BR112016022798B1 - USES OF CELLS EXPRESSING CAR19, METHOD OF PRODUCING CELLS EXPRESSING CAR19, REACTIONAL MIXTURES, AND COMPOSITIONS AND THEIR USES - Google Patents

Abstract

CANCER TREATMENT USING ANTICD19 CHIMERIC ANTIGEN RECEPTOR. The invention provides compositions and methods for treating diseases associated with CD19 expression, for example, by administering a recombinant T cell comprising the CD19 CAR as described herein, in combination with a kinase inhibitor, e.g. of kinase described here. The invention also provides kits and compositions described herein.

Description

[001] This Application claims priority to applications US Serial No. 61/976,396, filed April 7, 2014, US Serial No. 62/007,309, filed June 3, 2014, US Serial No. 62/036,493, filed August 12, 2014, US Serial No. 62/076,238, filed November 6, 2014, US Serial No. 62/087,888, filed December 5, 2014, and Serial No. US 62/097,278, filed December 29, 2014, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

[002] This application contains a sequence listing that was submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 6, 2015, is called N2067-7051WO_SL.txt and is 252,236 bytes in size.

FIELD OF INVENTION

[003] The present invention generally relates to the use of T cells created to express a Chimeric Antigen Receptor (CAR), for example, in combination with another agent such as, for example, a kinase inhibitor and/or a cytokine, to treat a disease associated with the expression of Cluster of Differentiation Protein 19 (CD19).

BACKGROUND OF THE INVENTION

[004] Many patients with B cell malignancies are incurable with standard therapy. Additionally, traditional treatment options often have serious side effects. Attempts have been made in cancer immunotherapy, however, several obstacles make this goal very difficult to achieve clinical efficacy. Although hundreds of so-called tumor antigens have been identified, these are generally self-derived and thus are weakly immunogenic. Furthermore, tumors use several mechanisms to make them hostile to the initiation and propagation of immune attack.

[005] Recent developments using chimeric antigen receptor (CAR)-modified autologous T cell therapy (CART), which rely on redirection of T cells to appropriate cell surface molecule on cancer cells such as B cell malignancies, present promising results in harnessing the power of the immune system to treat B-cell malignancies and other cancers (see, for example, Sadelain et al., Cancer Discovery 3:388-398 (2013)). Clinical results of murine-derived CART19 (i.e., "CTL019") have shown promise in establishing complete remissions in patients suffering from CLL as well as in childhood ALL (see, e.g., Kalos et al., Sci Transl Med 3 :95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM 368:1509-1518 (2013)). In addition to the ability of the chimeric antigen receptor on genetically modified T cells to recognize and destroy targeted cells, a successful therapeutic T cell therapy needs to have the ability to proliferate and persist over a period of time, and also monitor leukemic cell escapes. . Variable T cell quality, whether it results from anergy, suppression, or exhaustion, will have effects on the performance of CAR-transformed T cells, but practitioners have limited control over this time period. To be effective, CAR-transformed patient T cells need to persist and maintain the ability to proliferate in response to CAR antigen. It has been shown that ALL patient T cells that perform T cells can do this with CART19 comprising a murine scFv (see, for example, Grupp et al., NEJM 368:1509-1518 (2013)).

SUMMARY OF THE INVENTION

[006] The invention features, at least in part, compositions and methods of treating disorders such as cancer (e.g., hematological cancers or other B cell malignancies) using immune effector cells (e.g., T cells or NK cells) that express a Chimeric Antigen Receptor (CAR) molecule (e.g., a CAR that binds to a B cell antigen, e.g., Cluster of Differentiation 19 (CD19) protein (e.g., OMIM Acc. No. 107265, Swiss Prot. Acc No. P15391). The compositions include, and the methods include administering, immune effector cells (e.g., T cells or NK cells) expressing a B cell-targeting CAR, in combination with a kinase inhibitor (e.g., one or more of a CDK4/6 inhibitor, a BTK inhibitor, an mTOR inhibitor, an MNK inhibitor, a Pl3K/mTOR inhibitor, or a combination thereof). In some embodiments, the combination maintains, or has better clinical efficacy when compared to either therapy alone. Engineering also pertains to the use of engineered cells, e.g., immune effector cells (e.g., T cells or NK cells), to express a CAR molecule that binds to a B cell antigen, e.g., CD19, in combination with a kinase inhibitor (e.g., a kinase inhibitor chosen from one or more of a cyclin-dependent kinase 4 (CDK4) inhibitor, a Bruton's tyrosine kinase (BTK) inhibitor, an mTOR inhibitor, a mitogen-activated protein kinase (MNK)-interacting kinase, a dual phosphatidylinositol 3-kinase (Pl3K)/mTOR inhibitor, or a combination thereof) to treat a disorder associated with the expression of a B cell antigen, e.g. , CD19 (e.g. a cancer, e.g. a hematological cancer).

[007] Accordingly, in one aspect, the invention pertains to a method of treating an individual, for example, a mammal having a disease associated with the expression of a B cell antigen, for example, CD19. The method comprises administering to the mammal an effective amount of a cell, e.g., an immune effector cell (e.g., a T cell or NK cell) that expresses a CAR molecule that binds B cell antigen, in combination with an inhibitor. of kinase, for example, a kinase inhibitor described herein. In one embodiment, the CAR molecule binds to CD19, for example, a CAR molecule that binds CD19 described here. In other embodiments, the CAR molecule binds to one or more of CD20, CD22 or ROR1.

[008] In one embodiment, the disease associated with B cell antigen expression (e.g., expression of one or more of CD19, CD20, CD22 or ROR1), is selected from a proliferative disease such as a cancer, a malignancy, or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia, or is a non-cancer related indication associated with B cell antigen expression, for example, one or more of CD19, CD20, CD22 or ROR1. In one embodiment, the disease is a solid or liquid tumor. In one embodiment, the cancer is pancreatic cancer. In one embodiment, the disease is a hematological cancer. In one embodiment, the hematologic cancer is leukemia. In one embodiment, the cancer is selected from the group consisting of one or more acute leukemias including, but not limited to, B-cell acute lymphocytic leukemia (BALL), T-cell acute lymphocytic leukemia (TALL), small lymphocytic lymphoma (SLL) , acute lymphocytic leukemia (ALL); one or more chronic leukemias including, but not limited to, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL). Additional hematologic cancers or hematologic conditions include but are not limited to lining cell lymphoma (MCL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL) , follicular lymphoma, hair cell leukemia, large follicular cell or small cell lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenstrom macroglobulinemia. In certain embodiments, the disease associated with B cell antigen expression (e.g., one or more of CD19, CD20, CD22, or ROR1) is a "preleukemia" which is a diverse collection of hematologic conditions united by ineffective production ( or dysplasia) of myeloid blood cells. In some embodiments, disease associated with B cell antigen expression (e.g., one or more of CD19, CD20, CD22, or ROR1) includes, but is not limited to, atypical and/or non-classical cancers, malignancies, pre-cancerous conditions. or proliferative diseases expressing B cell antigen (e.g., one or more of CD19, CD20, CD22, or ROR1). Any combination of the diseases associated with B cell antigen expression (e.g., one or more of CD19, CD20, CD22 or ROR1) described herein can be treated with the methods and compositions described herein.

[009] In one embodiment, the disease associated with B cell antigen expression (e.g., one or more of CD19, CD20, CD22, or ROR1) is a lymphoma, e.g., MCL, Hodgkin's lymphoma, or DLBCL. In one embodiment, the disease associated with B cell antigen expression (e.g., one or more of CD19, CD20, CD22, or ROR1) is leukemia, e.g., SLL, CLL, and/or ALL. In one embodiment, the disease associated with B cell antigen expression is multiple myeloma (e.g., a multiple myeloma that is CD19 negative, e.g., having a large majority (99.95%) of neoplastic plasma cells having a phenotype of negative CD19, for example, as detected by both flow cytometry and RT-PCR.

[0010] In one embodiment, the kinase inhibitor is a CDK4 inhibitor, for example, a CDK4 inhibitor described herein, for example, a CD4/6 inhibitor, such as, for example, 6-acetyl-8-cyclopentyl -5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one hydrochloride (also referred to as palbociclib or PD0332991). In one embodiment, the kinase inhibitor is a BTK inhibitor, for example, a BTK inhibitor described herein, such as, for example, ibrutinib. In one embodiment, the kinase inhibitor is an mTOR inhibitor, for example, an mTOR inhibitor described herein, such as, for example, rapamycin, a rapamycin analogue, OSI-027. The mTOR inhibitor may be, for example, an mTORC1 inhibitor and/or an mTORC2 inhibitor, for example, an mTORC1 inhibitor and/or mTORC2 inhibitor described herein. In one embodiment, the kinase inhibitor is an MNK inhibitor, for example an MNK inhibitor described herein, such as, for example, 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine . The MNK inhibitor may be, for example, an inhibitor of MNK1a, MNK1b, MNK2a and/or MNK2b. In one embodiment, the inhibitor may be a Pl3K/mTOR inhibitor, e.g., PF-04695102.

[0011] In one embodiment, the kinase inhibitor is a CDK4 inhibitor selected from alloysin 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-pyrazol-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); XL281 (BMS908662); and ribociclib.

[0012] In one embodiment, the kinase inhibitor is a CDK4 inhibitor, for example, palbociclib (PD0332991), and the 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 over a period of time, e.g. daily for 14-21 days of a 28-day cycle, or 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.

[0013] 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 a preferred embodiment, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin 2-inducible kinase (ITK), and is selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13.

[0014] In one embodiment, the kinase inhibitor is a BTK inhibitor, for example, ibrutinib (PCI-32765), and the 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 over a period of time, for example, daily during a 21-day cycle, or daily during 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.

[0015] In one embodiment, the kinase inhibitor is an mTOR inhibitor selected from temsirolimus; ridaforolimus (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-dimethylphosphinate azatricyclo[30.3.1.04.9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl, also known as AP23573 and MK8669; everolimus (RAD001); rapamycin (AY22989); semapimod; (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); and N2- [1,4-dioxo-4- [ [4-(4-oxo-8-phenyl-4 H-1-benzopyran-2-yl)morpholinium4-yl]methoxy]butyl]-L-arginylglycyl-L-a - aspartyl-serine-, internal salt (SF1126); and XL7 65.

[0016] In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., rapamycin, and the rapamycin is administered in 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 over 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 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 over a period of time, e.g., daily during 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.

[0017] 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-17804452; and 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine.

[0018] In one embodiment, the kinase inhibitor is a dual phosphatidylnositol 3-kinase (PI3K) and mTOR inhibitor selected from 2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6- (6-methoxy-3-pyridinyl)-4-methyl-pyrido [2,3-d]pyrimidin-7(8H)-one (PF-04691502); N-[4-[[4-(Dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N'-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl )phenyl]urea (PF-05212384, PKI-587); 2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1- yl]phenyl}propanenitrile (BEZ-235); apitolisib (GDC-0980, RG7422); 2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide (GSK2126458); 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo [4,5-c] maleic acid quinolin-2(3H)-one (NVP-BGT226); 3- [4-(4- Morpholinylpyrido [3',2':4,5]furo [3,2-d]pyrimidin-2-yl]phenol (PI-103); 5-(9- isopropyl-8- methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine (VS-5584, SB2343); and N-[2-[(3,5-Dimethoxyphenyl)amino]quinoxalin-3-yl]- 4-[(4-methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide (XL765).

[0019] In one embodiment, the cell expresses a CAR molecule comprising an anti-CD19 binding domain (e.g., a humanized or murine antibody or antibody fragment that specifically binds to CD19), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a co-stimulatory domain and/or a primary signaling domain). In one embodiment, the CAR comprises an antibody or antibody fragment that includes an anti-CD19 binding domain described herein (e.g., a humanized or murine antibody or antibody fragment that specifically binds CD19 as described herein), a transmembrane domain described herein, and an intracellular signaling domain described herein (e.g., an intracellular signaling domain comprising a co-stimulatory domain and/or a primary signaling domain described herein).

[0020] In one embodiment, the CAR molecule is capable of binding CD19 (e.g., wild-type mutant human CD19). In one embodiment, the CAR molecule comprises an anti-CD19 binding domain comprising one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (CDR2 of LC), and light chain complementary determining region 3 (LC CDR3) of an anti-CD19 binding domain described herein, and one or more (e.g., all three) heavy chain complementary determining region 1 (CDR1 of HC), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of an anti-CD19 binding domain described herein, e.g., an anti-CD19 binding domain comprising one or more, for example, all three, LC CDRs and one or more, for example, all three, HC CDRs. In one embodiment, the anti-CD19 binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of an anti-CD19 binding domain described herein, for example, the anti-CD19 binding domain has two variable heavy chain regions, each comprising an HC CDR1, a CDR2 of HC and an HC CDR3 described here. In one embodiment, the anti-CD19 binding domain comprises a light chain variable region described herein (e.g., in Table 7) and/or a murine heavy chain variable region described herein (e.g., in Table 7). In one embodiment, the anti-CD19 binding domain is a scFv comprising a murine light chain and a murine heavy chain of a Table 7 amino acid sequence. In one embodiment, the anti-CD19 binding domain (e.g., a scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g. substitutions), but not more than 30, 20 or 10 modifications (e.g. substitutions) of an amino acid sequence of a light chain variable region given in Table 7, or a sequence with 95-99% identity to an amino acid sequence of Table 7; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of one amino acid sequence of a heavy chain variable region given in Table 7, or a sequence with 95-99% identity to an amino acid sequence of Table 7. In one embodiment, the anti-CD19 binding domain comprises a sequence of SEQ ID NO:59, or a sequence with 95-99% identity thereof. In one embodiment, the anti-CD19 binding domain is a scFv, and a light chain variable region comprising an amino acid sequence described herein, for example, in Table 7, is linked to a heavy chain variable region comprising an amino acid sequence. amino acid described herein, for example in table 7, via a linker, for example a linker described herein. In one embodiment, the anti-CD19 binding domain includes a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 53) . The light chain variable region and heavy chain variable region of an scFv can be, for example, in any of the following orientations: light chain linker heavy chain variable region or light chain linker light chain variable region. heavy chain variable region.

[0021] In one embodiment, the CAR molecule comprises a humanized anti-CD19 binding domain that includes one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 1 (LC CDR1), light chain 2 (LC CDR2), and light chain 3 complementary determining region (LC CDR3) of a humanized anti-CD19 binding domain described here, and one or more (e.g., all three) complementary determining region of heavy chain 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized anti-CD19 binding domain described herein, e.g. a humanized anti-CD19 binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized anti-CD19 binding domain described herein, for example, the humanized anti-CD19 binding domain has two variable heavy chain regions, each comprising an HC CDR1 , an HC CDR2 and an HC CDR3 described here. In one embodiment, the humanized anti-CD19 binding domain comprises at least HC CDR2. In one embodiment, the light chain variable region comprises one, two, three, or all four germline sequence framework regions of VK3_L25. In one embodiment, the light chain variable region has a modification (e.g., substitution, e.g., a substitution of one or more amino acids found at the corresponding position in the variable chain variable region of SEQ ID NO: 58, e.g., a substitution in one or more of positions 71 and 87). In one embodiment, the heavy chain variable region comprises one, two, three, or all four germline sequence framework regions of VH4_4-59. In one embodiment, the heavy chain variable region has a modification (e.g., substitution, e.g., a substitution of one or more amino acids found at the corresponding position in the murine heavy chain variable region of SEQ ID NO: 58, e.g. , a substitution in one or more of positions 71, 73 and 78). In one embodiment, the humanized anti-CD19 binding domain comprises a light chain variable region described herein (e.g., in Table 3) and/or a light chain variable region described herein (e.g., in Table 3). In one embodiment, the humanized anti-CD19 binding domain is a scFv comprising a light chain and a heavy chain of a Table 3 amino acid sequence. In one embodiment, the humanized anti-CD19 binding domain (e.g., a scFv ) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g. substitutions), but not more than 30, 20 or 10 modifications (e.g. substitutions) of a sequence amino acid sequence of a light chain variable region given in Table 3, or a sequence with 95-99% identity to an amino acid sequence of Table 3; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of one amino acid sequence of a heavy chain variable region provided in Table 3, or a sequence with 95-99% identity to an amino acid sequence of Table 3. In one embodiment, the humanized anti-CD19 binding domain comprises a selected sequence of a group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, or a sequence with 95-99% identity thereof. In one embodiment, the humanized anti-CD19 binding domain is a scFv, and a light chain variable region comprising an amino acid sequence described herein, for example, in Table 3, is linked to a heavy chain variable region comprising a sequence of amino acid described herein, for example in table 3, by means of a linker, for example a linker described herein. In one embodiment, the humanized anti-CD19 binding domain includes a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 53 ). The light chain variable region and heavy chain variable region of an scFv can be, for example, in any of the following orientations: light chain linker heavy chain variable region or light chain linker light chain variable region. heavy chain variable region.

[0022] In one embodiment, the CAR molecule comprises a transmembrane domain of a protein 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 and CD154. In one embodiment, the transmembrane domain comprises a sequence of SEQ ID NO: 15. In one embodiment, the transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions), but no more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 15, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO: 15.

[0023] In one embodiment, the anti-CD19 binding domain is connected to the transmembrane domain by a hinge region, for example, a hinge region described here. In one embodiment, the encoded hinge region comprises SEQ ID NO:14 or SEQ ID NO:45, or a sequence with 95-99% identity thereof.

[0024] In one embodiment, the CAR molecule also comprises a sequence encoding a costimulatory domain, for example, a costimulatory domain described here. In one embodiment, the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), and 4-1BB (CD137 ). In one embodiment, the costimulatory domain comprises a sequence of SEQ ID NO: 16. In one embodiment, the costimulatory domain comprises a sequence of SEQ ID NO: 51. In one embodiment, the co-stimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions), but not more than 20, 10, or 5 modifications (e.g., substitutions) of an amino acid sequence. of SEQ ID NO: 16 or SEQ ID NO:51, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO: 16 or SEQ ID NO:51.

[0025] In one embodiment, the CAR molecule also comprises a sequence encoding an intracellular signaling domain, for example, an intracellular signaling domain described here. In one embodiment, the intracellular signaling domain comprises a functional 4-1BB signaling domain and/or a functional CD3 zeta signaling domain. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 and/or the sequence of SEQ ID NO: 17. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO:16 and/or the sequence of SEQ ID NO:43. In one embodiment, the intracellular signaling domain comprises a functional CD27 signaling domain and/or a functional CD3 zeta signaling domain. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 51 and/or the sequence of SEQ ID NO: 17. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO:51 and/or the sequence of SEQ ID NO:43. In one embodiment, the intracellular signaling domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but not more than 20, 10, or 5 modifications (e.g., substitutions) of a sequence. amino acid sequence of SEQ ID NO:16 or SEQ ID NO:51 and/or an amino acid sequence of SEQ ID NO:17 or SEQ ID NO:43, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO:16 or SEQ ID NO:51 and/or an amino acid sequence of SEQ ID NO:17 or SEQ ID NO:43. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO:16 or SEQ ID NO:51 and the sequence of SEQ ID NO:17 or SEQ ID NO:43, wherein the consequences comprising the intracellular signaling domain are expressed in the same structure and as a single polypeptide chain.

[0026] In one embodiment, the CAR molecule also comprises a leader sequence, for example, a leader sequence described here. In one embodiment, the leader sequence comprises an amino acid sequence of SEQ ID NO: 13, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO: 13.

[0027] In one embodiment, the CAR molecule comprises a leader sequence, for example, a leader sequence described herein, for example, a leader sequence of SEQ ID NO: 13, or having 95-99% identity thereof; an anti-CD19 binding domain described herein, for example, an anti-CD19 binding domain comprising an LC CDR1, an LC CDR2, an LC CDR3, an HC CDR1, an HC CDR2 and an HC CDR3 described herein, for example, a murine anti-CD19 binding domain described in Table 7, a humanized anti-CD19 binding domain described in Table 3, or a sequence with 95-99% identity thereof; a hinge region, for example, a hinge region described herein, for example, a hinge region of SEQ ID NO:14 or having 95-99% identity thereof; a transmembrane domain, for example, a transmembrane domain described herein, for example, a transmembrane domain having a sequence of SEQ ID NO:15 or a sequence having 95-99% identity thereof; an intracellular signaling domain, e.g., an intracellular signaling domain described herein (e.g., an intracellular signaling domain comprising a co-stimulatory domain and/or a primary signaling domain). In one embodiment, the intracellular signaling domain comprises a costimulatory domain, e.g., a costimulatory domain described herein, e.g., a 4-1BB costimulatory domain having a sequence of SEQ ID NO:16 or SEQ ID NO:51, or having 95-99% of the same identity, and/or a primary signaling domain, e.g., a primary signaling domain described herein, e.g., a CD3 zeta stimulatory domain having a sequence of SEQ ID NO:17 or SEQ ID NO:43, or having 95-99% of its identity.

[0028] In one embodiment, the CAR molecule comprises (e.g., consisting of) an amino acid sequence of SEQ ID NO:58, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO :42, or an amino acid sequence having at least one, two, three, four, five, 10, 15, 20, or 30 modifications (e.g., substitutions), but not more than 60, 50, or 40 modifications (e.g., , substitutions) of an amino acid sequence of SEQ ID NO:58, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42, or an amino acid sequence having 85%, 90%, 95 %, 96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO:58, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42.

[0029] In one embodiment, the cell expressing the CAR molecule comprises a vector that includes a nucleic acid sequence encoding the CAR molecule. In one embodiment, the vector is selected from the group consisting of a DNA, an RNA, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector. In one embodiment, the vector is a lentivirus vector. In one embodiment, the vector also comprises a promoter. In one embodiment, the promoter is an EF-1 promoter. In one embodiment, the EF-1 promoter comprises a sequence of SEQ ID NO: 100. In one embodiment, the vector is an in vitro transcribed vector, for example, a vector that transcribes RNA from a nucleic acid molecule described herein. In one embodiment, the nucleic acid sequence in the in vitro vector also comprises a poly(A) tail, e.g., a polyA tail described herein, e.g., comprising about 150 adenosine bases (SEQ ID NO:104). . In one embodiment, the nucleic acid sequence in the in vitro vector also comprises a 3'UTR, for example, a 3'UTR described herein, for example, comprising at least one repeat of a 3'UTR derived from human beta-globulin. In one embodiment, the nucleic acid sequence in the in vitro vector also comprises promoter, for example, a T2A promoter.

[0030] In certain embodiments of the compositions and methods described herein, the cell expressing the CAR molecule (also referred to herein as a "CAR-expressing cell") is a cell or population of cells as described herein, for example, an immune effector cell human or population of cells (e.g., a human T cell or a human NK cell, e.g., a human T cell described herein or a human NK cell described herein). In one embodiment, a human T cell is a CD8+ T cell. In one embodiment, the cell is an autologous T cell. In one embodiment, the cell is an allosteric T cell. In one embodiment, the cell is a T cell and the T cell is diaglycerol kinase (DGK) deficient. In one embodiment, the cell is a T cell and the T cell is Ikaros deficient. In one embodiment, the cell is a T cell and the T cell is deficient in both DGK and Ikaros. It should be understood that the compositions and methods described herein reciting the term "cell" encompass compositions and methods comprising one or more cells, e.g., a population of cells.

[0031] In another embodiment, the cell expressing the CAR molecule, for example, as described here, may also express another agent, for example, an agent that enhances the activity of a CAR-expressing cell.

[0032] In one embodiment, the method also includes administering a cell expressing the CAR molecule, as described herein, optionally in combination with a kinase inhibitor, for example, a BTK inhibitor such as ibrutinib, in combination with an agent that enhances the activity of a CAR-expressing cell. In certain embodiments, the agent is a cytokine, e.g., IL-7, IL-15, IL-21, or a combination thereof. In one embodiment, the method includes administering IL-7 to the subject. The cytokine can be released in combination with, for example, simultaneously or immediately after, administration of the CAR-expressing cell. Alternatively, the cytokine may be released after a prolonged period of time after administration of the CAR-expressing cell, for example, after evaluating the individual's response to the CAR-expressing cell.

[0033] In other embodiments, the agent that enhances the activity of a CAR-expressing cell may be an agent that inhibits an immune inhibitory molecule. Examples of immune 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. In one embodiment, the agent that inhibits an immune inhibitory molecule comprises a first polypeptide, e.g., an immune inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, for example, from an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide that is a intracellular signaling described here (e.g., comprising a co-stimulatory domain (e.g., 41BB, CD27, or CD28, e.g., as described here) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described here ).In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described here and/or a CD3 zeta signaling domain described here).

[0034] In one embodiment, lymphocyte infusion, e.g., allogeneic lymphocyte infusion, is used in the treatment of cancer, wherein the lymphocyte infusion comprises at least one CAR-expressing cell that binds a B cell antigen (e.g., example, CD19) (also referred to herein as a CD19 CAr-expressing cell), as described herein. In one embodiment, autologous lymphocyte infusion is used in the treatment of cancer, wherein the autologous lymphocyte infusion comprises at least one cell expressing CD19.

[0035] In one embodiment, the CD19 CAR-expressing cell, e.g., T cell, is administered to an individual who has received a prior stem cell transplant, e.g., autologous stem cell transplant.

[0036] In one embodiment, the CD19 CAR-expressing cell, e.g., T cell, is administered to an individual who has received a prior dose of melphalan.

[0037] In one embodiment, the cell expressing the CAR molecule, for example, a CAR molecule described here, is administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a CAR molecule. , for example, an agent described herein.

[0038] In one embodiment, the kinase inhibitor is administered in combination with an agent that ameliorates one or more side effects associated with administration of the kinase inhibitor, for example, an agent described herein.

[0039] In one embodiment, the cell expressing the CAR molecule, e.g., a CAR molecule described herein, and the kinase inhibitor are administered in combination with an additional agent that treats the disease associated with CD19, e.g., a additional agent described here.

[0040] In one embodiment, cells expressing a CAR molecule, e.g., a CAR molecule described here, are administered at a dose and/or dosage schedule described here.

[0041] In one embodiment, the CAR molecule is introduced into T cells, for example, using in vitro transcription, and the individual (e.g., human) receives an initial administration of cells comprising a CAR molecule, and one or more subsequent administrations of cells comprising a CAR molecule, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, more than one administration of cells comprising a CAR molecule are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of cells comprising a CAR molecule are administered per week. . In one embodiment, the subject (e.g., human subject) receives more than one administration of cells comprising a CAR molecule per week (e.g., 2, 3, or 4 administrations per week) (also referred to herein as a cycle), followed by a week without any administration of cells comprising a CAR molecule, and then one or more additional administrations of cells comprising a CAR molecule (e.g., more than one administration of the cells comprising a CAR molecule per week) is administered to the individual. In another embodiment, the subject (e.g., human subject) receives more than one cycle of cells comprising a CAR molecule, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4 , or 3 days. In one embodiment, cells comprising a CAR molecule are administered every other day for 3 administrations per week. In one embodiment, cells comprising a CAR molecule are administered for at least two, three, four, five, six, seven, eight or more weeks.

[0042] In one embodiment, the combination of the kinase inhibitor and cells expressing a CAR molecule, e.g., a CAR molecule described herein, is administered as a first-line treatment for disease, e.g., cancer, for example, the cancer described here. In another embodiment, the combination of the kinase inhibitor and cells expressing a CAR molecule, e.g., a CAR molecule described herein, is administered as a second, third, or fourth line treatment for disease, e.g., cancer. , for example, the cancer described here.

[0043] In one embodiment, a cell (e.g., a population of cells) described herein is administered to the individual.

[0044] In one embodiment, the method includes administering a population of cells, a plurality of which comprise a CAR molecule described herein. In some embodiments, the CAR-expressing cell populations comprise a mixture of cells expressing different CARs. For example, in one embodiment, the cell populations expressing CAR may include a first cell expressing a CAR having an anti-CD19 binding domain described herein, and a second cell expressing a CAR having a different CD19 binding domain, e.g. , an anti-CD19 binding domain described here that differs from the anti-CD19 binding domain in the CAR expressed by the first cell. As another example, CAR-expressing cell populations may include a first cell expressing a CAR that includes an anti-CD19 binding domain, for example, as described herein, and a second cell expressing a CAR that includes an antigen-binding domain. to a target other than CD19 (e.g., CD123 or mesothelin). In one embodiment, the CAR-expressing cell populations include, for example, a first cell expressing a CAR that includes a primary intracellular signaling domain, and a second cell expressing a CAR that includes a secondary signaling domain.

[0045] In one embodiment, the method includes administering cell populations in which at least one cell in the population expresses a CAR having an anti-CD19 domain described herein, and an agent that enhances the activity of a CAR-expressing cell, e.g. , a second cell expressing the agent that enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an immune inhibitory molecule. Examples of immune inhibitory molecules include PD1, PD-L1, CTLA-4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. In one embodiment, the agent that inhibits an immune inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, for example, from an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide that is a intracellular signaling domain described herein (e.g., comprising a co-stimulatory domain (e.g., 41BB, CD27 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described here).In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g. , a CD28 signaling domain described here and/or a CD3 zeta signaling domain described here).

[0046] In another aspect, the invention pertains to a cell expressing a CAR molecule described herein for use as a medicament in combination with a kinase inhibitor, e.g., a kinase inhibitor described herein (e.g., a BTK inhibitor). such as ibrutinib). In another aspect, the invention pertains to a kinase inhibitor described herein (e.g., a BTK inhibitor such as ibrutinib) for use as a medicament in combination with a cell expressing a CAR molecule described herein.

[0047] In another aspect, the invention pertains to a cell expressing a CAR molecule described herein for use in combination with a kinase inhibitor, e.g., a kinase inhibitor described herein (e.g., a BTK inhibitor such as ibrutinib ), in the treatment of a disease expressing B cell antigen (e.g., CD19). In another aspect, the invention pertains to a kinase inhibitor described herein (e.g., a BTK inhibitor such as ibrutinib), for use in combination with a cell expressing a CAR molecule described herein, in treating a disease expressing the antigen of B cells (e.g. CD19). A disease may be, for example, a cancer such as a hematological cancer. The cancer may be, for example, lymphoma, CLL, MCL, ALL, DLBCL, multiple myeloma, or other cancer described herein.

[0048] In another aspect, the invention pertains to a cell expressing a CAR molecule described herein for use as a medicament in combination with a cytokine, for example, IL-7, IL-15 and/or IL-21 as described herein. . In another aspect, the invention pertains to a cytokine described herein for use as a medicament in combination with a cell expressing a CAR molecule described herein.

[0049] In another aspect, the invention pertains to a cell expressing a CAR molecule described herein for use in combination with a cytokine, for example, IL-7, IL-15 and/or IL-21 as described herein, in the treatment of a disease expressing CD19. In another aspect, the invention pertains to a cytokine described herein for use in combination with a cell expressing a CAR molecule described herein, in the treatment of a disease expressing CD19.

[0050] In another aspect, the invention pertains to a method of treating a mammal having Hodgkin's lymphoma, comprising administering to the mammal an effective amount of the cell (e.g., cells) expressing a CAR molecule, e.g., a CAR molecule described here.

[0051] In one embodiment, the cell expressing a CAR molecule, e.g., a CAR molecule described herein, is administered in combination with an agent that increases the effectiveness of a cell expressing a CAR molecule, e.g., an agent described here.

[0052] In one embodiment, the cell expressing a CAR molecule, for example, a CAR molecule described herein, is administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a CAR molecule. , for example, an agent described herein.

[0053] In one embodiment, the cell expressing a CAR molecule, for example, a CAR molecule described here, is administered in combination with an agent that treats Hodgkin's lymphoma, for example, an agent described here.

[0054] In one embodiment, the cell expressing a CAR molecule, e.g., a CAR molecule described herein, is administered in combination with a low immune boosting dose of an mTOR inhibitor, e.g., an mTOR inhibitor described here. While not wishing to be bound by theory, it is believed that treatment with a low dose of immune booster (e.g., a dose that is insufficient to completely suppress the immune system but sufficient to improve function) is accompanied by a decrease in PD-1 positive T cells or an increase in PD-1 negative T cells. PD-1 positive T cells, but not PD-1 negative T cells, can be depleted by engagement with cells expressing a PD-1 ligand, for example, PD-L1 or PD-L2.

[0055] In one embodiment, this method can be used to optimize the performance of a CAR cell described herein in the individual. While not wishing to be bound by theory, it is believed that, in one embodiment, the performance of endogenous, unmodified immune effector cells, e.g., T cells, is improved. While not wishing to be bound by theory, it is believed that, in one embodiment, the performance of a cell expressing CD19 CAR is improved. In other embodiments, cells, e.g., T cells, that have or will be engineered to express a CAR, can be treated ex vivo by contact with an amount of an mTOR inhibitor that increases the number of PD1-negative immune effector cells, e.g. 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.

[0056] In one embodiment, administration of a low immune boosting dose of an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or a catalytic inhibitor, is initiated prior to administration to a CAR-expressing cell described herein , for example, T cells. In one embodiment, the mTOR inhibitor is RAD001 or rapamycin. In one embodiment, the CAR cells are administered after a sufficient period of time, or sufficient dosage, of an mTOR inhibitor, such that the level of PD1-negative immune effector cells, e.g., T cells, or the cell ratio PD1-negative immune effector cells, e.g., T cells/ PD1-positive immune effector cells, e.g., T cells, have been, at least transiently, increased.

[0057] In one embodiment, the cell, for example, an immune effector cell (e.g., a T cell or NK cell), to be created to express a CAR, is harvested after a sufficient period of time, or after sufficient dosage of a low immune-boosting 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/effector cells PD1-positive immune cells, e.g., T cells, in the individual or harvested from the individual, have been, at least transiently, increased.

[0058] In embodiments, any of the methods described herein also comprise performing lymphodepletion in an individual, for example, prior to administration of one or more cells that express a CAR molecule described herein, for example, a CAR molecule that binds CD19 . Lymphodepletion may comprise, for example, administering one or more of melphalan, cytoxan, cyclophosphamide, and fludarabine.

[0059] In some embodiments, the CAR-expressing cell that is administered comprises a regulatable CAR (RCAR), for example, an RCAR as described herein. The RCAR may comprise, for example, an intracellular signaling member comprising an intracellular signaling domain and a first switch domain, an antigen-binding member comprising an antigen-binding domain that binds CD19 and a second switch domain; and a transmembrane domain. The method may also comprise administering a dimerization molecule, for example, in an amount sufficient to cause dimerization of the first and second permutation domains.

[0060] In some embodiments, the CAR-expressing cell and the kinase inhibitor are administered simultaneously or substantially simultaneously, for example, as a first line of therapy. In some embodiments, the method comprises administering a combination of the BTK inhibitor (e.g., ibrutinib) and the CAR-expressing cell (e.g., a CAR19-expressing cell) to the subject, as a first-line therapy.

[0061] In other embodiments, the CAR-expressing cell and the kinase inhibitor are administered sequentially. For example, the kinase inhibitor is administered before the CAR-expressing cell, or the CAR-expressing cell is administered before the kinase inhibitor.

[0062] In some embodiments, the disease associated with CD19 expression is a hematological cancer (e.g., a hematological cancer described herein such as CLL, MCL, or ALL) and the individual is, or is identified as, a partial responder, unresponsive, or relapsed to one or more therapies for hematological cancer, for example, to a BTK inhibitor such as ibrutinib. In some embodiments, the individual has, or is identified as having, a BTK mutation. The mutation may be, for example, a point mutation, an insertion, or a deletion. The mutation may be, for example, a mutation in the binding site for the BTK inhibitor, for example in or near the ATP binding pocket. The mutation may confer a diminished response (e.g., resistance) to the BTK inhibitor.

[0063] In some embodiments of any of the methods described herein, the method comprises administering the BTK inhibitor (e.g., ibrutinib) to the subject, reducing the amount (e.g., cessation administration) of the BTK inhibitor, and subsequently administering the CAR-expressing cell (e.g., a CAR19-expressing cell) to the individual.

[0064] In some embodiments, the method comprises administering the BTK inhibitor (e.g., ibrutinib) to the subject and subsequently administering a combination of the BTK inhibitor and the CAR-expressing cell (e.g., a CAR19-expressing cell) to the subject.

[0065] In some embodiments, the method comprises administering the BTK inhibitor (e.g., ibrutinib) to the subject, reducing the amount (e.g., cessation or discontinuity administration) of the BTK inhibitor, and subsequently administering a combination of the cell expressing CAR (e.g., a cell expressing CAR19) and a second BTK inhibitor (e.g., a BTK inhibitor other than the first BTK inhibitor, e.g., other than ibrutinib) to the individual. In some embodiments, the second BTK inhibitor is chosen from one or more of GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM. -A13, or a combination thereof.

[0066] In some embodiments, the disease associated with B cell antigen expression (e.g., CD19) is a hematologic cancer (e.g., a hematologic cancer described herein, e.g., CLL, MCL, or ALL), and the method delays or decreases resistance to the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the CAR-expressing cell (e.g., a CAR19-expressing cell) to the individual, or both. In some embodiments, the disease associated with CD19 expression is a hematologic cancer (e.g., a hematologic cancer described herein, e.g., CLL, MCL, or ALL), and wherein the method prolongs remission or delays recurrence of the cancer. hematological. For example, remission may be prolonged, remission may be delayed, resistance may be delayed, or resistance may be decreased, compared to the expected course of disease when treated with a kinase inhibitor or CAR-expressing cell monotherapy.

[0067] Exemplary treatment regimens that can be used in any of the aforementioned methods include one or more of the following:

[0068] In one embodiment, the kinase inhibitor and the CAR-expressing cell (e.g., the CAR19-expressing cell) are administered to the individual, e.g., mammal, as a first-line therapy.

[0069] In another embodiment, the CAR-expressing cell (e.g., the CAR19-expressing cell) is administered to the individual, e.g., mammal, after administration of the kinase inhibitor.

[0070] In other embodiments, the CAR-expressing cell (e.g., the CAR19-expressing cell) is administered after ceasing administration of the kinase inhibitor.

[0071] In other embodiments, administration of the kinase inhibitor is initiated prior to administration of the CAR19-expressing cell, and the CAR19-expressing cell is administered in combination with continued administration of the kinase inhibitor.

[0072] In one embodiment, an individual is administered a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), for example, as a first-line therapy. After a predetermined time interval (for example, 1 or 2 months, but also 2 weeks, 3 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 6 months, 9 months, 12 months, 15 months, or 18 months), a CAR-expressing cell (e.g., a CAR19-expressing cell) is administered to the individual alone, or in combination with the kinase inhibitor. In some embodiments, the individual's response to treatment is assessed at predetermined time intervals, for example, before or during treatment with the kinase inhibitor and/or CAR-expressing cell. If the evaluation shows that the individual is a complete responder, the CAR-expressing cell (e.g., a CAR19-expressing cell) will not be administered. If evaluation shows that the individual is a partial responder, or has stable disease in response, to the kinase inhibitor, the CAR-expressing cell (e.g., a CAR19-expressing cell) will be administered in combination with the kinase inhibitor, e.g. as described here. If evaluation shows that the individual is a non-responder or relapser, the CAR-expressing cell (e.g., a CAR19-expressing cell) will be administered in combination with the kinase inhibitor or a second kinase inhibitor, e.g., a second kinase inhibitor. kinase as described here.

[0073] In other embodiments, the individual, e.g., mammal, is, or is identified as being, a complete or partial responder to the BTK inhibitor (e.g., ibrutinib), or a complete or partial responder to the CAR19-expressing cell.

[0074] In some embodiments, when an individual is (or is identified as being) a complete kinase inhibitor responder (e.g., a BTK inhibitor such as ibrutinib), the individual is not administered a CAR-expressing cell (e.g. , a cell expressing CAR19) during the period of complete response. In other embodiments, when an individual is (or is identified as being) a complete responder (e.g., a complete responder to ibrutinib) to the kinase inhibitor, the individual is administered a CAR-expressing cell (e.g., a CAR19-expressing cell). during the complete response period. In one embodiment, after the CAR-expressing cell (e.g., a CAR19-expressing cell), the individual experiences a prolonged response or delayed relapse (e.g., compared to the expected course of the disease when treated without CAR therapy).

[0075] In some embodiments, when an individual is (or is identified as being) a partial responder to the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the individual is not administered a CAR-expressing cell (e.g. , a cell expressing CAR19) during the partial response period. In other embodiments, when an individual is (or is identified as being) a partial responder to the kinase inhibitor, the individual is administered a CAR-expressing cell (e.g., a CAR19-expressing cell) (alone or in combination with the BTK inhibitor ) during the partial response period. In one embodiment, following CAR therapy, the individual experiences complete response and/or prolonged response or delayed relapse (e.g., compared to the expected course of disease when treated without CAR therapy).

[0076] In some embodiments, when an individual has (or is identified as having) stable disease following treatment with the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the individual is not administered CAR therapy during the period of stable illness. In other embodiments, when an individual has (or is identified as having) stable disease following treatment with the kinase inhibitor, the individual is administered CAR therapy during the period of stable disease. In one embodiment, following CAR therapy, the individual experiences a partial response, a complete response, and/or prolonged response or delayed relapse (e.g., compared to the expected course of disease when treated without CAR therapy).

[0077] In some embodiments, when an individual has (or is identified as having) progressive disease following treatment with the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the individual is not administered a CAR-expressing cell ( for example, a cell expressing CAR19) during the period of progressive disease. In other embodiments, when an individual has (or is identified as having) progressive disease following treatment with the kinase inhibitor, the individual is administered a CAR-expressing cell (e.g., a CAR19-expressing cell) during the period of progressive disease. In one embodiment, following CAR therapy, the subject experiences stable disease, a partial response, a complete response, and/or prolonged response or delayed relapse (e.g., compared to the expected course of disease when treated without CAR therapy). .

[0078] In other embodiments, the CAR-expressing cell is administered in combination with a second kinase inhibitor, wherein the second kinase inhibitor is other than ibrutinib, when the mammal is, or is identified as being, a non-responder or relapser. to ibrutinib. The second kinase inhibitor can be chosen from one or more of GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13, or a combination thereof.

[0079] In other embodiments, the subject, e.g., the mammal, is (or is identified as being) a partial responder to the kinase inhibitor, and the subject is administered the CAR-expressing cell (e.g., the CAR19-expressing cell). , alone or in combination with the BTK inhibitor, during the partial response period.

[0080] In other embodiments, the subject, e.g., the mammal, is (or has been identified as being) a non-responder having progressive or stable disease following treatment with ibrutinib, and the subject is administered the CAR-expressing cell (e.g., the CAR19-expressing cell), alone or in combination with a second BTK inhibitor, during the period of progressive or stable disease, where the second kinase inhibitor is other than ibrutinib.

[0081] In another aspect, provided herein is a method of treating an individual, e.g., a mammal, having a disease associated with expression of B cell antigen (e.g., CD19). The method comprises administering to the subject an effective amount of a kinase inhibitor as described herein (e.g., a BTK kinase inhibitor described herein, e.g., ibrutinib) and a CAR-expressing cell (e.g., a CAR19-expressing cell) in combination with (e.g. simultaneously (or substantially simultaneously), or sequentially).

[0082] In some embodiments, the kinase inhibitor and the CAR-expressing cell (e.g., a CAR19 cell) are administered, in combination, for example, as a first-line therapy,

[0083] In some embodiments, the kinase inhibitor is administered initially, for example, as a monotherapy or first line of therapy; After reducing the amount (e.g., cessation or discontinuation administration) of the kinase inhibitor, administer the CAR-expressing cell (e.g., a CAR19-expressing cell) to the subject.

[0084] In other embodiments, the kinase inhibitor is administered initially, for example, as a monotherapy or first line of therapy; and subsequently administering a combination of the kinase inhibitor and the CAR-expressing cell (e.g., a CAR19-expressing cell) to the subject.

[0085] In other embodiments, the kinase inhibitor is administered initially, for example, as a monotherapy or first line of therapy; After reducing the amount (e.g., cessation or discontinuation administration) of the kinase inhibitor, administer a combination of a second kinase inhibitor and the CAR-expressing cell (e.g., a CAR19-expressing cell) to the subject.

[0086] In some embodiments, the individual's response to treatment is assessed at predetermined time intervals, for example, before or during treatment with the kinase inhibitor and/or CAR-expressing cell. If the evaluation shows that the individual is a complete responder, the CAR-expressing cell (e.g., a CAR19-expressing cell) will not be administered. If evaluation shows that the individual is a partial responder, or has stable disease in response, to the kinase inhibitor, the CAR-expressing cell (e.g., a CAR19-expressing cell) is administered in combination with the kinase inhibitor, e.g. as described here. If evaluation shows that the individual is a non-responder or relapser, the CAR-expressing cell (e.g., a CAR19-expressing cell) will be administered in combination with the kinase inhibitor or a second kinase inhibitor, e.g., a second kinase inhibitor. kinase as described here.

[0087] In some embodiments, the disease associated with B cell antigen expression (e.g., CD19) is a hematological cancer, leukemia, lymphoma, MCL, CLL, ALL, Hodgkin's lymphoma, or multiple myeloma.

[0088] In some embodiments, the kinase inhibitor is a BTK inhibitor chosen from ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX- 774, or LFM-A13; a CDK4 inhibitor chosen from palbociclib, aloisine A, flavopiridol, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl] -4- cromenone; crizotinib (PF-02341066, P276-00, RAF265, indisulam, roscovitine, dinaciclib, BMS 387032, MLN8054, AG-024322, AT7519, AZD5438, BMS908662; or ribociclib; an mTOR inhibitor chosen from rapamycin, a rapamycin analog just like everolimus, temsirolimus, ridaforolimus, semapimod, AZD8055, PF04691502, SF1126, (4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine.

[0089] In some aspects, the invention features a method of treating or providing anti-tumor immunity in an individual, e.g., mammal, having Hodgkin's lymphoma. The method comprises administering to the subject an effective amount of a cell expressing a CAR molecule that binds CD19, alone or in combination with a second therapy.

[0090] In another aspect, the invention features a method of treating, or providing anti-tumor immunity to an individual, e.g., a mammal, having a multiple myeloma (e.g., a CD19-positive multiple myeloma, or a CD19-positive myeloma). negative). In one embodiment, the multiple myeloma is CD19 negative, e.g., has a vast majority (99.95%) of neoplastic plasma cells with a CD19 negative phenotype, e.g., as detected by both flow cytometry and RT-PCR. . The method comprises administering to the subject an effective amount of a cell expressing a CAR molecule that binds CD19, alone or in combination with a second therapy (e.g., a standard of care therapy for multiple myeloma). The method may also comprise administering a kinase inhibitor as described herein.

[0091] In embodiments of the methods related to Hodgkin's lymphoma or multiple myeloma, the CAR molecule is a humanized CAR molecule, for example, as described here. In embodiments, the CAR molecule is a CAR molecule as described herein. For example, in embodiments the CAR molecule comprises an anti-CD19 binding domain comprising one or more of (e.g., 2, 3, 4, 5, or all of) LC CDR1 of SEQ ID NO: 5, a CDR2 of LC of SEQ ID NO: 26, and a CDR3 of LC of SEQ ID NO: 27; an HC CDR1 of SEQ ID NO: 19, an LC CDR2 of any of SEQ ID NOS: 2023, and a HC CDR3 of SEQ ID NO: 24.

[0092] In some embodiments of methods related to Hodgkin's lymphoma or multiple myeloma, the CAR molecule (e.g., CART19 or CTL019) is administered as a monotherapy. In some embodiments, the method also comprises administering a kinase inhibitor, for example, a BTK inhibitor (such as ibrutinib), a CDK4 inhibitor, an mTOR inhibitor, or an MNK inhibitor.

[0093] In some embodiments of methods related to multiple myeloma, the CAR molecule (e.g., CART19 or CTL019) is administered in combination with a standard of care therapy for multiple myeloma, e.g., with myeloablative chemotherapy and/or autologous stem cell transplant rescue (e.g., after administration of melphalan (e.g., high-dose melphalan)).

[0094] In another aspect, the invention features a composition comprising a cell that expresses a CAR molecule that binds a B cell antigen (for example, one or more of CD19, CD20, CD22 or ROR1), and one or more inhibitors of kinase, wherein the kinase inhibitor is chosen from a Bruton's tyrosine kinase (BTK) inhibitor, a cyclin-dependent kinase 4 (CDK4) inhibitor, an mTOR inhibitor, or a protein kinase-interacting kinase inhibitor mitogen-activated (MNK). The CAR-expressing cell and one or more kinase inhibitors may be present in a single dose form, or as two or more dose forms.

[0095] In embodiments, the compositions described here are for use as a medicine.

[0096] In embodiments, the compositions described here are for use in treating a disease associated with the expression of B cell antigen (e.g., CD19).

Methods and compositions for producing CAR-expressing cells

[0097] The present invention also provides, in certain aspects, a method of preparing immune effector cell populations (e.g., T cells or NK cells) that can be created to express a CAR (e.g., a CAR described herein ), the method comprising: providing immune effector cell populations; and contacting the immune effector cells with a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) under conditions sufficient to inhibit a target of the kinase inhibitor (e.g., BTK and/or ITK). The method may also comprise contacting, for example transducing, immune effector cells with a nucleic acid encoding a CAR molecule.

[0098] In some aspects, the invention provides a method of preparing a CAR-expressing cell (e.g., a CAR-expressing immune effector cell or population of cells), comprising: contacting the cell or population of cells with a kinase inhibitor, for example, a BTK inhibitor such as ibrutinib; and introducing (e.g., transducing) a nucleic acid encoding a CAR molecule into the cell or population of cells under conditions such that the CAR molecule is expressed.

[0099] In certain embodiments of methods of producing CAR-expressing cells, the CAR molecule encoded by the nucleic acid is a CAR molecule that binds CD19. In embodiments, the method also comprises culturing the cell or cells under conditions that allow the cell or at least a subpopulation of the cells to express the CAR molecule. In embodiments, the cell is a T cell or NK cell, or the cell populations include T cells, NK cells, or both. In embodiments, the method comprises contacting the cell or cells with the kinase inhibitor (e.g., for 10-20, 20-30, 3040, 40-60, or 60-120 minutes) and subsequently removing most or all of the kinase inhibitor of the cell or cells. In embodiments, the kinase inhibitor is added after the cell or cells are harvested or before the cell or cells are stimulated. In embodiments, the kinase inhibitor is a BTK inhibitor, a CDK4 inhibitor, an mTOR inhibitor, or an MNK inhibitor. In embodiments, the kinase inhibitor is ibrutinib. In embodiments, the cell populations also comprise cancer cells, e.g., leukemia or lymphoma cells. Cancer cells can be, for example, CLL, MCL, or ALL cells. In embodiments, the kinase inhibitor inhibits a target (e.g., BTK) in cancer cells, e.g., reduces its activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70% , 80%, 90%, 95%, or 99%. In embodiments, the kinase inhibitor inhibits a target (e.g., ITK) on immune effector cells, e.g., reduces their activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70% , 80%, 90%, 95%, or 99%.

