtained from the subject prior to introduction of the CAR molecule; thereby treating said hematological cancer or providing anti-tumor immunity against said hematological cancer.

4. 4. The method of any of claims 1-3, wherein the immune effector cell population exhibits an increase in one or more of ex vivo expansion of the immune cell population, efficacy of the immune cell population for a therapy, or yield of the immune cell population when any of (i)-(v) is optimized.

5. 5. The method of any of claims 2-4, wherein the CAR-expressing cell comprises a nucleic acid encoding a CAR, e.g., a CAR molecule described herein, e.g., a CD19 CAR described herein (e.g., CTL019).

6. 6. The method of any of claims 2-5, further comprising introducing into said immune effector cell population a nucleic acid encoding a CAR, e.g., a presently claimed CAR molecule, e.g., a presently claimed CD19 CAR (e.g., CTL019).

7. 7. The method of claim 5 or 6, wherein the CAR molecule comprises a sequence according to Table 1 or Table 4.

8. The method according to any one of claims 1 to 7, wherein the subject from which immune cells are obtained and / or the subject to be treated is a human cancer patient.

9. 9. The method of claim 8, wherein the subject is 18 years of age or younger (e.g., 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or younger).

10. 10. The method of claim 8, wherein the subject is over 18 years of age.

11. The method according to any one of claims 1 to 10, wherein the subject has a disease associated with expression of a tumor-associated antigen or a cancer-associated antigen.

12. The blood cancers include 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, 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, and leukemia.

12. The method of any of claims 1 to 11, wherein the cancer is selected from one or more of the following: myeloma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma (HL), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenstrom's macroglobulinemia.

13. The method of any of claims 1 to 11, wherein the hematological cancer is a leukemia, e.g., CLL, ALL, or a lymphoma, e.g., MCL, NHL, or HL.

14. The method of any of claims 1-9 or 11-13, wherein the subject has leukemia and is a pediatric patient, for example aged 18 years or younger.

15. The method of any of claims 1 to 14, wherein the subject is classified as having standard risk, high risk, or very high risk ALL.

16. The method of any of claims 1 to 14, wherein the subject does not have lymphoma, for example, the subject does not have non-Hodgkin's lymphoma (NHL).

17. The method of any of claims 1 to 16, wherein the subject does not have recurrent cancer.

18. The method according to any one of claims 1 to 17, wherein the immune effector cell population is obtained prior to administering the chemotherapy to the subject.

19. The method of any of claims 1 to 17, wherein the immune effector cell population is obtained before the subject has undergone 2, 3, 4, or 5 chemotherapy cycles.

20. 18. The method of any of claims 1-17, wherein the obtaining of the immune effector cell population occurs after the subject has undergone one chemotherapy cycle but before the subject has undergone more than one (e.g. more than one, more than two, more than three, more than four, or more than five) chemotherapy cycles.

21. The method of any of claims 1 to 20, 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.

22. 22. The method of claim 21, wherein the immune effector cell population is obtained from the subject before the subject undergoes a consolidation chemotherapy cycle or an induction chemotherapy cycle.

23. 22. The method of claim 21, wherein the immune effector cell population is obtained from the subject before the subject undergoes a consolidation chemotherapy cycle.

24. 22. The method of claim 21, wherein 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.

25. 22. The method of claim 21, wherein the immune effector cell population is obtained from the subject before the subject undergoes a later potentiation cycle.

26. 22. The method of claim 21, wherein 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 late intensification cycle.

27. The method of any of claims 1 to 26, wherein the one or more chemotherapy cycles or drugs are selected from Table 8.

28. The method of any of claims 1-27, wherein the chemotherapy comprises the drugs and dosing regimens set forth in Table 8.

29. 28. The method of any of claims 1-27, 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.

30. The method of any of claims 1 to 27, wherein the immune effector cell population is obtained from the subject before the subject receives cyclophosphamide and / or cytarabine.

31. 31. The method of any of claims 1 to 30, wherein the obtained immune effector cell population comprises one or more of a higher number of less differentiated T cells, e.g. a higher number 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 further rounds of chemotherapy).

32. The method of claim 231, wherein the obtained immune effector cell population comprises at least 20% naive T cells, at least 2% stem central memory T cells, and / or at least 4% central memory T cells.

33. The method of claim 31 or 32, wherein the obtained immune effector cell population comprises at least 50% central memory T cells.

34. 33. The method of claim 31 or 32, wherein the obtained immune effector cell population comprises less than 55% effector memory T cells and terminal effector T cells combined.

35. 32. The method of claim 31 , wherein the obtained immune effector cell population exhibits an increase in absolute T cell counts (ATC) 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).

36. 32. The method of claim 31 , wherein the obtained immune effector cell population comprises an absolute number of T cells of at least 400 per microliter, an absolute number of naive T cells of at least 200 per microliter, an absolute number of stem central memory T cells of at least 20 per microliter, and / or an absolute number of central memory T cells of at least 40 per microliter.

37. 37. The method of any of claims 1 to 36, wherein the obtained 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+.

38. 33. The method of claim 32, 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+.

39. 39. The method of any of claims 1 to 38, wherein the immune cell population is obtained from a subject with a hematological cancer, such as a leukemia, such as CLL, ALL, or a lymphoma, such as MCL, NHL, or HL.

40. 40. The method of any of claims 1 to 39, further comprising removing regulatory T cells, such as CD25+ T cells, from the obtained immune cell population, thereby obtaining a regulatory T cell depleted cell population, such as a CD25+ depleted cell population.

41. 41. The method of claim 40, wherein 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%, less than 1% CD25+ cells.

42. 42. The method of any of claims 1 to 41, wherein the obtained immune effector cell population is cells of a subject with cancer, such as a subject with a CD25-expressing cancer, such as chronic lymphocytic leukemia (CLL).

43. 42. The method of claim 41 , wherein 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.

44. 41. The method of claim 40, wherein the regulatory T cells, e.g., CD25+ T cells, are depleted from the obtained immune effector cell population using an anti-CD25 antibody or a fragment thereof.

45. 45. The method of claim 44, wherein the anti-CD25 antibody or fragment thereof is conjugated to a substrate, e.g., a bead.

46. 44. The method of claim 43, wherein 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%, less than 1% of the leukemia cells, e.g., CLL cells, ALL cells, or lymphoma cells, e.g., MCL cells, HL cells.

47. 44. The method of claim 43, wherein 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., CLL cells.

48. 44. The method of claim 43, wherein 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., CLL cells.

49. 49. The method of any of claims 1 to 48, further comprising removing cells expressing a tumor antigen, such as a tumor antigen free of CD25, such as CD19, CD30, CD38, CD123, CD20, CD14, or CD11b, from said obtained immune effector cell population, thereby obtaining a regulatory T cell depleted cell population, such as a CD25+ depleted cell population, and a population of tumor antigen depleted cells suitable for expression of a CAR, such as a CAR as described in the present claims.

50. 49. The method of any of claims 1 to 48, further comprising removing cells expressing a check point inhibitor, such as a check point inhibitor as described in the present claims, e.g. one or more of PD1+ cells, LAG3+ cells, and TIM3+ cells, from the obtained immune effector cell population, thereby obtaining a population of regulatory T cell depleted cells, such as CD25+ depleted cells, and check point inhibitor depleted cells, such as PD1+, LAG3+, and / or TIM3+ depleted cells.

51. 49. The method of any of claims 1 to 48, wherein the obtained immune effector cell population is selected based on expression of one or more markers, e.g. CD3, CD28, CD4, CD8, CD45RA and CD45RO, e.g. the obtained immune effector cell (e.g. T cell) population is CD3+ and / or CD28+.

52. The method of any of claims 1 to 51, further comprising activating said regulatory T cell depleted cell population, such as a CD25+ depleted cell population.

53. 53. The method of any of claims 1-52, further comprising transducing cells from the immune effector cell population with a vector comprising a nucleic acid encoding a CAR, such as a presently claimed CAR, such as a presently claimed CD19 CAR.

54. 54. The method of claim 53, further comprising expanding the immune effector cell population engineered to express a CAR, e.g., a CAR described in the present claims, e.g., a CD19 CAR described in the present claims, e.g., by a method described in the present claims.

55. 55. The method of any of claims 1 to 54, further comprising transducing cells from said regulatory T cell depleted cell population, such as said CD25+ depleted cell population, with a vector comprising a nucleic acid encoding a CAR, such as a presently claimed CAR, such as a presently claimed CD19 CAR.

56. 56. The method of claim 55, further comprising expanding said regulatory T cell depleted cell population 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.

57. 57. The method of claim 54 or 56, wherein the cell population is expanded for a period of 8 days or less, such as 7 days, 6 days, or 5 days.

58. 57. The method of claim 54 or 56, wherein the cell population is 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.

59. 59. The method of claim 58, wherein the cells expanded for 5 days exhibit at least a 1-fold, 2-fold, 3-fold, or 4-fold increase in cell doublings upon antigenic stimulation compared to the same cells expanded in culture for 9 days under the same culture conditions.

60. 59. The method of claim 58, wherein the cells are expanded in culture for 5 days, and the resulting cells exhibit higher pro-inflammatory cytokine production, e.g., IFN-γ and / or GM-CSF levels, as compared to the same cells cultured in culture for 9 days under the same culture conditions.

61. The method of claim 54 or 56, wherein the cell population is expanded by culturing the cells in the presence of an agent that stimulates a CD3 / TCR complex associated signal, e.g., as described in the present claims, 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 a fragment thereof, and / or an anti-CD28 antibody or a fragment thereof.

62. 56. The method of any of claims 54-61-55, wherein the cell population is expanded in an appropriate medium (e.g. a medium as described in the present claims) 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 expansion period as measured by a method as described in the present claims, e.g. flow cytometry.

63. 63. The method of claim 62, wherein the cell population is expanded in the presence of a cytokine, such as IL-15 and / or IL-7 (e.g., IL-15 and IL-7).

64. 64. The method of any of claims 54-63, wherein said population of cells is cryopreserved after said expansion period.

65. 65. The method of any of claims 1-64, further comprising contacting the immune effector cell population with a nucleic acid encoding a telomerase subunit, e.g., hTERT.

66. For example, 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) generated by any of the presently claimed methods.

67. (i) said 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) the immune effector cell population comprises an absolute number of T cells per microliter of at least 400, an absolute number of naive T cells per microliter of at least 200, an absolute number of stem central memory T cells per microliter of at least 20, and / or an absolute number of central memory T cells per microliter of at least 40. The immune effector cell preparation (e.g., reaction mixture) of claim 66, selected from:

68. The immune effector cell preparation (e.g., reaction mixture) of claim 66, 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+.

69. 68. The immune effector cell preparation (e.g., reaction mixture) of claim 67, 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+.

70. 70. An immune effector cell preparation (e.g. a reaction mixture) according to any of claims 67 to 69, comprising a nucleic acid encoding a CAR, e.g. a CAR as claimed herein.

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