[00100] In some aspects, the present invention also provides a reaction mixture comprising a kinase inhibitor (e.g., a BTK inhibitor) and a CAR molecule or a nucleic acid encoding a CAR molecule. In some embodiments, the reaction mixture also comprises immune effector cell populations.

[00101] In some embodiments, one or more immune effector cells express the CAR molecule or comprise the nucleic acid encoding the CAR molecule. In some embodiments, the kinase inhibitor is chosen from a BTK inhibitor, a CDK4 inhibitor, an mTOR inhibitor, or an MNK inhibitor. In some embodiments, the BTK inhibitor is chosen from: ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13 . In embodiments, the reaction mixture comprises cancer cells, e.g., hematological cancer cells. Cancer cells may be, for example, cells that were harvested from the individual when immune effector cells were harvested from the individual.

[00102] In certain aspects, the present invention also provides a reaction mixture comprising immune effector cell populations, and a CAR molecule or a nucleic acid encoding a CAR molecule, wherein the immune effector cells comprise covalently inactivated lTK. In embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of ITK are covalently inactivated. In some embodiments, the reaction mixture also comprises cancer cells. In embodiments, the cancer cells comprise covalently inactivated BTK. In embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of BTK is covalently inactivated. In embodiments, a BTK or ITK forms a covalent bond at or near its ATP-binding domain to a small molecule such as ibrutinib. In embodiments, the BTK forms a covalent bond at or near its cysteine-481 to a small molecule such as ibrutinib.

[00103] In embodiments, a reaction mixture as described herein also comprises a buffer or other reagent, for example, a solution containing PBS. In embodiments, the reaction mixture also comprises an agent that activates and/or expands to the cells of the population, for example, an agent that stimulates a signal associated with the CD3/TCR complex and/or a ligand that stimulates a co-stimulatory molecule in the cell surface. In embodiments, the agent is an antibody-conjugated bead with anti-CD3, or a fragment thereof, and/or anti-CD28 antibody, or a fragment thereof. In embodiments, the reaction mixture also comprises one or more factors for proliferation and/or viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-Y, IL- 4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFß, and TNF-a or any of the other additives for cell growth. In embodiments, the reaction mixture also comprises IL-15 and/or IL-7. In embodiments, a plurality of the cell population in the reaction mixture comprises a nucleic acid molecule, e.g., a nucleic acid molecule described herein, that comprises a CAR encoding sequence, e.g., a CD19 CAR encoding sequence, e.g. as described here. In embodiments, a plurality of the cell 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 embodiments, the vector is a vector described herein, for example, a vector selected from the group consisting of a DNA, an RNA, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector. In embodiments, the reaction mixture also comprises a cryoprotectant or stabilizer such as, for example, a saccharide, an oligosaccharide, a polysaccharide and a polyol (e.g., trehalose, mannitol, sorbitol, lactose, sucrose, glucose and dextran), salts and crown ethers. In one embodiment, the cryoprotectant is dextran.

[00104] In some embodiments, the preparation method described here also comprises contacting immune effector cell populations with a nucleic acid encoding a telomerase subunit, for example, hTERT. The nucleic acid encoding the telomerase subunit may be DNA.

[00105] In some embodiments, the preparation method described here also comprises culturing immune effector cell populations in serum comprising 2% hAB serum.

[00106] Titles, subtitles or numbered or lettered elements, for example, (a), (b), (i) etc., are presented simply for ease of reading. The use of titles or numbered or lettered elements in this document does not require that the steps or elements be performed in alphabetical order or that the steps or elements are necessarily discrete from each other.

[00107] All publications, patent applications, patents, and other references mentioned here are incorporated herein by reference in their entirety.

[00108] Other characteristics, objectives and advantages of the invention will be evident from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[00109] Figures 1A, 1B and 1C are graphic representations of cytotoxicity as tested on ND317 T cell (normal donor) transduced with mouse anti-CD19 CAR or humanized anti-CD19 CARs of the invention and cultured with control K562 cells that do not express CD19 (K562cc) as shown in Figure 1A, K562 cells transformed with CD19 (K562.CD19) as shown in Figure 1B, or malignant B cells isolated from a B cell isolate patient (Pt 14) as shown in Figure 1C.

[00110] Figures 2A and 2B are graphs showing the proliferative response of humanized anti-CD-19 and mouse CAR-expressing cells to CD19+ cells, where the highest number of viable CAR+ T cells correlates with populations showing CD4+ cell proliferation and maximum CD8+ for primary CLL cells.

[00111] Figure 3 is a graphical representation of the deconvoluted HPLC mass spectra for scFvs of the invention, where the top row depicts untreated scFv and the bottom row depicts the cognate deglycolyzed scFv.

[00112] Figure 4 is a graphical representation of conformational stability as measured by Differential Scanning Fluorimetry. The Tm of mouse scFv was 57°C (thick line). All humanized scFv variants show elevated Tm around 70°C compared to mouse scFv. Waste introduced by humanization has improved Tm by more than 10° C.

[00113] Figure 5 is a graphical representation of CD19 CAR-transduced T cell proliferation, in which CART19 cells are targeted to (a) a chronic myelogenous leukemia ("CML") cell line that is negative for CD19. expression of CD19, and consequently used; (b) recombinant K562 cells positive for CD19 expression, and consequently used as a positive control; or (c) Pt14 B cells isolated from a CLL patient and expressing CD19 on a cell surface.

[00114] Figures 6A and 6B are schematics of representative CARs.

[00115] Figure 7 depicts the progression of HALLX5447 primary ALL disease in NSG mice after treatment with CD19-transduced CAR T cells. Growth of primary human ALL cells in NSG mice after treatment with CD19-specific CAR T cells demonstrated control of disease progression. The mean percentage of CD19+ human ALL cells was an indicator of peripheral blood disease burden in NSG mice for day 65 after tumor implantation. Black circles: mice treated with 100ul of PBS via the tail vein; red squares: mice treated with mock transduced T cells; blue triangles: mice treated with murine CD19 CAR-transduced T cells; and inverted purple triangles: mice treated with humanized CD19 CAR-transduced T cells. Significance calculated by ANOVA; * means P<0.01.

[00116] Figure 8 depicts the expression of CD19 on the patient's tumor cells. CD138+ CD45dim tumor cells were stained for CD19 (x-axis) and CD38 (y-axis). Approximately 1-2% of tumor cells expressed CD19 antigen.

[00117] Figure 9 is two graphs showing cell proliferation and cell size of CART19 cells when treated with increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM).

[00118] Figures 10A and 10B show the proliferation of CART19 cells stimulated with MCL cell lines, at the same time in the presence or absence of ibrutinib. Figure 10A is a series of histograms showing the proliferation of CART19 cells stimulated with MOLM14, JEKO-1, and RL tumor cell lines, in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100nM, and 1000 nM). Cells were stained by CFSE and analyzed by flow cytometry to determine the percentage of proliferating cells, designated by the bar in each histogram. Figure 10B is a quantification of representative histograms in Figure 10A.

[00119] Figures 11A and 11B show CD107a degranulation of CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. Figure 11A is a series of flow cytometry profiles showing CD107a degranulation of CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM , and 1000 nM). CD107a expression is measured on the y axis. Figure 11B is the quantification of the results from Figure 11A.

[00120] Figure 12 is a series of flow cytometry profiles showing intra-cytoplasmic lL-2 production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of concentrations increasing amounts of ibrutinib (10 nM, 100nM, and 1000 nM). The y-axis represents the expression of lL-2.

[00121] Figure 13 is a series of flow cytometry profiles showing intracytoplasmic TNF-a production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100nM, and 1000 nM). The y axis represents the expression of TNF-a.

[00122] Figure 14 is a series of flow cytometry profiles showing intracytoplasmic IFN-g production by CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100nM, and 1000 nM). The y axis represents the expression of IFN-g.

[00123] Figure 15 is a series of graphs showing cytokine secretion from CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM).

[00124] Figures 16A, 16B, 16C, 16D, 16E, and 16F are graphs showing killing by CART19 of tumor cells, MOLM14 (FIG. 16A and 16D), JEKO (FIG. 16B and 16E), and RL ( FIG. 16C and 16F), alone or in the presence of increasing concentrations of ibrutinib. Non-transduced (UTD) or CART19 cells were incubated with tumor cells at varying ratios and total cell flow (FIG. 16A, 16B, and 16C) and percentage of dead cells were assessed (16D, 16E, and 16F).

[00125] Figures 17A, 17B, and 17C are graphic representations of CART19 killing of tumor cells after 24 hours as measured by flow cytometry to count the total number of cells. Tumor cell lines MOLM14 (FIG. 17A), JEKO (FIG. 17B), and RL (FIG. 17C) were incubated with untransduced cells (UTD) or CART19 alone (ALONE), or in combination with varying concentrations of ibrutinib. .

[00126] FIG. 18A, 18B, 18C, and 18D are graphical representations of CART19 dose finding in the MCL RL mouse model. Tumor burden was monitored by bioluminescence imaging (BLI) over time (FIG. 18A and 18B). Overall survival was monitored over time (FIG. 18C).

[00127] Figures 19A and 19B are graphic representations of CART19 dose finding in the JEKO-1 MCL mouse model. Tumor size is monitored by bioluminescence imaging (BLI) over time (FIG. 19A) and overall survival is also monitored over time (FIG. 19B).

[00128] Figure 20 is a schematic showing the protocol for administering and evaluating CART19 and ibrutinib combination therapy in in vivo mouse models.

[00129] Figure 21A, 21B, 21C, and 21D is a graphical representation showing the transduction efficiency of PBMCs in terms of generating CART19 T cells when cells are treated with ibrutinib prior to transduction. Untreated PBMCs were analyzed for CAR19 expression (FIG. 21A) and after transduction (FIG. 21C). Ibrutinib-treated PBMCs were analyzed for CAR19 expression before transduction (FIG. 21B) and after transduction (FIG. 21D).

[00130] Figure 22A, 22B, 22C, 22D, and 22E is a graphical representation of the effect of ibrutinib treatment on CD3/CD28-stimulated T cell proliferation. Increasing concentrations of ibrutinib were evaluated: Untreated (FIG. 22A); 0.1 µM ibrutinib (FIG. 22B); 0.5 µM ibrutinib (FIG. 22C), 1 µM ibrutinib (FIG. 22D), and 5 µM ibrutinib (FIG. 22E).

[00131] Figure 23 is a graphical representation demonstrating that ibrutinib does not affect the cytotoxicity of CART19.

[00132] Figure 24 is a series of graphic representations that demonstrate that treatment with ibrutinib does not promote distortion of TH1/TH2 cytokines in CART19 cells.

[00133] Figures 25A and 25B are graphical representations showing that continuous administration of ibrutinib does not affect the function of CART19 in clearing tumor cells in vivo. Figure 25A shows Nalm/6 cells detected in peripheral blood during each treatment regimen. Figure 25B shows a Kaplan-Meier survival curve comparing the survival of mice receiving CART19 with or without ibrutinib dosing.

[00134] Figures 26A, 26B, 26C, 26D, 26E, and 26F are graphical representations showing the transduction efficiency of CAR19 in CLL patient cells at the indicated time points during treatment with ibrutinib. Cells were not transduced (FIG. 26A, 26B, and 26C), or were transduced with CAR19 (FIG. 26D, 26E, and 26F). GAM-stained cells express CAR19 and are present in boxes in each profile.

[00135] FIG. 27 is a series of graphical representations depicting the proliferation rate of untransduced cells compared to cells that were transduced with CAR19 at the indicated time points during ibrutinib treatment from a panel of patients.

[00136] FIGs 28A, 28B, and 28C are graphical representations demonstrating that treatment with ibrutinib in CLL patients induces lymphocytosis. Patient cells were isolated at the indicated time points: baseline (FIG. 28A); cycle 2, day 1 (FIG. 28B); and cycle 12, day 1 (FIG. 28C).

[00137] FIGs 29A, 29B, and 29C are graphical representations demonstrating that ibrutinib treatment in three CLL patients induces CD200 expression on tumor cells over time in CLL. Each profile contains an overlay of CD200 expression histograms from cells isolated at the indicated time points: baseline (screen); cycle 2, day 1; and cycle 12, day 1.

[00138] FIGs 30A, 30B, and 30C are graphical representations demonstrating that ibrutinib treatment in CLL patients decreases the frequency of PD1+ T cells over time. Patient cells were isolated at the indicated time points: baseline (FIG. 30A); cycle 2, day 1 (FIG. 30B); and cycle 12, day 1 (FIG. 30C).

[00139] FIGs 31A and 31B are a graphical representation demonstrating the sensitivity of the MCL RL (FIG. 31A) and JEKO-1 (FIG. 31B) cell lines to ibrutinib treatment.

[00140] Figure 32 is a graphical representation demonstrating the effect of ibrutinib treatment in an in vivo model of MCL.

[00141] FIGs 33A and B are images from immunohistochemical analysis of a Hogdkin's lymphoma showing CD19-expressing cells present in the tumor. Figure 33A is at 1X magnification and Figure 33B is at 20X magnification.

[00142] Figure 34 is a schematic diagram of the experimental structure during a study to evaluate the therapeutic efficacy of CART19 treatment in patients with Hodgkin's lymphoma.

[00143] FIGs 35A, 35B, 35C, and 35D show flow cytometry analysis of PD1 and CAR19 expression on T cells. Figure 35A and 35B are representative flow cytometry profiles demonstrating the distribution of PD-1 expression and CAR19 on CD4+ T cells from individuals who are complete responders (CR) or non-responders (NR) to CART therapy. Figure 35C is a graph showing the percentage of PD1 cells in the CD4+ T cell population of groups of individuals with different responses to CART therapy. FIG. 35D is a graph showing the percentage of PD1 cells in the CD8+ T cell population of groups of individuals with different responses to CART therapy.

[00144] FIGs 36A and 36B show the distribution of PD1 expression in CD4 2 CAR19 expressing cells (FIG. 36A) or CD8 and CAR19 expressing cells (FIG. 36B) from groups of individuals with different responses to therapy of CART.

[00145] Figure 37 shows flow cytometry analysis of expression of PD1, CAR 19, LAG3, and TIM3 on T cells from individuals who are complete responders (CR) or non-responders (NR) to CART therapy.

[00146] FIGs 38A and 38B show the distribution of PD1 and LAG3 expression (FIG. 38A) or PD1 and TIM3 expression (FIG. 38B) of groups of individuals with different responses to CART therapy.

[00147] Figure 39 shows plasma cell IgA immunophenotyping of a myeloma patient who received CART19, demonstrating the response to CART19 therapy.

[00148] FIGs 40A and 40B are graphs showing an increase in titers for influenza vaccine lines as compared to placebo. In Figure 40A, the increase above baseline in geometric mean titers for each of the 3 influenza vaccine lines (H1N1 A/California/07/2009, H3N2 A/Victoria/210/2009, B/Brisbane/60/2008 ) relative to the increase in the placebo cohort 4 weeks after vaccination is shown for each of the RAD001 dosing cohorts in the intention-to-treat population. The bold black line indicates the 1.2-fold increase in titers relative to placebo that is required to be met by 2 of 3 influenza vaccine strains to meet the study's primary endpoint. The star "*" indicates that the increase in GMT titer relative to placebo exceeds 1 with a posterior probability of at least 80%. FIG 40B is a graph of the same data as FIG. 40A for the subset of subjects with baseline influenza titers <= 1:40.

[00149] Figure 41 shows a scatterplot of RAD001 concentration versus fold increase in geometric mean titer for each influenza vaccine strain 4 weeks after vaccination. RAD001 concentrations (1 hour post dose) were measured after subjects had been dosed for 4 weeks. All subjects who had pharmacokinetic measurements were included in the analysis set. The fold increase in geometric mean titers at 4 weeks post-vaccination relative to baseline is shown on the axis.

[00150] Figure 42 is a graphical representation showing the increase in titers for heterologous influenza strains as compared to placebo. The increase above baseline in geometric mean influenza titers for 2 heterologous influenza strains (A/H1N1 strain A/New Jersey/8/76 and A/H3N2 strain A/Victoria/361/11) not contained in the vaccine against influenza relative to the increase in the placebo cohort 4 weeks after vaccination is shown for each of the RAD001 dosing cohorts in the intention-to-treat population. * indicates that the increase in titer relative to placebo exceeds 1 with a posterior probability of at least 80%.

[00151] Figure 43A and 43B are graphic representations of IgG and IgM levels before and after influenza vaccination. Anti-A/H1N1/California/07/2009 influenza IgG and IgM levels were measured in serum obtained from individuals before and 4 weeks after influenza vaccination. No significant difference in change from baseline to 4 weeks post-vaccination in anti-H1N1 influenza IgG and IgM levels was detected between the RAD001 and placebo cohorts (all p-values > 0.05 by test sum test Kruskal-Wallis rank sum method).

[00152] FIGs 44A, 44B, and 44C are graphical representations of the decrease in percentage of CD4 and CD8 positive in PD-1 and increase in CD4 negative T cells in PD-1 after RAD001 treatment. The percentage of PD-1-positive CD4, CD8, and PD-1-negative CD4 T cells was determined by FACS analysis of PBMC samples at baseline, after 6 weeks of study drug treatment (Week 6), and 6 weeks after study drug discontinuation and 4 weeks after influenza vaccination (Week 12). Figure 44A shows that there was a significant decrease (-37.1 - - 28.5%) in PD-1 positive CD4 T cells at week 12 in cohorts receiving RAD001 at dose levels 0.5 mg/Day (n =25), 5 mg/Week (n=29) and 20 mg/Week (n=30) as compared to the placebo cohort (n=25) with p=0.002 (0.02), p=0.003 ( q=0.03), and p= 0.01 (q=0.05) respectively. Figure 44B shows that there was a significant decrease (-43.3 - -38.5%) in PD-1 positive CD8 T cells at week 12 in cohorts receiving RAD001 (n=109) at dose levels of 0. 5 mg/Day (n=25), 5 mg/Week (n=29) and 20 mg/Week (n=30) as compared to the placebo cohort (n=25) with p=0.01 (0 .05), p=0.007 (q=0.04), and p= 0.01 (q=0.05) respectively. Figure 44C shows that there was a significant increase (3.0 - 4.9%) in PD-1 negative CD4 T cells at week 12 in cohorts receiving RAD001 (n=109) at dose levels 0.5 mg/Day (n=25), 5 mg/Week (n=29) and 20 mg/Week (n=30) as compared to the placebo cohort (n=25) with p=0.0007 (0.02), p=0.03 (q=0.07), and p= 0.03 (q=0.08) respectively.

[00153] Figure 45A and 45B are graphical representations of the decrease in percentage of PD-1 positive CD4 and CD8 and increase in PD-1 negative CD4 T cells after RAD001 treatment adjusted for differences in PD-1 expression in baseline. The percentage of PD-1-positive CD4, CD8, and PD-1-negative CD4 T cells was determined by FACS analysis of PBMC samples at baseline, after 6 weeks of study drug treatment (Week 6), and 6 weeks after study drug discontinuation and 4 weeks after influenza vaccination (Week 12). Figure 45A shows a significant 30.2% decrease in PD-1+ CD4 T cells at week 6 in the pooled RAD cohort (n=84) compared to placebo cohort (n=25) with p=0.03 (q=0.13). The decrease in PD-1 positive CD4 T cells at week 12 in the mixed RAD as compared to the placebo cohort is 32.7% with p= 0.05 (q=0.19). Figure 45B shows a significant 37.4% decrease in PD-1 positive CD8 T cells at week 6 in the pooled RAD001 cohort (n=84) compared to placebo cohort (n=25) with p=0.008 ( q=0.07). The decrease in PD-1 positive CD8 T cells at week 12 in the RAD001 pooled as compared to the placebo cohort is 41.4% with p=0.066 (q=0.21). FIGS. 45A and 45B represent the data in FIGs 44A, 44B, and 44C, but with the different RAD001 dosage groups of FIGs 44A, 44B, and 44C mixed into the RAD001 treated group in FIGs 45A and 45B.

[00154] Figure 46 depicts increases in exercise and energy in elderly individuals in response to RAD001.

[00155] FIGs 47A and 47B describe the predicted effect of RAD001 on P70 S6K activity in cells. Figure 47A depicts inhibition of P70 S6 kinase with higher weekly and daily doses of RAD001; Figure 47B depicts inhibition of P70 S6 kinase with lower weekly doses of RAD001.

[00156] FIGs 48A and 48B show the expression of IL-7 receptor (CD127) in cancer cell lines and CART cells. CD127 expression was measured by flow cytometry analysis in three cancer cell lines: RL (lining cell lymphoma), JEKO (also known as Jeko-1, lining cell lymphoma), and Nalm-6 (B -ALL) (Fig. 48A). CD127 expression was measured by flow cytometry analysis on CD3-positive cells (CART) that were infused and circulated into NSG mice (Fig. 48B).

[00157] FIGS. 49A, 49B, and 49C show the antitumor response after CART19 treatment and subsequent IL-7 treatment. NSG mice engrafted with a luciferase-expressing coated lymphoma cell line (RL-luc) on day 0 were treated with varying dosages of CART19 cells on day 6, and tumor burden was monitored. The mice were divided into 4 groups and received no CART19 cells, 0.5x106CART19 cells (CART19 0.5E6), 1x106CART19 cells (CART19 1E6), or 2x106CART19 cells (CART19 2E6). Tumor burden after CART treatment was measured by bioluminescence detection (mean BLI) (Fig. 49A). Mice receiving 0.5x106CART19 cells (CART19 0.5E6) or 1x106CART19 cells (CART19 1E6) were randomized to receive recombinant human IL-7 (rhIL-7) or not. Tumor burden, represented here by mean bioluminescence (BLI), was monitored for the three mice (#3827, #3829, and #3815, receiving the indicated starting CART19 dose) of Figure 49A that were treated with IL-7, starting at day 85 (Fig. 49B). IL-7 was administered via IP injection 3 times weekly. Tumor burden, represented here by mean bioluminescence (BLI) before day 85 (PRE) and after day 115 (POST), was compared between mice that did not receive IL-7 (CTRL) and mice that received IL-7 treatment (IL-7) (Fig. 49C).

[00158] FIGS. 50A and 50B show T cell dynamics after IL-7 treatment. The level of human T cells detected in the blood was monitored for each of the mice receiving IL-7 or control mice (Fig. 50A). The level of CART19 cells (CD3+ cells) detected in the blood was measured before (PRE) and 14 days after (Day 14) the initiation of IL-7 treatment (Fig. 50B).

[00159] Figure 51 depicts the structures of two exemplary RCAR configurations. Antigen binding numbers comprise an antigen binding domain, a transmembrane domain, and a switch domain. The intracellular binding members comprise a switch domain, a co-stimulatory signaling domain, and a primary signaling domain. The two configurations demonstrate that the first and second switch domains described here can be in different orientations relative to the antigen-binding member and the intracellular binding member. Other RCAR configurations are also described here.

[00160] Figure 52A is an image of an RL cell line.

[00161] Figure 52B is a group of flow cytometry scatterplots showing the expression of CD19 and CD5 in primary RL and RL cell lines.

[00162] Figure 52C is an image showing t(11;14) translocation by fluorescence in situ hybridization (FISH).

[00163] Figure 52D is a graph showing the IC50 (per percentage MTT conversion) of ibrutinib inhibition in different cell lines.

[00164] Figure 52E is a group of images and graphs showing the engraftment of RL cells into NOD-SCID-gamma chain knockout (NSG) mice and the resulting tumor burden.

[00165] Figure 52F is a group of histological images showing the localization of MCL cells to various organs in mice.

[00166] Figure 52G is a group of histological images of mice that were injected with MCL-RL cells.

[00167] Figure 53A is a group of graphs showing the number of CART19 CD107a+ cells when exposed to various MCL cell lines.

[00168] FIG. 53B is a group of graphs showing the amount of IL-2 and TNF-alpha produced by CART19 cells when exposed to various MCL cell lines.

[00169] Figure 53C is a graph showing the percentage of killing of various MCL cell lines by CART19 cells at various effector:target cell ratios.

[00170] Figure 53D is a graph showing the amount of carboxyfluoroscein succinimidyl ester (CFSE), a measurement of proliferation, in CART19 cells exposed to various MCL cell lines.

[00171] Figure 53E is a group of graphs showing the percentage of T cells before and after expansion.

[00172] Figure 53F is a group of graphs showing the percentage of CAR-19 transduced or non-transduced T cells that express or produce various biomolecules (e.g., cytokines).

[00173] Figure 54A is a group of images showing the activation of T cell interleukin-2 inducible kinase (ITK) when CART19 cells were specifically or non-specifically stimulated.

[00174] FIG. 54B is a group of graphs showing surface expression of CD107a (a measurement of degranulation), IL-2 production, and TNF-alpha production by CART19 cells at various concentrations with ibrutinib.

[00175] Figure 54C is a group of histograms showing the amount of CFSE in CART19 cells with various concentrations of ibrutinib and expressed in various MCL cell lines.

[00176] Figure 54D is a group of graphs showing the expression or production of various cytokines and biomarkers as indicators of the Th1 or Th2 status of CART19 cells when combined with different concentrations of ibrutinib.

[00177] Figure 54E is a group of graphs showing the percentage death by CART19 cells of various MCL cell lines when combined with different concentrations of ibrutinib.

[00178] Figure 54F is a bar graph showing the expression of various markers of intrinsic cytotoxic function of CART19 cells when combined with various concentrations of ibrutinib.

[00179] Figure 55 is a schematic representation of an in vivo mouse model experimental setup for testing the effect of CART19 and/or ibrutinib on MCL-RL injected mice, with one readout being luminescence (a measurement of tumor number cells).

[00180] Figure 56 is a schematic of an in vivo mouse model experimental setup for testing the effect of CART19 and/or ibrutinib on MCL-RL injected mice, with one readout being luminescence (a measurement of tumor number cells).

[00181] Figure 57 is a group of graphs showing luminescence (a measurement of tumor cell number measurement) in mice treated with ibrutinib at different concentrations and their overall survival after treatment.

[00182] Figure 58 is a group of graphs showing luminescence (a measurement of tumor cell number) in mice treated with ibrutinib or CART19 cells as well as their overall survival after treatment.

[00183] Figure 59 is a graph showing luminescence (a measurement of tumor cell number) in mice after treatment with ibrutinib, non-transduced T cells, ibrutinib with non-transduced T cells, CART19 cells, and CART19 cells with ibrutinib .

[00184] Figure 60 is a graph showing luminescence (a measurement of tumor cell number) in mice after treatment with ibrutinib alone, CART19 cells alone, or the combination of ibrutinib with CART19 cells.

[00185] Figure 61A is a group of graphs showing the level of Th1 cytokines produced in mice treated with ibrutinib and/or CART19 cells. Figure 61B is a group of graphs showing the level of Th2 cytokines produced in mice treated with ibrutinib and/or CART19 cells.

[00186] Figure 61C is a graph showing the percentage of cells expressing the Ki67 proliferation marker in mice treated with CART19 cells or CART19 cells plus ibrutinib.

[00187] Figure 61D is a graph showing the percentage of cells expressing the antiapoptotic marker BCL-2 in mice treated with CART19 cells or CART19 cells plus ibrutinib.

DETAILED DESCRIPTION Definitions

[00188] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the invention belongs.

[00189] The term "one" and "one" refers to one or more than one (that is, at least one) of the grammatical objective of the article. By way of example, "an element" means one element or more than one element.

[00190] The term "about" when referring to a measurable value such as a quantity, a temporal duration, and the like, is intended to encompass variations of ±20% or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value, such that the variations are appropriate to carry out the methods described.

[00191] The term "Chimeric Antigen Receptor" or alternatively "CAR" refers to a group of polypeptides, typically two in the simplest embodiments, that when on an immune effector cell, provide the cell with specifically for a target cell, typically a cancer cell, and with intracellular signal generation. In some embodiments, a CAR comprises at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as "an intracellular signaling domain") comprising a functional signaling domain derived from a molecule stimulatory and/or costimulatory molecule as defined below. In some aspects, the groups of polypeptides are contiguous to each other, e.g., they are on the same polypeptide chain (e.g., they comprise a chimeric fusion protein). In some embodiments, the groups of polypeptides are not contiguous to each other, for example, they are on different polypeptide chains. In some embodiments, the polypeptide groups include a dimerization exchange that, in the presence of a dimerization molecule, can couple the polypeptides to each other, for example, can couple an antigen-binding domain to an intracellular signaling domain. In one aspect, the stimulatory molecule is the zeta chain associated with a T cell receptor complex. In one aspect, the cytoplasmic signaling domain also comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one aspect, the costimulatory molecule is chosen from the costimulatory molecules described herein, for example, 4-1BB (i.e., CD137), CD27 and/or CD28. In one aspect, 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 aspect, 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 co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, 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 co-stimulatory molecule(s) and a functional signaling derived from a stimulatory molecule. In one aspect, 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 co-stimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence at the amino terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR also comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen-binding domain (e.g., a scFv) during CAR processing and cellular localization to the cell membrane.

[00192] The term "binding domain" refers to the functional portion of a protein that acts by transmitting information within the cell to regulate cellular activity through defined signaling series generating second messengers or functioning as effectors responding to such messengers.

[00193] As used herein, the term "CD19" refers to the Cluster of Differentiation 19 protein, which is an antigenic determinant detectable in leukemia precursor cells. Human and murine amino acid and nucleic acid sequences can be found in a public database such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found as UniProt/Swiss-Prot accession no. P15391 and the nucleotide sequence encoding human CD19 can be found in accession no. NM_001178098. As used herein, "CD19" includes proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions, and full-length wild-type CD19 binding variants. CD19 is expressed in most B-lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. Other cells with expressed CD19 are provided below in the definition of "disease associated with CD19 expression." It is also an early marker of B cell progenitors. See, for example, Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one aspect, the antigen-binding portion of CART recognizes and binds an antigen within the extracellular domain of the CD19 protein. In one aspect, the CD19 protein is expressed on a cancer cell.

[00194] As used herein, the term "CD20" refers to an antigenic determinant known to be detectable on B cells. Human CD20 is also called 4 membrane-spanning domains, subfamily A, membrane 1 (MS4A1). Human and murine amino acid and nucleic acid sequences can be found in a public database such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD20 can be found in Accession Nos. NP_690605.1 and NP_068769.2, and the nucleotide sequence encoding human CD20 transcript variants 1 and 3 can be found in Accession Nos. NM_152866.2 and NM_021950.3, respectively. In one aspect, the antigen-binding portion of the CAR recognizes and binds an antigen within the extracellular domain of the CD20 protein. In one aspect, the CD20 protein is expressed on a cancer cell.

[00195] As used herein, the term "CD22," refers to an antigenic determinant known to be detectable in leukemia precursor cells. Human and murine amino acid and nucleic acid sequences can be found in a public database such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequences of human CD22 isoforms 1-5 can be found in Accession Nos. NP 001762.2, NP 001172028.1, NP 001172029.1, NP 001172030.1, and NP 001265346.1, respectively, and the nucleotide sequence encoding human CD22 variants 15 can be found in Accession Nos. NM 001771.3, NM 001185099 .1, NM 001185100.1, NM 001185101.1, and NM 001278417.1, respectively. In one aspect, the antigen-binding portion of the CAR recognizes and binds an antigen within the extracellular domain of the CD22 protein. In one aspect, the CD22 protein is expressed on a cancer cell.

[00196] As used herein, the term "ROR1" refers to an antigenic determinant known to be detectable in leukemia precursor cells. Human and murine amino acid and nucleic acid sequences can be found in a public database such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequences of precursor isoforms 1 and 2 of human ROR1 can be found in Accession Nos. NP_005003.2 and NP_001077061.1, respectively, and the mRNA sequences encoding them can be found in Accession Nos. NM_005012.3 and NM_001083592.1, respectively. In one aspect, the antigen-binding portion of the CAR recognizes and binds an antigen within the extracellular domain of the ROR1 protein. In one aspect, the ROR1 protein is expressed in a cancer cell.

[00197] The term "antibody," as used herein, refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule that specifically binds with an antigen. Antibodies may be polyclonal or monoclonal, multiple- or single-chain, or intact immunoglobulins, and may be derived from natural sources or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[00198] The term "antibody fragment" refers to at least a portion of an antibody, which retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution) an epitope of a antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), an Fd fragment consisting of the VH and CH1 domains, linear antibodies, single-domain antibodies such as sdAb (or VL or VH), VHH domains, multispecific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region, and a Isolated CDR or other epitope-binding fragments of an antibody. An antigen-binding fragment can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv (see, for example, Hollinger and Hudson, Nature Biotechnology 23 :1126-1136, 2005). Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds, such as a fibronectin type III (Fn3) (see United States Patent No.: 6,703,199, which describes fibronectin polypeptide minibodies).

[00199] 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, wherein the regions light and heavy chain variables are contiguously linked, for example by means of a synthetic linker, e.g. a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains specificity of the intact antibody from which it is derived. Unless specified, as used herein a scFv may have the VL and VH variable regions in any order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-ligand-VL.

[00200] The CAR portion of the invention comprising an antibody or antibody fragment thereof can exist in a variety of forms where the antigen-binding domain is expressed as part of a contiguous polypeptide chain including, for example, a fragment of single-domain antibody (sdAb), a single-chain antibody (scFv), a humanized antibody, or 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 aspect, the antigen-binding domain of a CAR composition of the invention comprises an antibody fragment. In another aspect, the CAR comprises an antibody fragment comprising an scFv. The precise amino acid sequence boundaries of a given CDR can be determined using any of several well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest,"5a. Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 ("Chotia" numbering scheme), or a combination of same.

[00201] As used herein, the term "binding domain" or "antibody molecule" refers to a protein, for example, an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragment. In one embodiment, an antibody molecule is a multispecific antibody molecule, for example, it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope. and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In one embodiment, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[00202] The CAR portion of the invention comprising an antibody or antibody fragment thereof can exist in a variety of forms where the antigen-binding domain is expressed as part of a contiguous polypeptide chain including, for example, a fragment of single-domain antibody (sdAb), a single-chain antibody (scFv), a humanized antibody, or 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 aspect, the antigen-binding domain of a CAR composition of the invention comprises an antibody fragment. In another aspect, the CAR comprises an antibody fragment comprising an scFv.

[00203] The term "antibody heavy chain," refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which typically determines the class to which the antibody belongs.

[00204] The term "antibody light chain," refers to the shorter of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (K) and lambda (X) light chains refer to the two largest light chain isotypes of antibodies.

[00205] The term "recombinant antibody" refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody that has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or antibody sequence amino acid was obtained using recombinant DNA or amino acid sequence technology that is available and well known in the art.

[00206] The term "antigen" or "Ag" refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response, therefore encodes an "antigen" as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence from a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences from more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Furthermore, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily evident that an antigen can be generated synthesized or can be derived from a biological sample, or can be a macromolecule in addition to a polypeptide. Such a biological sample may include, but is not limited to, a tissue sample, a tumor sample, a cell, or a fluid with other biological components.

[00207] The term "anticancer effect" refers to a biological effect that can be manifested in various ways, including, but not limited to, for example, a decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, decrease in cancer cell proliferation, decrease in cancer cell survival, or improvement in various physiological symptoms associated with the cancerous condition. An "anticancer effect" may also be manifested by the ability of peptides, polynucleotides, cells and antibodies to prevent cancer from occurring in the first place. The term "antitumor effect" refers to a biological effect that can be manifested by various methods, including, but not limited to, for example, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in proliferation of tumor cell, or a decrease in tumor cell survival.

[00208] The term "autologous" refers to any material derived from the same individual to which it must later be reintroduced.

[00209] 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 aspects, allogeneic material from individuals of the same species may be sufficiently different genetically to interact antigenically.

[00210] The term "xenogenic" refers to a graft derived from an animal of a different species.

[00211] The term "cancer" refers to a disease characterized by the uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include, but are 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 here, for example, both terms encompass solid and liquid tumors, e.g., diffuse or circulating. As used herein, the term "cancer" or "tumor" includes premalignant as well as malignant cancers and tumors.

[00212] The phrase "disease associated with expression of CD19" includes, but is not limited to, a disease associated with expression of CD19 or condition associated with cells that express, or at some time expressed, CD19 including, for example, proliferative diseases such as a cancer or malignancy or a pre-cancerous condition such as myelodysplasia, myelodysplastic syndrome or pre-leukemia; or a non-cancer indication associated with CD19-expressing cells. For the avoidance of doubt, a disease associated with CD19 expression may include a condition associated with cells that do not presently express CD19, for example, because CD19 expression has been down-regulated, for example, due to treatment with a molecule targeting CD19. for example, a CD19 CAR, but which at one point expressed CD19. In one aspect, a cancer associated with expression of CD19 is a hematological cancer. In one aspect, hematologic cancer is a leukemia or a lymphoma. In one aspect, a cancer associated with expression of CD19 includes cancers and malignancies including, but not limited to, for example, one or more acute leukemias including, but not limited to, for example, B-cell acute lymphoblastic leukemia (BALL). , T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL); one or more chronic leukemias including, but not limited to, for example, chronic myelogenous leukemia (CML), Chronic Lymphoid Leukemia (CLL). Additional cancers or hematologic conditions associated with CD19 expression include, but are not limited to, for example, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma , hairy cell leukemia, follicular large cell or small cell lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, lining cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, lymphoma Hodgkin's, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemia" which are a diverse collection of hematologic conditions united by the ineffective production (or dysplasia) of myeloid blood cells, and the like. Other diseases associated with the expression of CD19 expression include, but are not limited to, for example, atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases associated with the expression of CD19. Non-cancer indications associated with CD19 expression include, but are not limited to, for example, autoimmune disease (e.g., lupus), inflammatory disorders (allergy and asthma), and transplantation. In some embodiments, cells expressing tumor antigen express, or at some point expressed, mRNA encoding the tumor antigen. In one embodiment, cells expressing tumor antigen produce the tumor antigen protein (e.g., wild type or mutant), and the tumor antigen protein may be present at normal levels or reduced levels. In one embodiment, cells expressing tumor antigen produced detectable levels of a tumor antigen protein at one point, and subsequently produced substantially no detectable tumor antigen protein.

[00213] The phrase "disease associated with the expression of a B cell antigen" includes, but is not limited to, a disease associated with the expression of one or more of CD19, CD20, CD22 or ROR1, or the condition associated with cells that express, or have at some time expressed, one or more of CD19, CD20, CD22 or ROR1, including, for example, proliferative diseases such as a cancer or malignancy or a precancerous condition such as myelodysplasia, myelodysplastic syndrome or pre-leukemia; or a non-cancer indication associated with cells expressing one or more of CD19, CD20, CD22 or ROR1. For the avoidance of doubt, a disease associated with B-cell antigen expression may include a condition associated with cells that do not currently express B-cell antigen, for example, because antigen expression has been down-regulated, e.g. due to treatment with a molecule targeting the B cell antigen, for example, a CAR targeting B cells, but which at one point expressed the antigen. The phrase "disease associated with the expression of a B cell antigen" includes a disease associated with the expression of CD19, as described herein.

[00214] The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter 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 an antibody or antibody fragment of the 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 the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and side chains aromatics (e.g. tyrosine, phenylalanine, tryptophan, histidine). In this way, one or more amino acid residues within a CAR of the invention can be replaced by other amino acid residues from the same side chain family and the altered CAR can be tested using the functional assays described herein.

[00215] The term "stimulation," refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex or CAR) with its cognate ligand (or tumor antigen in the case of a CAR) thereby mediating a signal transduction event, such as, but not limited to, signal transduction through the TCR/CD3 complex or signal transduction through the appropriate NK signaling or receptor domains of the CAR. Stimulation can mediate altered expression of certain molecules.

[00216] The term "stimulatory molecule," refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides the cytoplasmic signaling sequence(s) that regulate immune cell activation in a stimulatory manner during at least some aspect of the immune cell signaling reaction series. In one aspect, the signal is a primary signal that is initiated, for example, by binding of a TCR/CD3 complex with a peptide-loaded MHC molecule, and that leads to the mediation of a T cell response, including, but not limited to, limited to, proliferation, activation, differentiation, and the like. A primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif that is known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of an ITAM-containing cytoplasmic signaling sequence which is of particular use in the invention include, but are not limited to, those derived from CD3 zeta, FcR common gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon R1b) , CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In a specific CAR of the invention, the intracellular signaling domain in any one or more CARS of the invention comprises an intracellular signaling sequence, for example, a CD3-zeta primary signaling sequence. In a specific CAR of the invention, the CD3-zeta primary signaling sequence is the sequence provided as SEQ ID NO: 17, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, monkey (ape) and similar. In a specific CAR of the invention, the CD3-zeta primary signaling sequence is the sequence as provided in SEQ ID NO: 43, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, monkey (ape) and similar.

[00217] The term "antigen presenting cell" or "APC" refers to an immune system cell such as an accessory cell (e.g., a B cell, a dendritic cell, and the like) that presents a foreign antigen complexed with complexes of major histocompatibility (MHC's) on its surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[00218] An "intracellular signaling domain," as the term is used here, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the CAR-containing cell, e.g., a CART cell. Examples of immune effector function, for example in a CART cell, include cytolytic activity and auxiliary activity, including cytokine secretion.

[00219] In one embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Examples of primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen-dependent simulation. In one embodiment, the intracellular signaling domain may comprise a costimulatory intracellular domain. Examples of costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen-independent stimulation. For example, in the case of a CART, a primary intracellular signaling domain may comprise a cytoplasmic sequence of a T cell receptor, and a costimulatory intracellular signaling domain may comprise cytoplasmic sequence of a coreceptor or costimulatory molecule.

[00220] A primary intracellular signaling domain may comprise a signaling motif that is known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR common gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a , CD79b, DAP10, and DAP12.

[00221] The term "zeta" or alternatively "zeta chain", "CD3-zeta" or "TCR-zeta" is defined as a protein provided as GenBan Acc. No. BAG36664.1, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, and the like, and a "zeta stimulatory domain" or alternatively a "CD3-stimulatory domain" zeta" or a "TCR-zeta stimulatory domain" is defined as the amino acid residues of the cytoplasmic domain of the zeta chain, or functional derivatives thereof, that are sufficient to functionally transmit an initial signal necessary for T cell activation. , the cytoplasmic domain of zeta comprises residues 52 to 164 of GenBank Acc. No. BAG36664.1 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, which are functional orthologs thereof. In one aspect, the "zeta stimulatory domain" or a "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO:17. In one aspect, the "zeta stimulatory domain" or a "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO:43.

[00222] The term "costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds with 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, but are not limited to, an MHC class I molecule, BTLA, and a Toll ligand receptor, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) , and 4-1BB (CD137). Other examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, 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, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, 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, CD19a, and a ligand that specifically binds to CD83.

[00223] A costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. A costimulatory molecule can be represented in the following protein families: THF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like.

[00224] The intracellular signaling domain may comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment or derivative thereof.

[00225] The term "4-1BB" refers to a membrane of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2, or equivalent waste from a non-human species, e.g., mouse, rodent, monkey, ape, and the like; and a "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank accno. AAA62478.2, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, and the like. In one aspect, the "4-1BB costimulatory domain" is a sequence provided as SEQ ID NO:16 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, and the like. .

[00226] "Immune effector cells," when that term is used here, refers to a cell that is involved in an immune response, for example, in promoting an immune effector response. Examples of immune effector cells include T cells, for example, alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[00227] "Immune effector function or immune effector response," when that term is used here, refers to the function or response, for example, of an immune effector cell, which enhances or promotes an immune attack by a target cell. For example, an immune effector function or response refers to a property of a T or NK cell that promotes the death or inhibition of growth or proliferation of a target cell. In the case of a T cell, primary stimulation and costimulation are examples of immune effector function or response.

[00228] The term "encoding" refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for the synthesis of other polymers and macromolecules in biological processes having or a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties that result from it. Thus, a gene, cDNA, or RNA, encodes a protein if the transcription and translation of mRNA that corresponds to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually given in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[00229] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron.

[00230] The term "effective amount" or "therapeutically effective amount" is used interchangeably here, and refers to an amount of a compound, formulation, material, or composition, as described here effective to obtain a particular biological result.

[00231] The term "endogenous" refers to any material from or produced within an organism, cell, tissue or system.

[00232] The term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[00233] The term "expression" refers to the transcription and/or translation of a particular nucleotide sequence directed by a promoter.

[00234] The term "transfer vector" refers to a composition of substance that comprises an isolated nucleic acid and that can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to also include non-viral and non-plasmid compounds that facilitate the transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[00235] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[00236] The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are the only ones among retroviruses to be capable of infecting non-dividing cells; They can release a significant amount of genetic information into the host cell's DNA, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.

[00237] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors, which can be used in the clinic, include, but are not limited to, for example, the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Non-clinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[00238] The term "homolog" or "identity" refers to the subunit sequence identity between two polymeric molecules, for example, between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both two molecules is occupied by the same monomeric subunit; for example, if one position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of pairings or homologous positions; for example, if half (e.g., five positions in a ten-subunit-long polymer) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10) are paired or homologous, the two sequences are 90% homologous.

[00239] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other DNA-binding subsequences antibody antigen) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (receptor antibody or antibody fragment) in which residues of a complement determining region (CDR) of the receptor are replaced by residues of 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, human immunoglobulin Fv framework region (FR) residues are replaced with corresponding non-human residues. Furthermore, a humanized antibody/antibody fragment may comprise residues that are found neither in the recipient antibody nor in the imported framework or CDR sequences. These modifications can also refine and optimize antibody or antibody fragment performance. In general, the humanized antibody or antibody fragment thereof will comprise substantially all or at least one, and typically two, variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[00240] "Fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, where the entire molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.

[00241] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," however, the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in substantially purified form, or may exist in a non-native environment, such as, for example, a host cell.

[00242] In the context of the present invention, the following abbreviations for commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[00243] The term "operably linked" or "transcriptional control" refers to the functional link between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in functional linkage with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects transcription or expression of the coding sequence. Operably linked DNA sequences may be contiguous to each other and, for example, where necessary to join two protein-coding regions, are in the same reading frame.

[00244] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal, intratumoral injection, or infusion techniques.

[00245] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in single or double stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring 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 explicitly indicated sequence. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected codons (or all) is replaced by mixed base residues and/or deoxyinosine (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)).

[00246] The terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no simulation is placed on the maximum number of amino acids that can comprise a protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked together by peptide bonds. As used herein, the term refers both to short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which are commonly referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[00247] The term "promoter" refers to a DNA sequence recognized by the cell's synthetic mechanism, or introduced synthetic mechanism, required to initiate specific transcription of a polynucleotide sequence.

[00248] The term "promoter/regulatory sequence" refers to a nucleic acid sequence that is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some cases, this sequence may be the core promoter sequence and in other cases, this sequence may also include an enhancer sequence and other regulatory elements that are 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.

[00249] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked with a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in the cell under most or all physiological conditions of the cell.

[00250] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked with a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer that corresponds the promoter is present in the cell.

[00251] The term "tissue-specific" promoter refers to a nucleotide sequence that, when operably linked with a polynucleotide encoded 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.

[00252] The term "flexible polypeptide linker" or "linker" as used in the context of an scFv refers to a peptide linker consisting of amino acids, such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions to each other. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n, 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 (SEQ ID NO:105). In one embodiment, the flexible polypeptide linkers include, but are not limited to (Gly4 Ser)4 (SEQ ID NO:106) or (Gly4 Ser)3 (SEQ ID NO:107). In another embodiment, the linkers include multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO:108). Also included within the scope of the invention are binders described in WO 2012/138475, incorporated herein by reference).

[00253] As used herein, a 5' cap (also called an RNA cap, an RNA 7-methylguanosine cap, or an m7G RNA cap) is a modified guanine nucleotide that has been added to the "front" or 5' end of a eukaryotic messenger RNA immediately after the start of transcription. The 5' cap consists of a terminal group that is attached to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. The addition of buffering is coupled to transcription, and occurs cotranscriptionally, so that each influences the other. Immediately after the start of transcription, the 5' end of the mRNA being synthesized is ligated by an end-synthesizing complex associated with RNA polymerase. This enzyme complex catalyzes the chemical reactions that are required for mRNA buffering. The synthesis proceeds as a multistep biochemical reaction. The buffer portion can be modified to modulate the functionality of mRNA, such as its stability or translational efficiency.

[00254] As used herein, "in vitro transcribed RNA" refers to an RNA, preferably mRNA, that has been synthesized in vitro. Generally, in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector comprises a template that is used to generate in vitro transcribed RNA.

[00255] As used here, a "poly(A)" is a series of adenosines linked by polyadenylation to the mRNA. In the preferred embodiment of a construct for transient expression, the polyA is between 50 and 5000 (SEQ ID NO: 109), preferably greater than 64, more preferably greater than 100, more preferably greater than 300 or 400. Poly( A) can be chemically or enzymatically modified to modulate mRNA functionality, such as localization, stability, or translational efficiency.

[00256] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase. In larger eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the protein attached to it help protect mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally it can also occur later in the cytoplasm. After transcription has been completed, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is generally characterized by the presence of the base sequence AAUAAA close to the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3' end at the cleavage site.

[00257] As used herein, "transient" refers to the expression of a non-integrated transgene over a period of hours, days, or weeks, wherein the time period of expression is shorter than the time period for expression of the gene whether integrated into the genome or contained in a stable plasmid replicon in the host cell.

[00258] The term "signal transduction reaction series" refers to the biochemical link between a variety of signal transduction molecules that play a role in transmitting a signal from one portion of a cell to another portion of a cell. The phrase "cell surface receptor" includes molecules and complexes of molecules capable of receiving a signal and transmitting a signal across a cell's membrane.

[00259] The term "individual" is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human).

[00260] The term, a "substantially purified" cell refers to a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that has been separated from other cell types with which it is normally associated in its naturally occurring state. In some cases, a substantially purified cell population refers to a homogeneous population of cells. In other cases, this term simply refers to the cell that has been separated from the cells with which it is naturally associated in its natural states. In some aspects, the cells are grown in vitro. In other respects, the cells are not cultured in vitro.

[00261] The term "therapeutic", as used herein, means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.

[00262] The term "prophylaxis", as used herein, means the prevention of protective treatment for a disease or disease state.

[00263] In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to antigens that are common to specific hyperproliferative disorders. In certain aspects, the hyperproliferative disorder antigens of the present invention are derived from cancers including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemias. , uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas, such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.

[00264] The term "transfected" or "transformed" or "transduced" refers to a process by which exogenous nucleic acid is transferred or introduced into the 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 object cell and its offspring.

[00265] The term "specifically binds," refers to an antibody, or a ligand, that recognizes and binds with a binding partner protein (e.g., a stimulatory tumor antigen) present in a sample, but whose antibody or ligand substantially does not recognize or bind to other molecules in the sample.

[00266] "Regtable chimeric antigen receptor (RCAR)," when that term is used herein, refers to a group of polypeptides, typically two in the simplest embodiments, that when in an RCARX cell, provide the RCARX cell with specificity for a target cell, typically a cancer cell, and with tunable intracellular signal generation or proliferation, which can optimize an immune effector property of the RCARX cell. An RCARX cell relies, at least in part, on an antigen-binding domain to provide specificity to a target cell comprising the antigen bound by the antigen-binding domain. In one embodiment, an RCAR includes a dimerization switch that, in the presence of a dimerization molecule, can couple an intracellular signaling domain to the antigen-binding domain.

[00267] "Membrane anchor" or "membrane tethering domain", when that term is used herein, refers to a polypeptide or moiety, for example, a myristoyl group, sufficient to anchor an extracellular or intracellular domain to the plasma membrane .

[00268] "Switch domain," when that term is used herein, for example, when referring to an RCAR, refers to an entity, typically a polypeptide-based entity, that, in the presence of a dimerization molecule, associates with another switching domain. The association results in a functional coupling of a first entity linked to, e.g., fused to, a first switching domain, and a second entity linked to, e.g., fused to, a second switching domain. A first and second switching domain are collectively referred to as a dimming switch. In embodiments, the first and second switching domains are the same as each other, for example, they are polypeptides having the same primary amino acid sequence, and are collectively referred to as a homodimerization switch. In embodiments, the first and second switching domains are different from each other, for example, they are polypeptides having different primary amino acid sequences, and are collectively referred to as a heterodimerization switch. In embodiments, the switching is intracellular. In embodiments, the switching is extracellular. In embodiments, the switching domain is a polypeptide-based entity, e.g., FKBP or FRB-based, and the dimerization molecule is small molecule, e.g., a rapalog. In embodiments, the switching domain is a polypeptide-based entity, e.g., a scFv that binds a myc peptide, and the dimerization molecule is a polypeptide, a fragment thereof, or a multimer of a polypeptide, e.g. a myc ligand or multimers of a myc ligand that bind to one or more myc scFvs. In embodiments, the switching domain is a polypeptide-based entity, e.g., myc receptor, and the dimerization molecule is an antibody or fragments thereof, e.g., myc antibody.

[00269] "Dimerization molecule," when that term is used here, for example, when referring to an RCAR, refers to a molecule that promotes the association of a first switch domain with a second switch domain. In embodiments, the dimerization molecule does not occur naturally in the individual, or does not occur in concentrations that would result in significant dimerization. In embodiments, the dimerization molecule is a small molecule, e.g., rapamycin or a rapalog, e.g., RAD001.

[00270] The term "bioequivalent" refers to an amount of an agent other than the reference compound (e.g., RAD001), required to produce an effect equivalent to the effect produced by the reference dose or reference amount of the reference compound ( for example, RAD001). In one embodiment the effect is the level of mTOR inhibition, e.g., as measured by P70 S6 kinase inhibition, e.g., as assessed in an in vivo or in vitro assay, e.g., as measured by an assay described herein. for example, the Boulay assay, or measurement of phosphorylated S6 levels by western blot. In one embodiment, the effect is to alter the ratio of PD-1 positive/PD-1 negative T cells, as measured by cell sorting. In one embodiment a bioequivalent amount or dose of an mTOR inhibitor is the amount or dose that achieves the same level of P70 S6 kinase inhibition as does the reference dose or reference amount of a reference compound. In one embodiment, a bioequivalent amount or dose of an mTOR inhibitor is the amount or dose that achieves the same level of change in a PD-1 positive/PD-1 negative T cell ratio as does the reference dose or amount. reference compound.

[00271] The term "low dose, immune enhancing" when used in conjunction with an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 or rapamycin, or a catalytic mTOR inhibitor, refers to a dose of mTOR inhibitor that partially, but not completely, inhibits mTOR activity, for example, as measured by inhibition of P70 S6 kinase activity. Methods for assessing mTOR activity, for example by inhibition of P70 S6 kinase, are described here. The dose is insufficient to result in complete immune suppression, but it is sufficient to enhance the immune response. In one embodiment, the low dose, immune enhancing, mTOR inhibitor results in a decrease in the number of PD-1 positive T cells and/or an increase in the number of PD-1 negative T cells, or an increase in a ratio of PD-1 negative T cells/PD-1 positive T cells. In one embodiment, the low dose, immune enhancing, mTOR inhibitor results in an increase in the number of naïve T cells. In one embodiment, the low dose, immune enhancing, mTOR inhibitor results in one or more of the following:

[00272] an increase in the expression of one or more of the following markers: CD62Lhigh, CD127high, CD27+, and BCL2, for example, on memory T cells, for example, memory T cell precursors;

[00273] a decrease in the expression of KLRG1, for example, in memory T cells, for example, memory T cell precursors; It is

[00274] an increase in the number of memory T cell precursors, for example, cells with any one or a combination of the following characteristics: increased CD62L, increased CD127elevated, increased CD27+, decreased KLRG1, and increased BCL2;

[00275] wherein any of the changes described above occur, for example, at least transiently, for example, when compared to an untreated individual.

[00276] "Refractory", as used herein, refers to a disease, for example, cancer, that does not respond to treatment. In embodiments, a refractory cancer may be resistant to a treatment before or at the start of treatment. In other embodiments, refractory cancer may become refractory during treatment.

[00277] A "complete responder", as used herein, refers to an individual having a disease, e.g., cancer, who exhibits a complete response, e.g., a complete remission, to a treatment. A complete response can be identified, for example, using the Cheson criteria as described here.

[00278] A "partial responder", as used herein, refers to an individual having a disease, e.g., cancer, who exhibits a partial response, e.g., a partial remission, to a treatment. A partial response can be identified, for example, using the Cheson criteria.

[00279] A "non-responder", as used herein, refers to an individual having disease, e.g., a cancer, who does not exhibit a response to a treatment, e.g., the patient has stable disease or progressive disease. A non-responder can be identified, for example, using the Cheson criteria as described here.

[00280] The term "relapse", as used herein, refers to the reappearance of a disease (e.g., cancer) after an initial period of responsiveness (e.g., complete response or partial response). The initial period of responsiveness may involve the level of cancer cells falling below a certain threshold, for example, below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. . Reappearance may involve the level of cancer cells arising above a certain threshold, for example, above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Recidivism can be identified, for example, using the Cheson criteria as described here.

[00281] Stripes: Throughout this description, various aspects of the invention may be presented in a strip format. It should be understood that the strip format description is merely for convenience and brevity and should not be construed as an inflexible simulation of the scope of the invention. Consequently, the description of a range should be considered to have specifically described all possible sub-ranges as well as individual numerical values within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically described sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95 to 99% of identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes sub-ranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the range width.

Description

[00282] Provided herein are compositions of matter and methods of use for treating a disease such as cancer (e.g., hematological cancers or other B cell malignancies) using immune effector cells (e.g., T cells or NK cells) that express a chimeric antigen receptor (CAR) (e.g., a CAR that targets a B cell marker such as CD19). Methods include, among other things, administering immune effector cells (e.g., T cells or NK cells) expressing a B cell targeting CAR described herein in combination with another agent such as a kinase inhibitor, e.g., a kinase inhibitor described herein. here.

[00283] The present invention provides, at least in part, experiments supporting the high efficacy of a combination of a CAR therapy (e.g., a CAR B-cell targeting therapy) and a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib. Combining a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, with CAR therapy may increase the efficacy of the combination therapy relative to a kinase inhibitor monotherapy, or a dose of CAR-expressing cells, or both. These beneficial effects may, for example, allow for a lower dose of the kinase inhibitor or CAR-expressing cells, or both, while maintaining efficacy. The results are applicable to a wide variety of cancers, for example, hematological cancers and other B-cell malignancies. For example, ibrutinib inhibits BTK, which is elevated in most lymphomas. An immune effector cell (e.g., T cell or NK cell) that expresses CAR19 targeting cancers with surface expression of CD19, which is expressed on most B cell malignancies. Alternatively or in combination with CAR19, any other CAR targeting B cell (e.g., a CAR targeting one or more of: CD20, CD22, or ROR1) can be used in the combination therapies described here. Therefore, the combination of a CAR therapy (e.g., one or more of CD19 CAR, CD20 CAR, CD22 CAR, or ROR1 CAR therapy) with a BTK inhibitor (e.g., ibrutinib) is suitable for the treatment of a wide range of cancers involving the overproliferation of B cells, including lymphomas (e.g., Hodgkin's lymphoma), MCL, CLL, DLBCL, and multiple myeloma.

[00284] According to the present invention, ibrutinib can reduce tumor masses and mobilize neoplastic B cells in peripheral blood (see, for example, Example 8 here). Without wishing to be bound by theory, certain lymphomas, such as MCL, are characterized by masses of cancerous cells in centers of proliferation in lymph nodes. Immune effector cells expressing CAR sometimes have difficulty penetrating these densely packed masses. Thus, a BTK inhibitor, such as ibrutinib, can reduce tumor masses and mobilize neoplastic B cells in peripheral blood, making lymphoma cells more vulnerable to CAR-expressing cells.

[00285] Alternatively or in combination, BTK inhibitors, such as ibrutinib, can also affect CAR-expressing cells. The present invention demonstrates that ibrutinib treatment increases the level of circulating CART19 cells (see, for example, the data shown in Example 8). Without wishing to be bound by theory, the increase in the level of circulating CART19 cells could be a result of, for example, increased proliferation, altered T cell phenotype, or other factors. For example, ibrutinib can inhibit ITK, a kinase with homology to BTK. ITK is expressed on T cells, and its inhibition can alter the T cell phenotype. Treatment with a kinase inhibitor, such as ibrutinib, can alter the T cell phenotype from a Th2 phenotype to a Th1 phenotype, and thereby the T cell proliferative capacity. Pretreatment, or coadministration, to an individual, of a BTK inhibitor can increase the T cell proliferative capacity in the individual, thereby increasing the level of circulating CAR-expressing cells. Furthermore, an individual pretreated with a BTK inhibitor, e.g., ibrutinib, may have a T cell population with an elevated proliferative capacity in their apheresis for CAR manufacturing.

[00286] In one aspect, the invention provides a plurality of chimeric antigen receptors (CAR) comprising an antibody or antibody fragment designed for specific binding to a B cell antigen (e.g., chosen from one or more of CD19, CD20, CD22 or ROR1 protein). In one aspect, the invention provides a cell (e.g., T cell) engineered to express a CAR, wherein the CAR T cell ("CART") exhibits an anti-cancer property. In one aspect, a cell is transformed with the CAR and the CAR is expressed on a cell surface. In some embodiments, the cell (e.g., T cell) 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 can stably express the CAR. In another embodiment, the cell (e.g., T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR. In some such embodiments, the cell may transiently express the CAR.

[00287] In one aspect, the anti-CD19 protein binding portion of the CAR is an scFv antibody fragment. In one aspect, such antibody fragments are functional in that they maintain equivalent binding affinity, for example, they bind the same antigen with comparable affinity as the IgG antibody from which it is derived. In one aspect, such antibody fragments are functional in that they provide a biological response that may include, but is not limited to, activation of an immune response, inhibition of signal transduction origination of their target antigen, inhibition of kinase, and the like, as will be understood by the skilled artisan. In one aspect, the anti-CD19 antigen binding domain of the CAR is an scFv antibody fragment that is humanized in comparison to the murine sequence of the scFv from which it is derived. In one aspect, the parental murine scFv sequence is the CAR19 construct provided in PCT publication WO2012/079000 (incorporated herein by reference) and provided here as SEQ ID NO:59. In one embodiment, the anti-CD19 binding domain is a scFv described in WO2012/079000 and provided in SEQ ID NO:59.

[00288] In some aspects, the antibodies of the invention are incorporated into a chimeric antigen receptor (CAR). In one aspect, the CAR comprises the polypeptide sequence provided as SEQ ID NO: 12 in PCT publication WO2012/079000, and provided herein as SEQ ID NO: 58, wherein the scFv domain is replaced by one or more selected sequences of SEQ ID NOS: 1-12. In one aspect, the scFv domains of SEQ ID NOS:1-12 are humanized variants of the scFv domain of SEQ ID NO:59, which is a scFV fragment of murine origin that specifically binds a human CD19. Humanization of this mouse scFv may be desired for clinical setting, where mouse-specific residues may induce a human anti-mouse antigen (HAMA) response in patients receiving CART19 treatment, e.g., treatment with T cells transduced with the construct from CAR19.

[00289] In one aspect, the anti-CD19 binding domain, e.g., humanized scFv, portion of a CAR of the invention is encoded by a transgene whose sequence has been codon optimized for expression in a mammalian cell. In one aspect, the complete CAR construct of the invention is encoded by a transgene whose entire sequence has been codon optimized for expression in a mammalian cell. Codon optimization refers to the observation that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in DNA coding is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimization methods are known in the art, and include, for example, methods described in at least United States Patent Numbers 5,786,464 and 6,114,148.

[00290] In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:1. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:2. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:3. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:4. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:5. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:6. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:7. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:8. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:9. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:10. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:11. In one aspect, the humanized CAR19 comprises the scFv portion provided in SEQ ID NO:12.

[00291] In one aspect, the CARs of the invention combine an antigen-binding domain of a specific antibody with an intracellular signaling molecule. For example, in some aspects, the intracellular signaling molecule includes, but is not limited to, the CD3-zeta chain, 4-1BB signaling molecules and CD28 combinations thereof. In one aspect, the CD19 CAR comprises a CAR selected from the sequence provided in one or more of SEQ ID NOS: 31 - 42. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO: 31. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO:32. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO:33. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO:34. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO:35. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO:36. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO:37. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO:38. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO:39. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO:40. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO:41. In one aspect, the CD19 CAR comprises the sequence provided in SEQ ID NO:42.

[00292] Furthermore, the present invention provides CD19 CAR compositions and their use in medicines or methods for treating, among other diseases, cancer or any malignancy or autoimmune diseases involving cells or tissues that express CD19.

[00293] In one aspect, the CAR of the invention can be used to eradicate normal cells expressing CD19, thereby applicable for use as a cell conditioning therapy prior to cell transplantation. In one aspect, the normal cell expressing CD19 is a stem cell expressing CD19 and a cell transplant is a stem cell transplant.

[00294] In one aspect, the invention provides a cell (e.g., T cell) engineered to express a chimeric antigen receptor (CAR), wherein the CAR-expressing cell, e.g., CAR T cell ("CART") , exhibits an anti-cancer property. A preferred antigen is CD19. In one aspect, the antigen-binding domain of the CAR comprises a partially humanized anti-CD19 antibody fragment. In one aspect, the antigen-binding domain of the CAR comprises a partially humanized anti-CD19 antibody fragment comprising an scFv. Accordingly, the invention provides a CD19-CAR that comprises a humanized anti-CD19 binding domain and is created in an immune effector cell, for example, a T cell or an NK cell, and methods of using it for adoptive therapy.

[00295] In one aspect, the CD19 CAR comprises at least one intracellular domain selected from the group of a CD13 signaling domain (4-1BB), a CD28 signaling domain, a CD3 zeta signal domain, and any combination thereof. In one aspect, the CD19 CAR comprises at least one intracellular signaling domain and one or more costimulatory molecules other than CD137 (4-1BB) or CD28.

Chimeric Antigen Receptor (CAR)

[00296] The present invention encompasses a recombinant DNA construct comprising sequences encoding a CAR, wherein the CAR comprises an antibody or antibody fragment that specifically binds to a B cell antigen (e.g., CD19, e.g., human CD19 ), wherein the sequence of the antibody fragment is contiguous with and in the same frame in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. The intracellular signaling domain may comprise a co-stimulatory signaling domain and/or a primary signaling domain, for example, a zeta chain. The costimulatory signaling domain refers to a portion of the CAR comprising at least a portion of the intracellular domain of a costimulatory molecule. In one embodiment, the antigen-binding domain is a murine antibody or antibody fragment described herein. In one embodiment, the antigen-binding domain is a humanized antibody or antibody fragment.

[00297] In specific aspects, a CAR construct of the invention comprises a scFv domain selected from the group consisting of SEQ ID NOS:1-12 or a scFv domain of SEQ ID NO:59, in which the scFv may be preceded by an optional leader sequence as provided in SEQ ID NO: 13, and followed by an optional hinge sequence as provided in SEQ ID NO:14 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49 , a transmembrane region as provided in SEQ ID NO:15, an intracellular signaling domain including SEQ ID NO:16 or SEQ ID NO:51 and a CD3 zeta sequence including SEQ ID NO:17 or SEQ ID NO :43, wherein the domains are contiguous with and in the same reading frame to form a simple fusion protein. Also included in the invention is a nucleotide sequence encoding the polypeptide of each of the scFv fragments selected from the group consisting of SEQ is NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IS NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:59. Also included in the invention is a nucleotide sequence encoding the polypeptide of each of the scFv fragments selected from the group consisting of SEQ is NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IS NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:59, and each of the domains of SEQ ID NOS: 13-17, plus the encoded CD19 CAR fusion protein of the invention. In one aspect, exemplary CD19 CAR constructs comprise an optional leader sequence, an extracellular antigen-binding domain, a hinge, a transmembrane domain, and an intracellular stimulatory domain. In one aspect, an exemplary CD19 CAr construct comprises an optional leader sequence, an extracellular antigen-binding domain, a hinge, a transmembrane domain, an intracellular costimulatory domain, and an intracellular stimulatory domain. Specific CD19 CAR constructs containing humanized scFv domains of the invention are provided as SEQ ID NOS: 31-42, or a murine scFv domain is provided as SEQ ID NO: 59.

[00298] Full-length CAR sequences are also provided here as SEQ ID NOS: 31-42 and 58, as shown in Table 7 and Table 3.

[00299] An exemplary leader sequence is provided as SEQ ID NO: 13. An exemplary hinge/spacer sequence is provided as SEQ ID NO: 14 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49. An exemplary transmembrane domain sequence is provided as SEQ ID NO:15. An exemplary sequence of the intracellular signaling domain of the 4-1BB protein is provided as SEQ ID NO: 16. An exemplary sequence of the intracellular signaling domain of CD27 is provided as SEQ ID NO:51. An exemplary CD3zeta y domain sequence is provided as SEQ ID NO: 17 or SEQ ID NO:43.

[00300] In one aspect, the present invention encompasses a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises the nucleic acid sequence encoding an anti-CD19 binding domain, e.g. example, described herein, that is contiguous with and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. In one aspect, the anti-CD19 binding domain is selected from one or more of SEQ ID NOS:1-12 and 58. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of the sequence provided in one or more of SEQ ID NOS:61-72 and 59. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:61. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:62. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:63. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:64. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:65. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:66. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:67. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:68. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:69. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:70. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:71. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:72.

[00301] In one aspect, the present invention encompasses a recombinant nucleic acid construct comprising a transgene encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an anti-CD19 binding domain selected from one or more of SEQ ID NOS:61-72, wherein the sequence is contiguous with and in the same reading frame as the nucleic acid sequence encoding an intracellular signaling domain. An exemplary intracellular signaling domain that can be used in CAR includes, but is not limited to, one or more intracellular signaling domains of, for example, CD3-zeta, CD28, 4-1BB, and the like. In some cases, the CAR may comprise any combination of CD3-zeta, CD28, 4-1BB, and the like. In one aspect, the nucleic acid sequence of a CAR construct of the invention is selected from one or more of SEQ ID NOS:85-96. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:85. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:86. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:87. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:88. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:89. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:90. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:91. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:92. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:93. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:94. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:95. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:96. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:97. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:98. In one aspect, the nucleic acid sequence of a CAR construct is SEQ ID NO:99.

[00302] The nucleic acid sequence encoding the desired molecules can be obtained using recombinant methods known in the art, such as, for example, analyzing libraries of cells expressing the gene, deriving the gene from a vector known to include the same, or isolating directly from cells and tissues containing it, using standard techniques. Alternatively, the nucleic acid of interest can be synthetically produced, rather than cloned.

[00303] The present invention includes retroviral and lentiviral vector constructs expressing a CAR that can be directly transduced into a cell.

[00304] The present invention also includes an RNA construct that can be directly transferred into a cell. One method for generating mRNA for use in transfection involves in vitro transcription (IVT) of a template with specially designed primers, followed by addition of polyA, to produce a construct containing 3' and 5' untranslated sequence ("UTR"), a 5' cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length (SEQ ID NO:118). RNA thus produced can effectively transfect different species of cells. In one embodiment, the model includes sequences for the car CAR. In one embodiment, an RNA CAR vector is transduced into a T cell by electroporation.

Antigen Binding Domain

[00305] In one aspect, the CAR of the invention comprises a target-specific binding element otherwise referred to as an antigen-binding domain. The choice of moiety depends on the type and number of ligands defining the surface of a target cell. For example, the antigen-binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of surface markers that can act as ligands for the antigen-binding domain in a CAR of the invention include those associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells.

[00306] In one aspect, the CAR-mediated T cell response may be directed to an antigen of interest by creating an antigen-binding domain that specifically binds a desired antigen on the CAR.

[00307] In one aspect, the portion of the CAR comprising the antigen-binding domain comprises an antigen-binding domain that targets CD19. In one aspect, the antigen-binding domain targets human CD19. In one aspect, the antigen-binding domain of the CAR has the same or one binding specificity as the FMC63 scFV fragment described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one embodiment, the antigen-binding domain of the CAR includes the scFV fragment described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997).

[00308] The antigen-binding domain can be any domain that binds to antigen including, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a murine antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a single domain antibody such as a heavy chain variable domain (VH), a variable chain domain (VL) and a camelid-derived nanobody variable domain (VHH), and to a negative scaffold known in the art to function as an antigen-binding domain, such as a recombinant fibronectin domain, and the like.

[00309] In one embodiment, the CAR molecule comprises an anti-CD19 binding domain comprising one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of an anti-CD19 binding domain described herein, and one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of an anti-CD19 binding domain described herein, e.g. anti-CD19 comprising one or more, for example, all three, LC CDRs and one or more, for example, all three, HC CDRs. In one embodiment, the anti-CD19 binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of an anti-CD19 binding domain described herein, for example, the anti-CD19 binding domain has two variable heavy chain regions, each comprising an HC CDR1, a CDR2 of HC and an HC CDR3 described here. In one embodiment, the anti-CD19 binding domain comprises a light chain variable region described herein (e.g., in Table 7) and/or a murine heavy chain variable region described herein (e.g., in Table 7). In one embodiment, the anti-CD19 binding domain is a scFv comprising a murine light chain and a murine heavy chain of a Table 7 amino acid sequence. In one embodiment, the anti-CD19 binding domain (e.g., a scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g. substitutions), but not more than 30, 20 or 10 modifications (e.g. substitutions) of an amino acid sequence of a light chain variable region given in Table 7, or a sequence with 95 to 99% identity to an amino acid sequence of Table 7; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of one amino acid sequence of a heavy chain variable region given in Table 7, or a sequence with 95 to 99% identity to an amino acid sequence of Table 7. In one embodiment, the anti-CD19 binding domain comprises a sequence of SEQ ID NO:59, or a sequence with 95-99% identity thereof. In one embodiment, the anti-CD19 binding domain is a scFv, and a light chain variable region comprising an amino acid sequence described herein, for example, in Table 7, is linked to a heavy chain variable region comprising an amino acid sequence. amino acid described herein, for example in table 7, via a linker, for example a linker described herein. In one embodiment, the anti-CD19 binding domain includes a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 53) . The light chain variable region and heavy chain variable region of an scFv can be, for example, in any of the following orientations: light chain linker heavy chain variable region or light chain linker light chain variable region. heavy chain variable region.

[00310] In some cases, it is beneficial for the antigen-binding domain to be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, it may be beneficial for the antigen-binding domain of the CAR to comprise human or humanized residues as the antigen-binding domain of an antibody or antibody fragment.

[00311] Thus, in one aspect, the antigen-binding domain comprises a humanized antibody or a fragment antibody. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a murine or humanized anti-CD19 binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 ( HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a murine or humanized anti-CD19 binding domain described herein, e.g., a humanized anti-CD19 binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a murine or humanized anti-CD19 binding domain described herein, for example, the humanized anti-CD19 binding domain has two variable heavy chain regions, each comprising a HC CDR1, an HC CDR2 and an HC CDR3 described here. In one embodiment, the humanized anti-CD19 binding domain comprises a humanized light chain variable region described herein (e.g., in Table 3) and/or a humanized heavy chain variable region described herein (e.g., in Table 3). . In one embodiment, the humanized anti-CD19 binding domain comprises a humanized heavy chain variable region described herein (e.g., in Table 3), e.g., at least two humanized heavy chain variable regions described herein (e.g., in table 3). In one embodiment, the anti-CD19 binding domain is a scFv comprising a light chain and a heavy chain of a Table 3 amino acid sequence. In one embodiment, the anti-CD19 binding domain (e.g., a scFv) comprises : a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g. substitutions) but not more than 30, 20 or 10 modifications (e.g. substitutions) of an amino acid sequence of a light chain variable region given in Table 3, or a sequence with 95-99% identity to an amino acid sequence of Table 3; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but not more than 30, 20, or 10 modifications (e.g., substitutions) of one amino acid sequence of a heavy chain variable region provided in Table 3, or a sequence with 95-99% identity to an amino acid sequence of Table 3. In one embodiment, the humanized anti-CD19 binding domain comprises a selected sequence of a group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, or a sequence with 95-99% identity thereof. In one embodiment, the nucleic acid sequence encoding the humanized anti-CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:72, or a sequence with 95- 99% of its identity. In one embodiment, the humanized anti-CD19 binding domain is a scFv, and a light chain variable region comprising an amino acid sequence described herein, for example, in Table 3, is linked to a heavy chain variable region comprising a sequence of amino acid described herein, for example in table 3, by means of a linker, for example a linker described herein. In one embodiment, the humanized anti-CD19 binding domain includes a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO:53 ). The light chain variable region and heavy chain variable region of an scFv can be, for example, in any of the following orientations: light chain linker heavy chain variable region or light chain linker light chain variable region. heavy chain variable region.

[00312] In one aspect, the antigen-binding domain portion comprises one or more sequences selected from SEQ ID NOS:1-12. In one aspect, the humanized CAR is selected from one or more sequences selected from SEQ ID NOS: 31-42. In some aspects, a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to raise similarity to a naturally produced human antibody or fragment thereof.

[00313] A humanized antibody can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (see, for example, European Patent No. EP 239,400; International Publication No. WO 91/09967; and United States Patent Nos. 5,225,539, 5,530,101, and 5,585,089, each of which are incorporated herein in their entirety by reference), coating and resurfacing (see, for example, European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS , 91:969-973, each of which is incorporated herein in its entirety by reference), chain shuffling (see, e.g., United States Patent No. 5,565,332, which is incorporated herein in its entirety by reference) , and techniques described in, for example, United States Patent Application Publication No. US2005/0042664, United States Patent Application Publication No. US2005/0048617, United States Patent No. 6,407,213, U.S. Patent United No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678- 84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp) :5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al ., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein in its entirety by reference. Often, framework residues in the framework regions will be replaced with the corresponding residue from the CDR donor antibody to alter, e.g., improve antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues in particular positions. (See, for example, Queen et al., United States Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.)

[00314] A humanized antibody or antibody fragment has one or more amino acid residues that remain in it from a source that is non-human. These amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. As provided herein, humanized antibodies or antibody fragment comprise one or more CDRs of non-human immunoglobulin molecules and framework regions in which the amino acid residues comprising the framework are derived completely or mainly from the human germline. Multiple techniques for humanizing antibodies or antibody fragments are known in the art and can essentially be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), replacing rodent CDR sequences or CDRs with the corresponding CAR sequences from a human antibody, i.e., graft of CDR (EP 239,400; PCT Publication No. WO 91/09967; and United States Patent Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference herein in their entirety). In such humanized antibodies and antibody fragment, substantially less than one intact human variable domain has been replaced by the corresponding sequence from a non-human species. Humanized antibodies are often human antibodies in which some CDR residues and possibly some structural residues (FR) are replaced by residues from analogous sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by coating or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., Protein Engineering, 7 (6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (United States Patent No. 5,565,332), the contents of which are incorporated here by reference herein in its entirety.

[00315] The choice of human variable domains, both light and heavy, to be used in the preparation of humanized antibodies is to reduce antigenicity. According to the so-called "best fit" method, the variable domain sequence and the variable domain of a rodent antibody are analyzed in comparison with the library of known human variable domain sequences. The human sequence that is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chotia et al., J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference herein in their entirety). Another method uses a particular structure derived from the consensus sequence of all human antibodies from a particular subgroup of light and heavy chains. The same structure can be used for several different humanized antibodies (see, for example, Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference herein in their entirety). In some embodiments, the framework region, e.g., all four framework regions, of the heavy chain variable region are derived from the germline sequence of VH4_4-59. In one embodiment, the structural region may comprise one, two, three, four or five modifications, e.g., substitutions, for example, of the amino acid in the corresponding murine sequence (e.g., of SEQ ID NO:59). In one embodiment, the framework region, for example, all four framework regions of the light chain variable region are derived from a germline sequence of VK3_1.25. In one embodiment, the structural region may comprise one, two, three, four or five modifications, e.g., substitutions, for example, of the amino acid in the corresponding murine sequence (e.g., of SEQ ID NO:59).

[00316] In some aspects, the portion of a CAR composition of the invention that comprises an antibody fragment is humanized with retention of high affinity for the target antigen and other favorable biological properties. According to one aspect of the invention, humanized antibodies and antibody fragments are prepared by a process of analyzing the parenteral sequences and various conceptual humanized products using three-dimensional models. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these views allows analysis of more similar roles of residues in the functioning of the candidate immunoglobulin sequence, for example, analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody or antibody fragment characteristics, such as increased affinity for the target antigen, are obtained. In general, CDR residues are directly and most substantially involved in influencing antigen binding.

[00317] A humanized antibody or antibody fragment can maintain a similar antigenic specificity as the original antibody, for example, in the present invention, the ability to bind human CD19. In some embodiments, a humanized antibody or antibody fragment may have improved binding affinity and/or specificity to human CD19.

[00318] In one aspect, the anti-CD19 binding domain is characterized by particular functional characteristics and properties of an antibody or antibody fragment. For example, in one aspect, the portion of a CAR composition of the invention that comprises an antigen-binding domain specifically binds human CD19. In one aspect, the antigen-binding domain has the same or similar binding specificity to human CD19 as the FMC63 scFv described in Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one aspect, the invention relates to an antigen-binding domain comprising an antibody or antibody fragment, wherein the antibody-binding domain specifically binds to a CD19 protein or fragment thereof, wherein the antibody or The antibody fragment comprises a variable light chain and/or a variable heavy chain that includes an amino acid sequence of SEQ ID NO: 1-12 or SEQ ID NO: 59. In one aspect, the antigen-binding domain comprises an amino acid sequence of a scFv selected from SEQ ID NOs: 1-12 or SEQ ID NO:59. In certain respects, the scFv is contiguous with and in the same reading frame as a leader sequence. In one aspect, the leader sequence is the polypeptide sequence provided as SEQ ID NO:13.

[00319] In one aspect, the anti-CD19 binding domain is a fragment, for example, a single-chain variable fragment (scFv). In one aspect, the anti-CD19 binding domain is a Fv, Fab, (Fab')2, or bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17 , 105 (1987)). In one aspect, the antibodies and fragments thereof of the invention bind an affinity-enhanced or wild-type CD19 protein.

[00320] In some cases, scFvs can be prepared according to the method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. The length of the linker can greatly affect how the variable regions of an scFv fold and interact. In fact, if a short polypeptide linker is employed (e.g., between 5 to 10 amino acids) intrachain duplication will be prevented. Intrachain duplication is also required to drive the two variable regions together to form a functional epitope binding site. For examples of ligand orientation and size see, for example, Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, United States Patent Application Publications Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT Publications Nos. WO2006/020258 and WO2007/024715, are incorporated herein by reference.

[00321] An scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises glycine and serine amino acids. In another embodiment, the linker sequence comprises groups of glycine and serine repeats such as (Gly4Ser)n, where n is a positive integer equal to or greater than 1 (SEQ ID NO:18). In one embodiment, the linker may be (Gly4Ser)4 (SEQ ID NO:106) or (Gly4Ser)3 (SEQ ID NO:107). Variation in linker length can maintain or enhance activity, giving rise to superior efficacy in activity studies.

[00322] In some embodiments, the amino acid sequence of the antigen-binding domain (or other portions or the entire CAR) may be modified, for example, an amino acid sequence described herein may be modified, for example, by a conservative substitution . Families of amino acid residues having similar side chains have been defined in the art, including 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), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine).

[00323] Percent identity in the context of two or more polypeptide or nucleic acid sequences refers to two or more sequences that are the same. Two sequences are "substantially identical" if two sequences have a specific percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 70%, 71%. 72%. 73%, 74%, 75% , 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over a specified region, or, when unspecified, over the entire sequence), when compared and aligned for maximum correspondence during a comparison window, or region designated as measured using one of the sequence comparison algorithms or manual alignment or visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[00324] For sequence comparison, typically a sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are assigned, if necessary, and sequence algorithm program parameters are assigned. Predetermined program parameters can be used, or alternative parameters can be designed. The sequence comparison algorithm then calculates the percentage of sequence identities for the test sequences relative to the reference sequence, based on program parameters. Sequence alignment methods for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, through research using the similarity method Pearson and Lipman, (1988) Proc. Nat'l. Academic. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology).

[00325] Two examples of algorithms that are suitable for determining percentage sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAS analyzes is publicly available through the National Center for Biotechnology Information.

[00326] The percentage of identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4:11-17) which was incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a void extension penalty of 12, and an empty space penalty of 4. Additionally, the percentage of identity between two amino acid sequences can be determined using the algorithm Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453) which was incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossom 62 array or a PAM250 array, and a void weight of 16.14. 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[00327] In one aspect, the present invention contemplates modifications of the amino acid sequence of a starting antibody or fragment (e.g., scFv) that generate functionally equivalent molecules. For example, the VH or VL of an anti-CD19 binding domain, e.g., scFv, comprised in the CAR can be modified to maintain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the VH or VL structural region starting from the anti-CD19 binding domain, by example, scFv. The present invention contemplates modifications of complete CAR constructs, for example, modifications in one or more amino acid sequences of the various domains of the CAR construct in order to generate functionally equivalent molecules. The CAR construct can be modified to maintain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting CAR construction.

Bispecific CARs

[00328] In one embodiment, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, on the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In one embodiment, a bispecific antibody molecule comprises a heavy chain variable domain sequence and a chain variable domain sequence that have binding specificity for a first epitope and a heavy chain variable domain sequence and a chain variable that have binding specificity for a second epitope. In one embodiment, a bispecific antibody molecule comprises an antibody half having binding specificity for a first epitope and an antibody half having binding specificity for a second epitope. In one embodiment, a bispecific antibody molecule comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In one embodiment, a bispecific antibody molecule comprising an scFv, or fragment thereof, has binding specificity for a first epitope and a scFv, or fragment thereof, has binding specificity for a second epitope.

Transmembrane Domain

[00329] With respect to the transmembrane domain, in various embodiments, a CAR may be designated to comprise a transmembrane domain that is linked to the extracellular domain of the CAR. A transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10 to 15 amino acids from the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids from the intracellular region). In one aspect, the transmembrane domain is one that is associated with one of the other domains of the CAR, for example, in one embodiment, the transmembrane domain may be from the same protein as the signaling domain, co-stimulatory domain, or the hinge domain. It is derived from. In another aspect, the transmembrane domain is not derived from the same protein that any other CAR domain is derived from. In some cases, the transmembrane domain may be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, for example, to minimize interactions with other members of the membrane complex. receiver. In one aspect, the transmembrane domain is capable of homodimerization with another CAR on a cell surface of a CAR-expressing cell. In a different aspect, the amino acid sequence of the transmembrane domain can be modified or replaced in order to minimize interactions with the binding domains of the native binding pattern present in the same CAR-expressing cell.

[00330] The transmembrane domain can be derived from a natural or recombinant source. Where the source is natural, the domain may be derived from any transmembrane or membrane-bound protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain(s) if the CAR binds to a target. A transmembrane domain of particular use in this invention may include at least the transmembrane region(s), for example, 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. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB ( CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, 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, NKG2D, NKG2C.

[00331] In some cases, the transmembrane domain can be linked to the extracellular region of the CAR, for example, the antigen-binding domain of the CAR, through a hinge, for example, a hinge of a human protein. For example, in one embodiment, the hinge may be a human Ig (immunoglobulin) hinge, e.g., an IgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a CD8a hinge. In one embodiment, the hinge or spacer comprises (e.g., consisting of) the amino acid sequence of SEQ ID NO:14. In one aspect, the transmembrane domain comprises (e.g., consisting of) a transmembrane domain of SEQ ID NO: 15.

[00332] In one aspect, the hinge or spacer comprises an IgG4 hinge. For example, in one embodiment, the hinge or spacer comprises a hinge of the amino acid sequence ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ ID NO:45). In some embodiments, the hinge or spacer comprises a hinge encoded by a nucleotide sequence of GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGA GTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCA AGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTG GTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTG GTACGTGGACGGCGTGGAGG TGCACAACGCCAAGACCAAGCCCC GGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTG ACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTG TAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCAT CAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACC CTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCT GACCTG CCTGGTGAAGGGCTTCTACCCCAGGCGACATCGCCGTGG AGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACC CCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCG GCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTA GCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAG AAGAGCCTGAGCCTGTCCCTGGGCAAGATG (SEQ ID NO:46).

[00333] In one aspect, the hinge or spacer comprises an IgD hinge. For example, in one embodiment, the hinge or spacer comprises a hinge of the amino acid sequence RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKK EKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCF VVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLP RSLWNAGTSVTCTLNHPSLPPQRLMA LREPAAQAPVKLSLNLLASSD PPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPG STTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSY VTDH (SEQ ID NO:47). In some embodiments, the hinge or spacer comprises a hinge encoded by a nucleotide sequence of AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGGTGTTCCTACT GCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGC ACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGA AAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAG ACCCCTGAATGTCCATCCCATACCCAGCC GCTGGGCGTCTATCTC TTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACC TTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTG ACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGA AGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACT CAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCT GTCACATGTACT CTAAATCATCCTAGCCTGCCCCCACAGCGTCTG ATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAG CCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCT GGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGC TCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTC GCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACC ACATTCTG GGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGC CAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCC TGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACC ATT (SEQ ID NO:48).

[00334] In one aspect, the transmembrane domain can be recombinant, where it will comprise predominantly hydrophobic residues such as leucine and valine. In one aspect, a triplet of phenylalanine, tryptophan and valine, can be found at each end of a recombinant transmembrane domain.

[00335] Optionally, a short oligo- or polypeptide linker, between 2 and 10 amino acids in length, can form the link between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine doublet provides a particularly suitable linker. For example, in one aspect, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO:49). In some embodiments, the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO:50).

[00336] In one aspect, the joint or spacer comprises a KIR2DS2 joint.

cytoplasmic domain

[00337] The cytoplasmic domain or region of the CAR includes an intracellular signaling domain. An intracellular signaling domain is generally responsible for activating at least one of the normal effector functions of the immune cell into which the CAR has been introduced. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell, for example, may be cytolytic activity or helper activity or activity including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function. While the intracellular signaling domain can generally 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 as long as it transduces the effector function signal. The term intracellular signaling domain is therefore intended to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[00338] Examples of intracellular signaling domains for use in the CAR of the invention include the cytoplasmic sequences of a T cell receptor (TCR) receptor and coreceptors that act in concert to initiate signal transduction following antigen receptor engagement. , as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capacity.

[00339] It is known that the signals generated through the TCR alone are not sufficient for complete activation of the T cell and that the secondary and/or costimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an independent manner. of antigen to provide a secondary or costimulatory signal (secondary cytoplasmic domain, e.g., a costimulatory domain).

[00340] A primary signaling domain regulates the primary activation of the TCR complex in a stimulatory manner, or in an inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs that are well known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[00341] Examples of ITAM containing primary intracellular signaling domains that are of particular use in the invention include those of CD3 zeta, FcR common gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In one embodiment, a CAR of the invention comprises an intracellular signaling domain, for example, a CD3-zeta primary signaling domain.

[00342] In one embodiment, a primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain that has altered activity (e.g., increased or decreased) compared to the native ITAM domain. In one embodiment, a primary signaling domain comprises a primary intracellular signaling domain containing modified ITAM, e.g., a primary intracellular signaling domain containing optimized and/or truncated ITAM. In one embodiment, a primary signaling domain comprises one, two, three, four or more ITAM motifs.

[00343] Other examples of molecules containing a primary intracellular signaling domain that are of particular use in the invention include those of DAP10, DAP12, and CD32.

[00344] The CAR intracellular signaling domain may comprise the CD3-zeta signaling domain by itself or it may be combined with any other desired intracellular signaling domain(s) useful in the context of a CAR of the invention. For example, the intracellular signaling domain of the CAR may comprise a CD3 zeta chain portion and a co-stimulatory signaling domain. The costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 , and a ligand that specifically binds with CD83, and the like. For example, costimulation of CD27 has been shown to enhance the expansion, effector function, and survival of human CAR T cells in vitro and to increase human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012; 119(3 ):696-706). Other examples of costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, 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, CD11b, ITGAX, CD11c, ITGB1, CD29 , ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, 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, and CD19a.

[00345] The intracellular signaling sequences within the cytoplasmic portion of the CAR of the invention can be linked together in a random or specific order. Optionally, a short oligo or polypeptide linker, e.g., between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length can form the link between the intracellular signaling sequence. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a simple amino acid, for example, an alanine, a glycine, can be used as a suitable ligand.

[00346] In one aspect, the intracellular signaling domain is designed to comprise two or more, for example, 2, 3, 4, 5, or more co-stimulatory signaling domains. In one embodiment, the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains, are separated by a linker molecule, e.g., a linker molecule described herein. In one embodiment, the intracellular signaling domain comprises two co-stimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[00347] In one aspect, the intracellular signaling domain is designed to comprise the CD3-zeta signaling domain and the CD28 signaling domain. In one aspect, the intracellular signaling domain is designed to comprise the CD3-zeta signaling domain and the 4-1BB signaling domain. In one aspect, the 4-1BB signaling domain is a signaling domain of SEQ ID NO: 16. In one aspect, the CD3-zeta signaling domain is a signaling domain of SEQ ID NO: 17.

[00348] In one aspect, the intracellular signaling domain is designed to comprise the CD3-zeta signaling domain and the CD27 signaling domain. In one aspect, the CD27 signaling domain comprises an amino acid sequence of QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACS P (SEQ ID NO:51). In one aspect, the CD27 signaling domain is encoded by a nucleic acid sequence of AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACAT GACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCT ATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO:52).

[00349] In one aspect, the CAR-expressing cell described here may also comprise a second CAR, e.g., a second CAR that includes a different antigen-binding domain, e.g., to the same target (CD19) or a different target ( e.g. CD123 or mesothelin). In one embodiment, when the CAR-expressing cell comprises two or more different CARs, the antigen-binding domain of the different CARs may be such that the antigen-binding domains do not interact with each other. For example, a cell expressing a first and second CAR may have an antigen-binding domain of the first CAR, e.g., as a fragment, e.g., a scFv, that does not form an association with the antigen-binding domain of the second. CAR, for example, the antigen-binding domain of the second CAR is VHH.

[00350] In another aspect, the CAR-expressing cell described here may also express another agent, for example, an agent that enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Inhibitory molecules, e.g., PD1, can, in some embodiments, decrease the ability of a CAR-expressing cell to mount an immune effector response. 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. In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, for example, from an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide that is a intracellular signaling domain described herein (e.g., comprising a co-stimulatory domain (e.g., 41BB, CD27, or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described here). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of a PD1 extracellular domain), and a second polypeptide of an intracellular signaling domain described herein (e.g. example, a CD28 signaling domain described here and/or a CD3 zeta signaling domain described here). PD1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2 have been shown to down-regulate T cell activation upon binding to PD1 (Freeman et a. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2: 261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094) . Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.

[00351] In one embodiment, the agent comprises the extracellular domain (ECD) of an inhibitory molecule, for example, Programmed Death 1 (PD1), can be fused to a transmembrane domain and intracellular signaling domains such as 41BB and CD3 zeta (also referred to here as a PD1 CAR). In one embodiment, the PD1 CAR, when used in combinations with a CD19 CAR described herein, improves T cell persistence. In one embodiment, the CAR is a PD1 CAR comprising the extracellular domain of PD1 indicated as underlined in SEQ ID NO: 121. In one embodiment, the PD1 CAR comprises the amino acid sequence of SEQ ID NO: 121. Malpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrrnspsnqtdk laafpedrsqpgqdcrf'rvtqlpngrdfhmsvvrarmdsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtl ytttpaprpptpaptiasq please glstatkdtydalhmqalppr (SEQ ID NO: 121).

[00352] In one embodiment, the PD1 CAR comprises the amino acid sequence provided below (SEQ ID NO:119). pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvt qlpngrdfhmsvvrarmdsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqpl slrpeacrpaa ggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcel rvkfsrsadapaykqgqnqlynclnlgrrccydvldkrrgrdpcmggkprrknpqcglynclqkdkmacayscigmkgcrrrgk ghdglyqglstatkdty dalhmqalppr (SEQ ID NO:119).

[00353] In one embodiment, the agent comprises a nucleic acid sequence encoding the PD1 CAR, for example, the PD1 CAR described here. In one embodiment, the nucleic acid sequence for the PD1 CAR is shown below, with the PD1 ECD underlined below in SEQ ID NO: 120 atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagaccacccggatggtttctggac tctccggatcgcccgtggaatcccccaaccitctcaccggcacicttggtigtgactgae ggceataatgcgaccttcacgtgctcgttctc caacacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggt cgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgc gctaggcgaaa cgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgac cgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtcacgaccactccg gcgccgcgcccacc gactccggccccaactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccctgccgccggagg tgctgtgcatacccggggattggacttcgcatgcgacatctacatttgggctcctctcgccggaacttgtggcgtgctccttctgtccctggt catcaccctgtactgcaagcggggtcggaaaagcttct gtacattttcaagcagcccttcatgaggcccgtgcaaaccacccaggagga ggacggttgctcctgccggpccccgaagaggaagaaggaggttgcgagctgcgcgtgaagttctcccggagcgccgacgcccccgc ctataagcagggccagaaccagctgtacaacgaactgaacctgggacggcgggaagagtacgatg tgctggacaagcggcgcggcc gggaccccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgagctgcagaaggacaagatggccga ggcctactccgaaattgggatgaagggagagcggcggaggggaaaggggcacgacggcctgtaccaaggactgtccaccgccacc aaggacacatacgatgccctgcacat gcaggcccttccccctcgc (SEQ ID NO: 120).

[00354] In another aspect, the present invention provides cell populations expressing CAR, for example, CART cells. In some embodiments, the CAR-expressing cell populations comprise a mixture of cells expressing different CARs. For example, in one embodiment, the cell populations expressing CAR may include a first cell expressing a CAR having an anti-CD19 binding domain described herein, and a second cell expressing a CAR having a different CD19 binding domain, e.g. , an anti-CD19 binding domain described here, which differs from the anti-CD19 binding domain in the CAR expressed by the first cell. As another example, CAR-expressing cell populations may include a first cell expressing a CAR that includes an anti-CD19 binding domain, for example, as described herein, and a second cell expressing a CAR that includes an antigen-binding domain. to a target other than CD19 (e.g., CD123). In one embodiment, the CAR-expressing cell populations include, for example, a first cell expressing a CAR that includes a primary intracellular signaling domain, and a second cell expressing a CAR that includes a secondary signaling domain.

[00355] In another aspect, the present invention provides cell populations in which at least one cell in the population expresses a CAR having an anti-CD19 binding domain described herein, and a second cell expressing another agent, e.g. which enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Inhibitory molecules, for example, can, in some embodiments, decrease the ability of a CAR-expressing cell to mount an immune effector response. 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, or TGFR beta. In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, for example, from an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide that is a intracellular signaling domain described herein (e.g., comprising a co-stimulatory domain (e.g., 41BB, CD27, or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described here).In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g. , a CD28 signaling domain described here and/or a CD3 zeta signaling domain described here).

Regulable chimeric antigen receptors

[00356] In some embodiments, a adjustable CAR (RCAR) where CAR activity can be controlled is desirable to optimize the safety and efficacy of a CAR therapy. There are many ways in which CAR activities can be regulated. For example, inducible apoptosis using, for example, a caspase fused to a dimerization domain (see, e.g., Di Stasa et al., N Engl. J. Med. 2011 Nov. 3; 365(18):1673-1683 ), can be used as a safety key in the CAR therapy of the present invention. In one embodiment, cells (e.g., T cells or NK cells) expressing a CAR of the present invention also comprise an inducible apoptosis switch, wherein a human caspase (e.g., caspase 9) or a modified version is fused to a modification of the human FKB protein that allows conditional dimerization. In the presence of a small molecule, such as rapalog (e.g., AP 1903, AP20187), the inducible caspase (e.g., caspase 9) is activated and induces rapid apoptosis and death of cells (e.g., T cells or NK) expressing a CAR of the present invention. Examples of a caspase-based inducible apoptosis switch (or one or more aspects of such a switch) have been described in, for example, US2004040047; US20110286980; US20140255360; WO1997031899; WO2014151960; WO2014164348; WO2014197638; WO2014197638; all of which are incorporated by reference herein.

[00357] In one aspect, an RCAR comprises a group of polypeptides, typically two in the simplest embodiments, wherein the components of a standard CAR described herein, e.g., an antigen-binding domain and an intracellular signaling domain, are divided into separate polypeptides or members. In some embodiments, the polypeptide groups include a dimerization switch that, in the presence of a dimerization molecule, can couple the polypeptides to each other, for example, can couple an antigen-binding domain to an intracellular signaling domain. In one embodiment, the CARs of the present invention utilize a dimming switch such as those described, for example, in WO2014127261, which is incorporated by reference herein.

[00358] In one aspect, an RCAR comprises two polypeptides or members: 1) an intracellular signaling member comprising an intracellular signaling domain, e.g., a primary intracellular signaling domain described herein, and a first switch domain; 2) an antigen-binding member comprising an antigen-binding domain, for example, that targets CD19, as described herein and a second switch domain. Optionally, the RCAR comprises a transmembrane domain described herein. In one embodiment, a transmembrane domain may be disposed in the intracellular signaling member, the antigen-binding member, or both. (Unless otherwise noted, when members or elements of an RCAR are described here, the order may be as given, but other orders are also included. In other words, in one embodiment, the order is as given in the text, but In other embodiments, the order may be different. For example, the order of elements on one side of a transmembrane region may be different from the example, for example, the placement of a switch domain with respect to an intracellular signaling domain may be different, e.g. reversed).

[00359] In one embodiment, the first and second switch domains can form an intracellular or extracellular dimerization switch. In one embodiment, the dimming switch may be a homodimerization switch, for example, where the first and second switching domains are the same, or a heterodimerization switch, for example, where the first and second switching domains are different from each other.

[00360] In embodiments, an RCAR may comprise a "multiswitch." A multiswitch may comprise heterodimerization switch domains or homodimerization switch domains. A multiswitch comprises a plurality of, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10, switch domains, independently, in a first member, for example, an antigen-binding member, and a second member, for example, an intracellular signaling member. In one embodiment, the first member may comprise a plurality of first switch domains, e.g., FKBP-based switch domains, and the second member may comprise a plurality of second switch domains, e.g., FRB-based switch domains. In one embodiment, the first member may comprise a first and a second switch domain, e.g., FKBP-based switch domain and an FRB-based switch domain, and the second member may comprise a first and a second switch domain, e.g. For example, an FKBP-based switching domain and an FRB-based switching domain.

[00361] In one embodiment, the intracellular signaling member comprises one or more intracellular signaling domains, for example, a primary intracellular signaling domain and one or more co-stimulatory signaling domains.

[00362] In one embodiment, the antigen-binding member may comprise one or more intracellular signaling domains, for example, one or more co-stimulatory signaling domains. In one embodiment, the antigen-binding member comprises a plurality, e.g., 2 or 3 costimulatory signaling domains described herein, e.g., selected from 41BB, CD28, CD27, ICOS, and OX40, and in embodiments, the primary intracellular signaling. In one embodiment, the antigen-binding member comprises the following co-stimulatory signaling domains, from the extracellular to intracellular direction: 41BB-CD27; 41BB-CD27; CD27-41BB; 41BB-CD28; CD28-41BB; OX40-CD28; CD28-OX40; CD28-41BB; or 41BB-CD28. In such embodiments, the intracellular binding member comprises a CD3 zeta domain. In such an embodiment the RCAR comprises (1) an antigen binding member comprising an antigen binding domain, a transmembrane domain, and two co-stimulatory domains and a first switch domain; and (2) an intracellular signaling domain comprising a transmembrane domain or a membrane-binding domain and at least one primary intracellular signaling domain, and a second switch domain.

[00363] One embodiment provides RCARs in which the antigen-binding member is not bound to the CAR cell surface. This allows a cell having an intracellular signaling member to be conveniently paired with one or more antigen-binding domains, without transforming the cell with a sequence encoding the antigen-binding member. In such embodiments, the RCAR comprises: 1) an intracellular signaling member comprising: a first switch domain, a transmembrane domain, an intracellular signaling domain, e.g., a primary intracellular signaling domain, and a first switch domain; and 2) an antigen-binding member comprising: an antigen-binding domain, and a second switch domain, wherein the antigen-binding member does not comprise a transmembrane domain or membrane-binding domain, and, optionally, does not comprises an intracellular signaling domain. In some embodiments, the RCAR may also comprise 3) a second antigen-binding member comprising: a second antigen-binding domain, e.g., a second antigen-binding domain that binds a different antigen than that bound by the antigen-binding domain. antigen binding; and a second switching domain.

[00364] Also provided here are RCARs in which the antigen-binding member comprises bispecific activation and targeting capacity. In this embodiment, the antigen-binding member may comprise a plurality, e.g., 2, 3, 4, or 5 antigen-binding domains, e.g., scFvs, wherein each antigen-binding domain binds to an antigen target, for example, different antigens or the same antigen, for example, the same or different epitopes on the same antigen. In one embodiment, the plurality of antigen-binding domains are in tandem, and optionally, a linker or hinge region is disposed between each of the antigen-binding domains. Suitable linkers and hinge regions are described here.

[00365] One embodiment provides RCARs having a configuration that allows proliferation switching. In this embodiment, the RCAR comprises: 1) an intracellular signaling member comprising: optionally, a transmembrane domain or membrane binding domain; one or more costimulatory signaling domains, for example, selected from 41BB, CD28, CD27, ICOS, and OX40, and a switch domain; and 2) an antigen-binding member comprising: an antigen-binding domain, a transmembrane domain, and a primary intracellular signaling domain, e.g., CD3 zeta domain, wherein the antigen-binding member does not comprise a switch domain, or does not comprise a switch domain that dimerizes with a switch domain in the intracellular signaling member. In one embodiment, the antigen-binding member does not comprise a costimulatory signaling domain. In one embodiment, the intracellular signaling member comprises a switch domain of a homodimerization switch. In one embodiment, the intracellular signaling member comprises a first switch domain of a heterodimerization switch and the RCAR comprises a second intracellular signaling member that comprises a second switch domain of the heterodimerization switch. In such embodiments, the second intracellular signaling member comprises the same intracellular signaling domains as the intracellular signaling member. In one embodiment, the dimming switch is intracellular. In one embodiment, the dimerization switch is extracellular.

[00366] In any of the RCAR configurations described here, the first and second switch domains comprise an FKBP-FRB-based switch as described here.

[00367] Also provided here are cells comprising an RCAR described here. Any cell that is created to express an RCAR can be used as an RCARX cell. In one embodiment the RCARX cell is a T cell, and is referred to as an RCART cell. In one embodiment, the RCARX cell is an NK cell, and is referred to as an RCARN cell.

[00368] Nucleic acids and vectors comprising sequences encoding RCAR are also provided herein. Sequence encoding several elements of an RCAR can be arranged on the same nucleic acid molecule, e.g. the same plasmid or vector, e.g. viral vector, e.g. lentiviral vector. In one embodiment, (i) sequence encoding an antigen-binding member and (ii) sequence encoding an intracellular signaling member, may be present in the same nucleic acid, e.g., vector. The production of proteins can be obtained, for example, by the use of separate promoters, or by the use of a bicistronic transcription product (which can result in the production of two proteins by cleavage of a single translation production or by the translation of two separate products ). In one embodiment, a sequence encoding a cleavable peptide, for example, a P2A or F2A sequence, is disposed between (i) and (ii). In one embodiment, a sequence encoding an IRES, for example, an EMCV or EV71 IRES, is disposed between (i) and (ii). In these embodiments, (i) and (ii) are transcribed as a simple RNA. In one embodiment, a first promoter is operably linked to (i) and a second promoter is operably linked to (ii), such that (i) and (ii) are transcribed as separate mRNAs.

[00369] Alternatively, the sequence encoding various elements of an RCAR can be arranged on different nucleic acid molecules, for example, different plasmids or vectors, for example, viral vector, for example, lentiviral vector. For example, the sequence (i) encoding an antigen-binding member may be present in a first nucleic acid, e.g., a first vector, and (ii) the sequence encoding an intracellular signaling member may be present in the second nucleic acid. , for example, the second vector.

Dimming Switches

[00370] Dimming switches can be non-covalent or covalent. In a noncovalent dimerization switch, the dimerization molecule promotes a noncovalent interaction between the switch domains. In a covalent dimerization switch, the dimerization molecule promotes a covalent interaction between the switch domains.

[00371] In one embodiment, the RCAR comprises a FKBP/FRAP, or FKBP/FRB-based dimming switch. FKBP12 (FKBP-binding protein, or FK506) is an abundant cytoplasmic protein that functions as an initial intracellular target for the natural product immunosuppressive drug, rapamycin. Rapamycin binds to FKBP and the large PI3K homologue FRAP (RAFT, mTOR). FRB is a 93 amino acid portion of FRAP, which is sufficient to bind the complete FKBP-rapamycin (Chen, J., Zheng, X. F., Brown, E. J. & Schreiber, S. L. (1995) Identification of an 11-kDa FKBP12-rapamycin - binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc Natl Acad Sci U S A 92: 4947-51.)

[00372] In embodiments, a FKBP/FRAP, e.g., an FKBP/FRB-based switch may use a dimerization molecule, e.g., rapamycin or a rapamycin analog.

[00373] The amino acid sequence of FKBP is as follows: Y A Y G A T G H P G I I P P H A T L V F D V E L L K L E T S Y (SEQ ID NO: 122)

[00374] In embodiments, an FKBP switch domain may comprise a fragment of FKBP having the ability to bind with FRB, or a fragment or analogue thereof, in the presence of rapamycin or a rapalog, e.g., the underlined portion of SEQ ID NO: 122, which is: SEQ ID NO: 123)

[00375] The amino acid sequence of FRB is as follows: ILWHEMWHEG LEEASRLYFG ERNVKGMFEV LEPLHAMMER GPQTLKETSF NQAYGRDLME AQEWCRKYMK SGNVKDLTQA WDLYYHVFRR ISK (SEQ ID NO: 124)

[00376] "FKBP/FRAP, for example, a switch based on FKBP/FRB" when such term is used herein, refers to a dimerization switch comprising: a first switch domain, which comprises a fragment of FKBP or analogue of even though it has the ability to bind with FRB, or a fragment or analogue thereof, in the presence of rapamycin or a rapalog, e.g., RAD001, and has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by, no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from the FKBP sequence of SEQ ID NO: 122 or 123; and a second switch domain, which comprises a fragment of FRB or an analogue thereof having the ability to bind with FRB, or a fragment or analogue thereof, in the presence of rapamycin or a rapalogue thereof, and has at least 70, 75 , 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by, no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 residues of the FRB sequence of SEQ ID NO: 124. In one embodiment, an RCAR described herein comprises a switch domain comprising amino acid residues described in SEQ ID NO: 122 (or SEQ ID NO: 123), and a switch domain comprising amino acid residues described in SEQ ID NO: 124.

[00377] In embodiments, the FKBP/FRB dimerization switch comprises a modified FRB switch domain that exhibits altered complex formation, e.g., enhanced between an FRB-based switch domain, e.g., the modified FRB switch domain, a switch domain based on FKBP, and the dimerization molecule, e.g., rapamycin or a rapalog, e.g., RAD001. In one embodiment, the modified FRB switch domain comprises one or more mutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, selected from mutations at amino acid position(s) L2031 , E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105, and F2108, where the wild-type amino acid is mutated by any other naturally occurring amino acid. In one embodiment, a mutant FRB comprises a mutation in E2032, where E2032 is mutated by phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I), e.g. , SEQ ID NO: 125, or leucine (E2032L), for example, SEQ ID NO: 126. In one embodiment, a mutant FRB comprises a mutation in T2098, where T2098 is mutated by phenylalanine (T2098F) or leucine (T2098L), e.g., SEQ ID NO: 127. In one embodiment, a mutant FRB comprises a mutation in E2032 and in T2098, where E2032 is mutated by any amino acid, and where T2098 is mutated by any amino acid, e.g., SEQ ID NO: 128 In one embodiment, a mutant FRB comprises a mutation of E2032I and T2098L, e.g., SEQ ID NO: 129. In one embodiment, a mutant FRB comprises a mutation of E2032L and T2098L, e.g., SEQ ID NO: 130.

[00378] Table 13. Exemplary mutant FRB having increased affinity for a dimerization molecule.

[00379] Other dimming switch based on GyrB-GyrB, a dimming switch based on Gibberellin, a label/binder dimming switch, and a halo-label/snap-label dimming switch. Following the guidance provided herein, such relevant switches and dimerization molecules will be evident to one skilled in the art.

Dimerization Molecule

[00380] The association between the switch domains is promoted by the dimerization molecule. In the presence of dimerization molecule interaction or association between switch domains allows signal transduction between a polypeptide associated with, for example, fused to, a first switch domain, and a polypeptide associated with, for example, fused to, a second switching domain. In the presence of non-limiting levels of dimerization molecule, signal transduction is increased by 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 5, 10, 50, 100 times, for example, as measured in a system described here.

[00381] Rapamycin and rapamycin analogs (sometimes referred to as rapalogs), for example, RAD001, can be used as dimerization molecules in an FKBP/FRB-based dimerization switch described here. In one embodiment, the dimerization molecule can be selected from rapamycin (sirolimus), RAD001 (everolimus), zotarolimus, temsirolimus, AP-23573 (ridaforolimus), biolimus and AP21967. Additional rapamycin analogs suitable for use with FKBP/FRB-based dimerization switches are also described in the section titled "Combination Therapies", or in the subsection titled "Exemplary mTOR Inhibitors".

Spin-off CAR

[00382] In some embodiments, the CAR expressing cell uses a fission CAR. The CAR scission method is described in greater detail in publications WO2014/055442 and WO2014/055657. Briefly, a fission CAR system comprises a cell expressing a first CAR having a first antigen-binding domain and a co-stimulatory domain (e.g., 41BB), and the cell also expressing a second CAR having a second antigen-binding domain. and an intracellular signaling domain (e.g., CD3 zeta). When the cell encounters the first antigen, the co-stimulatory domain is activated, and the cell proliferates. When the cell encounters the second antigen, the intracellular signaling domain is activated and cell killing activity begins. Thus, the CAR-expressing cell is only fully activated in the presence of both antigens.

RNA transfection

[00383] Methods for producing an in vitro transcribed NRA CAR are described here. The present invention also includes a CAR encoding RNA construct that can be directly transferred into a cell. One method for generating mRNA for use in transfection may involve in vitro transcription (IVT) of a template with specially designed primers, followed by addition of polyA, to produce a construct containing 3' and 5' untranslated sequence ("UTR"). ), a 5' cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length (SEQ ID NO:118). RNA thus produced can effectively transfect different species of cells. In one aspect, the model includes sequences for the CAR.

[00384] In one aspect, the anti-CD19 CAR is encoded by a messenger RNA (mRNA). In one aspect, the mRNA encoding the anti-CD19 CAr is introduced into an immune effector cell, e.g., a T cell or an NK cell, to produce a CAR-expressing cell, e.g., a CART cell or a c' NK squid from CAR.

[00385] In one embodiment, the in vitro transcribed RNA CAR can be introduced into a cell as a form of transient infection. RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate DNA source DNA. The desired model for in vitro transcription is a CAR of the present invention. For example, the template for the RNA CAR comprises an extracellular region comprising a single-chain variable domain of an antitumor antibody; a hinge region, a transmembrane domain (e.g., a CD8a transmembrane domain); and a cytoplasmic region that includes an intracellular signaling domain, for example, comprising the CD3-zeta signaling domain and the 4-1BB signaling domain.

[00386] In one embodiment, the DNA to be used for PCR contains an open reading frame. DNA can be from a naturally occurring DNA sequence from an organism's genome. In one embodiment, the nucleic acid may include some or all of the 5' and/or 3' translated regions (UTRs). Nucleic acid can include exons and introns. In one embodiment, the DNA to be used for PCR is a human nucleic acid sequence. In another embodiment, the DNA to be used for PCR is a human nucleic acid sequence including the 5' and 3' UTRs. The DNA may alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are linked together to form an open reading frame. The portions of DNA that are linked together can be from a single organism or from more than one organism.

[00387] PCR is used to generate a template for in vitro transcription of mRNA that is used for transfection. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR. "Substantially complementary", as used herein, refers to nucleotide sequences where most or all of the bases in the primer are complementary, or one or more bases are non-complementary, or mismatched. Substantially complementary sequences are capable of annealing or hybridizing to the intended target DNA under annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5' and 3' UTRs. Primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest. In one embodiment, the primers are designed to extend the codon region of a human cDNA, including all or portions of the 5' and 3' UTRs. Useful primers for PCR can be generated by synthetic methods that are well known in the art. "Forward primers" are primers that contain a region of nucleotides that are substantially complementary to nucleotides in the DNA template that are upstream of the DNA sequence that is to be amplified. "Upstream" is used here to refer to a location 5, the DNA sequence to be amplified, with respect to the amplified strand. "Reverse primers" are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified. "Downstream" is used here to refer to a location 3' to the DNA sequence to be amplified, with respect to the coding strand.

[00388] Any DNA polymerase useful for PCR can be used in the methods described here. The reagents and polymerase are commercially available from several sources.

[00389] Chemical structures with the ability to promote stability and/or translational efficiency can also be used. RNA preferably has 5' and 3' UTRs. In one embodiment, a 5' UTR is between one and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designating primers for PCR that anneal to different regions of the UTRs. Using this method, one skilled in the art can modify the 5' and 3' UTR lengths required to obtain optimal translational efficiency after transfection of the transcribed RNA.

[00390] The 5' and 3' UTRs may be the naturally occurring endogenous 5' and 3' UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful to modify the stability and/or translational efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease mRNA stability. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs that are well known in the art.

[00391] In one embodiment, a 5' UTR may contain the endogenous nucleic acid Kozak sequence. Alternatively, when a 5' UTR that is not endogenous to the nucleic acid of interest is being added by PCR as described above, a consensus Kozak sequence can be redesignated by addition of a 5' UTR sequence. Kozak sequences can increase the translational efficiency of some RNA transcripts, but do not appear to be required for all RNAs to ensure efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments a 5' UTR may be the 5'UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3' UTR or 5' UTR to prevent exonuclease degradation of the mRNA.

[00392] To ensure RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter must be linked to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In a preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[00393] In a preferred embodiment, the mRNA has both a 5' end cap and a 3' poly(A) tail that determines ribosome binding, translation initiation, and stability of the mRNA in the cell. In a circular DNA template, for example, plasmid DNA, RNA polymerase produces a long concatemeric product that is not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the end of the 3' UTR results in normal-sized mRNA that is not effective in eukaryotic transfection even if it is polyadenylated after transcription.

[00394] In a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[00395] The conventional method of polyA/T stretch interaction on a DNA template is molecular cloning. However, polyA/T sequence integrated into plasmid DNA can cause plasmid instability, which is why template plasmid DNA obtained from bacterial cells is often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time-consuming, but often unsafe. Which is therefore a method that allows the construction of DNA models with polyA/T 3' stretch in highly desirable cloning.

[00396] The polyA/T segment of the transcriptional DNA pattern can be produced during PCR using a reverse primer containing a polyT tail, such as 100T tail (SEQ ID NO: 110) (size can be from 50 to 5000 T (SEQ ID NO: 111)), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines (SEQ ID NO: 112).

[00397] Poly(A) tails of RNAs can also be extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP). In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides (SEQ ID NO: 113) results in about a two-fold increase in RNA translation efficiency. Additionally, the attachment of different chemical groups to the 3' end can increase mRNA stability. Such linkage may contain modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogues can also increase RNA stability.

[00398] 5' buffers also provide stability for RNA molecules. In a preferred embodiment, RNAs produced by the methods described herein include a 5' cap. The 5' buffer is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[00399] RNAs produced by the methods described here may also contain an Internal Ribosome Entry Site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designated sequence that initiates buffer-independent ribosome binding to mRNA and facilitates translation initiation. Any solutes suitable for electroporation, which may contain factors facilitating viability and permeability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants, may be included.

[00400] RNA can be introduced into target cells using any of several different methods, for example, commercially available methods that include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, mediated transfection by peptide, or biolistic particle delivery systems such as "gene guns" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).

Non-viral Delivery Methods

[00401] In some aspects, non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein into a cell or tissue or individual.

[00402] In some embodiments, the non-viral method includes the use of a transposon (also called a transposable element). In some embodiments, a transposon is a piece of DNA that can self-insert into a location in a genome, e.g., a piece of DNA that is capable of self-replicating and inserting its copy into a genome, or a piece of DNA that it can be spliced from a larger nucleic acid and inserted elsewhere in a genome. For example, the transposon comprises a DNA sequence prepared from inverted repeats flanking genes for transposition.

[00403] Exemplary nucleic acid delivery methods using a transposon include a Sleeping Beauty transposon system (SBTS) and a piggyBac transposon system (PB). See, for example, Aronovich et al. Hum. Mol. Genet. 20.R1(2011):R14-20; Singh et al. Cancer Res. 15(2008):2961-2971; Huang et al. Mol. Ther. 16(2008):580-589; Grabundzija et al. Mol. Ther. 18(2010):1200-1209; Kebriaei et al. Blood. 122.21(2013):166; Williams. Molecular Therapy 16.9(2008):1515-16; Bell et al. Nat. Protoc. 2.12(2007):3153-65; and Ding et al. Cell. 122.3(2005):473-83, all of which are incorporated herein by reference.

[00404] The SBTS includes two components: 1) a transposon containing a transgene and 2) a transposase enzyme source. The transposase can transpose the transposon from a carrier plasmid (or other donor DNA) to a target DNA, such as a host cell chromosome/genome. For example, the transposase binds to the carrier plasmid/donor DNA, cuts the transposon (including transgene(s)) out of the plasmid, and inserts it into the host cell genome. See, for example, Aronovich et al. above.

[00405] Exemplary transposons include a pT2-based transposon. See, for example, Grabundzija et al. nucleic acids Res 41.3(2013):1829-47; and Singh et al. Cancer Res. 68.8(2008): 2961-2971, all of which are incorporated herein by reference. Exemplary transposases include a Tc1/mariner-type transposase, for example, the SB10 transposase or the SB11 transposase (a hyperactive transposase that can be expressed, for example, from a cytomegalovirus promoter). See, for example, Aronovich et al.; Kebriaei et al.; and Grabundzija et al., all of which are incorporated herein by reference.

[00406] Use of SBTS allows efficient integration and expression of a transgene, for example, a nucleic acid encoding a CAR described here. Provided herein are methods of generating a cell, e.g., T cell or NK cell, that stably expresses a CAR described herein, e.g., using a transposon system such as SBTS.

[00407] According to the methods described here, in some embodiments, one or more nucleic acids, e.g., plasmids, containing the SBTS components are delivered to a cell (e.g., T or NK cell). For example, the nucleic acid(s) are released by standard nucleic acid delivery methods (e.g., plasmid DNA), e.g., methods described herein, e.g., electroporation, transfection, or lipofection. . In some embodiments, the nucleic acid contains a transposon comprising a transgene, for example, a nucleic acid encoding a CAR described herein. In some embodiments, the nucleic acid contains a transposon comprising a transgene (e.g., a nucleic acid encoding a CAR described herein) as well as a nucleic acid sequence encoding a transposase enzyme. In other embodiments, a system with two nucleic acids is provided, for example, a dual plasmid system, for example, where a first plasmid contains a transposon comprising a transgene, and a second plasmid contains a nucleic acid sequence encoding a transposase enzyme. For example, the first and second nucleic acids are co-released in a host cell.

[00408] In some embodiments, cells, e.g., T or NK cells, are generated that express a CAR described here using a combination of gene insertion using the SBTS and gene editing using a nuclease (e.g., zinc finger nucleases (ZFNs), Transcription Activator-like Effector Nucleases (TALENs), the CRISPR/Cas system, or the CRISPR/Cas system, or constructed meganuclease reconstructed homing endonucleases).

[00409] In some embodiments, use of a non-viral delivery method allows reprogramming of cells, for example, T or NK cells, and direct infusion of the cells into an individual. Advantages of non-viral vectors include, but are not limited to, the ease and relatively low cost of producing sufficient quantities required to serve a patient population, stability during storage, and lack of immunogenicity.

Nucleic Acid Constructs Encoding a CAR

[00410] The present invention also provides nucleic acid molecules encoding one or more CAR constructs described herein. In one aspect, the nucleic acid molecule is provided as an RNA transcription messenger. In one aspect, the nucleic acid molecule is provided as a DNA construct.

[00411] Accordingly, in one aspect, the invention pertains to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-CD19 binding domain (e.g., an anti-CD19 binding domain). -humanized CD19), a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain, e.g., a costimulatory signaling domain and/or a primary signaling domain, e.g., zeta chain. In one embodiment, the anti-CD19 binding domain is an anti-CD19 binding domain described herein, e.g., an anti-CD19 binding domain comprising a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO :10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:59, or a sequence with 95 to 99% identity thereof. In one embodiment, the transmembrane domain is the transmembrane domain of a protein selected from the group consisting of the T cell receptor alpha, beta, or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16 , CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, the transmembrane domain comprises a sequence of SEQ ID NO: 15, or a sequence with 95 to 99% identity thereof. In one embodiment, the anti-CD19 binding domain is connected to the transmembrane domain by a hinge region, e.g., a hinge described herein. In one embodiment, the hinge region comprises SEQ ID NO:14 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49, or a sequence with 95 to 99% identity thereof. In one embodiment, the isolated nucleic acid molecule also comprises a sequence encoding a co-stimulatory domain. In one embodiment, the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4 -1BB (CD137). In one embodiment, the costimulatory domain comprises a sequence of SEQ ID NO:16, or a sequence with 95 to 99% identity thereof. In one embodiment, the intracellular signaling domain comprises a functional 4-1BB signaling domain and a functional CD3 zeta signaling domain. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 or SEQ ID NO: 51, or a sequence with 95 to 99% identity thereof, and the sequence of SEQ ID NO: 17 or SEQ ID NO:43, or a sequence with 95 to 99% identity thereof, in which the sequences comprising the intracellular signaling domain are expressed in the same structure and as a single polypeptide chain.

[00412] In another aspect, the invention pertains to an isolated nucleic acid molecule encoding a CAR construct comprising a leader sequence of SEQ ID NO: 13, a scFv domain having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:59, (or a sequence with 95 to 99% identity thereof), a hinge region of SEQ ID NO:14 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49 (or a sequence with 95 to 99% identity thereof), a transmembrane domain having a sequence of SEQ ID NO: 15 (or a sequence with 95 to 99% identity thereof), a 4-1BB costimulatory domain having a sequence of SEQ ID NO:16 or a CD27 costimulatory domain having a sequence of SEQ ID NO:51 (or a sequence with 95-99 % identity thereof), and a CD3 zeta stimulatory domain having a sequence of SEQ ID NO:17 or SEQ ID NO:43 (or a sequence with 95 to 99% identity thereof).

[00413] In another aspect, the invention pertains to an isolated polypeptide molecule encoded by the nucleic acid molecule. In one embodiment, the isolated polypeptide molecule comprises a sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:59 or a sequence with 95 99% of its identity.

[00414] In another aspect, the invention pertains to a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule comprising an anti-CD19 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory, and wherein said anti-CD19 binding domain comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:59, or a sequence with 95-99% identity.

[00415] In one embodiment, the encoded CAR molecule also comprises a sequence encoding a costimulatory domain. In one embodiment, the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), and 4-1BB (CD137). In one embodiment, the costimulatory domain comprises a sequence of SEQ ID NO:16. In one embodiment, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the T cell receptor alpha, beta, or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16 , CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, the transmembrane domain comprises a sequence of SEQ ID NO:15. In one embodiment, the intracellular signaling domain comprises a 4-1BB functional signaling domain and a zeta functional signaling domain. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 16 and the sequence of SEQ ID NO: 17, wherein the outgrowths comprising the intracellular signaling domain are expressed in the same structure and as a single polypeptide chain. . In one embodiment, the anti-CD19 binding domain is connected to the transmembrane domain by a hinge region. In one embodiment, the hinge region comprises SEQ ID NO:14. In one embodiment, the hinge region comprises SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49.

[00416] In another aspect, the invention pertains to an encoded CAR molecule comprising a leader sequence of SEQ ID NO: 13, a scFv domain having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:59, or a sequence with 95 to 99% identity thereof, a hinge region of SEQ ID NO:14 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49, a transmembrane domain having a sequence of SEQ ID NO:15, a 4-1BB costimulatory domain having a sequence of SEQ ID NO:16 or a CD27 costimulatory domain having a sequence of SEQ ID NO:51, and a CD3 zeta stimulatory domain having a sequence of SEQ ID NO:17 or SEQ ID NO:43. In one embodiment, the encoded CAR molecule comprises a sequence selected from a group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:59, or a sequence with 95-99% identity.

[00417] Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as, for example, analyzing libraries of cells expressing the gene, deriving the gene from a vector known to include the same, or directly isolating of cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be synthetically produced, rather than cloned.

[00418] The present invention also provides vectors into which a DNA of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-lasting gene transfer since they allow stable, long-lasting integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from oncoretroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells such as hepatocytes. They also have the added advantage of low immunogenicity. A retroviral vector may also be, for example, a gamma-retroviral vector. A gamma-retroviral vector may include, for example, a promoter, a packaging signal (^), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTR), and a transgene of interest, for example, a gene encoding a CAR. A gamma-retroviral vector may lack viral structural genes such as gag, pol, and env. Exemplary gamma-retroviral vectors include Murine Leukemia Virus (MLV), Spleen Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, for example, in Tobias Maetzig et al., "Gammaretroviral vectors: Biology, Technology and Application"Viruses. June 2011 3(6): 677-713.

[00419] In another embodiment, the vector comprising the nucleic acid encoding the desired CAR of the invention is an adenoviral vector (A5/35). In another embodiment, expression of nucleic acids encoding CARs can be accomplished using transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See below, June et al. 2009Nature Reviews Immunology 9.10: 704-716, is incorporated herein by reference.

[00420] In brief summary, expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector. Vectors may be suitable for eukaryotic replication and integration. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

[00421] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, United States Patent Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In another embodiment, the invention provides a gene therapy vector.

[00422] The nucleic acid can be cloned into different types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[00423] Furthermore, the expression vector can be delivered to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1 -4, Cold Spring Harbor Press, NY), and in other virology manuals and molecular biology. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains a functional origin of replication in at least one organism, a promoter sequence, suitable restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and United States Patent No. 6,326,193).

[00424] Several viral-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and released into the individual's cells in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. Several adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

[00425] Additional promoter elements, e.g. enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although several promoters have been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is preserved when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function cooperatively or independently to activate transcription. Exemplary promoters include the CMV IE gene, EF-1a, ubiquitin C, or phosphoglycerokinase (PGK) promoters.

[00426] An example of a promoter that is capable of expressing a CAR transgene in a mammalian T cell is the EF1a promoter. The native EF1a promoter regulates the expression of the alpha subunit of the elongation factor 1 complex, which is responsible for the enzymatic release of aminoacyl tRNAs to the ribosome. The EF1a promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in regulating CAR expression from transgenes cloned into a lentiviral vector. See, for example, Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). In one aspect, the EF1a promoter is the sequence provided as SEQ ID NO:100.

[00427] Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence, capable of regulating the high levels of expression of polynucleotide sequence operably linked thereto. However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), immunodeficiency virus long terminal repeat (LTR) promoter. human (HIV), MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin, the myosin promoter, the elongation factor 1 promoter, the hemoglobin promoter, and the creatine kinase promoter. Furthermore, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence to which it is operably linked when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[00428] A vector may also include, for example, a signal sequence to facilitate secretion, a polyadenylation and transcription signal terminator (e.g., from the Bovine Growth Hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes (e.g., SV40 and ColE1 origin or others known in the art) and/or elements to allow selection (e.g., ampicillin resistance gene and/or zeocin marker).

[00429] In order to evaluate the expression of a CAR polypeptide or portion thereof, the expression vector to be introduced into a cell may also contain a selectable marker gene or a reporter gene or both to facilitate cell identification and selection. of expression of the researched cell populations being transfected or infected by means of viral vectors. In other aspects, the selectable marker can be continued on a separate piece of DNA and used in a cotransfection procedure. Both selectable markers and reporter genes can be flanked with appropriate regulatory sequences to ensure expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

[00430] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, for example, enzymatic activity. Expression of the reporter gene is assayed at a period of time after the DNA has been introduced into recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using commercially available or commercially available techniques. In general, the construct of the minimal 5' flanking region showing the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be linked to a reporter gene and used to evaluate agents for their ability to modulate promoter-regulated transcription.

[00431] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical or biological methods.

[00432] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). A preferred method for introducing a polynucleotide into a host cell is calcium phosphate transfection.

[00433] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, United States Patent Nos. 5,350,674 and 5,585,362.

[00434] Chemical methods for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle). Other prior art targeted release methods of nucleic acids are available, such as polynucleotide release with targeted nanoparticles or other suitable submicron sized release system.

[00435] In the case where a non-viral delivery system is used, an exemplary delivery system is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. Nucleic acid associated with a lipid can be encapsulated within the aqueous interior of a liposome, intercalated within the lipid bilayer of a liposome, linked to a liposome via a linker molecule that is associated with both the liposome and oligonucleotide, captured in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in or complexed with a micelle, or otherwise associated with a lipid. Compositions associated with lipid, lipid/DNA, or lipid/expression vector are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, such as micelles, or with a "collapsed" structure. They may also simply be interspersed into a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include fatty doublets that occur naturally in the cytoplasm as well as the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[00436] Lipids suitable for use can be obtained from commercial sources. For example, dimyrystil phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL.). Solutions of lipid feedstock in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform is used as the only solvent, as it is more easily evaporated than methanol. "Liposome" is a generic term encompassing a variety of single and multilamellar lipid carriers formed by the generation of closed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have lipid layers separated by aqueous media. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-recombination before the formation of closed structures and capture water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have structures different from those of normal vesicular structure in solution are also covered. For example, lipids may assume a micelle structure or merely exist as non-uniform aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[00437] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays can be carried out. Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical assays" such as detection of the presence or absence of a particular peptide, for example, by immunological methods (ELISAs and Western blots) or by assays described herein to identify agents that fall within the scope of the invention.

[00438] The present invention also provides a vector comprising a CAR encoding nucleic acid molecule. In one aspect, a CAR vector can be directly transduced into a cell, e.g., a T cell. In one aspect, the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plus plasmids (e.g., expression plasmids, cloning vectors, microcircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs. In one aspect, the vector is capable of expressing the CAR construct in mammalian T cells. In one aspect, the mammalian T cell is a human T cell.

Cell Sources

[00439] Prior to expansion and genetic modification or other modification, a source of cells, for example, T cells or natural killer (NK) cells, can be obtained from an individual. The term "subject" is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of individuals include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including mononuclear cells from peripheral blood, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.

[00440] In certain aspects of the present invention, immune effector cells, for example, T cells, can be obtained from a unit of blood collected from an individual using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In a 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 can be washed to provide the plasma fraction and, optionally, to place the cells in a buffer or appropriate media for subsequent processing steps. In one embodiment, cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the washing solution does not have calcium and may not have magnesium or may not have, if not all, divalent cations.

[00441] Initial activation steps in the absence of calcium can induce expanded activation. As those skilled in the art can readily appreciate, a washing step can be performed by methods known to those skilled in the art, such as using a semi-automated "full flow" centrifuge (e.g., the Cobe 2991 cell processor, the Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, cells can 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, undesirable components of the apheresis sample can be removed and the cells directly resuspended in culture media.

[00442] In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLLTM gradient or by counterflow centrifugal elutriation.

[00443] The methods described herein may include, for example, selection of a specific subpopulation of immune effector cells, for example, T cells, which are a population depleted by regulatory T cells, depleted CD25+ cells, using, for example, a technique of negative selection, for example, described here. Preferably, the regulatory depleted T cell populations contain less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.

[00444] In one embodiment, regulatory T cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody, or fragment thereof, or a CD25-binding ligand, IL-2. In one embodiment, the anti-CD25 antibody, or fragment thereof, or CD25 binding ligand is conjugated to a substrate, e.g., a bead, or is otherwise coated on a substrate, e.g., a bead. In one embodiment, the anti-CD25 antibody, or fragment thereof, is conjugated to a substrate as described herein.

[00445] In one embodiment, regulatory T cells, e.g., CD25+ T cells, are removed from the population using MiltenyiTM CD25 depletion reagent. In one embodiment, the ratio of cells to CD25 depletion reagent is 1e7 cells to 20 uL, or 1e7 cells to 15 uL, or 1e7 cells to 10 uL, or 1e7 cells to 5 uL, or 1e7 cells to 2.5 uL, or 1e7 cells for 1.25 uL. In one embodiment, for example, for regulatory T cells, e.g. CD25+ depletion, greater than 500 million cells/mL is used. In another aspect, a cell concentration of 600, 700, 800, or 900 million cells/mL is used.

[00446] In one embodiment, the immune effector cell populations to be depleted include about 6 x 109 CD25+ T cells. In other aspects, the immune effector cell populations to be depleted include about 1 x 109 to 1x 1010 CD25+ T cells, and any integer value in between. In one embodiment, the resulting population of regulatory depleted T cells has 2 x 109 regulatory T cells, e.g., CD25+ cells, or less (e.g., 1 x 109, 5 x 108, 1 x 108, 5 x 107, 1 x 107 , or fewer CD25+ cells).

[00447] In one embodiment, regulatory T cells, e.g., CD25+ cells, are removed from the solution using the CliniMAC system with a set of depletion tubes, such as, e.g., tube 162-01. In one embodiment, the CliniMAC system is performed in a depletion configuration such as, for example, DEPLETION2.1.

[00448] Without wishing to be bound by a particular theory, the decrease in the level of negative regulators of immune cells (e.g., decrease in the number of unwanted immune cells, e.g., TREG cells), in an individual before apheresis or during Manufacturing a CAR-expressing cell product may reduce an individual's risk of relapse. For example, methods of depleting TREG cells are known in the art. Methods of depleting TREG cells include, but are not limited to, cyclophosphamide, anti-GITR antibody (an anti-GITR antibody described herein), CD25 depletion, and combinations thereof.

[00449] In some embodiments, the manufacturing methods comprise reducing the number of (e.g., depleting) TREG cells prior to manufacturing the CAR-expressing cell. For example, manufacturing methods comprise contacting the sample, e.g. the apheresis sample, with an anti-GITR antibody and/or an anti-CD25 antibody (or fragment thereof, or a CD25 binding ligand), e.g. , to deplete TREG cells prior to manufacturing the CAR-expressing cell product (e.g., T cell, NK cell).

[00450] In one embodiment, an individual is pretreated with one or more therapies that reduce TREG cells prior to collecting cells for manufacturing a CAR-expressing cell product, thereby reducing the risk of the individual re-initiating treatment with a CAR-expressing cell. CAR. In one embodiment, methods of decreasing TREG cells include, but are not limited to, administering to the subject one or more of cyclophosphamide, anti-GITR antibody, CD25 depletion, or a combination thereof. Administration of one or more of cyclophosphamide, anti-GITR antibody, CD25 depletion, or a combination thereof, may occur before, during, or after an infusion of the CAR-expressing cell product.

[00451] In one embodiment, an individual is pretreated with cyclophosphamide prior to harvesting cells for the manufacture of a CAR-expressing cell product, thereby reducing the risk of the individual returning to CAR-expressing cell treatment. In one embodiment, an individual is pretreated with an anti-GITR antibody prior to collecting cells for manufacturing a CAR-expressing cell product, thereby reducing the risk of the individual re-initiating CAR-expressing cell treatment.

[00452] In one embodiment, the cell populations to be removed are neither regulatory T cells nor tumor cells, but cells that otherwise negatively affect the expansion and/or function of CART cells, e.g., cells expressing CD14 , CD11b, CD33, CD15, or other markers expressed by potentially immunosuppressive cells. In one embodiment, such cells are intended to be removed concurrently with regulatory T cells and/or tumor cells, or following said depletion, or in another order.

[00453] The methods described here may include more than one selection step, for example, more than one depletion step. Enrichment of a T cell population by negative selection can be accomplished, for example, with a combination of antibodies targeting surface markers unique to the negatively selected cells. One method is the sorting and/or selection of cells using negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies targeting cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail may include antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[00454] The methods described herein may also include removing cells from the population that express a tumor antigen, e.g., a tumor antigen that does not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14 or CD11b, to thereby provide depleted, e.g., CD25+-depleted, and tumor antigen-depleted, regulatory cell populations that are suitable for expression of a CAR, e.g., a CAR described herein. In one embodiment, cells expressing tumor antigen are removed simultaneously with regulatory T cells, e.g., CD25+ cells. For example, an anti-CD25 antibody, or fragment thereof, and an anti-tumor antigen antibody, or fragment thereof, can be bound to the same substrate, e.g., bead, which can be used to remove cells or an anti-CD25 antibody. CD25, or fragment thereof, or the antitumor antigen antibody, or fragment thereof, can be attached to the separate beads, a mixture of which can be used to remove the cells. In other embodiments, the removal of regulatory T cells, e.g., CD25+ cells, and the removal of cells expressing tumor antigen is sequential, and may occur, for example, in any order.

[00455] Methods are also provided that include removing cells from the population that express a checkpoint inhibitor, e.g., a checkpoint inhibitor described herein, e.g., one or more PD1+ cells, LAG3+ cells, and TIM3+ cells, to thereby provide depleted regulatory T cell populations, e.g., CD25+ depleted cells, and checkpoint inhibitor-depleted cells, e.g., PD1+, LAG3+ and/or TIM3+ depleted cells. Exemplary checkpoint inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, TIGIT, CTLA -4, BTLA and LAIR1. In one embodiment, cells expressing checkpoint inhibitor are removed simultaneously with regulatory T cells, e.g., CD25+. For example, an anti-CD25 antibody, or fragment thereof, and an anti-checkpoint inhibitor antibody, or fragment thereof, can be linked to the same bead that can be used to remove cells, or an anti-CD25 antibody. CD25, or fragment thereof, and the anti-checkpoint inhibitor antibody, or fragment thereof, can be attached to the separate beads, a mixture of which can be used to remove the cells. In other embodiments, the removal of regulatory T cells, e.g., CD25+ cells, and the removal of checkpoint inhibitor-expressing cells is sequential, and may occur, for example, in any order.

[00456] Methods described here may include a positive selection step. For example, T cells can be isolated by incubation with anti-CD3/anti-CD28 conjugated beads (e.g., 3x28), such as DYNABEADS® M-450 CD3/CD28 T, for a period of time sufficient for positive selection of cells. desired T cells. In one embodiment, the time period is about 30 minutes. In another embodiment, the time period ranges from 30 minutes to 36 hours or more and every integer value in between. In another embodiment, the period of time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the time period is 10 to 24 hours, e.g. 24 hours. Longer incubation time periods can be used to isolate T cells in any situation where there are few T cells compared to other cell types, such as in isolation of tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Furthermore, the use of longer incubation time periods may increase the capture efficiency of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as also described here), subpopulations of T cells can be preferentially selected for or against at the start of culture or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at the start of culture or at other desired time points.

[00457] In one embodiment, a T cell population can be selected that expresses one or more of IFN-v, TNFa, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10 , IL-13, granzyme B, and perforin, or other appropriate molecules, for example, other cytokines. Methods for cellular expression analysis can be determined, for example, by the methods described in PCT Publication No.: WO 2013/126712.

[00458] For isolation of a desired cell population by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain aspects, it may be desirable to significantly decrease the volume in which the beads and cells are mixed together (e.g., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one aspect, a concentration of 10 billion cells/mL, 9 billion/mL, 8 billion/mL, 7 billion/mL, 6 billion/mL, or 5 billion/mL is used. In one aspect, a concentration of 1 billion cells/mL is used. In still one aspect, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/mL is used. In other aspects, concentrations of 125 or 150 million cells/mL can be used.

[00459] The use of high concentrations can result in increased cell production, cell activation, and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or of samples where there are many tumor cells present (e.g., tissue, tumor ,leukemic blood, etc.). Such cell populations may have therapeutic value and would be desirable to obtain. For example, the use of a high concentration of cells allows for more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

[00460] In a related aspect, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between particles and cells are minimized. This selects cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express high levels of CD28 and are more efficiently captured than dilute concentrations of CD8+ T cells. In one aspect, the cell concentration used is 5 x 106/mL. In other aspects, the concentration used can be from about 1 x 105/mL to 1 x 106/mL, and any whole number value in between.

[00461] In other aspects, cells can be incubated in a rotor for varying lengths of time at varying speeds at 2 to 10oC or at room temperature.

[00462] T cells for stimulation can also be frozen after a washing step. Not wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing the granulocytes and to some extent, monocytes in the cell population. After the washing step that removes the plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves the use of PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% DMSO. Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% 5% Dextrose, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing e.g. Hespan and PlasmaLyte A, the cells are then frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a nitrogen storage tank. Other controlled freezing methods can be used as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.

[00463] In certain aspects, cryopreserved cells are thawed and washed as described herein and allowed to stand for one hour at room temperature before activation using the methods of the present invention.

[00464] Also contemplated in the context of the invention is the collection of blood samples or apheresis product from an individual at a period of time before when cells expanded as described herein may be needed. In this way, the source of cells to be expanded can be collected at any necessary time point, and desired cells, such as T cells, isolated and frozen for later use in immune effector cell therapy for any number of diseases or conditions that arise. would benefit from immune effector cell therapy, such as those described here. In one aspect, a blood sample or apheresis is taken from a generally healthy individual. In certain aspects, a blood sample or apheresis is taken from a generally healthy individual who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain aspects, T cells can be expanded, frozen and used at a later time. In certain aspects, samples are collected from a patient immediately after diagnosis of a particular disease as described herein, but before any treatments. In another aspect, cells are isolated from a blood sample or an apheresis from an individual prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents 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 irradiation.

[00465] In another aspect of the present invention, T cells are obtained from a patient directly after treatment that leaves the individual with functional T cells. In this regard, it has been observed that following certain cancer treatments, in particular treatments that damage the immune system, immediately after treatment during the period when patients would normally be recovering from treatment, the quality of T cells obtained may be ideal or improved in terms of its ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described here, these cells may be in a preferred state for enhanced engraftment and expansion in vivo. Thus, it is contemplated in the context of the present invention to collect blood cells, including T cells, dendritic cells, or other hematopoietic lineage cells, during this recovery phase. Furthermore, in certain aspects, mobilization regimens (e.g., mobilization with GM-CSF) and conditioning can be used to create a condition in an individual in which the repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time after therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[00466] In one embodiment, immune effector cells expressing a CAR molecule, for example, a CAR molecule described here, are obtained from an individual who has received a low immune boosting dose of an mTOR inhibitor. In one embodiment, the immune effector cell populations, e.g., T cells, to be created to express a CAR, are harvested after a sufficient period of time, or after sufficient dosing of a low booster dose of an mTOR inhibitor, of 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 the individual or collected from the individual has been, at least temporarily, increased.

[00467] In other embodiments, a population of immune effector cells, e.g., T cells, that have, or will be created to express a CAR, can be treated ex vivo by contact 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.

[00468] In one embodiment, a T cell population is deficient in diaglycerol kinase (DGK). DGK-deficient cells include cells that do not express DGK RNA or protein, or have reduced or inhibited DGK activity. DGK-deficient cells can be generated by genetic methods, e.g., administration of RNA interference agents, e.g., siRNA, shRNA, miRNA, to reduce or prevent expression of. Alternatively, DGK-deficient DGK cells can be generated by treatment with DGK inhibitors described here.

[00469] In one embodiment, a T cell population is Ikaros deficient. Ikaros-deficient cells include cells that do not express Ikaros RNA or protein, or have reduced or inhibited Ikaros activity, Ikaros-deficient cells can be generated by genetic methods, e.g., administration of RNA interference agents, e.g., siRNA , shRNA, miRNA, to reduce or prevent Ikaros expression. Alternatively, Ikaros-deficient cells can be generated by treatment with Ikaros inhibitors, e.g., lenalidomide.

[00470] In embodiments, a 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 and Ikaros deficient cells can be generated by any of the methods described here.

[00471] In one embodiment, NK cells are obtained from the individual. In another embodiment, the NK cells are an NK cell line, e.g., NK-92 cell line (Conkwest).

Allogenic CAR

[00472] In embodiments described here, the immune effector cell may be an allogeneic immune effector cell, e.g., T cell or NK cell. For example, the cell may be an allosteric T cell, e.g., an allosteric T cell lacking the expression of a functional T cell receptor (TCR) and/or human leukocyte antigen (HLA), e.g., HLA class I and /or HLA class II.

[00473] The T cell without a functional TCR can be, for example, created so that it does not express any functional TCR on its surface, created so that it does not express one or more subunits comprising a functional TCR, or created so that it produces very little functional TCR on its surface. Alternatively, the T cell may express a substantially compromised TCR, for example, by expressing mutated or truncated forms of one or more of the TCR subunits. The term "substantially compromised TCR" means that this TCR will not provoke an adverse immune reaction in a host.

[00474] The T cell described here can be, for example, created so that it does not express a functional HLA on its surface. For example, a T cell described herein can be created so that cell surface expression HLA, e.g., HLA class 1 and/or HLA class II, is downregulated.

[00475] In some embodiments, the T cell may not have a functional TCR and a functional HLA, e.g., HLA class I and/or HLA class II.

[00476] Modified T cells lacking the expression of a functional TCR and/or HLA can be obtained by any suitable methods, including a knockout or reduction of one or more TCR or HLA subunits. For example, the T cell can include TCR and/or HLA knockdown using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN), or zinc finger endonuclease (ZFN). ).

[00477] In some embodiments, the allogeneic cell may be a cell that does not express or expresses at low levels of an inhibitory molecule, for example, by any method described herein. For example, the cell may be a cell that expresses or expresses at low levels an inhibitory molecule, for example, which may decrease the ability of a CAR-expressing cell to mount an immune effector response. 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 an inhibitory molecule, for example by inhibition at the DNA, RNA, or protein level, can optimize a cell expressing CAR performance. In embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), a transcription activator-like effector nuclease (TALEN) ), or a zinc finger endonuclease (ZFN), for example, as described herein, can be used.

siRNA and shRNA to inhibit TCR or HLA

[00478] In some embodiments, TCR expression and/or HLA expression can be inhibited using siRNA or shRNA that targets a nucleic acid encoding a TCR and/or HLA in a T cell.

[00479] Expression of siRNA and shRNAs in T cells can be achieved using any conventional expression system, for example, such as a lentiviral expression system.

[00480] Exemplary shRNAs that down-regulate TCR component expression are described, for example, in United States Publication No.: 2012/0321667. Exemplary siRNA and shRNA that down-regulate the expression of HLA class I and/or HLA class II genes are described, for example, in United States Publication No.: US 2007/0036773.

CRISPR to inhibit TCR or HLA

[00481] "CRISPR" or "CRISPR for TCR and/or HLA" or "CRISPR to inhibit TCR and/or HLA" as used herein refers to a group of clustered and regularly interspaced short palindromic repeats, or a system comprising such a group of repetitions. "Cas", as used here, refers to a CRISPR-associated protein. A "CRISPR/Cas" system refers to a system derived from CRISPR and Cas that can be used to silence or mutate a TCR and/or HLA gene.

[00482] Naturally occurring CRISPR/Cas systems are found in approximately 40% of sequenced eubacterial and 90% of sequenced archaeal genomes. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843-1845.

[00483] The CRISPR/Cas system has been modified for use in gene editing (silencing, enhancing or changing specific genes) in eukaryotes such as mice or primates. Wiedenheft et al. (2012) Nature 482: 331-8. This is accomplished by introducing into the eukaryotic cell a plasmid containing a specifically designed CRISPR and one or more appropriate Cas.

[00484] The CRISPR sequence, sometimes called a CRISPR site, comprises alternating repeats and spacers. In a naturally occurring CRISPR, the spacers generally comprise sequences foreign to the bacteria such as a plasmid or phage sequence; In the TCR and/or HLA CRISPR/Cas system, the spacers are derived from the TCR or HLA gene sequence.

[00485] CRISPR site RNA is constitutively expressed and processed by Cas proteins into small RNAs. These comprise a spacer flanked by a repeat sequence. RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Horvath et al. (2010) Science 327: 167-170; Makarova et al. (2006) Biology Direct 1: 7. Spacers thus function as templates for RNA molecules, analogously to siRNAs. Pennisi (2013) Science 341: 833-836.

[00486] As these occur naturally in many different types of bacteria, the exact arrangement of CRISPR and the structure, function and number of Cas genes and their product differs somewhat from species to species. Haft et al. (2005) PLoS Comput. Biol. 1: e60; Kunin et al. (2007) Genome Biol. 8: R61; Mojica et al. (2005) J. Mol. Evol. 60: 174-182; Bolotin et al. (2005) Microbiol. 151: 2551-2561; Pourcel et al. (2005) Microbiol. 151: 653-663; and Stern et al. (2010) Trends. Genet. 28: 335-340. For example, Cse (Cas subtype, E. coli) proteins (e.g., CasA) form a functional complex Cascade, which processes CRISPR transcription of RNAs into spacer-repeat units that the Cascade maintains. Brouns et al. (2008) Science 321: 960-964. In other prokaryotes, Cas6 processes CRISPR transcription. CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 or Cas2. The Cmr (Cas RAMP module) proteins in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognize and cleave complementary target RNAs. A simpler CRISPR system relies on the Cas9 protein, which is a nuclease with two active cutting sites, one for each strand of the double helix. Combination of Cas9 and CRISPR site NRA can be used in a system for gene editing. Pennisi (2013) Science 341: 833-836.

[00487] The CRISPR/Cas system can thus edit a TCR and/or HLA gene (by adding or deleting a base pair), or introduce a premature interruption, thereby decreasing the expression of a TCR and/or HLA. The CRISPR/Cas system can alternatively be used as RNA interference, turning off the TCR and/or HLA gene in a reversible model. In a mammalian cell, for example, RNA can guide the Cas protein to a TCR and/or HLA promoter, sterically blocking RNA polymerases.

[00488] Artificial CRISPR/Cas systems can be generated that inhibit the TCR and/or HLA, using technologies known in the art, for example, those described in United States Publication No. 20140068797, and Cong (2013) Science 339: 819 -823. Other artificial CRISPR/Cas systems that are known in the art can also be generated that inhibit TCR and/or HLA, for example, those described in Tsai (2014) Nature Biotechnol., 32:6 569-576, United States Patent No. °s: 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359.

TALEN to inhibit TCR and/or HLA

[00489] "TALEN" or "TALEN for HLA and/or TCR" or "TALEN for inhibiting HLA and/or TCR" refers to a transcription activator-type effector nuclease, an artificial nuclease that can be used to edit the gene of HLA and/or TCR.

[00490] TALENs are artificially produced by fusing a TAL effector DNA binding domain to a DNA cleavage domain. Transcription activator-like effects (TALEs) can be created to bind any desired DNA sequence, including a portion of the HLA or TCR gene. By combining an engineered TALE with a DNA cleavage domain, a restriction enzyme can be produced that is specific to any desired DNA sequence, including an HLA or TCR sequence. These can then be introduced into a cell, where they can be used for genome editing. Boch (2011) Nature Biotech. 29: 135-6; and Boch et al. (2009) Science 326: 1509-12; Moscow et al. (2009) Science 326: 3501.

[00491] TALEs are proteins secreted by Xanthomonas bacteria. The DNA binding domain contains a highly conserved sequence of 33-34 amino acids, with the exception of the 12th and 13th amino acids. These two positions are highly variable, showing a strong correlation with specific nucleotide recognition. They can thus be created to bind a desired DNA sequence.

[00492] To produce a TALEN, a TALE protein is fused to a nuclease (N), which is a wild-type or mutated FokI endonuclease. Several FokI mutations have been made for its use in TALENs; these, for example, improve cleavage activity or specificity. Cermak et al. (2011) Nucl. Acids Res. 39: e82; Miller et al. (2011) Nature Biotech. 29: 143-8; Hockemeyer et al. (2011) Nature Biotech. 29: 731-734; Wood et al. (2011) Science 333: 307; Doyon et al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotech. 25: 786-793; and Guo et al. (2010) J. Mol. Biol. 200: 96.

[00493] The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains to target sites in the genome with appropriate orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for obtaining high levels of activity. Miller et al. (2011) Nature Biotech. 29: 143-8.

[00494] An HLA or TCR TALEN can be used within a cell to produce a double strand break (DSB). A mutation can be introduced into the break site if repair mechanisms improperly repair the break through non-homologous end ligation. For example, improper repair can introduce a frameshift mutation. Alternatively, foreign DNA can be introduced into the cell along with TALEN; Depending on the consequences of the foreign DNA and chromosomal sequence, this process can be used to correct a defect in the HLA or TCR gene or introduce such a defect into a wild-type TCR or HLA gene, thereby decreasing HLA or TCR expression. .

[00495] TALENs specific to HLA or TCR sequences can be constructed using any method known in the art, including various schemes using modular components. Zhang et al. (2011) Nature Biotech. 29: 149-53; Geibler et al. (2011) PLoS ONE 6: e19509.

Zinc finger nuclease to inhibit HLA and/or TCR

[00496] "ZFN" or "Zinc Finger Nuclease" or "ZFN for HLA and/or TCR" or "ZFN for inhibiting HLA and/or TCR" refers to a zinc finger nuclease, an artificial nuclease that can be used to edit the HLA and/or TCR gene.

[00497] Like a TALEN, a ZFN comprises a FokI nuclease domain (or derivative thereof) fused to a DNA binding domain. In the case of a ZFN, the DNA binding domain comprises one or more zinc fingers. Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Academic. Sci USA 93: 1156-1160.

[00498] A zinc finger is a small protein structural motif established by one or more zinc ions. A zinc finger may comprise, for example, Cys2His2, and may recognize an approximately 3-bp sequence. Several zinc fingers of known specificity can be combined to produce multifinger polypeptides of sequences of about 6, 9, 12, 15, or 18-bp. Various selection and modular assembly techniques are expected to generate zinc fingers (and combinations thereof) recognizing specific sequences, including phage display, yeast, one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells.

[00499] Like a TALEN, a ZFN must dimerize to cleave DNA. Thus, a pair of ZFNs is required to target non-palindromic DNA sites. The two individual ZFNs must bind opposite strands of DNA with their nucleases appropriately spaced apart. Bitinaite et al. (1998) Proc. Natl. Academic. Sci USA 95: 10570-5.

[00500] Also like a TALEN, a ZFN can create a double-strand break in DNA, which can create a frame-shift mutation if repaired improperly, inducing a decrease in the expression and quantity of HLA and/or TCR in a cell . ZFNs can also be used with homologous recombination to mutate the HLA or TCR gene.

[00501] ZFNs specific to HLA and/or TCR sequences can be constructed using any method known in the art. See, for example, Provasi (2011) Nature Med. 18: 807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al. (2008) Mol. Ther. 16: 1200-7; and Guo et al. (2010) J. Mol. Biol. 400: 96; United States Patent Publication 2011/0158957; and United States Patent Publication 2012/0060230.

Telomerase Expression

[00502] While not wishing to be bound by any particular theory, in some embodiments, a therapeutic T cell has short-term persistence in a patient, due to shortened telomeres in the T cell; consequently, transfection with a telomerase gene can lengthen T cell telomeres and improve T cell persistence in the patient. See, Carl June, "Adoptive T cell therapy for cancer in the clinic," Journal of Clinical Investigation, 117:1466-1476 (2007). Thus, in one embodiment, an immune effector cell, e.g., a T cell, ectopically expresses a telomerase subunit, e.g., the telomerase catalytic subunit, e.g., TERT, e.g., hTERT. In some aspects, this invention provides a method of producing a cell expressing CAR, comprising contacting a cell with a nucleic acid encoding a telomerase subunit, e.g., the telomerase catalytic subunit, e.g., TERT, e.g., hTERT. The cell can be contacted with the nucleic acid before, simultaneously with, or after being contacted with a construct encoding a CAR.

[00503] In one aspect, the invention features a method of preparing immune effector cell populations (e.g., T cells or NK cells). In one embodiment, the method comprises: providing immune effector cell populations (e.g., T cells or NK cells), contacting the immune effector cell populations with a nucleic acid encoding a CAR; and contacting immune effector cell populations with a nucleic acid encoding a telomerase subunit, e.g., hTERT, under conditions that allow expression of CAR and telomerase.

[00504] In one embodiment, the nucleic acid encoding the telomerase subunit is DNA. In one embodiment, the nucleic acid encoding the telomerase subunit comprises a promoter capable of regulating expression of the telomerase subunit.

[00505] In one embodiment, hTERT has the amino acid sequence of GenBank Protein ID AAC51724.1 (Meyerson et al., "hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cell and during Immortalization" Cell Volume 90, Issue 4, August 22, 1997, Pages 785-795) as follows: MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPA AFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCER GAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAW GLLLRRVGDDVLV HLLARCALFVLVAPSCAYQVCGPPLYQLGAATQA RPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSAS RSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVS PARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDT PCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFL L QKNRLFFYRKSVWSKLQSIGIRQHLKRVQL RELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTF RREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRT FVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCV RRYAVVQKAAHGHVRKAFKSHV STLTDLQPYMRQFVAHLQETSPLR DAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQG SILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTF LRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFP WCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLF D )

[00506] In one embodiment, the hTERT has a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 131. In an In one embodiment, hTERT has a sequence of SEQ ID NO: 131. In one embodiment, hTERT comprises a deletion (e.g., of no more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, at the C, or both. In one embodiment, the hTERT comprises a transgenic amino acid sequence (e.g., of no more than 5, 10, 15, 20, or 30 amino acids) at the N terminus, the terminus, or both.

[00507] In one embodiment, hTERT is encoded by the nucleic acid sequence from GenBank accession no. AF018167 (Meyerson et al., "hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cell and During Immortalization" Cell Volume 90, Issue 4, August 22, 1997, Pages 785-795): 1 caggcagcgt ggtcctgctg cgcacgtggg aagccctggc cccggccacc cccgcgatgc 61 cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcg cagcc actac cgcgaggtgc 121 tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg ctggcggctg gtgcagcgcg 181 gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtg ccctgggacg 241 cacggccgcc ccccgccgcc ccctccttcc gccaggtgtc ctgcctgaag gagctggtgg 301 cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaa cgtgctggcc ttcggcttcg 361 cgctgctgga cggggcccgc gggggccccc ccgaggcctt caccacca gc gtgcgcagct 421 acctgcccaa cacggtgacc gacgcactgc gggggagcgg ggcgtggggg ctgctgttgc 481 gccgcgtggg cgacgacgtg ctggttcacc tgctggcacg ctgcgcgctc tttgtgctgg 541 tggctcccag ctgcg cctac caggtgtgcg ggccgccgct gtaccagctc ggcgctgcca 601 ctcaggcccg gcccccgcca cacgctagtg gaccccgaag gcgtctggga tgcgaacggg 661 cctggaacca tagcgtcagg gaggccgggg tccccctggg cctgccagcc ccgggtgcga 721 ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcc caagaggccc aggcgtggcg 781 ctgcccctga gccggagcgg acgcccgttg ggcaggggtc ctgggcccac ccgggca gga 841 cgcgtggacc gagtgaccgt ggtttctgtg tggtgtcacc tgccagaccc gccgaagaag 901 ccacctcttt ggagggtgcg ctctctggca cgcgccactc ccacccatcc gtgggccgcc 961 agcaccacgc gggcccccca tccacatcgc ggccaccacg tccct gggac acgccttgtc 1021 ccccggtgta cgccgagacc aagcacttcc tctactcctc aggcgacaag gagcagctgc 1081 ggccctcctt cctactcagc tctctgaggc ccagcctgac tggcgctcgg aggctcgtgg 1141 agaccatctt tctgggttcc aggccctgga tgccagggac tccccgcagg ttgccccgcc 1201 tgccccagcg ctactggcaa atgcggcccc tgtttctgga gctgcttggg aaccacgcgc 1261 agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcg gtcaccccag 1321 cagccggtgt ctgtgcccgg gagaagcccc agggctctgt ggcggccccc gaggaggagg 1381 acacagaccc ccgtcgcctg gtgcagctgc tccgccagca cagcag cccc tggcaggtgt 1441 acggcttcgt gcgggcctgc ctgcgccggc tggtgccccc aggcctctgg ggctccaggc 1501 acaacgaacg ccgcttcctc aggaacacca agaagttcat ctccctgggg aagcatgcca 1561 agctctcgct gcaggagctg acgtggaaga tgagcgtgcg gggctgcgct tggctgcgca 1621 ggagcccagg ggttggctgt gttccggccg cagagcaccg tctgcgtgag gagatcctgg 1681 ccaagttcc t gcactggctg atgagtgtgt acgtcgtcga gctgctcagg tctttctttt 1741 atgtcacgga gaccacgttt caaaagaaca ggctcttttt ctaccggaag agtgtctgga 1801 gcaagttgca aagcattgga atcagacagc acttgaagag ggtgcagctg cgg gagctgt 1861 cggaagcaga ggtcaggcag catcgggaag ccaggcccgc cctgctgacg tccagactcc 1921 gcttcatccc caagcctgac gggctgcggc cgattgtgaa catggactac gtcgtgggag 1981 ccagaacgtt ccgcagagaa aagagggccg agcgtctcac ctcgagggtg aaggcactgt 2041 tcagcgtgct caactacgag cgggcgcggc gccccggcct cctgggcgcc tctgtgctgg 2101 gcctggacga tatccacagg gcctggcgca ccttcgtgct gcgtgtgcgg gcccaggacc 2161 cgccgcctga gctgtacttt gtcaaggtgg atgtgacggg cgcgtacgac accatccccc 2221 aggacaggct cacggaggtc atcgccagca tcatcaaacc ccagaacacg tactgcgtgc 22 81 gtcggtatgc cgtggtccag aaggccgccc atgggcacgt ccgcaaggcc ttcaagagcc 2341 acgtctctac cttgacagac ctccagccgt acatgcgaca gttcgtggct cacctgcagg 2401 agaccagccc gctgagggat gccgtcgtca tcgagcagag ctcctccctg aatgaggcca 2461 gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca ccacgccgtg cgcatcaggg 2521 gcaagtccta cgtccagtgc caggggatcc cgcagggctc catcctctcc acgctgctct 2581 gcagcctgtg ctacggcgac atggagaaca agctgtttgc ggggattcgg cgggacgggc 2641 tgctcctgcg tttggtggat gatttcttgt tggtgacacc tcacctcacc cacgcgaaaa 2701 ccttcctca g gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtg aacttgcgga 2761 agacagtggt gaacttccct gtagaagacg aggccctggg tggcacggct tttgttcaga 2821 tgccggccca cggcctattc ccctggtgcg gcctgctgct ggatacccgg accctggagg 2881 tgcagagcga ctactccagc tatgcccgga cctccatcag agccagtctc accttcaacc 2941 gcggcttcaa ggctgggagg aacatgcgtc gcaaactctt tgg ggtcttg cggctgaagt 3001 gtcacagcct gtttctggat ttgcaggtga acagcctcca gacggtgtgc accaacatct 3061 acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcag ctcccatttc 3121 atcagcaagt ttggaagaac cccacat ttt tcctgcgcgt catctctgac acggcctccc 3181 tctgctactc catcctgaaa gccaagaacg cagggatgtc gctgggggcc aagggcgccg 3241 ccggccctct gccctccgag gccgtgcagt ggctgtgcca ccaagcattc ctgctcaagc 3301 tgactcgaca ccgtgtcacc tacgtgccac tcctggggtc actcaggaca gcccagacgc 3361 agctgagtcg gaagctcccg gggacgacgc tgactgccct ggaggccgca gccaacccgg 3421 cactgccctc agacttcaag accatcctgg actgatggcc acccgcccac agccaggccg 3481 agagcagaca ccagcagccc tgtcacgccg ggctctacgt cccagggagg gaggggcggc 3541 ccacacccag gcccgcaccg ctgggagtct gaggcctgag tga gtgtttg gccgaggcct 3601 gcatgtccgg ctgaaggctg agtgtccggc tgaggcctga gcgagtgtcc agccaagggc 3661 tgagtgtcca gcacacctgc cgtcttcact tccccacagg ctggcgctcg gctccacccc 3721 agggccagct tttcctcacc aggagcccgg cttccactcc ccacatagga atagtccatc 3781 cccagattcg ccattgttca cccctcgccc tgccctcctt tgccttccac ccccaccatc 384 2 aaaaaaaa 4021 aaaaaaa (SEQ ID NO: 132)

[00508] In one embodiment, hTERT is encoded by a nucleic acid having a sequence at least 80%, 85%, 90%, 95%, 96, 97%, 98%, or 99% identical to the sequence of SEQ ID NO : 132. In one embodiment, hTERT is encoded by a nucleic acid of SEQ ID NO: 132.

Activation and Expansion of immune effector cells (e.g., T cells)

[00509] Immune effector cells such as T cells can be activated and expanded generally using methods as described, for example, in United States Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and United States Patent Application Publication No. 20060121005.

[00510] The ex vivo expansion procedure of hematopoietic stem and progenitor cells described in United States Patent No. 5,199,942, incorporated herein by reference, can be applied to the cells of the present invention. Other suitable methods are known in the art, therefore, the present invention is not limited to any particular method of ex vivo cell expansion. Briefly, ex vivo T cell culture and expansion may comprise: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from peripheral blood collections or bone marrow explants; and (2) expand such cells ex vivo. In addition to the cell growth factors described in United States Patent No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 and c-kit ligand can be used for cell culture and expansion.

[00511] Generally, immune effector cell populations, for example, cells depleted by regulatory T cells, can be expanded by contact with a surface having attached to it an agent that stimulates a signal associated with the CD3/TCR complex and a ligand that stimulates a costimulatory molecule on the surface of T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD3 antibody. CD2 immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of an accessory molecule on the surface of T cells, a ligand that binds the accessory molecule is used. For example, T cell populations can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under appropriate conditions to stimulate T cell proliferation. To stimulate the proliferation of CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besançon, France) can be used as other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975- 3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Met. 227(1-2):53-63, 1999).

[00512] In certain aspects, the primary stimulatory signal and the costimulatory signal regarding the T cell can be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, agents can be coupled to the same surface (i.e., in "cis" formation) or to separate surfaces (i.e., in "trans" formation). Alternatively, one agent can be coupled to a surface and to another agent in the solution. In one aspect, the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activating signal is in solution or coupled to a surface. In certain respects, both agents may be in solution. In one aspect, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent that will bind to the agents. In this regard, see, for example, United States Patent Application Publications Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present invention.

[00513] In one aspect, the two agents are immobilized in the accounts, either in the same account, i.e., "cis," or in separate accounts, i.e., "trans." By way of example, the agent providing the primary activating signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the co-stimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof. same; and both agents are co-immobilized at the same rate in equivalent molecular amounts. In one aspect, a 1:1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell growth is used. In certain aspects of the present invention, a ratio of anti-CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed compared to the expansion observed using a 1:1 ratio. In one particular aspect, an increase of about 1 to about 3 times is observed compared to the expansion observed using a 1:1 ratio. In one aspect, the ratio of CD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100 and every integer value in between. In one aspect, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, that is, the ratio of CD3:CD28 is lower than that. In certain aspects, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the beads is greater than 2:1. In a particular aspect, a 1:100 ratio of CD3:CD28 antibody bound to the beads is used. In one aspect, a 1:75 CD3:CD28 ratio of antibody bound to the beads is used. In another aspect, a 1:50 CD3:CD28 ratio of antibody bound to the beads is used. In one aspect, a 1:30 CD3:CD28 ratio of antibody bound to the beads is used. In a preferred aspect, a 1:10 ratio of CD3:CD28 antibody bound to the beads is used. In one aspect, a 1:3 ratio of CD3:CD28 antibody bound to the beads is used. In yet another aspect, a 3:1 ratio of CD3:CD28 antibody bound to the beads is used.

[00514] Particle-to-cell ratios of 1:500 to 500:1 and any integer value in between can be used to stimulate T cells or other target cells. As those skilled in the art can readily appreciate, the ratio of particles to cells may depend on the particle size relative to the target cell. For example, small size beads would only turn on a few cells, while larger beads would turn on many. In certain aspects, the ratio of cells to particles ranges from 1:100 to 100:1 and any integer value therebetween, and in other aspects, the ratio comprises 1:9 to 9:1 and any integer value therebetween. , can also be used to stimulate T cells. The ratio of anti-CD3- and anti-CD28-coupled particles to T cells that results in T cell stimulation can vary as noted above include 1:100, 1:50, 1 :40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1 , 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1 with a preferred ratio being at least 1 :1 particles per T cell. In one aspect, a particle to cell ratio of 1:1 or less is used. In a particular aspect, a preferred particle:cell ratio is 1:5. In other aspects, the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one aspect, the ratio of particles to cells is 1:1 to 10:1 on the first day and additional particles are added to the cells every day or every other day thereafter up to 10 days, at final ratios. from 1:1 to 1:10 (based on cell counts on the day of addition). In a particular aspect, the ratio of particles to cells is 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In one aspect, particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1:5 on the third and fifth days of stimulation. In one aspect, the particle to cell ratio is 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In one aspect, particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1:10 on the third and fifth days of stimulation. One skilled in the art will appreciate that a variety of other relationships may be suitable for use in the present invention. In particular, the ratios will vary depending on particle size and cell size and type. In one aspect, the most typical ratios for use are in the neighborhood of 1:1, 2:1 and 3:1 on the first day.

[00515] In other aspects, cells, such as T cells, are combined with agent-coated beads, the beads and cells are subsequently separated, and then the cells are cultured. In an alternative aspect, prior to culturing, the agent-coated beads and cells are not separated, but are cultured together. In another aspect, the beads and cells are first concentrated by applying a force, such as a magnetic force, resulting in increased binding of cell surface markers, thereby inducing cellular stimulation.

[00516] By way of example, cell surface proteins can be linked allowing the paramagnetic beads to which anti-CD3 and anti-CD28 are linked (3x28 beads) to contact T cells. In one aspect, the cells (e.g., 104 to 109 T cells) and beads (e.g., DYNABEADS® M-450 CD3/CD28 T paramagnetic beads in a 1:1 ratio) are combined in a buffer, e.g., PBS (without divalent cations such as calcium and magnesium) . Again, those skilled in the art can readily appreciate that any cell concentration can be used. For example, the target cell may be very rare in the sample and comprises only 0.01% of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the scope of the present invention. In certain aspects, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase cell concentration), to ensure maximum cell and particle contact. For example, in one aspect, a concentration of about 10 billion cells/mL, 9 billion/mL, 8 billion/mL, 7 billion/mL, 6 billion/mL, 5 billion/mL, or 2 billion cells/mL mL is used. In one aspect, more than 100 million cells/mL are used. In another aspect, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In still one aspect, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/mL is used. In other aspects, concentrations of 125 or 150 million cells/mL can be used. Use of high concentrations may result in increased cell production, cell activation, and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and would be desirable to obtain in certain respects. For example, using high concentration of cells allows for more efficient CD8+ T cell selection than normally weaker CD28 expression.

[00517] In one embodiment, cells transduced with a nucleic acid encoding a CAR, for example, a CAR described here, are expanded, for example, by a method described here. In one embodiment, cells are expanded in culture over a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days). In one embodiment, the cells are expanded over a period of 4 to 9 days. In one embodiment, the cells are expanded over a period of 8 days or less, e.g., 7, 6, or 5 days. In one embodiment, the cells, for example, a CD19 CAR cell described here, 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. . Potency can be defined, for example, by various T cell functions, e.g., proliferation, target cell killing, cytokine production, activation, migration, or combinations thereof. In one embodiment, cells, e.g., a CD19 CAR cell described herein, expanded for 5 days show at least a one-, two-, three-, or four-fold increase in cell doublings under antigen stimulation compared to the same cells expanded in culture for 9 days under the same culture conditions. In one embodiment, cells, e.g., cells expressing a CD19 CAR described here, are expanded in culture for 5 days, and the resulting cells exhibit elevated pro-inflammatory cytokine production, e.g., levels of IFN-Y and /or GM-CSF, compared to the same cells expanded in culture for 9 days under the same culture conditions. In one embodiment, cells, e.g., a CD19 CAR cell described herein, expanded for 5 days show at least a one-, two-, three-, four-, five-, ten-fold or greater increase in pg/mL of cytokine production. pro-inflammatory, e.g., levels of IFN-Y and/or GM-CSF, compared to the same cells expanded in culture for 9 days under the same culture conditions.

[00518] Several cycles of stimulation may also be desired so that T cell culture time may be 60 days or more. Appropriate conditions for T cell culture include appropriate media (e.g., Minimum Essential Media or RPMI 1640 Media or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., serum fetal bovine or human), interleukin-2 (IL-2), insulin, IFN-Y, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFß, and TNF- a or any other cell growth additives known to the skilled artisan. Other cell growth additives include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media may include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, or serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined group of hormones, and/or an amount of cytokine(s) sufficient for T cell growth and expansion. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cell cultures that should not be infused into an individual. Target cells are maintained under conditions necessary to support growth, e.g., an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).

[00519] In one embodiment, cells are expanded by appropriate media (e.g., the media described herein) that include one or more interleukins that results in at least a 200-fold increase (e.g., 200-fold, 250-fold, 300-fold , 350 times) in cells over a 14-day period of expansion, for example, as measured by a method described herein such as flow cytometry. In one embodiment, cells are expanded in the presence of IL-15 and/or IL-7 (e.g., IL-15 and IL-7).

[00520] In embodiments, methods described herein, e.g., CAR-expressing cell manufacturing methods, comprise removing regulatory T cells, e.g., CD25+ T cells, from a cell population, e.g., using an anti-antibody. - CD25, or fragment thereof, or a CD25-binding ligand, IL-2. Methods of removing regulatory T cells, e.g., CD25+ T cells, from a cell population are described herein. In embodiments, the methods, e.g., manufacturing methods, also comprise contacting a cell population (e.g., a cell population in which regulatory T cells, such as CD25+ T cells, have been depleted; or a cell population that an anti-CD25 antibody, fragment thereof, or CD25 binding ligand) was previously contacted with IL-15 and/or IL-7. For example, the cell population (e.g., which has previously been contacted with an anti-CD25 antibody, fragment thereof, or CD25 binding ligand) is expanded in the presence of IL-15 and/or IL-7.

[00521] In some embodiments, a CAR-expressing cell described herein is contacted with a composition comprising an interleukin-15 polypeptide (IL-15), an interleukin-15 alpha receptor polypeptide (IL-15Ra), or a combination of either an IL-15 polypeptide or an IL-15Ra polypeptide, e.g., hetIL-15, during manufacture of the CAR-expressing cell, e.g., ex vivo. In embodiments, a CAR-expressing cell described herein is contacted with a composition comprising an IL-15 polypeptide during manufacture of the CAR-expressing cell, e.g., ex vivo. In embodiments, a CAR-expressing cell described herein is contacted with a composition comprising a combination of both an IL-15 polypeptide and an IL-15 Ra polypeptide during manufacture of the CAR-expressing cell, e.g., ex vivo. In embodiments, a CAR-expressing cell described herein is contacted with a composition comprising hetIL-15 during manufacture of the CAR-expressing cell, e.g., ex vivo.

[00522] In one embodiment the CAR expressing cell described here is contacted with a composition comprising hetIL-15 during ex vivo expansion. In one embodiment, the CAR-expressing cell described herein is contacted with a composition comprising an IL-15 polypeptide during ex vivo expansion. In one embodiment, the CAR-expressing cell described herein is contacted with a composition comprising both an IL-15 polypeptide and an IL-15Ra polypeptide during ex vivo expansion. In one embodiment, the contact results in the survival and proliferation of a lymphocyte subpopulation, e.g., CD8+ T cells.

[00523] T cells that have been exposed to varying times of stimulation can exhibit different characteristics. For example, mononuclear cell products from typical blood or apheresis peripheral blood have a helper T cell population (TH, CD4+) that is larger than the cytotoxic or suppressor T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces T cell populations that before about 8 to 9 days consist predominantly of TH cells, while after about 8 to 9 days, T cell populations consist predominantly of TH cells. T cell comprises an ever-increasing population of TC cells. Consequently, depending on the treatment purpose, infusing an individual with a T cell population that predominantly comprises TH cells may be advantageous. Similarly, if a specific subset of TC cell antigen has been isolated, it may be beneficial to expand this subset to a greater degree.

[00524] Furthermore, in addition to the CD4 and CD8 markers, other phenotypes can vary significantly, but to a large extent, reproducibly during the course of a cell expansion process. Thus, such reproducibility allows for the ability to tailor an activated T cell product for specific purposes.

[00525] In other embodiments, the preparation method described here also comprises contacting immune effector cell populations with a nucleic acid encoding a telomerase subunit, for example, hTERT. The nucleic acid encoding the telomerase subunit may be DNA.

[00526] In some embodiments, a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) is added during the CAR cell manufacturing process. According to the non-limiting theory here, the kinase inhibitor can improve the quality of the cell populations produced. For example, CAR-expressing cells are often produced from a cancer patient's own plasma apheresis sample, which may contain cancer cells, and the kinase inhibitor may alter signaling in those cancer cells (e.g., a cancer expressing BTK such as CLL or MCL), for example, reducing their proliferation or increasing levels of apoptosis. As another example, the kinase inhibitor may alter signaling in cells expressing CAR (or immune effector cells before they express CAR), for example, by inhibiting ITK in T cells. The kinase inhibitor may shift the balance of T cells from TH2 cells to TH1 cells.

[00527] The kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) can be added to the reaction mixture at a level sufficient to inhibit its target, e.g., BTK. In some embodiments, the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) is added at a concentration of about 0.10.2, 0.2-0.5, 0.5-1, 1- 2, 2-5, or 5-10 µM. In some embodiments, the kinase inhibitor is a covalent inhibitor (such as ibrutinib) and a short pulse is sufficient to irreversibly inactivate the target while avoiding non-specific toxicity. Accordingly, the kinase inhibitor can be added, for example, 10 to 20, 20 to 30, 30 to 40, 40 to 60, or 60 to 120 minutes. The kinase inhibitor may also be added over longer periods of time, for example, if the kinase inhibitor has a non-covalent action. Thus, the kinase inhibitor can be added, for example, for 2 to 4, 4 to 6, 6 to 8, 8 to 12, 12 to 18, or 18 to 24 hours, or for 1 to 2, 2 to 3 , 3 to 4, 4 to 6, 6 to 8, 8 to 10 days, or for the entire length of time that the cells are being cultured. The kinase inhibitor can be added at various points during the manufacturing process, for example, after harvesting the cells, before bead stimulation, after bead stimulation, before transduction, after transduction, or before administration of the cells. to the patient. In some embodiments, the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) is added after harvesting the cells or prior to stimulation, e.g., with beads. Before and after in this context may refer to, for example, about 1, 5, 15, 30, 45, or 60 minutes before or after, or 1, 2, 3, 4, 5, or 6 hours before or after. after.

[00528] Once a CD19 CAR is constructed, various assays can be used to evaluate the activity of the molecule, such as, but not limited to, the ability to expand T cells upon antigen stimulation, sustain T cell expansion in the absence of re-stimulation, and anticancer activities in in vitro and animal models. Assays to evaluate the effects of a CD19 CAR are described in greater detail below.

[00529] Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, T cells (1:1 mixture of CD4+ and CD8+ T cells) expressing the CARs are expanded in vitro for more than 10 days followed by lysis and SDS-PAGE under reducing conditions. CARs containing the full-size TCR-Z cytoplasmic domain and the endogenous TCR-Z chain are detected by western blotting using an antibody to the TCR-Z chain. The same T cell subsets are used for SDS-PAGE analysis under non-reducing conditions to allow assessment of covalent dimer formation.

[00530] In vitro expansion of CAR+ T cells following antigen stimulation can be measured by flow cytometry. For example, a mixture of CD4+ and CD8+ T cells is stimulated with aCD3/aCD28 beads followed by transduction with lentiviral vectors expressing GFP under the control of the promoters to be analyzed. Exemplary promoters include the CMV IE, EF-1a, ubiquitin C, or phosphoglycerokinase (PGK) gene promoters. GFP fluorescence is assessed on day 6 of culture on CD4+ and/or CD8+ T cell subsets by flow cytometry. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Alternatively, a mixture of CD4+ and CD8+ T cells is stimulated with aCD3/aCD28-coated magnetic beads on day 0, and transduced with CAR on day 1 using a bicistronic lentiviral vector expressing CAR together with eGFP using a 2A ribosomal skipping sequence. . Cultures are restimulated with CD19+ K562 cells (K562-CD19), wild-type K562 cells (K562 wild-type), or K562 cells expressing hCD32 and 4-1BBL in the presence of anti-CD3 and anti-CD28 antibody (K562- BBL-3/28) after washing. Exogenous IL-2 is added to cultures every two days at 100 IU/mL. GFP+ T cells are enumerated by flow cytometry using bead-based counting. See, for example, Milone et al., Molecular Therapy 17(8): 14531464 (2009).

[00531] Sustained CAR+ T cell expansion in the absence of restimulation can also be measured. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, mean T cell volume (fl) is measured on day 8 of culture using a Coulter Multisizer particle counter, a Nexcelom Cellometer Vision, or Millipore Scepter, after stimulation with aCD3/aCD28-coated magnetic beads on day 0, and transduction with the CAR indicated on day 1.

[00532] Animal models can also be used to measure CART activity. For example, xenograft model using CD19-specific CAR+ T cells to treat primary human pre-B ALL in immunodeficient mice can be used. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, after establishment of ALL, mice are randomized to treatment groups. Different numbers of T cells created by aCD19-Z and aCD19-BB-Z are co-injected at a 1:1 ratio into NOD-SCID-Y-/- mice carrying B-ALL. The vector copy number of aCD19-Z and aCD19-BB-Z in mouse spleen DNA is assessed at various times after T cell injection. Animals are assessed for leukemia at weekly intervals. Peripheral blood CD19+ B-ALL immature cell counts are measured in mice that are injected with aCD19-Z CAR+ T cells or mock-transduced T cells. Survival curves for the groups are compared using the logrank test. In addition, absolute peripheral blood CD4+ and CD8+ T cell counts 4 weeks after T cell injection into NOD-SCID-Y-/- mice can also be analyzed. Mice are injected with leukemic cells and 3 weeks later are injected with T cells engineered to express CAR by a bicistronic lentiviral vector encoding CAR linked to eGFP. T cells are normalized to 45 to 50% GFP+ T cell input by mixing with mock-transduced cells prior to injection, and confirmed by flow cytometry. Animals are evaluated for leukemia at 1-week intervals. Survival curves for CAR+ T cell groups are compared using the logrank test.

[00533] The dose-dependent CAR treatment response can be evaluated. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). For example, peripheral blood is obtained 35 to 70 days after establishment of leukemia in mice injected on day 21 with CAR T cells, an equivalent number of sham-transduced T cells, or no T cells. Mice from each group are randomly bled for determination of peripheral blood CD19+ ALL blast counts and then killed on days 35 and 49. The remaining animals are evaluated on days 57 and 70.

[00534] Assessment of cell proliferation and cytokine production has been previously described, for example, in Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, assessment of CAR-mediated proliferation is performed in microtiter plates by mixing washed T cells with K562 cells expressing CD19 (K19) or CD32 and CD137 (KT32-BBL) for a final T cell:K562 ratio of 2:1. K562 cells are irradiated with gamma radiation before use. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultures with KT32-BBL cells to function as a positive control for stimulation of T cell proliferation as these signals support CD8+ T cell expansion long-term ex vivo. T cells are enumerated in cultures using CountBright™ fluorescent beads (Invitrogen, Carlsbad, CA) and flow cytometry as described by the manufacturer. CAR+T cells are identified by GFP expression using T cells that are created with antiviral vectors expressing CAR linked to eGFP-2A. For CAR+ T cells not expressing GFP, CAR+ T cells are detected with biotinylated recombinant CD19 protein and a secondary avidin-PE conjugate. Expression of CD4+ and CD8+ on T cells is also simultaneously detected with specific monoclonal antibodies (BD Biosciences). Cytokine assessments are performed on supernatants collected 24 hours after restimulation using the Human TH1/TH2 Cytometric Cytometric Bead Array Kit (BD Biosciences, San Diego, CA) according to the manufacturer's instructions. Fluorescence is assessed using a FACScalibur flow cytometer, and data is analyzed according to the manufacturer's instructions.

[00535] Cytotoxicity can be assessed by a standard 51Cr release assay. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, target cells (K562 lines and primary pro-B-ALL cells) are loaded with 51Cr (as NaCrO4, New England Nuclear, Boston, MA) at 37oC for 2 hours with frequent shaking, washed twice in complete RPMI, and placed in microtiter plates. Effector T cells are mixed with target cells in the wells in full RPMI at varying effector cell:target cell (E:T) ratios. Additional wells containing media alone (spontaneous release, SR) or a 1% solution of Triton-X 100 detergent (total release, TR) are also prepared. After 4 hours of incubation at 37oC, the supernatant from each well is collected. The released 51Cr is then measured using a gamma particle counter (Packard Instrument Co., Waltham, MA). Each condition is performed in at least triplicates, and the percentage of lysis is calculated using the formula: % Lysis = (ER- SR) / (TR - SR), where ER represents the average of 51Cr released for each experimental condition.

[00536] Imaging technologies can be used to evaluate the proliferation and specific trafficking of CARs in tumor-carrying animal models. Such assays have been described, for example, in Barrett et al., Human Gene Therapy 22:1575-1586 (2011). Briefly, NOD/SCID/Yc-/-(NSG) mice are injected IV with Nalm-6 cells after 7 days, then with T cells 4 hours after electroporation with the CAR constructs. T cells are stably transfected with a lentiviral construct to express firefly luciferase, and mice are imaged for bioluminescence. Alternatively, the therapeutic efficacy and specificity of a single injection of CAR+ T cells into a Nalm-6 xenograft model can be measured as follows: NSG mice are injected with cells transduced by Nalm-6 to stably express firefly luciferase, followed by a single tail vein injection of CD19 CAR-electroporated T cells 7 days later. Animals are imaged at various time points after injection. For example, photon density heatmaps of luciferase positive firefly leukemia in representative mice on day 5 (2 days before treatment) and day 8 (24 hours after CAR+ PBLs) can be generated.

[00537] Other assays, including those described in the Example section here, as well as those that are known in the art can also be used to evaluate the CD19 CAR constructs of the invention.

Kinase Inhibitor

[00538] In one embodiment, the kinase inhibitor is a CDK4 inhibitor, for example, a CDK4 inhibitor described herein, for example, a CDK4/6 inhibitor, such as, for example, 6-acetyl-8 hydrochloride - cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido [2,3-d]pyrimidin-7-one, (also referred to as palbociclib or PD0332991). In one embodiment, the kinase inhibitor is a BTK inhibitor, for example, a BTK inhibitor described herein, such as, for example, ibrutinib. In one embodiment, the kinase inhibitor is an mTOR inhibitor, for example, an mTOR inhibitor described herein, such as, for example, rapamycin, a rapamycin analogue, OSI-027. The mTOR inhibitor may be, for example, an mTORC1 inhibitor and/or an mTORC2 inhibitor, for example, an mTORC1 inhibitor and/or mTORC2 inhibitor described herein. In one embodiment, the kinase inhibitor is an MNK inhibitor, for example, an MNK inhibitor described herein, such as, for example, 4-amino-5-(4-fluoroanilino)-pyrazole [3,4-d] pyrimidine. The MNK inhibitor may be, for example, an inhibitor of MNK1a, MNK1b, MNK2a and/or MNK2b.

[00539] In one embodiment, the kinase inhibitor is a CDK4 inhibitor selected from alloysin 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- hydrochloride 1-benzopyran-4-one, (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 acid [ 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-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine (AZD5438); and XL281 (BMS908662).

[00540] In one embodiment, the kinase inhibitor is a CDK4 inhibitor, for example, palbociclib (PD0332991), and the 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 over a period of time, e.g. daily for 14 to 21 days of a 28-day cycle, or daily for 7 to 12 days of a 21-day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of palbociclib are administered.

[00541] An exemplary CDK4/6 inhibitor is LEE011 (also called ribociclib), the structure of which is shown below.

[00542] Without being bound by theory, it is believed that administering a CAR-expressing cell described here with a CDK4/6 inhibitor (e.g., LEE011 or another CDK4/6 inhibitor described here) can achieve elevated responsiveness, e.g. for example, with high remission rates and/or lower relapse rates, for example, compared to a CDK4/6 inhibitor alone.

[00543] While not wishing to be bound by theory, in some embodiments the CDK4/6 inhibitor acts to reduce cyclin D1 activity in cancer cells, for example, MCL cells. Some cancer cells are characterized by elevated cyclin D1 levels due to a translocation. CDK4 complexes with cyclin D to promote cell cycle progression. Accordingly, in some embodiments, administration of the CK4/6 inhibitor reduces cancer cell proliferation. See, for example, Marzec et al., "Mantle cell lymphoma cells express predominantly cyclin D1a isoform and are highly sensitive to selective inhibition of CDK4 kinase activity." Blood. 2006 September 1;108(5):1744-50. Epub May 11, 2006.

[00544] 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 BTK inhibitor does not reduce or inhibit the kinase activity of interleukin 2-inducible kinase (ITK), and is selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO- 4059; CNX-774; and LFM-A13.

[00545] In one embodiment, the kinase inhibitor is a BTK inhibitor, for example, ibrutinib (PCI-32765), and the 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 over a period of time, for example, daily during a 21-day cycle, or daily during 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.

[00546] In embodiments, the BTK inhibitor (e.g., ibrutinib) is administered to an individual who has CLL, lining cell lymphoma (MCL), or small lymphocytic lymphoma (SLL). For example, the individual to whom the BTK inhibitor is administered has a deletion on the short branch of chromosome 17 (del(17p), for example, in a leukemic cell). In other examples, the individual to whom the BTK inhibitor is administered does not have a del(17p). In embodiments, the individual to whom the BTK inhibitor is administered has relapsed into CLL or SLL, e.g., the individual has previously been administered a cancer therapy (e.g., has previously been administered one, two, three, or four cancer therapies). ). In embodiments, the individual to whom the BTK inhibitor is administered has refractory CLL or SLL. In other embodiments, the individual to whom the BTK inhibitor is administered has follicular lymphoma, e.g., relapsed or refractory follicular lymphoma.

[00547] The structure of ibrutinib (1- [(3R)-3- [4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo [3,4-d]pyrimidin-1-yl]piperidin-1- il]prop-2-en-1-one) is shown below.

[00548] Ibrutinib concentrations exceeding approximately 400 nM are not clinically relevant. In the study by Advani et al (J Clin Oncol 2012;31:88), the mean peak serum concentration of ibrutinib was about 130ng/mL, which is equivalent to 295 nM (based on the ibrutinib molecular weight of 440.5 ). Therefore, the data show the effect of ibrutinib on T cells at a concentration of 1uM that significantly exceeds the clinically relevant concentration in vivo.

[00549] In one embodiment, the kinase inhibitor is an mTOR inhibitor selected from temsirolimus; ridaforolimus (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-azatricycle [ 30.3.1.04.9] hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669; 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-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3- d]pyrimidin-7(8H )-one (PF04691502); and N2- [1,4-dioxo-4- [ [4-(4-oxo-8-phenyl-4 H-1-benzopyran-2-yl)morpholinium4-yl]methoxy]butyl]-L-arginylglycyl-L-a - aspartyl-serine-, internal salt (SF1126); and XL765.

[00550] In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., rapamycin, and the rapamycin is administered in 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 over 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 over a period of time, e.g. daily during 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.

[00551] 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-17804452; and 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine.

[00552] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, is administered to an individual in combination with a phosphoinositide 3-kinase (PI3K) inhibitor. ) (e.g., a PI3K inhibitor described herein, e.g., idelalisib or duvelisib) and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with idelalisib and rituximab. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with duvelisib and rituximab. Idelalisib (also called GS-1101 or CAL-101; Gilead) is a small molecule that blocks the delta isoform of PI3K. The structure of idelalisib (5-Fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinone) is shown below.

[00553] Duvelisib is a small molecule that blocks PI3K-d,Y. The structure of duvelisib (8-Chloro-2-phenyl-3- [(1S)-1-(9H-purin-6-ylamino)ethyl]-1(2H)-isoquinolinone) is shown below.

[00554] In embodiments, the individual has CLL. In embodiments, the individual has relapsed CLL, for example, the individual has previously been administered a cancer therapy (e.g., was previously administered an anti-CD20 antibody or was previously administered ibrutinib). For example, the individual has a deletion on the short branch of chromosome 17 (del(17p), for example, in a leukemic cell). In other examples, the individual does not have a del(17p). In embodiments, the individual comprises a leukemic cell comprising a mutation in the immunoglobulin heavy chain variable region (IgVH) gene. In other embodiments, the individual does not comprise a leukemic cell comprising a mutation in the immunoglobulin heavy chain variable region (IgVH) gene. In embodiments, the individual has a deletion on the long branch of chromosome 11 (del(11q)). In other embodiments, the individual does not have a del(11q). In embodiments, idelalisib is administered at a dosage of about 100 to 400 mg (e.g., 100 to 125, 125 to 150, 150 to 175, 175 to 200, 200 to 225, 225 to 250, 250 to 275, 275 to 300, 325 to 350, 350 to 375, or 375 to 400 mg), for example, BID. In embodiments, duvelisib is administered at a dosage of about 15 to 100 mg (e.g., about 15 to 25, 25 to 50, 50 to 75, or 75 to 100 mg), for example, twice daily. In embodiments, rituximab is administered at a dosage of about 350 to 550 mg/m2 (e.g., 350 to 375, 375 to 400, 400 to 425, 425 to 450, 450 to 475, or 475 to 500 mg/m2) , for example, intravenously.

[00555] In one embodiment, the kinase inhibitor is a dual phosphatidylnositol 3-kinase (PI3K) and mTOR inhibitor selected from 2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6- (6-methoxy-3-pyridinyl)-4-methyl-pyrido [2,3-d]pyrimidin-7(8H)-one (PF-04691502); N-[4-[[4-(Dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N'-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl )phenyl]urea (PF-05212384, PKI-587); 2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1- yl]phenyl}propanenitrile (BEZ-235); apitolisib (GDC-0980, RG7422); 2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide (GSK2126458); 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo [4,5-c] maleic acid quinolin-2(3H)-one (NVP-BGT226); 3- [4-(4- Morpholinylpyrido [3',2':4,5]furo [3,2-d]pyrimidin-2-yl]phenol (PI-103); 5-(9- isopropyl-8- methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine (VS-5584, SB2343); and N-[2-[(3,5-Dimethoxyphenyl)amino]quinoxalin-3-yl]- 4-[(4-methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide (XL765).

[00556] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, is administered to an individual in combination with an anaplastic lymphoma kinase (ALK) inhibitor. . Exemplary ALK kinase inhibitors include, but are not limited to (Pfizer), ceritinib (Novartis), alectinib (Chugai), brigatinib (also called AP26113; Ariad), entrectinib (Ignyta), PF-06463922 (Pfizer), TSR-011 (Tesaro) (see, for example, Clinical Trial Identifier No. NCT02048488), CEP-37440 (Teva), and X-396 (Xcovery). In some embodiments, the individual has a solid cancer, e.g., a solid cancer described herein, e.g., lung cancer.

[00557] The chemical name of crizotinib is 3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2 -the mine. The chemical name of ceritinib is 5-Chloro-N2-[2-isopropoxy-5-methyl-4-(4-piperidinyl)phenyl]-N4-[2-(isopropylsulfonyl)phenyl]-2,4-pyrimidinediamine. The chemical name of alectinib is 9-ethyl-6,6-dimethyl-8-(4-morpholinopiperidin-1-yl)-11-oxo-6,11-dihydro-5H-benzo[b]carbazol-3- carbonitrile. The chemical name of brigatinib is 5-Chloro-N2-{4-[4-(dimethylamino)-1-piperidinyl]-2-methoxyphenyl}-N4-[2-(dimethylphosphoryl)phenyl]-2,4-pyrimidinediamine. The chemical name of entrectinib is N-(5-(3,5-difluorobenzyl)-1H-indazol-3-yl)-4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H- pyran-4-yl)amino)benzamide. The chemical name of PF-06463922 is (10R)-7-Amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4- (methene)pyrazole [4,3-h] [2,5,11]-benzoxadiazacyclotetradecin-3-carbonitrile. The chemical structure of CEP-37440 is (S)-2-((5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-1-methoxy-6,7,8 ,9-tetrahydro-5H-benzo[7]annulen-2-yl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide. The chemical name of X-396 is (R)-6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-N-(4-(4-methylpiperazine-1-carbonyl)phenyl )pyridazine-3-carboxamide.

[00558] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, is administered to an individual in combination with an indoleamine 2,3-dioxygenase inhibitor. (GONE). IDO is an enzyme that catalyzes the degradation of the amino acid, L-tryptophan, into kynurenine. Many cancers overexpress IDO, for example, prostate, colorectal, pancreatic, cervical, gastric, ovarian, head, and lung cancer. pDCs, macrophages, and dendritic cells (DCs) can express IDO. Without being bound by theory, it is believed that a decrease in L-tryptophan (e.g., catalyzed by IDO) results in an immunosuppressive environment inducing T cell anergy and apoptosis. Thus, without being bound by theory, it is believed that an IDO inhibitor may enhance the efficacy of a CAR-expressing cell described here, for example, by decreasing the suppression or death of a CAR-expressing immune cell. In embodiments, the individual has a solid tumor, e.g., a solid tumor described herein, e.g., prostate, colorectal, pancreatic, cervical, gastric, ovarian, head, or lung cancer. Exemplary IDO inhibitors include, but are not limited to, 1-methyl-tryptophan, indoximod (NewLink Genetics) (see, e.g., Clinical Trial Identifier Nos. NCT01191216; NCT01792050), and INCB024360 (Incyte Corp.) (see, for example, Clinical Trial Identifiers Nos. NCT01604889; NCT01685255)

[00559] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, is administered to an individual in combination with a myeloid-derived suppressor cell modulator ( MDSCs). MDSCs accumulate at the periphery and tumor site of many solid tumors. These cells suppress T cell responses, thereby preventing the effectiveness of CAR-expressing cell therapy. Without being bound by theory, administration of an MDSC modulator is believed to enhance the efficacy of a CAR-expressing cell described here. In one embodiment, the subject has a solid tumor, e.g., a solid tumor described herein, e.g., glioblastoma. Exemplary modulators of MDSCs include, but are not limited to, MCS110 and BLZ945. MCS110 is a monoclonal antibody (mAb) against macrophage colony-stimulating factor (M-CSF). See, for example, Clinical Trial Identifier # NCT00757757. BLZ945 is a small molecule inhibitor of colony stimulating factor receptor (CSF1R). See, for example, Pyonteck et al. Nat. Med. 19(2013):1264-72. The structure of BLZ945 is shown below.

[00560] In some embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, is administered to an individual in combination with an interleukin-15 polypeptide (IL -15), an interleukin-15 alpha receptor polypeptide (IL-15Ra), or a combination of both an IL-15 polypeptide and an IL-15Ra polypeptide, e.g., hetIL-15 (Admune Therapeutics, LLC) . hetIL-15 is a heterodimeric noncovalent complex of IL-15 and IL-15Ra. hetIL-15 is described in, for example, U.S. 8,124,084, U.S. 2012/0177598, U.S. 2009/0082299, U.S. 2012/0141413, and U.S. 2011/0081311, incorporated herein by reference. In embodiments, het-IL-15 is administered subcutaneously. In embodiments, the individual has a cancer, e.g., solid cancer, e.g., melanoma or colon cancer. In embodiments, the individual has a metastatic cancer.

Therapeutic Application Diseases and/or Disorders Associated with CD19

[00561] In one aspect, the invention provides methods for treating a disease associated with the expression of CD19. In one aspect, the invention provides methods for treating a disease in which part of the tumor is CD19 negative and part of the tumor is CD19 positive. For example, the CAR of the invention is useful for treating individuals who have undergone treatment for a disease associated with elevated CD19 expression, wherein the individual who has undergone treatment for elevated levels of CD19 exhibits a disease associated with elevated levels. of CD19.

[00562] The therapies described here can be used to treat, for example, individuals who respond to a kinase inhibitor such as ibrutinib (e.g., partial response or complete response) or individuals who do not respond (e.g., non-responders or relapsers ). Without wishing to be bound by theory, many patients undergoing treatment with kinase inhibitors (e.g., BTK inhibitors such as ibrutinib) may show a reduced response to treatment (e.g., they are partial responders or non-responders to treatment, or recur during treatment). Consequently, administration of the CAR therapies described here in combination with kinase inhibitors may result in beneficial effects.

[00563] Exemplary therapeutic regimens for these individuals are described below.

[00564] In some cases, when the individual is a non-responder or relapser to a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the kinase inhibitor is withdrawn and CAR therapy is administered. In other cases, when individuals do not respond to a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), kinase inhibitor therapy is continued and CAR therapy is added to the regimen. This use is supported, for example, by experiments in Example 8 herein, which indicate that CAR therapy is effective as a monotherapy in ibrutinib-resistant cells. Without wishing to be bound by theory, continuation of kinase inhibitor therapy may improve the efficacy of CAR therapy, for example, by increasing the number of CAR-expressing cells in the bloodstream (see, Example 8 here).

[00565] Without being bound by theory, an individual who is a non-responder or relapser to a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) may be a non-responder for at least two reasons: Individuals may have a mutation in the target drug (e.g., BTK; e.g., a C481S mutation) that prevents target inhibition, or may have alterations in other sets of reactions that regulate proliferation even when the target is adequately inhibited (e.g., a mutation in PLCY, such as an activating mutation in PLCY resulting in constitutive BTK-independent cell signaling). Treatment may be changed depending on the reason for non-responsiveness. For example, in the first situation (in some embodiments), if individuals have (or are identified as having) a mutation that prevents the kinase inhibitor from inhibiting its target, a second kinase inhibitor (e.g., directed against the same target) ) can be substituted for (or administered in combination with) the kinase inhibitor. More specifically, in some embodiments where the patient has (or is identified as having) a mutation that prevents ibrutinib from inhibiting BTK, a second BTK inhibitor, e.g., a BTK inhibitor described herein such as GDC-0834, RN-486 , CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13) can be substituted for ibrutinib. Without wishing to be bound by theory, the second kinase inhibitor may act in a region of the target that is not disrupted by the mutation, and therefore the individual is sensitive to the second kinase inhibitor. In other embodiments, the original kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) is maintained. According to the non-limiting theory here, the original kinase inhibitor may have useful activity in CAR-expressing cells, for example, promoting a TH1 phenotype, promoting proliferation, or otherwise increasing the levels or activity of the cells.

[00566] As noted above, in some cases an individual is non-responsive because the individual has a change (e.g., a mutation) in another reaction series that can regulate proliferation even when the target is adequately inhibited. Consequently, if the individual has (or is identified as having) a change in a reaction series that renders the kinase activity inhibitor ineffective, kinase inhibitor therapy may be continued. Without wishing to be bound by theory, the activity of a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) may promote useful biological changes in cancer cells even if the kinase inhibitor alone is not sufficient to dampen proliferation. For example, the kinase inhibitor may be enough to mobilize cancer cells outside of lymph nodes, making it more vulnerable to CAR therapy.

[00567] Turning now to individuals who respond to a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), several therapeutic regimens are now described. In some embodiments, when an individual is (or is identified as being) a complete responder to the kinase inhibitor, the individual is not administered CAR therapy during the period of complete response. In other embodiments, when an individual is (or is identified as being) a complete responder to the kinase inhibitor, the individual is administered CAR therapy during the period of complete response. In one embodiment, following CAR therapy, the individual experiences prolonged response or delayed recurrence (e.g., compared to the expected course of disease when treated without CAR therapy). For example, MCL treated with ibrutinib monotherapy has a median duration of response of about 17.5 months.

[00568] In some embodiments, when an individual is (or is identified as being) a partial responder to the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the individual is not administered CAR therapy during the period partial response. In other embodiments, when an individual is (or is identified as being) a partial responder to the kinase inhibitor, the individual is administered CAR therapy during the partial response period. In one embodiment, following CAR therapy, the individual experiences complete response and/or prolonged response or delayed relapse (e.g., compared to the expected course of disease when treated without CAR therapy).

[00569] In some embodiments, when an individual has (or is identified as having) stable disease after initiating treatment with the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the individual is not administered a therapy with CAR during the period of stable disease. In other embodiments, when an individual has (or is identified as having) stable disease after initiating treatment with the kinase inhibitor, the individual is administered CAR therapy during the period of stable disease. In one embodiment, following CAR therapy, the subject experiences a partial response, a complete response, and/or prolonged response or delayed relapse (e.g., compared to the expected course of disease when treated without CAR therapy).

[00570] In some embodiments, when an individual has (or is identified as having) progressive disease after initiating treatment with the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), the individual is not administered a therapy with CAR during the period of progressive disease. In other embodiments, when an individual has (or is identified as having) progressive disease after initiating treatment with the kinase inhibitor, the individual is administered CAR therapy during the period of progressive disease. In one embodiment, following CAR therapy, the subject experiences stable disease, a partial response, complete response, and/or prolonged response or delayed relapse (e.g., compared to the expected course of disease when treated without CAR therapy). .

[00571] In this way, one or more disease assessment steps can be performed before or during treatment, to determine which course of treatment is appropriate for a given patient. For example, an individual may be administered a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) as a first-line therapy. Then, after a period of time (e.g. 1 or 2 months, but also 2 weeks, 3 weeks, 1 month, 1.5 months, 2 months, 3 months, 4 months, 6 months, 9 months, 12 months , 15 months, or 18 months) the patient's response can be evaluated. If the assessment shows that the individual is a complete responder, in some modalities, CAR therapy will not be administered, for example, as described above. If evaluation shows that the individual is a partial responder or has stable disease, in some embodiments, CAR therapy will be administered in combination with the kinase inhibitor, for example, as described above. If evaluation shows that the individual is a non-responder or relapser, in some embodiments, CAR therapy will be administered in combination with the kinase inhibitor or a second kinase inhibitor, for example, as described above. In some embodiments, the kinase inhibitor controls the disease at the same time that a CAR-expressing cell is being manufactured, for example, at the same time that the patient's own T cells are being created to express a CAR and/or other factors.

[00572] Clinical standards for classifying a patient's responsive state or relapsed state are known in the art. As an example, for malignant lymphoma, standardized response criteria are 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 by reference herein in their entirety). Accordingly, in some embodiments, an individual is considered a complete responder, partial responder, having stable disease, a non-responder, or a relapser according to Cheson criteria or modified Cheson criteria. Criteria for classifying other hematological malignancies are known in the art.

[00573] According to the criteria in table 2 of Cheson 2007, a complete responder has disappearance of all evidence of disease; a partial responder has measurable disease regression and no new sites; a patient with stable disease has difficulty achieving CR/PR or PD; and a patient with relapsing disease or progressive disease has any new lesion or enlargement at greater than or equal to 50% of previously involved sites from nadir. Assessment may involve a determination of whether the disease is FDG avid, PET positive or negative, whether nodules are present, for example palpable in the liver or spleen, whether the bone marrow is cleared or shows involvement.

[00574] CAR therapy and the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) can be administered, for example, simultaneously or sequentially. In some embodiments, CAR therapy is initiated at substantially the same time as kinase inhibitor therapy is initiated. In some embodiments, CAR therapy is initiated before kinase inhibitor therapy begins. In some embodiments, CAR therapy is initiated after kinase inhibitor therapy begins. For example, CAR therapy may be initiated, e.g., at least 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 6, 9, 12, 15, 18, or 24 months after the start kinase inhibitor therapy. In some embodiments, CAR therapy is initiated at the same time that a patient has physiologically relevant levels of the kinase inhibitor in his or her body.

[00575] When administered in combination, CAR therapy and the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), or both, can be administered in an amount or dose that is equal to, less than or greater than the amount or dosage of each agent used individually, for example, as a monotherapy. In certain embodiments, the administered amount or dosage of CAR therapy, the kinase inhibitor, or both, is less (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than that the amount or dosage of each agent used individually, for example, as a monotherapy. In other embodiments, the amount or dosage of CAR therapy, the kinase inhibitor, or both, that results in a desired effect (e.g., cancer treatment) is less (e.g., at least 20%, at least 30% , at least 40%, or at least 50% less) than the amount or dosage of each agent used individually, for example, as a monotherapy, required to obtain the same therapeutic effect.

[00576] When administered in combination, CAR therapy and the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), or both, can be administered for a duration that is equal to, less than, or greater than the duration of each agent used individually, for example, as a monotherapy. In certain embodiments, the duration of administration of CAR therapy, the kinase inhibitor, or both, is shorter (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than that the duration of each agent used individually, for example, as a monotherapy. In other embodiments, the duration of administration of the CAR therapy, the kinase inhibitor, or both, that results in a desired effect (e.g., cancer treatment) is shorter (e.g., at least 20%, at least 30% %, at least 40%, or at least 50% shorter) than the duration of each agent used individually, for example, as a monotherapy, required to obtain the same therapeutic effect. In some embodiment, the patient is administered an abbreviated course of the kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib). For example, the abbreviated course of the kinase inhibitor may last about 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-15, 15-18, 18-21, or 21-24 months in total or it can last about 02, 2-4, 4-6, 6-8, 8-10, 10-12, 12-15, 15-18, 18-21, or 21-24 months after administration of CAR therapy. In embodiments, the abbreviated course of the kinase inhibitor ends before recurrence. In embodiments, the kinase inhibitor is administered at normal levels (e.g., monotherapy) during the abbreviated course.

[00577] In embodiments, a single dose of CAR-expressing cells comprises about 5 x 108 CD19 CART cells. A dose of CAR expressing cells may also comprise about 5 x 106, 1 x 107, 2 x 107, 5 x 107, 1 x 108, 2 x 108, 5 x 108, 1 x 109, 2 x 109, or 5 x 109cells, e.g., CD19 CAR cells, e.g., CD19 CART cells.

[00578] In one aspect, the invention pertains to a vector comprising CD19 CAR operably linked to the promoter for expression in mammalian cells, e.g., T cells. In one aspect, the invention provides a recombinant cell, e.g., a T cell. , expressing the CD19 CAR for use in treating tumors expressing CD19, wherein the recombinant T cell expressing the CD19 CAR is called a CD19T CAR. In one aspect, the CD19 CART described herein is capable of contacting a tumor cell with at least one CD19 CART expressed on its surface so that the CART targets the tumor cell and tumor growth is inhibited.

[00579] In one aspect, the invention pertains to a method of inhibiting the growth of a tumor cell expressing CD19, comprising contacting the tumor cell with one expressing CD19 CAR, for example, a CD19 CAR T cell, described herein so that CART is activated in response to the antigen and targets the cancer cell, whereby tumor growth is inhibited. The CD19 CAR-expressing cell, e.g., T cell, is administered in combination with a kinase inhibitor, e.g., a kinase inhibitor described herein.

[00580] Administered "in combination", as used herein, means that two (or more) different treatments are delivered to the individual during the course of the individual's affliction with the disorder, e.g., the two or more treatments are delivered after the individual have been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the release of one treatment is still occurring when the release of the second begins, so that there is overlap in terms of administration. This is sometimes referred to here as "simultaneous" or "concurrent" release. In other embodiments, the release of one treatment ends before the release of the other treatment begins. In some embodiments of any case, treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent than would be observed if the second treatment were administered in the absence of the first treatment. , or the analogous situation is observed with the first treatment. In some embodiments, the release is such that the reduction in a symptom, or other parameter related to the disorder is greater than that which would be observed with one treatment released in the absence of the other. The effect of the two treatments may be partially additive, fully additive, or greater than additive. The release may be such that an effect of the first released treatment is still detectable when the second is released. In one embodiment, the CAR-expressing cell is administered at a dose and/or dosage schedule described herein, and the kinase inhibitor or agent that enhances the activity of the CAR-expressing cell is administered at a dose and/or dosage schedule described herein. .

[00581] The invention includes a type of cell therapy where T cells are genetically modified to express a chimeric antigen receptor (CAR) and the CAR T cell is infused to a recipient in need thereof. The infused cell is capable of killing tumor cells in the recipient. Unlike antibody therapies, CAR-modified T cells are capable of replication in vivo resulting in long-term persistence that can induce prolonged tumor control. In various aspects, the T cells administered to the patient, or their descendants, persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months , fourteen months, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after administration of the T cell to the patient.

[00582] The invention also includes a type of cell therapy wherein T cells are modified, for example, by in vitro transcribed RNA, to transiently express a chimeric antigen receptor (CAR) and the CAR T cell is infused to a receptor in need for it. The infused cell is capable of killing tumor cells in the recipient. Thus, in many respects, the T cells administered to the patient are present for less than a month, e.g., three weeks, two weeks, one week, after administration of the T cell to the patient.

[00583] Without wishing to be bound by any particular theory, the anti-tumor immunity response elicited by CAR-modified T cells may be an active or passive immune response, or alternatively may be due to a direct vs indirect immune response. In one aspect, CAR-transduced T cells exhibit specific pro-inflammatory cytokine secretion and potent cytolytic activity in response to human cancer cells expressing CD19, resist inhibition of soluble CD19, mediate bystander killing, and mediate regression of a established human tumor. For example, antigen-free tumor cells within a heterogeneous field of CD19-expressing tumor may be susceptible to indirect destruction by CD19-redirected T cells that have previously been reacted against adjacent antigen-positive cancer cells.

[00584] In one aspect, the fully human CAR-modified T cells of the invention can be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. In one respect, the mammal is a human.

[00585] With respect to ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cell into a mammal: i) expansion of the cells, ii) introduction of a nucleic acid encoding a CAR into the cells, or iii) cryopreservation of the cells.

[00586] Ex vivo procedures are well known in the art and are described more fully below. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR described here. The CAR-modified cell can be administered to a recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the CAR-modified cell may be autologous with respect to the recipient. Alternatively, the cells may be allogeneic, syngeneic or xenogeneic with respect to the recipient. In addition to using a cell-based vaccine in terms of ex vivo immunization, also included in the methods described here are compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.

[00587] Generally, activated and expanded cells, as described here, can be used in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, the CAR-expressing cells described here are used in the treatment of diseases, disorders and conditions associated with CD19 expression. In certain aspects, the cells are used to treat patients at risk of developing diseases, disorders and conditions associated with CD19 expression. Accordingly, the present invention provides methods for treating or preventing diseases, disorders and conditions associated with CD19 expression comprising administering to an individual in need thereof, a therapeutically effective amount of the CAR expressing cells described herein, in combination with an inhibitor. of kinase, for example, a kinase inhibitor described herein.

[00588] The present invention also provides methods for inhibiting the proliferation or reduction of a cell population expressing CD19, the methods comprising contacting a cell population comprising a cell expressing CD19 with an anti-CD19 CAR expressing cell described herein that binds to cell expressing CD19, and contacting the cell populations expressing CD19 with a kinase inhibitor, for example, a kinase inhibitor described herein. In a specific aspect, the present invention provides methods for inhibiting the proliferation or reduction of CD19-expressing cancer cell populations, the methods comprising contacting the CD19-expressing cancer cell population with an anti-CD19 CAR-expressing cell described herein that binds to the cell expressing CD19, and contacting the cell expressing CD19 with a kinase inhibitor, for example, a kinase inhibitor described herein. In one aspect, the present invention provides methods for inhibiting the proliferation or reducing CD19-expressing cancer cell populations, the methods comprising contacting the CD19-expressing cancer cell population with an anti-CD19 CAR-expressing cell described herein that binds to cell expressing CD19 and contacting the cell expressing CD19 with a kinase inhibitor, e.g., a kinase inhibitor described herein. In certain aspects, the combination of the anti-CD19 CAR expressing cell described herein and the kinase inhibitor, e.g., a kinase inhibitor described herein, reduces the amount, number, amount or percentage of cells and/or cancer cells in at least 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 an individual with or model animal for a hematological cancer or other cancer associated with cells expressing CD19 relative to a negative control. In one respect, the individual is a human.

[00589] The present invention also provides methods for preventing, treating and/or controlling a disease associated with cells expressing CD19 (for example, a hematological cancer or atypical cancer expressing CD19), the methods comprising administering to an individual in need thereof a cell expressing anti-CD19 CAR that binds to the cell expressing CD19 and administering a kinase inhibitor, e.g., a kinase inhibitor described herein. In one respect, the individual is a human. Non-limiting examples of disorders associated with cells expressing CD19 include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma) and cancers (such as hematological cancers or atypical cancers expressing CD19).

[00590] The present invention also provides methods for preventing, treating and/or controlling a disease associated with cells expressing CD19, the methods comprising administering to an individual in need thereof an anti-CD19 CART cell of the invention that binds to the cell expressing CD19. In one respect, the individual is a human.

[00591] The present invention provides methods for preventing recurrence of cancer associated with cells expressing CD19, the methods comprising administering to an individual in need thereof an anti-CD19 expressing cell (such as an anti-CD19 CART cell) of the invention that binds to the cell expressing CD19. In one aspect, the methods comprise administering to the individual in need thereof an effective amount of an anti-CD19 expressing cell (such as an anti-CD19 CART cell) described herein that binds to the CD19 expressing cell in combination with an effective amount of other therapy.

[00592] In one aspect, the invention pertains to a method of treating cancer in an individual. The method comprises administering to the subject a cell (e.g., an immune effector cell) expressing a CAR targeting B cell, e.g., a T cell or NK cell, described herein, in combination with a kinase inhibitor, e.g., an inhibitor of kinase described here, so that the cancer is treated in the individual. An example of a cancer that is treatable by the methods described here is a cancer associated with B cell antigen expression, for example, CD19. In one embodiment, the disease is a solid or liquid tumor. In one embodiment, the disease is a hematological cancer. In one embodiment, the hematologic cancer is leukemia. In one embodiment, the hematological cancer is a mature B-cell neoplasm, for example, according to WHO classification. In one embodiment, the hematologic cancer is a CD19+ B lymphocyte-derived malignancy. In one embodiment, the cancer is selected from the group consisting of one or more acute leukemias including, but not limited to, B-cell acute lymphocytic leukemia (BALL), T-cell acute lymphocytic leukemia (TALL), small lymphocytic lymphoma (SLL) , acute lymphocytic leukemia (ALL); one or more chronic leukemias including, but not limited to, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); added hematologic cancers or hematologic conditions including but not limited to lining cell lymphoma (MCL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL) ( e.g., histiocyte/T-cell-rich large B-cell lymphoma, primary DLBCL of the CNS, primary cutaneous "leg" type DLBCL, or EBV+ DLBCL of the elderly), DLBCL associated with chronic inflammation, follicular lymphoma, pediatric follicular lymphoma, hair cell, follicular large cell or small cell lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma/leukemia (e.g., unclassifiable), splenic diffuse small pulp small B-cell lymphoma, capillary cell leukemia- variant, lymphoplasmacytic lymphoma, a heavy chain disease (e.g., alpha heavy chain disease, gamma heavy chain disease, or mu heavy chain disease), plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, zona nodal lymphoma marginal, pediatric marginal zone nodal lymphoma, primary cutaneous follicular center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymus) large B-cell lymphoma, intravascular l large B-cell lymphoma, ALK+ large B-cell lymphoma, B-cell lymphoma large arising in HHV8-associated Castleman disease, primary effusion lymphoma, unclassifiable B-cell lymphoma (e.g., with features intermediate between DLBCL and Burkitt's lymphoma or intermediate between DLBCL and classic Hodgkin's lymphoma), and "preleukemia "which are a diverse collection of hematologic conditions united by ineffective production (or dysplasia) of myeloid blood cells, and disease associated with expression of B cell antigen (e.g., CD19-) include, but are not limited to, atypical cancers and /or non-classical, malignancies, precancerous conditions or proliferative diseases expressing B cell antigen (e.g. CD19); and any combinations thereof.

[00593] In some embodiments, the cancer is Hodgkin's lymphoma, and the patient is treated with CAR-expressing cells, for example, as a monotherapy, or in combination with one or more additional therapeutic products. In embodiments, Hodgkin's lymphoma is stage I, II, III, or IV. The additional therapeutic product may comprise, for example, a kinase inhibitor such as a BTK inhibitor such as ibrutinib. The additional therapeutic product may comprise a treatment for Hodgkin's lymphoma. The additional therapeutic product may comprise, for example, radiation therapy, MOPP (Mustargen, Oncovin, Prednisone, and Procarbazine), ABVD (Adriamycin, bleomycin, vinblastine, and dacarbazine), Stanford V (a regimen with chemotherapy and radiation treatment) , or BEACOPP (Bleomycin, Etoposide, Adriamycin, Cyclophosphamide, Oncovin, Procarbazine, Prednisone). In some embodiments, the individual has previously been treated with, or is resistant to, or is refractory to, one or more of radiation therapy, MOPP, Stanford V, or BEACOPP.

[00594] Non-cancer-related indications associated with B cell antigen expression, e.g., one or more of CD19, CD20, CD22, or ROR1, include, but are not limited to, e.g., autoimmune disease, (e.g., lupus), inflammatory disorders (allergy and asthma) and transplantation.

[00595] In some embodiments, a cancer that can be treated with the combination described here is multiple myeloma. Multiple myeloma is a blood cancer characterized by the accumulation of a plasma cell clone in the bone marrow. Current therapies for multiple myeloma include, but are not limited to, treatment with lenalidomide, which is a thalidomide analog. Lenalidomide has activities that include antitumor activity, inhibition of angiogenesis, and immunomodulation. In some embodiments, a CD19 CAR, for example, as described herein, can be used to target myeloma cells. In some embodiments, the combination described herein can be used with one or more additional therapies, e.g., lenalidomide treatment.

[00596] The CAR-expressing cells described here can be administered alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.

[00597] In embodiments, a lymphodepletion chemotherapy is administered to the individual before, concurrently with, or after administration (e.g., infusion) of CAR cells, e.g., CAR-expressing cells described herein. In one example, lymphodepletion chemotherapy is administered to the subject prior to administration of CAR cells. For example, lymphodepletion chemotherapy ends 1 to 4 days (e.g., 1, 2, 3, or 4 days) before CAR cell infusion. In embodiments, multiple doses of CAR cells are administered, for example, as described herein. For example, a single dose comprises about 5 x 108CAR cells. In embodiments, a lymphodepletion chemotherapy is administered to the subject before, concurrently with, or after administration (e.g., infusion) of a CAR-expressing cell described herein. Hematological Cancer

[00598] Hematological cancer conditions are types of cancer such as leukemia, lymphoma and malignant lymphoproliferative conditions that affect the blood, bone marrow and lymphatic system.

[00599] Leukemia can be classified as acute leukemia and chronic leukemia. Acute leukemia can also be classified as acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL). Chronic leukemia includes chronic myelogenous leukemia (CML) and chronic lymphocytic leukemia (CLL). Other related conditions include myelodysplastic syndromes (MDS, formerly known as "preleukemia") which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells and risk of transformation into AML.

[00600] Lymphoma is a group of blood cell tumors that develop from lymphocytes. Exemplary lymphomas include non-Hodgkin's lymphoma and Hodgkin's lymphoma.

Combination therapies

[00601] The combination of a CAR-expressing cell described here (e.g., and a kinase inhibitor described here) can be used in combination with other known agents and therapies.

[00602] A CAR-expressing cell described herein, the kinase inhibitor and/or at least one additional therapeutic agent can be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the CAR-expressing cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.

[00603] CAR therapy and/or other therapeutic agents, procedures or modalities can be administered during periods of active disorder, or during a period of remission or less active disease. CAR therapy can be administered before other treatment, concurrently with treatment, after treatment, or during remission of the disorder.

[00604] When administered in combination, CAR therapy and one or more additional agents (e.g., kinase inhibitor and/or a third agent), or all, may be administered in an amount or dose that is the same, less or greater than the amount or dosage of each agent used individually, for example, as a monotherapy. In certain embodiments, the administered amount or dosage of the CAR therapy, the additional agent (e.g., kinase inhibitor and/or third agent), or all, is less (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, for example, as a monotherapy. In other embodiments, the amount or dosage of CAR therapy, additional agent (e.g., kinase inhibitor and/or third agent), or all, that results in a desired effect (e.g., cancer treatment) is less ( for example, at least 20%, at least 30%, at least 40%, or at least 50% less) than the amount or dosage of each agent used individually, for example, as a monotherapy, required to obtain the same effect therapeutic.

[00605] In other aspects, the combination of the CAR expressing cell described herein (e.g., and the kinase inhibitor) can be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporine, azathioprine , methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation, peptide vaccine, such as that described in Izumoto et al. 2008 J Neurosurg 108:963-971.

[00606] In one embodiment, the combination of a CAR-expressing cell described here (e.g., and a kinase inhibitor described here) can be used in combination with another chemotherapeutic agent. Exemplary chemotherapeutic agents include an anthracycline (e.g., doxorubicin (e.g., liposomal doxorubicin)); a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine); an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide); an immune cell antibody (e.g., alemtuzamab, gentuzumab, rituximab, ofatumumab, tositumomab, brentuximab); an antimetabolite (including, for example, folic acid antagonists, pyrimidine analogues, purine analogues and adenosine deaminase inhibitors (e.g. fludarabine)); a glucocorticoid-induced TNFR-related protein (GITR) agonist; a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin, or bortezomib); an immunomodulator such as thalidomide or a thalidomide derivative (e.g., lenalidomide).

[00607] General chemotherapeutic agents considered for use in combination therapies 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®), liposomal cytarabine injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), injection of liposomal daunorubicin citrate (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (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 (Purinetol®), methotrexate (Folex®), mitoxantrone (Novantrone®), milotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin, polyfeprosan 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®).

[00608] Exemplary alkylating agents include, without simulation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes: uracil mustard (Aminouracil Mustard®, Chloretaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamina®, Nordopan®, Uracil nitrogen mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, RevimmuneTM), ifosfamide (Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, Temozolomide (Temodar®), thiotepa (Tioplex®), busulfan (Busilvex ®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and Dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, without simulation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®); Melphalan (also known as L-PAM, L-sarcolysine, 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 (also known as hexamethylmelamine (HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine (Matulane®); Mechlorethamine (also known as nitrogen mustard, mustine and mechloroethamine hydrochloride, Mustargen®); Streptozocin (Zanosar®); Thiotepa (also known as thiophosphoamide, TESPA and TSPA, Tioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and Bendamustine HCl (Treanda®).

[00609] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, for example, a BTK inhibitor such as ibrutinib, is administered to an individual in combination with fludarabine, cyclophosphamide, and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with fludarabine, cyclophosphamide, and rituximab (FCR). In embodiments, the individual has CLL. For example, the individual has a deletion on the short branch of chromosome 17 (del(17p), for example, in a leukemic cell). In other examples, the individual does not have a del(17p). In embodiments, the individual comprises a leukemic cell comprising a mutation in the immunoglobulin heavy chain variable region (IgVH) gene. In other embodiments, the individual does not comprise a leukemic cell comprising a mutation in the immunoglobulin heavy chain variable region (IgVH) gene. In embodiments, fludarabine is administered at a dosage of about 10 to 50 mg/m2 (e.g., about 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 mg/m2), for example, intravenously. In embodiments, cyclophosphamide is administered at a dosage of about 200-300 mg/m2 (e.g., about 200-225, 225-250, 250-275, or 275-300 mg/m2), e.g., intravenously. . In embodiments, rituximab is administered at a dosage of about 400-600 mg/m2 (e.g., 400-450, 450500, 500-550, or 550-600 mg/m2), for example, intravenously.

[00610] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, for example, a BTK inhibitor such as ibrutinib, is administered to an individual in combination with bendamustine and rituximab. In embodiments, the individual has CLL. For example, the individual has a deletion on the short branch of chromosome 17 (del(17p), for example, in a leukemic cell). In other examples, the individual does not have a del(17p). In embodiments, the individual comprises a leukemic cell comprising a mutation in the immunoglobulin heavy chain variable region (IgVH) gene. In other embodiments, the individual does not comprise a leukemic cell comprising a mutation in the immunoglobulin heavy chain variable region (IgVH) gene. In embodiments, bendamustine is administered at a dosage of about 70-110 mg/m2 (e.g., 70-80, 80-90, 90-100, or 100-110 mg/m2), for example, intravenously. In embodiments, rituximab is administered at a dosage of about 400-600 mg/m2 (e.g., 400-450, 450-500, 500-550, or 550-600 mg/m2), for example, intravenously.

[00611] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, is administered to an individual in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and /or a corticosteroid (e.g. prednisone). In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). In embodiments, the individual has diffuse large B-cell lymphoma (DLBCL). In embodiments, the individual is non-bulky limited stage DLBCL (e.g., comprises a tumor having a size/diameter less than 7 cm). In embodiments, the individual is treated with radiation in combination with R-CHOP. For example, the individual is administered R-CHOP (e.g., 1 to 6 cycles, e.g., 1, 2, 3, 4, 5, or 6 cycles of R-CHOP), followed by radiation. In some cases, the individual is administered R-CHOP (e.g., 1 to 6 cycles, e.g., 1, 2, 3, 4, 5, or 6 cycles of R-CHOP) followed by radiation.

[00612] In embodiments, a CAR expressing cell described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, is administered to an individual in combination with etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin , and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (EPOCH-R). In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with dose-adjusted EPOCH-R (DA-EPOCH-R). In embodiments, the individual has B-cell lymphoma, e.g., an aggressive Myc-rearranged B-cell lymphoma.

[00613] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, for example, a BTK inhibitor such as ibrutinib, is administered to an individual in combination with rituximab and/or lenalidomide. Lenalidomide ((RS)-3-(4-Amino-1-oxo 1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione) is an immunomodulator. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with rituximab and lenalidomide. In embodiments, the individual has follicular lymphoma (FL) or lining cell lymphoma (MCL). In embodiments, the individual has FL and has not previously been treated with a cancer therapy. In embodiments, lenalidomide is administered at a dosage of about 10 to 20 mg (e.g., 10 to 15 or 15 to 20 mg), e.g., daily. In embodiments, rituximab is administered at a dosage of about 350 to 550 mg/m2 (e.g., 350 to 375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m2) , for example, intravenously.

[00614] Exemplary immunomodulators include, for example, afutuzumab (available from Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), pomelidomide, actimide (CC4047); and IRX-2 (mixture of human cytokines including interleukin 1, interleukin 2, and interferon Y, CAS 951209-71-5, available from IRX Therapeutics).

[00615] Exemplary anthracyclines include, for example, doxorubicin (Adriamycin® and Rubex®); bleomycin (lenoxane®); daunorubicin (dauorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®); liposomal daunorubicin (liposomal daunorubicin citrate, DaunoXome®); mitoxantroae (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and deacetylravidomycin.

[00616] Exemplary vinca alkaloids include, for example, vinorelbine tartrate (Navelbine®), Vincristine (Oncovin®), and Vindesine (Eldisine®)); vinblastine (also known as vinblastine sulfate, vincaleukoblastine and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®).

[00617] Exemplary proteasome inhibitors include 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-phenylbutanamido)-pentanamide); marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-Methyl-N- [(2-methyl-5-thiazolyl)carbonyl]-L- seryl-O-methyl-N- [(1S)-2- [(2R)-2-methyl-2-oxiranyl] -2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[00618] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, for example, a BTK inhibitor such as ibrutinib, is administered to an individual in combination with brentuximab. Brentuximab is an antibody-drug conjugate of anti-CD30 antibody and monomethyl auristatin E. In embodiments, the subject has Hodgkin's lymphoma (HL), e.g., relapsed or refractory HL. In embodiments, the subject comprises CD30+ HL. In embodiments, the individual has undergone an autologous stem cell transplant (ASCT). In embodiments, the individual has not undergone an ASCT. In embodiments, brentuximab is administered at a dosage of about 1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg), e.g., intravenously, for example, every 3 weeks.

[00619] In embodiments, a CAR-expressing cell described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, is administered to an individual in combination with brentuximab and dacarbazine or in combination with brentuximab and bendamustine. Dacarbazine is an alkylating agent with a chemical name of 5-(3,3-Dimethyl-1-triazenyl)imidazole-4-carboxamide. Bendamustine is an alkylating agent with a chemical name of 4-[5-[Bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid. In embodiments, the individual has Hodgkin's lymphoma (HL). In embodiments, the individual has not previously been treated with a cancer therapy. In embodiments, the individual is at least 60 years of age, e.g., 60, 65, 70, 75, 80, 85, or older. In embodiments, dacarbazine is administered at a dosage of about 300-450 mg/m2 (e.g., about 300-325, 325-350, 350-375, 375-400, 400-425, or 425-450 mg/m2) , for example, intravenously. In embodiments, bendamustine is administered at a dosage of about 75-125 mg/m2 (e.g., 75-100 or 100-125 mg/m2, e.g., about 90 mg/m2), for example, intravenously. In embodiments, brentuximab is administered at a dosage of about 1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg) , for example, intravenously, for example, every 3 weeks.

[00620] In some embodiments, a CAR-expressing cell described herein is administered to an individual in combination with a CD20 inhibitor, e.g., an anti-CD20 antibody (e.g., a mono- or bispecific anti-CD20 antibody). or a fragment thereof. Anti-CD20 antibodies include, but are not limited to, rituximab, ofatumumab, ocrelizumab, veltuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals), ocaratuzumab, and Pro131921 (Genentech). See, for example, Lim et al. Haematologica. 95.1(2010):135-43.

[00621] In some embodiments, the anti-CD20 antibody comprises rituximab. Rituximab is a chimeric human/mouse IgG1 kappa monoclonal antibody that binds to CD20 and causes cytolysis of a cell expressing CD20, for example, as described at www.accessdata.fda.gov/drugsatfda_docs/label/2010/103705s5311lbl. pdf. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with rituximab. In embodiments, the individual has CLL or SLL.

[00622] In some embodiments, rituximab is administered intravenously, for example, as an intravenous infusion. For example, each infusion provides about 500 to 2000 mg (e.g., about 500 to 550, 550 to 600, 600 to 650, 650 to 700, 700 to 750, 750 to 800, 800 to 850, 850 to 900, 900 to 950, 950 to 1000, 1000 to 1100, 1100 to 1200, 1200 to 1300, 1300 to 1400, 1400 to 1500, 1500 to 1600, 1600 to 1700, 1700 to 1800, 1800 to 1900, or 1900 to 2000 mg) of rituximab. In some embodiments, rituximab is administered at a dose of 150 mg/m2 to 750 mg/m2, e.g., about 150 to 175 mg/m2, 175 to 200 mg/m2, 200 to 225 mg/m2, 225 to 250 mg/m2, 250 to 300 mg/m2, 300 to 325 mg/m2, 325 to 350 mg/m2, 350 to 375 mg/m2, 375 to 400 mg/m2, 400 to 425 mg/m2, 425 to 450 mg /m2, 450 to 475 mg/m2, 475 to 500 mg/m2, 500 to 525 mg/m2, 525 to 550 mg/m2, 550 to 575 mg/m2, 575 to 600 mg/m2, 600 to 625 mg/m2 m2, 625 to 650 mg/m2, 650 to 675 mg/m2, or 675 to 700 mg/m2, where m2 indicates the individual's body surface area. In some embodiments, rituximab is administered at a dose interval of at least 4 days, e.g., 4, 7, 14, 21, 28, 35 days, or more. For example, rituximab is administered at a dose interval of at least 0.5 weeks, e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8 weeks, or more. In some embodiments, rituximab is administered at a dose and dose interval described herein over a period of time, e.g., at least 2 weeks, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks, or more. For example, rituximab is administered at a dose and dose range described here for a total of at least 4 doses per treatment cycle (e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, or more doses per treatment cycle).

[00623] In some embodiments, the anti-CD20 antibody comprises ofatumumab. Ofatumumab is a human IgG1k anti-CD20 monoclonal antibody with a molecular weight of approximately 149 kDa. For example, ofatumumab is generated using transgenic mouse and hybridoma technology and is expressed and purified from a recombinant murine cell line (NS0). See, for example, www.accessdata.fda.gov/drugsatfda_docs/label/2009/125326lbl.pdf; and Clinical Trial Identifier number NCT01363128, NCT01515176, NCT01626352, and NCT01397591. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with ofatumumab. In embodiments, the individual has CLL or SLL.

[00624] In some embodiments, ofatumumab is administered as an intravenous infusion. For example, each infusion provides about 150-3000 mg (e.g., about 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550- 600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1200, 1200-1400, 1400-1600, 1600-1800, 1800-2000, 2000-2200, 2200-2400, 2400-2600, 2600-2800, or 2800-3000 mg) of ofatumumab. In embodiments, ofatumumab is administered at a starting dose of about 300 mg, followed by 2000 mg, for example, for about 11 doses, for example, for 24 weeks. In some embodiments, ofatumumab is administered at a dose interval of at least 4 days, e.g., 4, 7, 14, 21, 28, 35 days, or more. For example, ofatumumab is administered at a dose interval of at least 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 26, 28, 20, 22, 24, 26, 28, 30 weeks, or more. In some embodiments, ofatumumab is administered at a dose and dose interval described herein over a period of time, e.g., at least 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 40, 50, 60 weeks or more, or 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12 months or more, or 1, 2, 3, 4, 5 years or more. For example, ofatumumab is administered at a dose and dose range described here for a total of at least 2 doses per treatment cycle (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 18, 20, or more doses per treatment cycle).

[00625] In some cases, the anti-CD20 antibody comprises ocrelizumab. Ocrelizumab is a humanized anti-CD20 monoclonal antibody, for example, as described in Clinical Trial Identifier Nos. NCT00077870, NCT01412333, NCT00779220, NCT00673920, NCT01194570, and Kappos et al. Lancet. 19.378(2011):1779-87.

[00626] In some cases, the anti-CD20 antibody comprises veltuzumab. Veltuzumab is a humanized monoclonal antibody against CD20. See, for example, Clinical Trial Identifier No. NCT00547066, NCT00546793, NCT01101581, and Goldenberg et al. Leuk Lymphoma. 51(5)(2010):747-55.

[00627] In some cases, the anti-CD20 antibody comprises GA101. GA101 (also called obinutuzumab or RO5072759) is a glyco-created humanized anti-CD20 monoclonal antibody. Take, for example, Robak. Curr. Opinion. Investigation. Drugs. 10.6(2009):588-96; Clinical Trial Identifier Numbers: NCT01995669, NCT01889797, NCT02229422, and NCT01414205; and www.accessdata.fda.gov/drugsatfda_docs/label/2013/125486s000lbl.p df.

[00628] In some cases, the anti-CD20 antibody comprises AME-133v. AME-133v (also called LY2469298 or ocaratuzumab) is a humanized IgG1 monoclonal antibody against CD20 with increased affinity for the FcYRIIIa receptor and enhanced antibody-dependent cellular cytotoxicity (ADCC) activity compared to rituximab. See, for example, Robak et al. BioDrugs 25.1(2011):13-25; and Forero-Torres et al. Clin Cancer Res. 18.5(2012):1395-403.

[00629] In some cases, the anti-CD20 antibody comprises PRO131921. PRO131921 is a humanized anti-CD20 monoclonal antibody created to have better binding to FcYRIIIa and enhanced ADCC compared to rituximab. See, for example, Robak et al. BioDrugs 25.1(2011):13-25; and Casulo et al. Clin Immunol. 154.1(2014):37-46; and Clinical Trial Identifier No. NCT00452127.

[00630] In some cases, the anti-CD20 antibody comprises TRU-015. TRU-015 is an anti-CD20 fusion protein derived from domains of an antibody against CD20. TRU-015 is smaller than monoclonal antibodies but maintains Fc-mediated effector functions. See, for example, Robak et al. BioDrugs 25.1(2011):13-25. TRU-015 contains an anti-CD20 single-chain variable fragment (scFv) linked to a human IgG1 joint, CH2, and CH3 domains, but not the CH1 and CL domains.

[00631] In some embodiments, an anti-CD20 antibody described herein is conjugated or otherwise linked to a therapeutic agent, e.g., a chemotherapeutic agent (e.g., cytoxan, fludarabine, histone deacetylase inhibitor, demethylating agent, vaccine peptide, anti-tumor antibiotic, tyrosine kinase inhibitor, alkylating agent, antimicrotubule or antimitotic agent), anti-allergic agent, anti-nausea (or anti-emetic) agent, pain reliever, or cytoprotective agent described herein.

[00632] In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with a B-cell lymphoma 2 (BCL-2) inhibitor (e.g., venetoclax, also called ABT-199 or GDC-0199 ;) and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with venetoclax and rituximab. Venetoclax is a small molecule that inhibits the antiapoptotic protein, BCL-2. The structure of venetoclax (4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3 -nitro-4- [(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide) is shown below .

[00633] In embodiments, the individual has CLL. In embodiments, the individual has relapsed CLL, for example, the individual has previously been administered cancer therapy. In embodiments, venetoclax is administered at a dosage of about 15 to 600 mg (e.g., 15-20, 20-50, 50-75, 75-100, 100-200, 200-300, 300-400, 400- 500, or 500-600 mg), for example, daily. In embodiments, rituximab is administered at a dosage of about 350-550 mg/m2 (e.g., 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m2) , for example, intravenously, for example, monthly.

[00634] In one embodiment, cells expressing a CAR, described herein, optionally in combination with a kinase inhibitor, for example, a BTK inhibitor such as ibrutinib, are administered to an individual in combination with a molecule that decreases the population of Treg cell. Methods that decrease the number of (e.g., deplete) Treg cells are known in the art and include, for example, depletion of CD25, administration of cyclophosphamide, modulation of GITR function. Without wishing to be bound by theory, it is believed that reducing the number of Treg cells in an individual prior to apheresis or prior to administration of a CAR-expressing cell described here reduces the number of unwanted immune cells (e.g., Tregs) in the microenvironment of tumor and reduces the individual's risk of recurrence. In one embodiment, cells expressing a CAR described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, are administered to an individual in combination with a molecule targeting GITR and/or modulating GITR functions. GITR, such as a GITR agonist and/or a GITR antibody that depletes regulatory T cells (Tregs). In embodiments, cells expressing a CAR described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, are administered to an individual in combination with cyclophosphamide. In one embodiment, GITR-binding molecules and/or molecules modulating GITR functions (e.g., GITR agonist and/or Treg-depleting GITR antibodies) are administered prior to administration of the CAR-expressing cell. For example, in one embodiment, the GITR agonist can be administered prior to apheresis of the cells. In embodiments, cyclophosphamide is administered to the subject prior to administration (e.g., infusion or reinfusion) of the CAR-expressing cell or prior to apheresis of the cells. In embodiments, cyclophosphamide and an anti-GITR antibody are administered to the subject prior to administration (e.g., infusion or re-infusion) of the CAR-expressing cell or prior to apheresis of the cells. In one embodiment, the individual has cancer (e.g., a solid cancer or a hematologic cancer such as ALL or CLL). In one embodiment, the individual has CLL. In embodiments, the individual has ALL. In embodiments, the individual has a solid cancer, e.g., a solid cancer described herein.

[00635] Exemplary GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as, for example, a GITR fusion protein described in United States Patent No.: 6,111,090, European Patent No.: 090505B1, United States Patent No.: 8,586,023, PCT Publication Nos.: WO 2010/003118 and 2011/090754, or an anti-GITR antibody described, e.g. , in United States Patent No: 7,025,62, European Patent No: 1947183B1, United States Patent No: 7,812,135, United States Patent No: 8,388,967, United States Patent No: 8,591 .886, European Patent No.: EP 1866339, PCT Publication No.: WO 2011/028683, PCT Publication No.: WO 2013/039954, PCT Publication No.: W02005/007190, PCT Publication No.: WO 2007/133822, PCT Publication No.: WO2005/055808, PCT Publication No.: WO 99/40196, PCT Publication No.: WO 2001/03720, PCT Publication No.: WO99/20758, PCT Publication No. No.: WO2006/083289, PCT Publication No.: WO 2005/115451, United States Patent No.: 7,618,632, and PCT Publication No.: WO 2011/051726.

[00636] In one embodiment, the combination of a CAR-expressing cell described herein and a kinase inhibitor described herein is administered to an individual in combination with a GITR agonist, e.g., a GITR agonist described herein. In one embodiment, the GITR agonist is administered prior to the CAR-expressing cell. For example, in one embodiment, the GITR agonist can be administered prior to apheresis of the cells. In one embodiment, the individual has CLL.

[00637] Drugs that inhibit the calcium-dependent calcineurin phosphatase (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor-induced signaling (rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993) also can be used. In another aspect, the cell compositions of the present invention can be administered to a patient in conjunction with (e.g., before, simultaneously with, or after) bone marrow transplantation, T cell ablative therapy using chemotherapy agents such as fludarabine. , external beam therapy (XRT), cyclophosphamide, and/or antibodies such as OKT3 or CAMPATH. In one aspect, the cell compositions of the present invention are administered following B cell ablative therapy such as agents that react with CD20, for example, Rituxan. For example, in one embodiment, individuals may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following transplantation, individuals receive an infusion of the expanded immune cells of the present invention. In an additional embodiment, expanded cells are administered before or after surgery.

[00638] In one embodiment, the individual may be administered an agent that reduces or improves side effects associated with the administration of a CAR-expressing cell. Side effects associated with the administration of a CAR-expressing cell include, but are not limited to CRS, and hemophagocytic lymphohistiocytosis (HLH), also called Macrophage Activation Syndrome (MAS). Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like. Accordingly, the methods described herein may comprise administering a CAR-expressing cell described herein to a subject and also administering an agent to control elevated levels of a soluble value resulting from treatment with a CAR-expressing cell. In one embodiment, the soluble factor elevated in the individual is one or more of IFN-Y, TNFa, IL-2 receptor and IL-6. Therefore, an agent administered to treat this side effect may be an agent that neutralizes one or more of these soluble factors. Examples of such agents include, but are not limited to, a steroid (e.g., corticosteroid), a TNFa inhibitor, and an IL-6 inhibitor. An example of a TNFa inhibitor is an anti-TNFα antibody molecule such as infliximab, adalimumab, Certolizumab pegol, and golimumab. Another example of a TNFα inhibitor is a fusion protein such as entanercept. Small molecule inhibitor of TNFa includes, but is not limited to, xanthine derivatives (e.g., pentoxifylline) and bupropion. An example of an IL-6 inhibitor is an anti-IL-6 antibody molecule or anti-IL-6 receptor antibody molecule such as tocilizumab (toc), sarilumab, elsilimab, CNTO 328, ALD518/BMS-945429, CNTO 136, CPSI-2364, CDP6038, VX30, ARGX-109, FE301, and FM101. In one embodiment, the anti-IL-6 receptor antibody molecule is tocilizumab. An example of an IL-1R-based inhibitor is anakinra.

[00639] In one embodiment, the individual may be administered an agent that enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule. Inhibitory molecules, e.g., Programmed Death 1 (PD1), can, in some embodiments, decrease the ability of a CAR-expressing cell to mount an immune effector response. 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 an inhibitory molecule, for example, by inhibition at the level of DNA, RNA or protein, can optimize CAR-expressing cell performance. In embodiments, an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), a transcription activator-like effector nuclease (TALEN), or a finger endonuclease zinc (ZFN), can be used to inhibit the expression of an inhibitory molecule in the CAR-expressing cell. In one embodiment, the inhibitor is an shRNA. In one embodiment, the inhibitory molecule is inhibited within a CAR-expressing cell. In these embodiments, a dsRNA molecule that inhibits expression of the inhibitory molecule is linked to the nucleic acid encoding a component, e.g., all components, of the CAR. In one embodiment, the inhibitor of an inhibitory signal may be, for example, an antibody or antibody fragment that binds to an inhibitory molecule. For example, the agent may be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2, or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX101, and marketed as Yervoy®; Bristol-Myers Squibb; Tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206). In one embodiment, the agent is an antibody or antibody fragment that binds to TIM3. In one embodiment, the agent is an antibody or antibody fragment that binds to LAG3. In one embodiment, the agent is an antibody or antibody fragment that binds to CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5) .

[00640] PD1 is an inhibitory member of the CD28 family of receptors which also includes CD28, CTLA-4, ICOS, and BTLA. PD1 is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2 have been shown to down-regulate T cell activation upon binding to PD1 (Freeman et al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2: 261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:2817; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immunosuppression can be reversed by inhibiting the local interaction of PD1 with PD-L1. Antibodies, antibody fragments, and other inhibitors of PD1, PD-L1, and PD-L2 are known and can be used in combination with a CD19 CAR described herein. For example, nivolumab (also referred to as BMS-936558 or MDX1106; Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody that specifically blocks PD1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are described in US 8,008,449 and WO2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD1. Pidilizumab and other humanized anti-PD1 monoclonal antibodies are described in WO2009/101611. Pembrolizumab (formerly known as lambrolizumab, and also referred to as Keytruda, MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD1. Pembrolizumab and other humanized anti-PD1 antibodies are described in US 8,354,509 and WO2009/114335. MEDI4736 (Medimmune) is a human monoclonal antibody that binds to PDL1, and inhibits the ligand's interaction with PD1. MDPL3280A (Genentech/Roche) is a human Fc-optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to PD-L1 are described in United States Patent No.: 7,943,743 and United States Publication No.: 20120039906. Other anti-PD-L1 binding agents include YW243.55.S70 (regions heavy and light chain variables are shown in SEQ ID NOs 20 and 21 in WO2010/077634) and MDX-1 105 (also referred to as BMS-936559, and, for example, anti-PD-L1 binding agent described in WO2007/ 005874). AMP-224 (B7-DCIg; Amplimmune; e.g., described in WO2010/027827 and WO2011/066342), is a soluble PD-L2 Fc fusion receptor that blocks the interaction between PD1 and B7-H1. Other anti-PD1 antibodies include AMP 514 (Amplimmune), among others, for example, anti-PD1 antibodies described in US 8,609,089, US 2010028330, and/or US 20120114649.

[00641] TIM3 (T cell immunoglobulin-3) also negatively regulates T cell function, particularly in CD8+ cytotoxic T 1 and CD4+ T helper 1 cells secreting IFN-g, and plays a critical role in T cell exhaustion Inhibition of the interaction between TIM3 and its ligands, for example, galectin-9 (Gal9), phosphotidylserine (PS), and HMGB1, can enhance the immune response. Antibodies, antibody fragments, and other inhibitors of TIM3 and its ligands are available in the art and can be used in combination with a CD19 CAR described herein. For example, antibodies, antibody fragments, small molecules, or peptide inhibitors that target TIM3 bind to the IgV domain of TIM3 to inhibit interaction with its ligands. Antibodies and peptides that inhibit TIM3 are described in WO2013/006490 and US20100247521. Other anti-TIM3 antibodies include humanized versions of RMT3-23 (described in Ngiow et al., 2011, Cancer Res, 71:3540-3551), and clone 8B.2C12 (described in Monney et al., 2002, Nature, 415 :536-541). Bispecific antibodies that inhibit TIM3 and PD-1 are described in US20130156774.

[00642] In other embodiments, the agent that enhances the activity of a CAR-expressing cell is a CEACAM inhibitor (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5 inhibitor). In one embodiment, the CEACAM inhibitor is an anti-CEACAM antibody molecule. Exemplary anti-CEACAM-1 antibodies are described in WO 2010/125571, WO 2013/082366 WO 2014/059251 and WO 2014/022332, for example, a monoclonal antibody 34B1, 26H7, and 5F4; or a recombinant form thereof, as described in, for example, US 2004/0047858, US 7,132,255 and WO 99/052552. In other embodiments, the anti-CEACAM antibody binds a CEACAM-5 as described in, for example, Zheng et al. PLoS One. September 2, 2010;5(9). pii: e12529 (DOI:10:1371/journal.pone.0021146), or cross-reacts with CEACAM-1 and CEACAM-5 as described in, for example, WO 2013/054331 and US 2014/0271618.

[00643] Without wishing to be bound by theory, it is believed that carcinoembryonic antigen cell adhesion molecules (CEACAM), such as CEACAM-1 and CEACAM-5, mediate, at least in part, inhibition of an antitumor immune response (see, for example, Markel et al. J Immunol. 2002 Mar 15;168(6):2803-10; Markel et al. J Immunol. 2006 Nov 1;177(9):6062-71; Markel et al. Immunology. February 2009;126(2):186-200; Markel et al. Cancer Immunol Immunother. February 2010;59(2):215-30; Ortenberg et al. Mol Cancer Ther. June 2012;11(6):1300-10; Stern et al. J Immunol. 1 June 2005;174(11):6692-701; Zheng et al. PLoS One. 2 September 2010;5(9) .pii:e12529). For example, CEACAM-1 has been described as a heterophilic ligand for TIM-3 and as playing a role in TIM-3-mediated T cell tolerance and exhaustion (see, e.g., WO 2014/022332; Huang, et al. (see, for example, WO 2014/022332; Huang, et al.). 2014) Nature doi:10.1038/nature13848). In embodiments, coblocking CEACAM-1 and TIM-3 has been shown to enhance an antitumor immune response in models of colorectal cancer (see, for example, WO 2014/022332; Huang, et al. (2014), supra). In other embodiments, coblocking CEACAM-1 and PD-1 reduces T cell tolerance as described, for example, in WO 2014/059251. Thus, CEACAM inhibitors can be used like other immunomodulators described herein (e.g., anti-PD-1 and/or anti-TIM-3 inhibitors) to enhance an immune response against a cancer, e.g., a melanoma, a lung cancer (e.g., NSCLC), bladder cancer, colon cancer, ovarian cancer, and other cancers as described herein.

[00644] LAG3 (lymphocyte activation gene-3 or CD223) is a cell surface molecule expressed on activated T cells and B cells that has been shown to play a role in CD8+ T cell exhaustion. Antibodies, antibody fragments, and other inhibitors of LAG3 and its ligands are available in the art and can be used in combination with a CD19 CAR described herein. For example, BMS-986016 (Bristol-Myers Squib) is a monoclonal antibody that targets LAG3. IMP701 (Immutep) is an antagonistic LAG3 antibody and IMP731 (Immutep and GlaxoSmithKline) is a LAG3-depleting antibody. Other LAG3 inhibitors include IMP321 (Immutep), which is a recombinant fusion protein of a portion of soluble LAG3 and Ig that binds to MHC class II molecules and activates antigen-presenting cells (APC). Other antibodies are described, for example, in WO2010/019570.

[00645] In some embodiments, CAR therapy and the kinase inhibitor are administered in combination with a bell fold-like receptor (TLR) agonist. The TLR agonist may be a TLR9 agonist. In some embodiments, the TLR agonist is an oligodeoxynucleotide, e.g., a CG-enriched oligodeoxynucleotide, e.g., an unmethylated CG-enriched oligodeoxynucleotide. See, for example, Sagiv-Barfi et al., "Ibrutinib enhances the antitumor immune response induced by intratumoral injection of a TLR9 ligand in syngeneic mouse lymphoma model." Blood. February 6, 2015. pii: blood-2014-08-593137, which is incorporated herein by reference in its entirety. In some embodiments, the TLR agonist is administered in combination with a CAR-expressing NK cell. Without being bound by theory, the TLR agonist may promote the activation of NK cells such as CAR-expressing NK cells. In some embodiments, the TLR agonist is administered by injection, e.g., intrahumoral injection.

[00646] In some embodiments, the agent that enhances the activity of a CAR-expressing cell may be, for example, a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule, or fragment of the same, and the second domain is a polypeptide that is associated with a positive signal, for example, a polypeptide comprising an intracellular signaling domain as described herein. In some embodiments, the polypeptide that is associated with a positive signal may include a costimulatory domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain of CD28, CD27, and/or ICOS, and/or a primary signaling domain. , for example, from CD3 zeta, for example, described here. In one embodiment, the fusion protein is expressed by the same cell that expresses the CAR. In another embodiment, the fusion protein is expressed by a cell, for example, a T cell that does not express an anti-CD19 CAR.

[00647] In one embodiment, the agent that enhances the activity of a CAR-expressing cell, described here, is miR-17-92.

[00648] In one embodiment, the agent that enhances the activity of a CAR described here is a cytokine. Cytokines have important functions related to T cell expansion, differentiation, survival, and homeostasis. Cytokines that can be administered to the individual receiving a CAR-expressing cell described here include: IL-2, IL-4, IL-7, IL-9, IL-15, IL-18, and IL-21, or a combination thereof. In preferred embodiments, an administered cytokine is IL-7, IL-15, or IL-21, or a combination thereof. The cytokine may be administered once a day or more than once a day, for example, twice a day, three times a day, or four times a day. The cytokine may be administered for more than one day, for example, the cytokine is administered for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. For example, the cytokine is administered once daily for 7 days.

[00649] In embodiments, the cytokine is administered in combination with CAR-expressing cells. The cytokine can be simultaneously administered concurrently with CAR-expressing cells, for example, administered on the same day. The cytokine can be prepared in the same pharmaceutical composition as the CAR-expressing cells, or it can be prepared in a separate pharmaceutical composition. Alternatively, the cytokine can be administered immediately after administration of the CAR-expressing T cells, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the CAR-expressing cells. In embodiments where the cytokine is administered in a dosing regimen that occurs over more than one day, the first day of the cytokine dosing regimen may be on the same day as administration with CAR-expressing cells, or the first day of the regimen. Cytokine dosing can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of CAR-expressing T cells. In one embodiment, on the first day, CAR-expressing cells are administered to the subject, and on the second day, a cytokine is administered once daily for the next 7 days. In a preferred embodiment, the cytokine to be administered in combination with CAR-expressing cells is IL-7, IL-15, and/or IL-21.

[00650] In other embodiments, the cytokine is administered for a sufficient period of time after administration of the CAR-expressing cells, for example, at least 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more after administration of CAR-expressing cells. In one embodiment, the cytokine is administered after evaluating the individual's response to CAR-expressing cells. For example, the individual is administered CAR-expressing cells in accordance with the dosage and regimens described herein. The individual's response to therapy such as CART is assessed at 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more after administration of CAR-expressing cells, using any of the methods described here, including inhibition of tumor growth, reduction of circulating tumor cells, or tumor regression. Individuals who do not exhibit a sufficient response to CART therapy may be administered a cytokine. Administration of the cytokine to the individual who has a suboptimal response to CART therapy improves CART efficacy and/or antitumor activity. In a preferred embodiment, the cytokine administered after administration of CAR-expressing cells is IL-7. Combination with a low dose of an mTOR inhibitor

[00651] In one embodiment, cells expressing a CAR molecule, for example, a CAR molecule described here, are administered in combination with a low immune boosting dose of an mTOR inhibitor.

[00652] In one embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 5, but not more than 90%, at least 10, but not more than 90%, at least 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%, or at least 70, but not more than 90%.

[00653] In one embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 5, but not more than 80%, at least 10, but not more than 80%, at least at least 15 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%, or at least 60, but not more than 80%.

[00654] In one embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 5, but not more than 70%, at least 10, but not more than 70%, at least 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%, or at least 50, but not more than 70%.

[00655] In one embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 5, but not more than 60%, at least 10, but not more than 60%, at least least 15 but not more than 60%, at least 20 but not more than 60%, at least 30 but not more than 60%, or at least 40 but not more than 60%.

[00656] In one embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 5, but not more than 50%, at least 10, but not more than 50%, at least least 15 but not more than 50%, at least 20 but not more than 50%, at least 30 but not more than 50%, or at least 40 but not more than 50%.

[00657] In one embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 5, but not more than 40%, at least 10, but not more than 40%, at least least 15 but not more than 40%, at least 20 but not more than 40%, at least 30 but not more than 40%, or at least 35 but not more than 40%.

[00658] In one embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 5, but not more than 30%, at least 10, but not more than 30%, at least least 15 but not more than 30%, at least 20 but not more than 30%, or at least 25 but not more than 30%.

[00659] In one embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 1, 2, 3, 4 or 5, but not more than 20%, at least 1, 2 , 3, 4 or 5, but not more than 30%, at least 1, 2, 3, 4 or 5, but not more than 35, at least 1, 2, 3, 4 or 5, but not more than than 40%, or at least 1, 2, 3, 4 or 5, but not more than 45%.

[00660] In one embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 1, 2, 3, 4 or 5, but not more than 90%.

[00661] As described herein, the extent of mTOR inhibition can be expressed as the extent of P70 S6 kinase inhibition, for example, the extent of mTOR inhibition can be determined by the level of decrease in P70 S6 kinase activity, for example, by a decrease in the phosphorylation of a P70 S6 kinase substrate. The level of extent of mTOR inhibition can be assessed by a method described here, for example, by the Boulay assay, or assessment of phosphorylated S6 levels by western blot.

EXEMPLARY MTOR INHIBITORS

[00662] As used herein, the term "mTOR inhibitor" refers to a compound or ligand, or a pharmaceutically acceptable salt thereof, that inhibits mTOR kinase in a cell. In one embodiment an mTOR inhibitor is an allosteric inhibitor. In one embodiment an mTOR inhibitor is a catalytic inhibitor.

[00663] Allosteric mTOR inhibitors include the tricyclic neutron compound rapamycin (sirolimus), rapamycin-related compounds, which are compounds having structure and functionality similar to rapamycin including, for example, rapamycin derivatives, rapamycin analogues (also referred to as rapalogs) and other macrolide compounds that inhibit mTOR activity.

[00664] Rapamycin is a macrolide antibiotic produced by streptomyces hygroscopicus having the structure shown in formula A.

[00665] See, for example, McAlpine, J.B., et al., J. Antibiotics (1991) 44: 688; Schreiber, S. L., et al., J. Am. Chem. Soc. (1991) 113: 7433; United States Patent No. 3,929,992. There are several numbering schemes proposed for rapamycin. To avoid confusion, when specific rapamycin analogues are named here, the names are provided with reference to rapamycin using the formula A numbering scheme.

[00666] Rapamycin analogs useful in the invention are, for example, O-substituted analogs in which the hydroxyl group on the cyclohexyl ring of rapamycin is replaced by OR1 in which R1 is hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl; for example, RAD001, also known as, everolimus as described in US 5,665,772 and WO94/09010 the contents of which are incorporated by reference. Other suitable rapamycin analogues include those substituted at position 26 or 28. The rapamycin analogue may be an epimer of an analogue mentioned above, particularly an epimer of an analogue substituted at position 40, 28 or 26, and may optionally be hydrogenated, e.g. example, as described in US 6,015,815, WO95/14023 and WO99/15530 the content of which is incorporated by reference, for example, ABT578 also known as zotarolimus or a rapamycin analog described in US 7,091,213, WO98/02441 and WO01/14387 the contents of which are incorporated by reference, for example, AP23573 also known as ridaforolimus.

[00667] Examples of rapamycin analogues suitable for use in the present invention of US 5,665,772 include, but are not limited to, 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'-en-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, 40-O-(2-acetaminoethyl)-rapamycin, 40-O-(2-nicotinamidoethyl)-rapamycin, 40-O-(2-(N-methyl-imidazo-2'-ylcarbetoxamido )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.

[00668] Other rapamycin analogues useful in the present invention are analogues where the hydroxyl group on the cyclohexyl ring of rapamycin and/or the hydroxy group at position 28 is replaced with a hydroxyester group are known, for example rapamycin analogues found U.S. RE44.768, for example temsirolimus.

[00669] Other rapamycin analogues useful in the present invention include those in which the methoxy group at position 16 is substituted with another substituent, preferably (optionally substituted by hydroxy) alkynyloxy, benzyl, orthomethoxybenzyl or chlorobenzyl and/or in which the methoxy group in the position 39 is deleted along with carbon 39 so that the cyclohexyl ring of rapamycin becomes a cyclopentyl ring without the methoxy group of position 39; for example, as described in WO95/16691 and WO96/41807, the contents of which are incorporated by reference. The analogues can also be modified so that the hydroxy at position 40 of rapamycin is alkylated and/or the carbonyl 32 is reduced.

[00670] Rapamycin analogs of WO95/16691 include, but are not limited to, 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-deoxy-39-formyl-42-nor-rapamycin, 39-demethoxy-40-deoxy-39-hydroxymethyl-42-nor-rapamycin, 39-demethoxy-40-deoxy-39-carboxy -42-nor-rapamycin, 39-demethoxy-40-deoxy-39-(4-methyl-piperazin-1-yl)carbonyl-42-nor-rapamycin, 39-demethoxy-40-deoxy-39- (morpholin-4 -yl)carbonyl-42-nor-rapamycin, 39-demethoxy-40-deoxy-39- [N-methyl,N-(2-pyridin-2-yl-ethyl)]carbamoyl-42-nor-rapamycin and 39- demethoxy-40-deoxy-39-(p-toluenesulfonylhydrazonomethyl)-42-nor-rapamycin.

[00671] Rapamycin analogs of WO96/41807 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.

[00672] Another suitable rapamycin analogue is umirolimus as described in US2005/0101624 the content of which is incorporated by reference.

[00673] RAD001, otherwise known as everolimus (Afinitor®), 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-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19, 30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-aza-tricycle [30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene- 2, 3,10,14,20-pentaone.

[00674] Other examples of allosteric mTOR inhibitors include sirolimus (rapamycin, AY-22989), 40-[3-hydroxy-2-(hydroxymethyl)-2-methyl-propanoate]-rapamycin (also called temsirolimus or CCI-779) and ridaforolimus (AP-23573/MK-8669). Other examples of allosteric mTOR inhibitors include zotarolimus (ABT578) and umirolimus.

[00675] Alternatively or additionally, catalytic APT-competitive mTOR inhibitors have been found to target the mTOR kinase domain directly and target both mTORC1 and mTORC2. These mTORC1 inhibitors are also more effective than such allosteric mTOR inhibitors as rapamycin, because they modulate rapamycin-resistant mTORC1 outputs such as 4EBP1-T37/46 phosphorylation and buffer-dependent translation.

[00676] 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, C.M., 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[2,3d]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]oxazol-5-yl)-1-isopropyl-1H-pyrazolo [3,4-d]pyrimidin-4-amine (WO10051043 and WO2013023184); N-(3-(N-(3-((3,5-dimethoxyphenyl)amino)quinoxaline-2-yl)sulfamoyl)phenyl)-3-methoxy-4-methylbenzamide (WO07044729 and WO12006552); PKI-587 (Venkatesan, A.M., 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).

[00677] Other 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 mammalian Target of Rapamicyn. Cancer Res. 69(15): 6232-6240).

[00678] mTOR inhibitors useful in accordance with the present invention also include prodrugs, derivatives, pharmaceutically acceptable salts, or analogues thereof of any of the foregoing.

[00679] mTOR inhibitors, such as RAD001, can be formulated for release based on methods well established in the art, based on the particular dosages described here. In particular, United States Patent 6,004,973 (incorporated herein by reference) provides examples of formulations usable with the mTOR inhibitors described herein.

MTOR INHIBITION ASSESSMENT

[00680] mTOR phosphorylates P70 S6 kinase, thereby activating P70 S6 kinase and allowing it to phosphorylate its substrate. The extent of mTOR inhibition can be expressed as the extent of P70 S6 kinase inhibition, for example, the extent of mTOR inhibition can be determined by the level of decrease in P70 S6 kinase activity, for example, by the decrease in phosphorylation of a P70 S6 kinase substrate. One can determine the level of mTOR inhibition by measuring P70 S6 kinase activity (the ability of P70 S6 kinase to phosphorylate a substrate), in the absence of inhibitor, for example, before inhibitor administration, and in the presence of inhibitor, or after administration of an inhibitor. The level of P70 S6 kinase inhibition determines the level of mTOR inhibition. Thus, if P70 S6 kinase is inhibited by 40%, mTOR activity, as measured by P70 S6 kinase activity, will be inhibited by 40%. The extent or level of inhibition referred to here is the average level of inhibition over the dosing interval. By way of example, if the inhibitor is provided once a week, the level of inhibition will be determined by the average level of inhibition during that interval, namely, one week.

[00681] Boulay et al., Cancer Res, 2004, 64:252-61, hereby incorporated by reference, teach an assay that can be used to evaluate the level of mTOR inhibition (referred to here as the Boulay assay). In one embodiment, the assay relies on evaluating the P70 S6 kinase activity of biological samples before and after administration of an mTOR inhibitor, e.g., RAD001. Samples can be taken at preselected times after treatment with an mTOR inhibitor, for example, 24, 48, and 72 hours after treatment. Biological samples, for example, from peripheral blood or skin mononuclear cells (PBMCs) can be used. Total protein extracts are prepared from the samples. P70 S6 kinase is isolated from protein extracts by immunoprecipitation using an antibody that specifically recognizes P70 S6 kinase. Isolated P70 S6 kinase activity can be measured in an in vitro kinase assay. The isolated kinase can be incubated with substrates of 40S ribosomal subunit (which is an endogenous substrate of P70 S6 kinase) and gamma-32P under conditions that allow phosphorylation of the substrate. Then, the reaction mixture can be resolved on an SDS-PAGE gel, and 32P signal analyzed using a PhosphorImager. A 32P signal corresponding to the size of the 40S ribosomal subunit indicates phosphorylated substrate and P70 S6 kinase activity. The increase and decrease in kinase activity can be calculated by quantifying the area and intensity of the 32P signal of the phosphorylated substrate (e.g., using ImageQuant, Molecular Dynamics), assigning arbitrary unit values to the quantified signal, and comparing the values after administration with values from before administration or with a reference value. For example, the percentage of inhibition of kinase activity can be calculated with the following formula: 1-(value obtained after administration/value obtained before administration) X 100. As described above, the extent or level of inhibition referred to here is the average level inhibition during the dose interval.

[00682] Methods for evaluating kinase activity, for example, P70 S6 kinase activity, are also provided in US 7,727,950, whereby incorporated by reference.

[00683] The level of mTOR inhibition can also be assessed by a change in the ratio of PD1-negative to PD1-positive T cells. Peripheral blood T cells can be identified as PD1 negative or positive by methods known in the art.

Low-Dose mTOR Inhibitors

[00684] Methods described here use immune-boosting low-dose mTOR inhibitors, for example, allosteric mTOR inhibitors, including rapalogues such as RAD001. In contrast, inhibitor levels that completely or almost completely inhibit the mTOR reaction series are immunosuppressive and are used, for example, to prevent organ transplant rejection. Furthermore, high doses of rapalogues that fully inhibit mTOR also inhibit tumor cell growth, and are used to treat a variety of cancers (see, for example, Antineoplastic effects of mammalian target of rapamycine inhibitors. Salvadori M. World J Transplant . 2012 Oct 24;2(5):74-83; Current and Future Treatment Strategies for Patients with Advanced Hepatocellular Carcinoma: Role of mTOR inhibition. Finn RS. Liver Cancer. 2012 Nov;1(3-4):247-256 ; Emerging pathways in Hepatocellular Carcinoma. Moeini A, Cornellà H, Villanueva A. Liver Cancer. 2012 Sep;1(2):83-93; Targeted cancer therapy - are days of systemic chemotherapy numbered? Joo WD, Visintin I, Mor G . Maturitas. 2013 Sep 20.; Role of natural and adaptive immunity in renal cell carcinoma response to VEGFR-TKIs and mTOR inhibitor. Santoni M, Berardi R, Amantini C, Burattini L, Santini D, Santoni G, Cascinu S. Int J Cancer. October 2, 2013).

[00685] The present invention is based, at least in part, on the surprising finding that doses of mTOR inhibitors well below those used in current clinical settings have a superior effect in increasing an immune response in an individual and increasing the ratio of T cells PD-1 negative/PD-1 positive T cells. It was surprising that low doses of mTOR inhibitors, producing only partial inhibition of mTOR activity, were able to effectively improve immune responses in human subjects and increase the ratio of PD-1 negative T cells/PD-1 positive T cells.

[00686] Alternatively, or in addition, without wishing to be bound by any theory, it is believed that a low immune boosting dose of an mTOR inhibitor may increase natural T cell numbers, e.g., at least transiently, e.g. , compared to an untreated individual. Alternatively or additionally, again while not wishing to be bound by theory, it is believed that treatment with an mTOR inhibitor after a sufficient amount of time or dosage results in one or more of the following:

[00687] an increase in the expression of one or more of the following markers: CD62Lelevated, CD127elevated, CD27+, and BCL2, for example, on memory T cells, for example, memory T cell precursors;

[00688] a decrease in the expression of KLRG1, for example, on memory T cells, for example, memory T cell precursors; It is

[00689] an increase in the number of memory T cell precursors, for example, cells with any one or combination of the following characteristics: increased CD62L, increased increased CD127, increased CD27+, decreased KLRG1, and increased BCL2;

[00690] and in which any of the changes described above occur, for example, at least transiently, for example, compared to an untreated individual (Araki, K et al. (2009) Nature 460:108-112). Memory T cell precursors are memory T cells that are early in the differentiation program. For example, memory T cells have one or more of the following characteristics: increased CD62L, increased CD127, increased CD27+, decreased KLRG1, and/or increased BCL2.

[00691] In one embodiment, the invention relates to a composition, or dosage form, of an mTOR inhibitor, for example, an allosteric mTOR inhibitor, for example, a rapalog, rapamycin, or RAD001, or an inhibitor of catalytic mTOR, which, when administered over a selected dosage regimen, e.g., once daily or once weekly, is associated with: a level of mTOR inhibition that is not associated with complete or significant immune suppression, but is associated with enhancement of the immune response.

[00692] An mTOR inhibitor, for example, an allosteric mTOR inhibitor, for example, a rapalog, rapamycin, or RAD001, or a catalytic mTOR inhibitor, can be provided in a sustained release formulation. Any of the compositions or unit dosage forms described herein may be provided in a sustained-release formulation. In some embodiments, a sustained-release formulation will have lower bioavailability than an immediate-release formulation. For example, in embodiments, to achieve a therapeutic effect similar to an immediate-release formulation, a sustained-release formulation will have from about 2 to about 5, about 2.5 to about 3.5, or about 3 times the amount of inhibitor provided in the immediate-release formulation.

[00693] In one embodiment, immediate release forms, for example, of RAD001, typically used for one administration per week, having 0.1 to 20, 0.5 to 10, 2.5 to 7.5, 3 to 6 , or about 5. mg per unit dosage form, are provided. For once-weekly administrations, these immediate-release formulations correspond to extended-release forms, having, respectively, 0.3 to 60, 1.5 to 30, 7.5 to 22.5, 9 to 18, or about 15 mg of an mTOR inhibitor, e.g. an allosteric mTOR inhibitor, e.g. rapamycin or RAD001. In embodiments, both forms are administered on a once/week basis.

[00694] In one embodiment, immediate release forms, for example, of RAD001, typically used for one administration per day, having 0.005 to 1.5, 0.01 to 1.5, 0.1 to 1.5, 0 .2 to 1.5, 0.3 to 1.5, 0.4 to 1.5, 0.5 to 1.5, 0.6 to 1.5, 0.7 to 1.5, 0.8 to 1.5, 1.0 to 1.5, 0.3 to 0.6, or about 0.5 mg per unit dosage form are provided. For once-daily administrations, these immediate-release forms correspond to the sustained-release forms, having, respectively, 0.015 to 4.5, 0.03 to 4.5, 0.3 to 4.5, 0.6 to 4 .5, 0.9 to 4.5, 1.2 to 4.5, 1.5 to 4.5, 1.8 to 4.5, 2.1 to 4.5, 2.4 to 4.5 , 3.0 to 4.5, 0.9 to 1.8, or about 1.5 mg of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin or RAD001. For once-weekly administrations, these immediate-release forms correspond to the sustained-release forms, having, respectively, 0.1 to 30, 0.2 to 30, 2 to 30, 4 to 30, 6 to 30, 8 to 30 , 10 to 30, 1.2 to 30, 14 to 30, 16 to 30, 20 to 30, 6 to 12, or about 10 mg of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g. rapamycin or RAD001.

[00695] In one embodiment, immediate release forms, for example, of RAD001, typically used for one administration per day, having 0.01 to 1.0 mg per unit dosage form, are provided. For once-daily administrations, these immediate-release forms correspond to the sustained-release forms, respectively having 0.03 to 3 mg of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin or RAD001. . For once-weekly administrations, these immediate-release forms correspond to the sustained-release forms, respectively taking 0.2 to 20 mg of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin or RAD001. .

[00696] In one embodiment, immediate release forms, for example, of RAD001, typically used for once per week administration, having 0.5 to 5.0 mg per unit dosage form, are provided. For once-weekly administrations, these immediate-release forms correspond to the sustained-release forms, respectively taking 1.5 to 15 mg of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin or RAD001. .

[00697] As described above, a target of the mTOR trail is the P70 S6 kinase. Therefore, doses of mTOR inhibitors that are useful in the methods and compositions described herein are those that are sufficient to activate no more than 80% inhibition of P70 S6 kinase activity relative to P70 S6 kinase activity in the absence of an inhibitor. of mTOR, for example, as measured by an assay described herein, e.g., the Boulay assay. In another aspect, the invention provides an amount of an mTOR inhibitor sufficient to activate no more than 38% inhibition of P70 S6 kinase activity relative to P70 S6 kinase activity in the absence of an mTOR inhibitor.

[00698] In one aspect, the dose of mTOR inhibitor useful in the methods and compositions of the invention is sufficient to activate, for example, when administered to a human subject, 90 +/-5% (i.e., 85-95%) , 89+/-5 %, 88+/-5 %, 87+/-5 %, 86+/-5 %, 85+/-5 %, 84+/-5 %, 83+/-5 %, 82+/-5%, 81+/-5%, 80+/-5%, 79+/-5%, 78+/-5%, 77+/5%, 76+/-5%, 75+ /-5%, 74+/-5%, 73+/-5%, 72 +/-5%, 71 +/-5%, 70 +/-5%, 69 +/-5%, 68 +/ -5%, 67 +/-5%, 66 +/-5%, 65 +/-5%, 64 +/-5%, 63 +/-5%, 62 +/-5%, 61 +/- 5%, 60 +/-5%, 59 +/-5%, 58 +/-5%, 57 +/-5%, 56 +/-5%, 55 +/-5%, 54 +/-5 %, 54 +/-5%, 53 +/-5%, 52 +/-5%, 51 +/-5%, 50 +/-5%, 49 +/-5%, 48 +/-5% , 47 +/-5%, 46 +/-5%, 45 +/-5%, 44 +/-5%, 43 +/-5%, 42 +/-5%, 41 +/-5%, 40 +/-5%, 39 +/-5%, 38 +/-5%, 37 +/-5%, 36 +/-5%, 35 +/-5%, 34 +/-5%, 33 +/-5%, 32 +/-5%, 31 +/-5%, 30 +/-5%, 29 +/-5%, 28 +/-5%, 27 +/-5%, 26 + /-5%, 25 +/-5%, 24 +/-5%, 23 +/-5%, 22 +/-5%, 21 +/-5%, 20 +/-5%, 19 +/ -5%, 18 +/-5%, 17 +/-5%, 16 +/-5%, 15 +/-5%, 14 +/-5%, 13 +/-5%, 12 +/- 5%, 11 +/-5%, or 10 +/-5%, inhibition of P70 S6 kinase activity, for example, as measured by an assay described herein, e.g., the Boulay assay.

[00699] P70 S6 kinase activity in an individual can be measured using methods known in the art, such as, for example, according to the methods described in United States Patent 7,727,950, by immunoblot analysis of phosphoP70 levels S6K and/or phosphoP70 S6 levels or by in vitro kinase activity assays.

[00700] As used herein, the term "about" in reference to a dose of mTOR inhibitor refers to up to +/- 10% variability in an amount of mTOR inhibitor, but may not include any variability around of the established dose.

[00701] In some embodiments, the invention provides methods comprising administering to an individual an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, at a dosage within a minimum target level. In some embodiments, the trough level is significantly lower than trough levels associated with dosage regimens used in organ transplant and cancer patients. In one embodiment, mTOR inhibitor, e.g., RAD001, or rapamycin, is administered to result in a trough level that is less than ^, 1/4, 1/10, or 1/20 of the trough level that results in immunosuppression. or an anti-cancer effect. In one embodiment, mTOR inhibitor, e.g., RAD001, or rapamycin, is administered to result in a trough level that is less than ^, 1/4, 1/10, or 1/20 of the trough level provided in the mTOR supplement. packaging approved by the FDA for use in immunosuppression or anti-cancer indications.

[00702] In one embodiment, a method described herein comprises administering to an individual an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, at a dosage that provides a target trough level of 0.1 to 10 ng/ mL, 0.1 to 5 ng/mL, 0.1 to 3ng/mL, 0.1 to 2 ng/mL, or 0.1 to 1 ng/mL.

[00703] In one embodiment, a method described herein comprises administering to an individual an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, at a dosage that provides a target trough level of 0.2 to 10 ng/ mL, 0.2 to 5 ng/mL, 0.2 to 3ng/mL, 0.2 to 2 ng/mL, or 0.2 to 1 ng/mL.

[00704] In one embodiment, a method described herein comprises administering to an individual an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, at a dosage that provides a target trough level of 0.3 to 10 ng/ mL, 0.3 to 5 ng/mL, 0.3 to 3ng/mL, 0.3 to 2 ng/mL, or 0.3 to 1 ng/mL.

[00705] In one embodiment, a method described herein comprises administering to an individual an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, at a dosage that provides a target trough level of 0.4 to 10 ng/ mL, 0.4 to 5 ng/mL, 0.4 to 3 ng/mL, 0.4 to 2 ng/mL, or 0.4 to 1 ng/mL.

[00706] In one embodiment, a method described herein comprises administering to an individual an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, at a dosage that provides a target trough level of 0.5 to 10 ng/ mL, 0.5 to 5 ng/mL, 0.5 to 3 ng/mL, 0.5 to 2 ng/mL, or 0.5 to 1 ng/mL.

[00707] In one embodiment, a method described herein comprises administering to an individual an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, at a dosage that provides a target trough level of 1 to 10 ng/mL, 1 to 5 ng/mL, 1 to 3 ng/mL, or 1 to 2 ng/mL.

[00708] As used herein, the term "trough level" refers to a concentration of a drug in plasma just before the next dose, or the minimum drug concentration between two doses.

[00709] In some embodiments, a target minimum level of RAD001 is in a range of between about 0.1 and 4.9 ng/mL. In one embodiment, the target minimum level is below 3 ng/mL, for example, it is between 0.3 or less and 3 ng/mL. In one embodiment, the target minimum level is below 3 ng/mL, for example, it is between 0.3 or less and 1 ng/mL.

[00710] In another aspect, the invention may utilize an mTOR inhibitor other than RAD001 in an amount that is associated with a target trough level that is bioequivalent to the specific target trough level for RAD001. In one embodiment, the target minimum level for an mTOR inhibitor other than RAD001, is a level that provides the same level of mTOR inhibition (e.g., as measured by a method described herein, e.g., inhibition of P70 S6 kinase ) as well as a minimum level of RAD001 described here.

Pharmaceutical Compositions: mTOR Inhibitors

[00711] In one aspect, the present invention relates to pharmaceutical compositions comprising an mTOR inhibitor, for example, an mTOR inhibitor as described herein, formulated for use in combination with CAR cells described herein.

[00712] In some embodiments, the mTOR inhibitor is formulated for administration in combination with an additional one, for example, as described here.

[00713] In general, the compounds of the invention will be administered in therapeutically effective amounts as described above by any of the usual and acceptable methods known in the art, alone or in combination with one or more therapeutic agents.

[00714] Pharmaceutical formulations can be prepared using conventional mixing and dissolving procedures. For example, the bulk drug substance (e.g., an mTOR inhibitor or stabilized form of the compound (e.g., complexed with a cyclodextrin derivative or other known complexing agent) is dissolved in a suitable solvent in the presence of one or more of the excipients described herein. The mTOR inhibitor is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to provide the patient with an elegant, easily handled product.

[00715] Compounds of the invention can be administered as pharmaceutical compositions by any conventional routine, in particular enterally, for example, orally, for example, in the form of tablets or capsules, or parenterally, for example, in the form of injectable suspensions or solutions, topically, for example, in the form of lotions, gels, ointments or creams, or in a nasal or suppository form. Where an mTOR inhibitor is administered in combination with (simultaneously with or separately from) another agent as described herein, in one aspect, both components may be administered by the same routine (e.g., parenterally). Alternatively, another agent can be administered in a different routine relative to the mTOR inhibitor. For example, one mTOR inhibitor can be administered orally and the other agent can be administered parenterally.

SUSTAINED RELEASE

[00716] mTOR inhibitors, e.g., allosteric mTOR inhibitors or catalytic mTOR inhibitors, described herein may be provided as pharmaceutical formulations in the form of solid oral dosage forms comprising an mTOR inhibitor described herein, e.g., rapamycin or RAD001 , which meets product stability requirements and/or has favorable pharmacokinetic properties over immediate-release (IR) tablets, such as reduced mean peak plasma concentrations, reduced inter- and intra-patient variability in the extent of drug absorption and in peak plasma concentration, reduced Cmax / Cmin ratio and/or reduced food effects. Provided pharmaceutical formulations may allow more precise dose adjustment and/or reduce frequency of adverse events, thereby providing safe treatments for patients with an mTOR inhibitor described here, for example, rapamycin or RAD001.

[00717] In some embodiments, the present invention provides stable extended-release formulations of an mTOR inhibitor described herein, for example, rapamycin or RAD001, which are multiparticulate systems and may have functional coatings and layers.

[00718] The term "extended-release multiparticulate formulation" as used herein refers to a formulation that allows release of an mTOR inhibitor described herein, e.g., rapamycin or RAD001, over an extended period of time, e.g., for at least least 1, 2, 3, 4, 5 or 6 hours. The extended release formulation may contain matrices and coatings prepared from special excipients, for example, as described herein, which are formulated in a manner to make the active ingredient available over an extended period of time following ingestion.

[00719] The term "extended release" can be used interchangeably with the terms "sustained release" (SR) or "extended release". The term "extended release" refers to a pharmaceutical formulation that does not release active drug substance immediately after oral dosing, but in an extended manner as defined in the monograph of the Ph. Eur. Pharmacopoeias (7th Ed.) for tablets and tablets. capsules and USP general chapter <1151> for pharmaceutical dosage forms. The term "immediate release" (IR) as used herein refers to a pharmaceutical formulation that releases 85% of the active drug substance in less than 60 minutes in accordance with the Industry Guide definition: "Dissolution Test of Drug Forms". Immediate-Release Solid Oral Dosage" (FDA CDER, 1997). In some embodiments, the term "immediate release" means release of everolismus from tablets within 30 minutes, for example, as measured in the dissolution assay described herein.

[00720] Stable extended release formulations of an mTOR inhibitor described here, for example, rapamycin or RAD001, can be characterized by an in vitro release profile using assays known in the art, such as a dissolution assay as described here: a dissolution vessel charged with 900 mL of pH 6.8 phosphate buffer containing 0.2% sodium dodecyl sulfate at 37°C and dissolution is carried out using a paddle method at 75 rpm in accordance with USP test monograph 711, and test monograph Ph.Eur. 2.9.3. respectively.

[00721] In some embodiments, stable extended-release formulations of an mTOR inhibitor described herein, e.g., rapamycin or RAD001, release the mTOR inhibitor in the in vitro release assay according to the following release specifications: 0.5h : <45%, or <40, e.g. <30% 1h: 20-80%, e.g. 30-60% 2h: >50%, or >70%, e.g. >75% 3h: >60 %, or >65%, e.g., >85%, e.g., >90%.

[00722] In some embodiments, stable extended-release formulations of an mTOR inhibitor described herein, e.g., rapamycin or RAD001, release 50% of the mTOR inhibitor no sooner than 45, 60, 75, 90, 105 min, or 120 min in the in vitro dissolution assay. Biopolymer Release Methods

[00723] In some embodiments, one or more cells expressing CAR as described herein, optionally in combination with a kinase inhibitor, e.g., a BTK inhibitor such as ibrutinib, may be administered or delivered to the subject via a scaffold. biopolymer, for example, a biopolymer implant. Biopolymer scaffolds can support or enhance the release, expansion, and/or dispersal of CAR-expressing cells described here. A biopolymer scaffold comprises a biocompatible (e.g., does not substantially induce an immune or inflammatory response) and/or biodegradable polymer that may be naturally occurring or synthetic.

[00724] Examples of suitable biopolymers include, but are not limited to, agar, agarose, alginate, alginate/calcium phosphate cement (CPC), beta-galactosidase (ß-GAL), (1,2,3,4,6 - pentaacetyl a-D-galactose), cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), poly(lactide), poly (caprolactone) (PCL), poly(lactide-co-glycolide) (PLG), polyethylene oxide (PEO), poly(lactic-co-glycolic acid) (PLGA), polypropylene oxide (PPO), polyvinyl alcohol) ( PVA), silk, soy protein, and soy protein isolate, alone or in combination with any other polymer composition, in any concentration and in any ratio. The biopolymer can be augmented or modified with molecules promoting migration or adhesion, e.g., collagen mimetic peptides that bind to the collagen receptor of lymphocytes, and/or stimulatory molecules to enhance release, expansion, or function, e.g., activity. of anticancer, of the cells to be released. The biopolymer scaffold may be an injectable, for example, a gel or a semisolid, or solid composition.

[00725] In some embodiments, cells expressing CAR described here are seeded onto the biopolymer scaffold prior to release to the individual. In embodiments, the biopolymer scaffold also comprises one or more additional therapeutic agents described herein (e.g., another CAR-expressing cell, an antibody, or a small molecule) or agents that enhance the activity of a CAR-expressing cell, e.g., incorporated or conjugated to the scaffold biopolymers. In embodiments, the biopolymer scaffold is injected, for example, intratumorally, or surgically implanted into the tumor or in sufficient proximity to the tumor to mediate an antitumor effect. Additional examples of biopolymer compositions and methods for their release are described in Stephan et al., Nature Biotechnology, 2015, 33:97-101; and WO2014/110591.

Pharmaceutical compositions and treatments

[00726] Pharmaceutical compositions of the present invention may comprise a CAR-expressing cell, for example, a plurality of CAR-expressing cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g. aluminum hydroxide); and preservatives. Compositions of the present invention are, in one aspect, formulated for intravenous administration.

[00727] Pharmaceutical compositions of the present invention can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the patient's condition, and the type and severity of the patient's illness, although appropriate dosages can be determined by clinical testing.

[00728] In one embodiment, the pharmaceutical composition is substantially free of, for example, there is no detectable level of a contaminant, for example, selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, clotted human serum, bovine serum albumin, bovine serum, culture medium components, vector packaging cell or plasmid components, a bacterium and a fungus. In one embodiment, the bacteria is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.

[00729] When "an immunologically effective amount," "an antitumor effective amount," "an effective tumor inhibiting amount," or "therapeutic amount" is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (individual). It can generally be established that a pharmaceutical composition comprising the T cells described herein can be administered at a dosage of 104 to 109 cells/kg of body weight, in some cases 105 to 106 cells/kg of body weight, including all integer values within those tracks. The T cell compositions can also be administered multiple times at these dosages. The cells can be administered using infusion techniques that are commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[00730] In certain aspects, it may be desired to administer activated T cells to an individual and then subsequently again draw blood (or have an apheresis performed), activate T cells from him in accordance with the present invention, and reinfuse the patient with these activated and expanded T cells. This process can be performed multiple times every few weeks. In certain aspects, T cells can be activated from 10cc to 400cc blood tests. In certain aspects, T cells are activated from 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc blood tests.

[00731] Administration of the individual compositions can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein can be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one aspect, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In one aspect, the T cell compositions of the present invention are administered by i.v. The T cell compositions can be injected directly into a tumor, lymph node, or site of infection.

[00732] In a particular exemplary aspect, individuals may undergo leukapheresis, in which leukocytes are collected, enriched or depleted ex vivo to select and/or isolate cells of interest, e.g., T cells. can be expanded by methods known in the art and treated so that one or more CAR constructs of the invention can be introduced, thereby creating a CAR T cell of the invention. Individuals in need may subsequently undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain aspects, following or concurrent with transplantation, individuals receive an infusion of the expanded CAR T cells of the present invention. In an additional aspect, expanded cells are administered before or after surgery.

[00733] The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage scaling for human administration can be achieved in accordance with accepted practices in the art. The dose for CAMPATH, for example, will generally be in the range of 1 to about 100 mg for an adult patient, generally administered daily for a period of between 1 and 30 days. The preferred daily dose is 1 to 10 mg per day, although in some cases higher doses of up to 40 mg per day may be used (described in United States Patent No. 6,120,766).

[00734] In one embodiment, the CAR is introduced into T cells, for example, using in vitro transcription, and the subject (e.g., human) receives an initial administration of CAR T cells of the invention, and one or more subsequent administrations of the CAR T cells of the invention, wherein one or more subsequent administrations are administered in less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after previous administration. In one embodiment, more than one administration of the CAR T cells of the invention is administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the CAR T cells of the invention are administered per week. In one embodiment, the subject (e.g., human subject) receives more than one administration of the CAR T cells per week (e.g., 2, 3, or 4 administrations per week) (also referred to herein as a cycle), followed by a week without any administration of CAR T cells, and then one or more additional administrations of CAR T cells (e.g., more than one administration of CAR T cells per week) is administered to the subject. In another embodiment, the subject (e.g., human subject) receives more than one cycle of CAR T cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the CAR T cells are administered every other day for 3 administrations per week. In one embodiment, the CAR T cells of the invention are administered for at least two, three, four, five, six, seven, eight or more weeks.

[00735] In one aspect, CAR-expressing cells are generated using lentiviral viral vectors, such as lentiviruses. Cells, e.g., CARTs generated in such a way will have stable CAR expression.

[00736] In one aspect, CAR-expressing cells, e.g., CARTs, are generated using a viral vector such as a gammaretroviral vector, e.g., a gammaretroviral vector described herein. CARTs generated using these vectors can have CAR expression.

[00737] In one aspect, CARTs transiently express CAR vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction. Transient expression of CARs can be accomplished by delivery of CAR vector RNA. In one aspect, CAR RNA is transduced into the T cell by electroporation.

[00738] A potential problem that may arise in patients who are being treated using transiently CAR-expressing T cells (particularly with murine scFv carrying CARTs) is anaphylaxis after multiple treatments.

[00739] Without being linked to this theory, it is believed that such an anaphylactic response would be caused by a patient developing a response to humoral anti-CAR, that is, anti-CAR antibodies having an anti-IgE isotype. It is believed that a patient's antibody producing cells undergo a class change from IgG isotype (which does not cause prophylaxis) to IgE isotype when there is an interval of ten to fourteen days in exposure to the antigen.

[00740] If a patient is at high risk of generating an anti-CAR antibody response during the course of transient CAR therapy (such as those generated by RNA transductions), CART infusion intervals should not last no longer than ten to fourteen days.

EXAMPLES

[00741] The invention is also described in detail by reference to the following experimental examples. These examples are provided for illustration purposes only, and are not intended to be limiting unless otherwise specified. Accordingly, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations that become apparent as a result of the teaching provided herein.

[00742] Without further description, it is believed that one skilled in the art can, using the preceding description and the following illustrative examples, prepare and use the compounds of the present invention and practice the claimed methods. The following working examples specifically highlight the various aspects of the present invention, and should not be construed as limiting in any way the remainder of the invention.

Example 1: Humanization of Murine Anti-CD19 Antibody

[00743] A CD19 antibody molecule may be, for example, an antibody molecule (e.g., a humanized anti-CD19 antibody molecule) described in WO2014/153270, which is incorporated herein by reference in its entirety. Humanization of murine CD19 antibody is desired for clinical setting, where mouse-specific residues can induce a human anti-mouse antigen (HAMA) response in patients receiving CART19 treatment, i.e., treatment with T cells transduced with the construct from CAR19. Hybridoma-derived murine CD19 antibody VH and VL sequences were extracted from published literature (Nicholson et al, 1997, supra). Humanization was performed by grafting murine CD19 antibody CDR regions onto human germline acceptor constructs VH4_4-59 and VK3_L25 (vBASE database). In addition to the CDR regions, five structural residues, i.e., VH #71, #73, #78 and VL #71 #87, believed to support the structural integrity of the CDR regions were retained from the murine sequence. Furthermore, the human J elements, JH4 and JK2, were used for heavy and light chain, respectively. The amino acid sequences of the humanized antibody were designated FMC63_VL_hz and FMC63_VH_hz1, respectively, and are shown below in Table 1. Residue numbering follows Kabat (Kabat E.A. et al, 1991, supra). For CDR definitions, both Kabat as well as Chotia et al, 1987 supra) were used. Residues from mouse CD19 are shown in bold/italics. Boxed positions #60/61/62 indicate the potential post-translational modification (PTM) site in CDR H2, also called HCDR2.

[00744] Table 1: Amino acid sequences of humanized CD19 variable domains (SEQ ID NOs:114-117, respectively, in the order of presentation).

[00745] These humanized CD19 IgGs were used to generate the soluble scFvs for expression and scFvs for the total CART CD19 constructs (see Examples below). Of interest was that during humanization, position 62 in the CDRH2 region preferred to be a serine residue rather than the alanine present in murine CDRH2. The murine sequence does not have a post-translational modification (PTM), and has asparagine-serine-alanine at positions 60/61/62, respectively in CDRH2. This generates potential PTM motifs (indicated as the boxed site in CDRH2) during the course of humanization. It was tested whether the PTM site generated during the humanization process was actually a "real" PTM or merely a theoretical site. It was hypothesized that the amino acid motif, asparagine followed by serine (NS) may be susceptible to post-translational deamination, however this was sometimes not readily apparent. It was also hypothesized that asparagine followed by any amino acid except proline and then serine (NxS, x#P) may be susceptible to post-translational N-glycolysation. To test this hypothesis, two IgG variants were generated in which the asparagine at position 60 (known to be a glycosylation site) was mutated to serine, or glutamine and designated FMC63_VH_hz2 (N60S) and FMC63_VH_hz2 (N60Q), respectively. These constructs were generated in order to eliminate the potential post-translational modification (PTM) site and test for maintained activity (see Example 2, below).

Cloning:

[00746] DNA sequences encoding humanized and mouse VL and VH domains were obtained, and the codons for the constructs were optimized for expression in Homo sapiens cells.

[00747] Sequences encoding VL and VH domains were subcloned from the cloning vectors into expression vectors suitable for secretion into mammalian cells. The light and heavy chains were cloned into individual expression vectors to allow cotransfection. Elements of the expression vector include a promoter (Cytomegalovirus (CMV) enhancer promoter), a signal sequence to facilitate secretion, a polyadenylation signal and transcription terminator (Bovine Growth Hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes (e.g. of SV40 and ColE1 origin or others known in the art) and elements to allow secretion (ampicillin resistant gene and zeocin marker).

Expression:

[00748] Chimeric and humanized IgG candidates were expressed in mammalian HEK293F cells on a 1mL scale. The cleared supernatants were used for FACS binding studies. More precisely, HEK293F cells were diluted to 5E5 cells/mL in FreeStyle medium supplemented with Pen/Strep and 1 mL transferred into a deep round-base 24-well plate. 0.5 µg of light chain and 0.5 µg of heavy chain mammalian expression plasmids were diluted in the same medium together with 4 µl of FuGENE HD (Roche REF 04709705001). After 15 minutes of incubation at RT, DNA/Fugene mixture was added dropwise to the cells and placed in a 5% CO2 incubator at 250 rpm, 37°C for five days. The supernatants were then separated from the cells by centrifugation. To measure IgG content, 200 µL aliquots were placed in the wells of 96-well microtiter plates. All samples and standards were measured in duplicate using Protein A Dip and readout biosensors (Fortebio Cat No 185010). The plate was placed in an Octet instrument (ForteBio) and allowed to equilibrate to 27°C in the thermostated chamber. Data were automatically processed using Octet User version 3.0 software and concentration determined by comparison to an IgG standard curve.

FACS Binding Analysis:

[00749] Humanized and chimeric antibodies were evaluated with a flow cytometry binding assay using 300.19-hsCD19FL cell line. This cell line was generated by transfecting the mouse preB cell line 300.19 with a vector (hCD19 FL/pEF4-myc-HisA) encoding the sequence encoding full-length CD19 and natural promoter as well as a Zeocin-resistant gene. Briefly, 300.19 cells were electroporated with the linearized plasmid and then cells expressing high levels of hsCD19 were identified using an APC-conjugated human anti-CD19 Ab (HIB19 clone from BD 555415) and subsequently sorted using a FACS Aria flow cytometer. Sorted hsCD19+ cells were cultured and confirmed to stably express high levels of hsCD19.

[00750] The binding assay can be performed directly with serum-free culture media containing an expressed IgG. All IgGs evaluated were normalized to the same concentration (85 nM), before being diluted by a serial dilution of up to 3 times 1.4pM. Then, in a 96-well plate, aliquots of 5x105 cells/cells were incubated for 30 minutes at 4°C with diluted IgGs. Cells were washed twice with FACS buffer (0.5% BSA in PBS) before addition of the detection antibody, an APC-conjugated goat anti-hu IgG, specific Fc fragment (Dianova #109-136- 098), diluted 1:1000 in FACS buffer. Cells were incubated for a further 30 minutes at 4°C, then washed twice in FACS buffer and assayed using FACS Calibur (BD Bioscience). Plotting of binding curves (mean fluorescence intensity versus IgG concentration) and EC50 determination were performed with GraphPad PrismTM 3.0 software with nonlinear regression analysis, sigmoidal dose response (variable slope).

[00751] FACS analyzes show that apparent binding to all IgGs evaluated can vary widely, with some constructs exhibiting a 5- to 10-fold change in EC50 as an IgG versus an scFv. Based on EC50 values, lead candidates are chosen that have a binding affinity within a factor of 2 or better compared to the chimeric reference.

Example 2: Characterization of anti-CD19 soluble scFv fragments derived from humanized CD19 IgG antibodies

[00752] Soluble scFv fragments were generated from the humanized CD19 IgGs described in Example 1 using standard molecular biology techniques. These soluble scFvs were used in characterization studies to examine the stability, cell surface expression, and binding properties of the scFvs. Additionally, experiments were also conducted to investigate the impact of potential PTM during the humanization process.

expression and purification of scFv

[00753] For transfection of each scFv construct, around 3e8 293F cells were transfected with 100 µg of plasmid using PEI as the transfection reagent in a ratio of 3:1 (PEI:DNA). Cells were cultured in 100 mL of EXPi293 expression media (Invitrogen) in a shaker flask at 37°C, 125 rpm, 8% CO2. The culture was harvested after six days and used for protein purification.

[00754] 293F cells were harvested by centrifugation at 3500g for 20 minutes. The supernatant was collected and filtered through a VacuCap90 PF Filter Unit (w/0.8/0.2µm Super Membrane, PALL). About 400 µl of 400ul Ni-NTA agarose beads (Qiagen) were added to the supernatant. The mixture was rotated and incubated for 4 hours at 4°C. It was loaded onto a purification column and washed with 20 mM Histidine wash buffer. The protein was eluted with 500µl of elution buffer with 300 mM Histidine. Samples were dialyzed against PBS buffer at 4C overnight. Protein samples were quantified using nanodrop 2000c.

scFv Conformation and Colloidal Stability Analysis

[00755] The thermostability of the scFv was determined by DSF: mixing 10-20 µl of protein sample with the Sypro Orange dye (Invitrogen Cat#S6650) of a final dilution of 1:1000, in a total volume of 25 µl in PBS, BioRad CFX1000 cycle (25°C for 2 minutes, then increment 0.5°C for 30 seconds, 25 to 95°C).

[00756] For analytical SEC experiment, about 15 to 20µg of scFv protein sample in 20µl of PBS were injected into TSKgel Super SW2000 at a flow rate of 0.3mL/minutes on an Agilent 1100 series.

EC50 per FACS Connection

[00757] 300.CD19 mouse cell lines were cultured in RPMI 1640 with 0.5 mg/mL Zeocin. Around 5.5 cells/per well were transferred to the BD Falcon 96 96-well plate. The cells were centrifuged at 900 rpm (Sorval Legend XT centrifuge) for 3 minutes. Supernatants were removed. Anti-CD19 scFv protein samples were diluted in DPBS with 5% FBS. The samples were added to the wells, mixed well with the cells and incubated for 1 hour. Cells were washed twice in DPBS with 5% FBS. Cells were incubated with anti-poly His PE (R&D) for 1 hour, washed twice before FACS analysis (LSRII from BD Biosciences).

Kinetic Analysis by Proteon

[00758] Kinetics were determined using Bio-Rad Proteon. Immobilization was performed using standard amine coupling on a GLC sensor chip. scFv samples were diluted to 0.03 mg/mL in acetate pH 4.5 and applied to the chip at a flow rate of 30 µL/minute for 300 seconds. The CD19 ligand was then serially diluted in PBS-Tween and injected at a flow rate of 50 µL/min for 120 seconds with a dissociation time of 480 seconds. The chip surface was regenerated with glycine pH 2.5. Data were fitted using a 1:1 Langmuir model.

CART19 surface expression and FACS staining

[00759] HEK293F suspension cells transiently transfected with different anti-hCD19 CARTs were harvested 2 days after transfection. About 1e6 cells were placed in each well of a 96-well V-shaped plate (Greiner Bioona, Germany) and washed three times with 0.2 mL of FACS buffer (1XPBS containing 4% bovine serum albumin (BSA). ) (BSA V fraction, Roche Diagnostics, Indianapolis, IN) Cells were resuspended in 0.2 mL of FCAS buffer with 0.2 µg of biotinylated protein L (GenScript, Piscataway, NJ) or 100 nM of hCD19( AA 1-291)-hIgG1 Fc (Generated in NIBRI) and incubated at 4°C for 30 minutes. Cells were then washed with 0.2 ml of FACS buffer three times, and incubated with 1 µl of Alexa Fluor streptavidin 488 (Life Technologies, Grand Island, NY) in 0.2 mL of FACS buffer for samples with L protein, or 2 µL of PE anti-human Fcx (Jackson ImmunoResearch Laboratories, West Grove, PA) in 0 .2 mL of FACS buffer for samples with hCD19-hIgG1 Fc for 30 minutes at 4°C in the dark. After washing with 0.2 mL of FACS buffer three times, cells were analyzed on an LSRII machine (BD Biosciences, San Jose, CA) using FACSDiva software (BD Biosciences, San Jose, CA). Immunofluorescence staining was analyzed as the relative log of cell fluorescence, and the percentage of Alexa Fluor 488-positive or PE-positive cells was measured.

Analysis of potential PMTs generated during the Humanization Process

[00760] Of interest was that during humanization, position 62 in the CDRH2 region prefers to be a serine residue, rather than the alanine present in murine CDRH2 as described in Example 1. It was tested whether the PTM site generated during the process of humanization was in fact a "real" PTM or merely a theoretical site. Two IgG variants were generated in which the asparagine at position 60 (known to be a glycosylation site) was mutated to serine, or glutamine and designated FMC63_VH_hz2 (N60S) and FMC63_VH_hz2 (N60Q), respectively. These constructs were generated in order to eliminate the potential post-translational modification (PTM) site and test for maintained activity.

Results

[00761] Humanized anti-CD19 scFv and mouse scFv were expressed in 293F cells and purified through His tag. The expression and production of fully humanized scFv were much higher than the original mouse scFv (data not shown).

[00762] To confirm identity and assess integrity, scFv constructs are analyzed with or without incubation with N-glycanase F (PNGaseF) followed by either high performance liquid chromatography-mass spectrometry (HPLC-MS) (See, Figure 3) and SDS-PAGE (data not shown). PNGaseF is an enzyme specific for the removal of N-linked glycan structures from the consensus sequence N-X-S/T/C where X is any amino acid except proline. Briefly, samples are diluted in water to 0.1 µg/µL and left untreated or incubated with PNGaseF at a ratio of 1:2 (w/w) PNGaseF: scFV for 3 hours at 37°C.

[00763] SDS-PAGE analysis is performed using a NuPAGE 4-12% Bis-Tris gel from Novex. Approximately 2 µg of scFV is loaded onto each column and electrophoresis is conducted at a constant 200 V for 40 minutes. After electrophoresis, the gel is stained using PhastGel Blue R 250 stain (Amersham Pharmacia) and the stain removed with 10% acetic acid, 30% methanol.

[00764] HPLC-MS analysis is performed on the Water’s Acquity UPLC system coupled to a Xevo-Tof mass spectrometer. Approximately 1 µg of each sample is loaded onto an R 1/10 2.1 x 100 mm 10 µm POROS column (Applied Biosciences) set at 60°C at a flow rate of 0.5 mL/min. The mobile phases are composed of 0.1% formic acid (A) and 0.1% formic acid, 75% isopropanol, 25% acetonitrile (B). The protein is eluted from the column with a reversed phase gradient of 25% to 90% B in 12 minutes. Acquisition is performed using positive electrospray scanning in the m/z range of 600-4000 Da with a 20-50V cone source voltage ramp. The resulting spectra are deconvolved using MaxEnt1.

[00765] The glycosylation site was introduced during the humanization process. The non-PTM variants (VH: N60S or N60Q) were left without this additional shape. The construct was the only one with a consensus N-linked glycosylation site in HC CDR2. From SDS-PAGE analysis, untreated samples migrated as bands consistent with the approximate molecular weights of the consequences for all constructs except 103101-WT (S/N) for which doublet is observed. This construct is the only one with a consensus N-linked glycosylation site on H-CDR2. When treated with PNGaseF, the high molecular weight band of the doublet is no longer present, suggesting partial occupancy of the site. Similarly, the molecular weights observed from the deconvoluted mass spectra are consistent with those predicted from the amino acid sequences. However, while the other constructs demonstrated a simple primary molecular species, 103101-WT (S/N) also had a population 1217 Daltons larger than that predicted from the sequence that is no longer present after PNGaseF treatment. This is consistent with the presence of a predominant simple N-linked glycoform, probably oligomannose 5 on a mass basis. The presence of the glycosylated form was confirmed by MS analysis as shown in Figure 3.

[00766] Conformational stability was measured by Differential Scanning Fluorimetry (DSF). As shown in Figure 4, the Tm of mouse scFv was 57°C, while the human variants showed higher Tm around 70°C. The Tm for fully humanized scFv is much better than murine scFv, clearly showing that fully humanized scFv is more stable than murine scFv. This stability will likely translate to the CART19 construct, likely leading to improved therapeutic properties.

[00767] The activity of purified scFv was measured by binding to hCD19 expressing cells as well as by binding to hCD19 antigen using SPR-based detection method. Mouse cell line 300 was used to determine binding of scFvs. The EC50 of mouse scFv to hCD19 was around 06 to 1.6 nM. The humanized variants showed EC50s of the same range in the low or sub nM EC50s range.

Example 3: CD19 CAR Constructs

[00768] ScFv to be used in the final CAR construction was derived from the humanized IgG described in Example 1. The order in which the VL and VH domains appear in the scFv was varied (i.e., VL-VH, or VH-VL orientation ), and where three or four copies of the "G4S" subunit (SEQ ID NO:18), wherein each subunit comprises the sequence GGGGS (SEQ ID NO:18) (e.g., (G4S)3 (SEQ ID NO:107 ) or (G4S)4(SEQ ID NO:106)), connect the variable domains to create the entire scFv domain, as shown in Table 2. Table 2. Humanized CD19 scFv constructs showing VH and VL orientation and size linker ("3G4S" is described as SEQ ID NO: 107 and "4G4S" is described as SEQ ID NO: 106).

[00769] The sequences of the humanized scFv fragments (SEQ ID NOS: 1-12) are provided below in table 3. Total CAR constructs were generated using SEQ ID NOs: 1-12 with additional sequences, SEQ ID NOs: 13- 17, shown below, to generate total CAR constructs with SEQ ID NOs: 31-42.

[00770] • leader (amino acid sequence) (SEQ ID NO: 13) MALPVTALLLPLALLLHAARP

[00771] • leader (nucleic acid sequence) (SEQ ID NO: 54) ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCT GCTGCATGCCGCTAGACCC

[00772] • CD8 joint (amino acid sequence) (SEQ ID NO: 14) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[00773] • CD8 articulation (nucleic acid sequence) (SEQ ID NO: 55) ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCAT CGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCA GCGGCGGGGGGCCAGTGCACACGAGGGGGCTGGACTTCGCCT GTGAT

[00774] • CD8 transmembrane (amino acid sequence) (SEQ ID NO: 15) IYIWAPLAGTCGVLLLSLVITLYC

[00775] • transmembrane (nucleic acid sequence) (SEQ ID NO: 56) ATCTACATCTGGGCGCCCTTGGCCGGACTTGTGGGGTCCTTCTC CTGTCACTGGTTATCACCCTTTACTGC

[00776] • 4-1BB intracellular domain (amino acid sequence) (SEQ ID NO: 16) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[00777] • 4-1BB Intracellular Domain (nucleic acid sequence) (SEQ ID NO: 60) AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTA TGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCC GATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[00778] • CD3 zeta domain (amino acid sequence) (SEQ ID NO: 17) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR

[00779] • CD3 zeta (nucleic acid sequence) (SEQ ID NO: 101) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCA GGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGA GGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGA TGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGT AC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATT GGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCC TTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGC

[00780] • CD3 zeta domain (amino acid sequence; NCBI Reference Sequence NM_000734.3) (SEQ ID NO:43) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHM QALPPR

[00781] • CD3 zeta (nucleic acid sequence; NCBI Reference Sequence NM_000734.3); (SEQ ID NO:44) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCA GGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGA GGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGA TGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATT GGGATGAAAGGCGAGCGCCCGGAGGGGCAAGGGGCACGATGGCC TTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGC

[00782] • IgG4 joint (amino acid sequence) (SEQ ID NO:102) ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV YTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM

[00783] • IgG4 articulation (nucleotide sequence) (SEQ ID NO:103) GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGA GTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCA AGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTG GTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCC GGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTG ACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTG TAAGGTGTCCAACAAGGGCCTGCCAGCAGCATCGAGAAAACCAT CAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACC CTGCCCCCTAGCCAAGAGGAGATGACCAAGA ACCAGGTGTCCCT GACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGG AGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACC CCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCG GCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTA GCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAG AAGAGCCTGAGCCTG TCCCTGGGCAAGATG

[00784] All of these clones contained a Q/K residue change in the signal domain of the costimulatory domain derived from 4-1BB. Table 3: Humanized CD19 CAR Constructs

[00785] Table 7: Murine CD19 CAR Constructs

[00786] The consequences of humanized CDR sequences of the scFv domains are shown in table 4 for the heavy chain variable domains and in table 5 for the light chain variable domains. "ID" means the respective SEQ ID NO for each CDR. Table 4. Heavy Chain Variable Domain (Kabat) CDRs

[00787] Table 5. Light Chain Variable Domain CDRs

[00788] Table 6 is a key identification correlating the numerical names of constructs to the specific orientation of the scFv light and heavy chains, the number of linker units (i.e., (G4S)3 (SEQ ID NO:107) or (G4S )4 (SEQ ID NO:106)), separating the heavy and light chains, and distinguishing the amino acid sequences in the heavy chain CDR2. Table 6: CD19 CAR Assignments.

[00789] The CAR scFv fragments were then cloned into lentiviral vectors to create a full-length CAR construct in a simple coding structure, and using the EF1 alpha promoter for expression (SEQ ID NO: 100). EF1 alpha promoter CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCC CACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCG GTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTC GTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTA TATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTT GCCGCCAG AACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCC TGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTC CACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGG AAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTT CGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCG CCGCGTGCGAATCTGGTGGCACCTTCGCGCC TGTCTCGCTGCTTT CGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACG CTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGC ACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCC CGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAG CGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCG GCCTGCTCTG GTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGC CCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGC GGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAAT GGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCAC ACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGA CTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATT AGTTCT CGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTT ATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTA GGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTT TGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAA AGTTTTTTTCTTCCATTTCAGGTGTCGTGA (SEQ ID NO: 100).

[00790] Analysis of the humanized CAR constructs was conducted as described in Example 4.

Example 4: Analysis of humanized CD19 constructs in CART

[00791] To evaluate the feasibility of targeting CD19 through a CAR technology, the single-chain variable fragments of an anti-CD19 antibody are cloned into a lentiviral CAR expression vector with the CD3zeta chain and the co-stimulatory molecule 4 -1BB in four different configurations and the optimal construct is selected based on the amount of effector T cell response from CD19 CAR-transduced T cells ("CART19" or "CART19 T cells") in response to CD19+ targets. Effector T cell responses include, but are not limited to, cell expansion, proliferation, duplication, cytokine production and target cell death or cytolytic activity (degranulation).

Materials and methods Generation of humanized CART19 T Cells

[00792] Humanized CART19 lentiviral transfer vectors are used to produce genomic material packaged into VSVg pseudotyped lentiviral particles. Lentiviral transfer vector DNA is mixed with the three packaging components of VSVg, gag/pol, and rev in combination with lipofectamine reagent to transfect them together into 293T cells. After 24 and 48 hours, the media are collected, filtered and concentrated by ultracentrifugation. The resulting viral preparation is stored at -80C. The number of transduction units is determined by titration in SupT1 cells. Redirected CART19 T cells are produced by activating fresh naïve T cells by committing to CD3x28 beads for 24 hours and then adding the appropriate number of transduction units to obtain the desired percentage of transduced T cells. These modified T cells are allowed to expand until they become rested and reduce in size at which point they are cryopreserved for later analysis. Cell numbers and sizes are measured using a multisizer III counter. Prior to cryopreservation, the percentage of transduced cells (expressing CART19 on a cell surface) and their relative fluorescence intensity of that expression are determined by flow cytometric analysis on an LSRII. From histogamma plots, the relative expression levels of CARs can be examined by comparing the percentage transduced with their relative fluorescent intensity.

Assessment of cytolytic activity, proliferation capabilities and cytokine secretion of humanized CART19-redirected T cells.

[00793] To evaluate the functional capabilities of humanized CAR19 T cells to kill, proliferate and secrete cytokines, the cells are thawed and allowed to recover overnight. In addition to humanized CART19, murine CART19 was used for comparative purposes while SS1-BBz was used as non-bleaching expressed CAR for baseline CAR/T cell effect. The “control” gold standard (GS) CART19 was used in all assays to compare assay variation. Importantly, GS CART19 are cells produced under research-grade (i.e., non-clinical grade) manufacturing conditions and include the addition of IL-2 to the growth culture. This likely impacts the overall viability and functionality of these cells and should not be evaluated as a direct comparison to the research grade output of the other transduced T cell populations. T cell killing was targeted to K562, a chronic myelogenous cell leukemia cell line expressing or not expressing CD19 or Pt14, B cells isolated from patients with CLL. For this flow-based cytotoxicity assay, target cells are stained with CSFE to quantify their presence. Target cells were stained for CD19 expression to confirm similar target antigen levels. The cytolytic activities of CAR19 T cells are measured in a titration of effector:target cell ratios of 10:1, 3:1, 1:1, 0.3:1 and 0:1 where effectors were defined as T cells expressing the chimeric anti-CD19 receptor. Assays were initiated by mixing an appropriate number of T cells with a constant number of target cells. After 16 hours, the entire volume of each mixture was removed and each well was washed and combined appropriately. T cells were stained for CD2 and all cells stained with live/dead marker 7AAD. After the final wash, the pelleted cells were resuspended in a specific volume with a predetermined number of bead counts. Cell staining data was collected by LSRII flow cytometry and analyzed with FloJo software using beads to quantify results.

[00794] To measure cell proliferation and cytokine production of humanized CAR19 T cells, the cells are thawed and allowed to recover overnight. In addition to humanized CART19, murine CART19 was used for comparative purposes while SS1-BBz was used as a non-bleaching expressed CAR for background CAR/T cell purposes. The “control” gold standard (GS) CART19 was used in all assays to compare assay variation. T cells were targeted to K562, a chronic myelogenous leukemia cell line expressing or not expressing CD19 or Pt14, B cells isolated from patients with CLL. Furthermore, CD3x28 beads were used to evaluate the potential of T cells to respond to endogenous immune signals. To analyze proliferation, T cells were stained with CSFE. Proliferation is the dilution of the CSFE stain that reflects the separation of the parental labels now into two daughter cells. The assay tests only an effector:target ratio of 1:1 and 1:0 where effectors were defined as T cells expressing the chimeric anti-CD19 receptor. The assay is performed in duplicate and 24h after cell mixing, 50% of the media is removed/replaced for cytokine analysis using the Luminex 10-plex human cytokine detection panel. After 5 days, T cells were stained for CAR expression, phenotyped as CD4 or CD8 cells, and stained for life/death with 7AAD. After the final wash, the pelleted cells were resuspended in a specific volume with a predetermined number of BD counting beads. Cell staining data was collected by LSRII flow cytometry and analyzed with FloJo software using beads to quantify results. Total cell counts were determined by number of cells counted relative to a specific number of beads multiplied by the fraction of beads yet to be counted.

[00795] To evaluate the potential for humanized CART19 cells to function similarly to the currently successful murine CART19, we wanted to evaluate in vitro the ability to kill target cells, to proliferate with respect to the targeted antigen and to show signs of persistence. By packaging each of the humanized CART19 lentiviral constructs and titrating them in SupT1 cells, we can determine the amount of virus to normalize transductions to be around 50%. This allows for more direct comparisons of activity starting with similar average integration sites per cell.

[00796] Therapeutic CAR19 T cells are generated by starting with blood from a normal apheresis donor whose untreated T cells are obtained by negative selection for T cells, CD4+ and CD8+ lymphocytes. These cells are activated by CD3x28 beads in 10% RPMI at 37C, 5% CO2.

[00797] After 24h, the T cells are bursting and the normalized amount of virus is added. T cells begin to divide in a logarithmic growth pattern that is monitored by measuring cell counts per mL and cell size. When T cells begin to rest, logarithmic growth slows and cell size shrinks. The combination of reduced growth rate and T cell size reaching ~300 fl determines the status for T cells to be cryopreserved or restimulated.

[00798] There is a very similar tendency for T cells to rest as seen by size. The pattern of near overlap between humanized CART cells with the current murine CART19 and UTD population indicates no unusual effect of humanized CAR19 on normal T cell expansion after activation. As a control, SS1-BBz is used to define unwanted antigen-independent CAR activity. The expansion profile in total cell numbers shows the differences in actual numbers in individual expansions is probably due mainly to the different starting number of cells. Normalizing the starting T cell numbers, a tight cluster is seen for all CART19 cells. Furthermore, the unwanted effect of antigen-independent CAR activation is detected in the lower and upper running line of the group.

[00799] The surface expression level for each of these CAR19 expressing cells was determined. Titrated virus normalized for transduction shows comparable expression levels correlating with transduction efficiency, percentage of cells transduced. Some CARs have had their titers extrapolated from previous packaging and, however, their transduced percentages are lower, their MFI is likewise reduced as expected. The results indicate that there is no detectable negative effect of humanized CAR19 on the ability of cells to expand normally when compared to UTD and murine CAR19 T cells.

[00800] The ability of humanized CART19 cells to selectively discern a specific cell surface epitope expressed on cells and destroy it is analyzed. Wild-type K562 cells do not express CD19, but can be transduced to express CD19. Comparing these killing curves, titrating the number of effector cells shows that those cells that express CD19 are destroyed. Redirected T cells from the same donor and modified with humanized CART19 cells or current clinical murine CART19 cells indicate no difference in their killing capacity. The killing curves show that a very similar killing capacity is found between humanized CART19 cells that target CD19+ CLL cells from patient 14. Interestingly, there is a decrease in overall cytolytic activity, in particularly GS CART19, suggesting that these cells can have specific inhibitory properties. The similar level of CD19 expressed on target cells indicates that expression level is not the reason for differences in cell killing.

[00801] The necessary property of humanized CART19 cells to proliferate after seeing target cells is found in all constructs after being stimulated by the control CD3x28 beads and the CD19 expression targets. Targeting Pt14 CLL cells appears to indicate a slightly higher proliferation rate with scFvs with a light to heavy chain orientation without shifts seen when having a 3x or 4x GGGGS binding (SEQ ID NOS 107 and 106, respectively). Proliferative results reflect the total number of cells accumulated over the 5 days, indicating that the humanized CART19s, 2146, 2144, 2136, 2141 and 2137 direct a more proliferative signal to T cells. Impressively, this was detected in CART19 cells humanized targeting CLL Pt14 cells.

[00802] In general, humanized CART19 constructs exhibit characteristics very similar to current murine CART19 in cytolytic activity, proliferative response and cytokine secretion for specific antigen targets. The potential of humanized CART19 cells, (2146, 2144, 2136, 2141 and 2137), to direct a more proliferative signal to T cells upon target activation appeared to be an extra benefit of these new constructs to potentially enhance therapeutic response.

Results

[00803] Using cytokine protection and degranulation assays alike, it is demonstrated that specifically engineered CART19 T cells target CD19+ cells.

[00804] ND317 cells transduced with humanized CD19CAR (a.k.a. "huCART19") constructs of the invention were analyzed. There was a close similarity in the size of T cells during their expansion after CD3x28 activation and transduction with the humanized CART19 candidates relative to unmodified T cells (UTD) and murine CART19.

[00805] Experiments showed little difference in the number of T cells that accumulated during their expansions after CD3x28 activation and transduction with different humanized CART19 candidates relative to murine CART19 and unmodified T cells (UTD).

[00806] The cell surface expressions of humanized CART19 are comparable and its expression level very similar to murine CART19. Overlay of histograms plotting the staining pattern of cell surface expression of each of the humanized CART19 transduced T cells and the mean fluorescent intensity (MFI) calculated from these profiles correlates well with the percentage of transduced cells.

[00807] Furthermore, humanized CART19 has similar specific cytotoxic activities in targeting CD19-expressing target cells and comparable to murine CART19. Plots of 16 hr flow-based killing assays using Effector to Target (E:T) titration ratios with effector humanized CART19 cells targeting CSFE labeled K562cc (FIG. 1A. not expressing CD19 controls), K562. CD19 (FIG. 1B, K562 cells transduced to express CD19) or Pt14 (FIG. 1C, CLL patient B cells). The cytolytic activities of all humanized CART19 cells are similar and comparable to murine CART19. The differences in cytolytic activity between different targets are similar and comparable indicating that murine CART19 activity is preserved in the humanized form of CART19.

[00808] Histogram overlay of CFSE-labeled humanized CART19 cells 6 days after being mixed with target cells shows their proliferative capacity (FIG. 5). The proliferative response released from CAR19 is a necessary response after commitment to and killing of target cells to develop a positive clinical response. The dilution of the SS1-BBz CSFE stain, an indicator of dividing daughter cells that dilute out of the parental cell stain, is a result of maintenance divisions of unrested T cells in a targeting-independent mechanism.

[00809] The overall ability of cell populations to proliferate is assessed with CD3x28 beads that mimic endogenous TCR and costimulatory CD28 engagement. The data indicate that each cell population has comparable proliferation potential. All murine and humanized CART19 cells proliferate strongly and comparably upon engagement with CD19-expressing K562 cells. Humanized CART19 cells similarly responded well to B cells obtained from a CLL patient, however, some appeared to respond slightly less. As shown in FIG. 2A and 2B, humanized CART19 cells 2136, 2137, 2140, 2141, 2144 and 2146 can be seen to have slightly stronger proliferation as evidenced by the greater dilution of the CSFE stain. These constructs have the same variable chain orientation from light to heavy, indicating that this is the orientation of choice. A closer look at the amino acid changes in the CDR2 heavy site (Table 1) reveals that each of the three variations YSSSL, YQSSL, and YNSSL (SEQ ID NOS: 28, 29, and 30, respectively) are represented in the constructs that appeared to have the stronger proliferations after observing targets. Furthermore, these observed constructs also have the G4S linker containing 3 copies of the subunit (3G4S) (SEQ ID NO: 107) and the G4S linker containing 4 copies of the subunit (4G4S) (SEQ ID NO: 106), indicating that ligand size did not influence function.

[00810] From the proliferative expansions described above, total cell numbers after 5 days of post-tumor impairment are determined. The cells show a decline in the numbers that were initially seeded, indicating that activation is required to maintain survival. An endogenous activation control is analyzed to show that the total cell count at the end of 6 days was similar. Humanized CART19 cells targeting CD19-expressing K562 cells show that the two murine CART19 cells end up with equally the highest cell numbers, with 2146 slightly above all other constructs with similar values. Total cell numbers were similarly analyzed 6 days after exposure to B cells from Patient 14 (pt14), and interestingly shows that the previously selected humanized CART19 constructs 2146, 2144, 2136, 2141 and 2137, all which have the light to heavy chain orientation and represent the three amino acid variations YSSSL, YQSSL and YNSSL (SEQ ID NOS: 28, 29 and 30, respectively), resulted in higher total cell numbers, higher than CART19s murine. This unexpected differentiation between the various humanized anti-CD19CAR clones may translate into the improved clinical efficacy of CART cells transduced with these constructs.

[00811] Baseline levels of cytokine produced from humanized CART19 cells after exposure to control K562 cells not expressing CD19 were analyzed. 24 hr supernatants were analyzed using a Luminex 30-plex panel. The potential cytokine profile of stimulation of the endogenous immune system with the CD3x28 beads indicates that each of the cell populations has a comparable cytokine profile.

[00812] The data also shows that humanized CART19 and murine CART19 produce similar cytokine profiles at similar levels when responding to the same targets. The cytokine profile was lower, however, similar to targeting Pt14 target cells.

Example 5: Humanized CD19 CAR T cell treatment in an in vivo ALL model.

[00813] Human primary ALL cells can be cultured in immunocompromised mice without having cultivated them in vitro. These mice can be used to test the efficacy of chimeric antigen receptor (CAR) T cells in a model that represents the patient population that will be encountered in the clinic. The model used here, HALLX5447, was passaged twice in NOD.Cg- PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice prior to use in studies testing the efficacy of CAR T cells.

[00814] Murine CD19 CAR T cells have previously been shown to target leukemia cells in an NSG mouse model of primary human ALL. The CD19 scFv (single-chain Fc variable fragment) was humanized and the present example compares the ability of T cells expressing a humanized CD19 CAR (CAR 2) to eliminate ALL tumor cells in vivo to that of CD19 CAR T cells. murine. Here, the efficacy of these cells was directly compared in mice with established primary human ALL, as assessed by peripheral blood FACS analysis of human CD19+ cells. After implantation of 1.5x106 primary ALL cells intravenously, a disease burden of 2.5 to 4% of human CD19+ cells in the blood was achieved within 2 weeks after tumor implantation. This percentage of CD19 is from total cells in the mice's blood. 100% of human cells in the mice before CAR T cell treatment are tumor cells. Percentages above 2% of human CD19+ cells in peripheral blood are considered to be established human ALL disease in this model. Mice carrying leukemia were treated with CAR T cells, as leukemia is established in mice approximately two to three weeks after tumor implantation. Mice in each group were treated with 5x106 total human T cells. The transduction efficiencies of human donor cells with CAR expressing lentivirus were between 40 to 60%. After treatment with T cells, mice were bled weekly to analyze the percentage of human CD19+ cells in the blood as a biomarker for disease progression.

[00815] Materials and Methods:

[00816] Human primary ALL cells: Primary cells were not cultured in vitro prior to implantation. These cells were harvested from an ALL patient and then transferred into mice for establishment and expansion. After the tumor cells were expanded in mice, the bone marrow and splenocytes were harvested and viably frozen in batches for reimplantation. Cells were frozen in 90% FBS and 10% DMSO at a minimum concentration of 5x106 cells per milliliter. For reimplantation, frozen ALL cells were thawed and then injected intravenously into NSG mice to generate mice with ALL that will be used to compare the antitumor efficacy of humanized CD19 CAR T cells and murine CD19 CAR T cells. .

[00817] Mice: 6-week-old NSg mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) were received from Jackson Laboratory (raw material number 005557). Animals were allowed to acclimate to the Novartis NIBRI vivarium for at least 3 days before the experiment. Animals were handled in accordance with Novartis ACUC regulations and guidelines.

[00818] Tumor Implantation: Human primary ALL cells seriously inoculated in vivo, model HALLX5447, were thawed in a water bath at 37°C. The cells were then transferred to a 15 mL conical tube and washed twice with cold sterile PBS. Primary ALL cells were then counted and resuspended at a concentration of 15x106 cells per milliliter of PBS. The cells were placed on ice and immediately (within an hour) implanted into the mice. ALL cells were injected intravenously via the tail vein in a volume of 100 µl, for a total of 1.5x106 cells per mouse.

[00819] CAR T cell dosage: mice were administered 5x106 T cells 16 days after tumor implantation. The cells were partially thawed in a water bath at 37 degrees Celsius and then completely thawed by adding 1 mL of cold sterile PBS to the tube containing the cells. Thawed cells were transferred to a 15 mL falcon tube and adjusted to a final volume of 10 mL with PBS. The cells were washed twice at 1000 rpm for 10 minutes each time and then counted on a hemocytometer. T cells were then resuspended at a concentration of 50x106 cells per mL of cold PBS and kept on ice until mice were dosed. Mice were injected intravenously via the tail vein with 100 µl of CAR T cells for a dose of 5x106 T cells per mouse. Five mice per group were treated with 100 µl of PBS alone (PBS), untransduced T cells (Mock), murine CD19 CAR T cells (muCTL019), or humanized CD19 CAR T cells (huCTL019). Untransduced T cells, muCTL019 T cells, and huCTL019 T cells were all prepared from the same human donor in parallel.

[00820] Animal Monitoring: The health status of the mice was monitored daily, including two weekly body weight assessments. The percentage of change in body weight was calculated as (BWcurrent - BWinitial)/(BWinitial) x 100%. Tumor burden was monitored weekly by FACS analysis of peripheral blood. Mice were bled weekly via tail vein into coated tubes and kept on ice. 10 to 20 µl of blood was seeded from the tubes into 96-well plates on ice. Red blood cells were lysed with ACK red blood cell lysis buffer (Life Technologies, catalog number A10492-01) and then washed twice with cold PBS. Cells were incubated with an Fc blocking mixture of human and mouse Fc block (Miltenyi Biotec, catalog numbers 130-059901 and 130-092-575) for 30 minutes and then incubated with an anti-human CD19 antibody for 30 minutes. Cells were fixed with a 2% paraformaldehyde solution for 20 minutes, washed, and stored in PBS + 2% FBS overnight before analysis on a BD Canto or Fortessa, followed by another analysis using FlowJo FACS software. Cells were analyzed to determine the percentage of human CD19+ cells in the blood of NSG HALLX5447 mice carrying human ALL tumor. Blood CD19 percentages are reported as the mean + standard error of the mean (SEM).

[00821] The percentage of treatment/control (T/C) values was calculated using the following formula: % T/C = 100 x ?T/?C if ?T > 0; % Regression = 100 x ?T/Tinitial if ?T < 0; where T = the mean percentage of peripheral blood CD19 from the drug-treated group on the final day of the study; Initial = percentage of CD19 in peripheral blood from the group treated with the drug on the initial day of dosing; ?T = mean percentage of CD19 in peripheral blood of the drug-treated group on the final day of the study - mean percentage of CD19 in peripheral blood of the drug-treated group on the initial day of dosing; C = mean percentage of peripheral blood CD19 from the control group on the final day of the study; and ?C = mean percentage of peripheral blood CD19 from the control group on the final day of the study - mean percentage of peripheral blood CD19 from the control group on the initial day of dosing.

[00822] T/C values in the range of 100% to 42% are interpreted to have no or minimal antitumor activity; T/C values that are < 42% and > 10% are interpreted to have antitumor activity or inhibition of tumor growth. T/C values < 10% or regression values > -10% are interpreted to be tumor stasis. Regression values < - 10% are reported as regression. Results:

[00823] The antitumor activity of murine and humanized CD19 CAR T cells was evaluated and directly compared in a primary model of human ALL. After tumor implantation on day 0, mice were randomized into treatment groups and treated with 5x106 T cells intravenously on day 16. ALL disease burden and animal health were monitored until animals reached target. Mice in all groups were euthanized on day 65 after tumor implantation when the disease burden in control groups was above 80% CD19+ cells in peripheral blood.

[00824] A clear difference in disease burden was observed between the control groups and the groups treated with the murine or humanized CD19 CAR T cells at P<0.01 from day 24 after tumor implantation, and continued until the end of the study on day 65. Murine or humanized CD19 CAR T cells demonstrate a similar ability to control human ALL HALLX5447 tumor cell growth in NSG mice. Both groups showed a maximum peripheral blood disease level of 12 to 15% human CD19+ cells on day 21 after HALLX5447 implantation. 42 days after tumor cell implantation, no human CD19+ cells were detectable in the huCTL019 group, while the percentage of human CD19+ cells in the muCTL019 group fell by about 1%. Both murine and humanized CD19 CAR T cells resulted in a comparable ability to control the expansion of primary human ALL cells in this model (P>0.05). The % T/C Values for the mock-transduced T cell group was 94.40%, demonstrating that mock-transduced T cells had no antitumor activity. The regression percentage of the muCTL019 group was -89.75% and the huCTL019 group was -90.46%, demonstrating that both of these treatments were capable of causing regression of the HALLX5447 tumor model. Peripheral blood human CD19+ cell percentages as a measure of disease burden in these mice are shown in Figure 7. The PBS treatment group, which did not receive any T cells, demonstrated tumor growth kinetics of primary ALL in reference mice. intravenously implanted NSG. The sham treatment group received non-transduced T cells that underwent the same in vitro expansion process as the CAR T cells. These cells function as a T cell control to show the non-specific T cell response in this tumor model. Both the PBS and mock-transduced T cell treatment groups demonstrated continued tumor progression throughout the experiment. Both murine and humanized CD19 CAR T cells control disease progression within one week of 5x106 T cell injections and demonstrate a similar ability to sustain disease control over the course of this 65-day study.

[00825] The antitumor activity of transduced murine and humanized CD19 CAR T cells was evaluated in an efficacy study in NSG mice carrying a primary model of human ALL, HALLX5447. This study demonstrated that both murine and humanized CD19 CAR T cells (muCTL019 and huCTL019) are capable of mounting an antitumor response in a primary model of human ALL. Furthermore, this response as assayed by peripheral blood disease burden is the same for muCTL019 and huCTL019 cells. Both murine and humanized CD19 CAR T cells control the growth of primary ALL within a week of mice being dosed with the T cells. Initially, after treatment, disease burden continued to increase before decreasing to virtually undetectable levels. Treatment with murine or humanized CAR T cells in a prolonged antitumor response over the course of 65-day disease progression in control-treated mice. Humanized CD19 CAR T cells demonstrated a similar ability to mount an effective anti-CD19 tumor response and control ALL disease burden as was observed with murine CD19 CAR T cells.

Example 6: CD19 CAR T cells for use in the treatment of multiple myeloma.

[00826] Even with current chemotherapy regimens, targeted therapies, and autologous bone marrow transplantation, myeloma is considered an incurable disease. The present example describes the treatment of multiple myeloma (MM) with autologous T cells targeting CD19 with a chimeric antigen receptor (lentivirus/CD19:4-1BB:CD3zeta; also known as "CART19" or CTL019). This example demonstrates that CD19-targeted CAR therapies have the potential to establish long-lasting, deep, durable remissions based on targeting myeloma stem cells and/or tumor cells that express very low levels (not detectable by most methods). ) of CD19.

[00827] In treating a patient with an aggressive secondary plasma cell leukemia, we found that CART19 administered two days after a salvage autologous stem cell transplant resulted in rapid plasma cell leukemia clearance and a very good partial response in one patient who has progressed through multiple lines of chemotherapy. This patient was transfusion dependent for months before treatment; Within two months of treatment, he had his blood counts recovered (with platelet counts in the normal range and white blood cell counts) and no transfusions were required, as he was discharged from hospital from his treatment.

[00828] Because myeloma cells naturally express CD19, the finding that CART19 treatment induced a rapid and significant tumor response was surprising. Without wishing to be bound by any theory, it was concluded that CART19 can be used to treat myeloma, because: (1) while myeloma cells are believed to be traditionally negative for CD19 expression by flow cytometry, there are data indicating that myeloma cells can express very low levels of CD19, such that expression is detectable by RNA, but not by flow cytometry or immunohistochemistry; and (2) the concept of targeting the clonotypic B cell, which is thought to be the cancer stem cell that gives rise to multiple myeloma, and is particularly resistant to chemotherapy. There is a clonal relationship between B cells and myeloma tumor cells, however traditional myeloma therapy targets malignant plasma cells other than B cells. CART19 for myeloma treatment therefore targets a different cell population more than conventional myeloma therapies. myeloma.

[00829] In the patient's only experience, the patient had circulating plasma cells, and we were able to test his tumor cells for CD19 expression. Approximately 1 to 2% of his tumor cells expressed the CD19 antigen. (FIG. 8). So, it was concluded that CART19 can have a direct effect on a very small population of his tumor cells; a very good partial response, although it was not predicted based on targeting only the very small population of CD19+ tumor cells.

[00830] In this case, CART19 was administered after transplantation with autologous stem cell rescue after a high dose of melphalan. Although this is standard therapy in myeloma, it is not curative. Additionally, this patient previously underwent tandem autologous stem cell transplants and relapsed early (<6 months) after transplant. Without wishing to be bound by a particular theory, the use of CART19 cells as described in the present example may have a non-overlapping mechanism in the treatment of myeloma when combined with a salvage autologous stem cell transplant.

[00831] A patient with refractory multiple myeloma was treated with CTL019 after myeloablative chemotherapy and ASCT. Remission was maintained despite loss of detectable CTL019 and reconstitution of normal CD19-positive B cells, indicating that this response does not require prolonged CTL019 activity. Furthermore, this patient response was accomplished even though the vast majority (99.95%) of neoplastic plasma cells were CD19 negative by both flow cytometry and RT-PCR.

[00832] The lack of detectable CD19 expression in this patient's dominant neoplastic plasma cell population suggests that the clinically relevant target of CTL019 resided outside this dominant CD19-negative population. Neoplastic plasma cells in multiple myeloma patients exhibit genetic, immunophenotypic, and functional heterogeneity. Particular subpopulations may be required for clone survival through anti-myeloma therapy. In the patient reported here, for example, the subset expressing small plasma cell CD19 was relatively resistant to melphalan but sensitive to CTL019. This finding suggests that therapeutically targeting a small subset of the clone may induce durable clinical benefit when coupled with conventional antimyeloma therapy.

[00833] Alternatively, the clinically relevant target of CTL019 in this patient may have resided outside the neoplastic plasma cell population. For example, CTL019 can target a stem cell population that is relatively small but gives rise to neoplastic plasma cells. Multiple myeloma may therefore be a disease of multiple late B lineage cell types, not quite terminally differentiated plasma cells, so therapies like CTL019 that target B lymphocytes could be useful adjuncts to therapies that directly target plasma cells.

[00834] Ten multiple myeloma patients will be treated with CART19 in a phase I trial, at least three patients have been treated to date.

Rational Dose and Risks/Benefits

[00835] It was decided to use fixed dosage through routine intravenous administration for this protocol. The primary objective of this protocol was to test the safety and feasibility of administering CART-19 cells to patients with multiple myeloma. The primary toxicities that have been anticipated are (I) cytokine release when CARs encounter their surrogate CD 19 antigen on malignant or normal B cells; (2) depletion of normal B cells, similar to rituximab therapy; (3) steroid-responsive skin and gastrointestinal syndromes similar to graft-versus-host disease, as previously observed when expanded/costimulated autologous T cells were coupled with ASCT to MM. One theoretical concept was whether uncontrolled transformation or proliferation of CART-19 T cells might occur in response to elevated levels of CD19. This was of lesser concern in this Application compared to another study of CLL patients, as the challenge of Clonotypic B cells in MM are believed to be much lower than the malignant B cell challenge in the refractory CLL patients treated under that study.

Rational Dose

[00836] With the first 3 patients, we observed clinical activity at doses ranging from 1.4 x l07 to l.l x l09 CART-19 cells. This observation demonstrates, at least in the first 3 patients treated, that there is no obvious dose response link. Complete response was observed in patients dosed with two log differences in dose doubling. Thus, unlike standard drugs that are metabolized, CAR T cells can have a wide range of dose response. This is most likely because CAR T cells are able to proliferate extensively in patients. Therefore, we established a dose range of l-5 x 108 CART-19 cells for infusion. In this single-patient study offered on a compassionate use basis, the patient was offered up to 5 x 108 CART19 cells, with no lower dose limit. For the ten patient trial, patients will be offered 1-5 x 107 CART-19 cells.

General Planning

[00837] This was a single patient study offered on a compassionate use basis; it was modeled after a Phase I study to determine whether infusion of autologous T cells transduced to express CART-19 is safe. The primary objectives of the study were to determine the safety, tolerability, and engraftment potential of CART-19 T cells in patients undergoing salvage ASCT after early relapse after the first ASCT. The protocol consisting of an open-label pilot study.

[00838] Upon entry individuals will undergo bone marrow biopsy and routine laboratory and imaging evaluation of their MM. Eligible individuals will undergo steady-state apheresis to obtain large numbers of peripheral blood mononuclear cells (PBMC) for the manufacture of CART-19. T cells will be purified from PBMC, transduced with the TCRZ/4-1BB lentiviral vector, expanded in vitro and then frozen for future administration. The number of patients who have inadequate T cell collections, expansion or manufacturing compared to the number of patients who have successfully manufactured T cells will be recorded; Product manufacturing feasibility is not believed to be problematic in this patient population.

[00839] Individuals generally have adequate peripheral blood stem cells leftover stored from the mobilization/collection performed in preparation for their first ASCT to conduct two additional ASCTs. Those who do will undergo a second collection mobilization procedure before or after their steady state apheresis with a regimen according to the attending physician's preference. Approximately two weeks after initial leukapheresis, subjects will be admitted to the hospital and receive high dose melphalan (day -2) followed by autologous stem cell infusion two days later (day 0), and all subjects will receive CART cell infusion. -19 twelve to fourteen days later (day +12-14). Up to 10 patients will be enrolled.

[00840] All subjects will have blood tests to evaluate the safety, engraftment and persistence of CART-19 cells at regular intervals until week 4 of the study. On day +42 and day +100, subjects will undergo bone marrow aspirations/biopsies to estimate bone marrow plasma cell burden and trafficking of CART-19 cells into the bone marrow. A formal response assessment will be done on day 100 according to International Myeloma Working Group (IMWG) 136 criteria, and TTP will be monitored according to routine clinical practice for patients with multiple myeloma. The primary efficacy outcome measured in this study will be a comparison of TTP after a patient's initial ASCT to TTP after ASCT in this study.

[00841] Since the primary purpose of this study is safety and feasibility of infusion of CART -19 ASCT cells, the study will employ an early cessation policy. Briefly, if fewer than 2 unexpected severe adverse events occur among the first five subjects treated, the study will then accrue an additional five subjects for a target enrollment of 10. We will observe treated subjects for 40 days after CART-19 infusion. (i.e., until the first official response assessment on day 42) before the enrollment of a subsequent individual until five individuals have been enrolled and thereby observed. For treatment of the second group of five patients, no waiting period will be required between individuals.

[00842] After 6 months of intense follow-up, individuals will be evaluated at least quarterly for two years with a medical history, physical examination, and blood tests. After this assessment, individuals will enter a roll-over study for annual telephone and questionnaire follow-up for up to thirteen additional years to estimate the diagnosis of long-term health problems, such as development of new malignancy.

Primary Study Purposes

[00843] The pilot experiment is designed to test the safety and feasibility of autologous T cells transduced with the CD19 TCRZ/4-lBB in patients undergoing salvage ASCT for MM after early relapse after first ASCT.

[00844] Primary safety and feasibility purposes include:

[00845] Occurrence of study-related adverse events, defined as NCJ CTC 2: grade 3 signs/symptoms, laboratory toxicities, and clinical events that are possibly, probably, or definitely related to study treatment at any time from infusion until week 24 This will include infusion toxicity and any toxicity possibly related to CART-19 cells including, but not limited to: a. Fevers b. Skin rash c. Neutropenia, thrombocytopenia, anemia, marrow aplasia d. Liver dysfunction e. Pulmonary infiltrates and other pulmonary toxicity f. GVHD-like syndromes affecting the gastrointestinal tract or skin.

[00846] The feasibility of manufacturing CART-19 cells from patient apheresis products. The number of manufactured products that do not meet release criteria for vector transduction efficiency, T cell purity, viability, sterility, and tumor contamination will be determined.

[00847] The depth and duration of response following autologous stem cell transplantation with CART19 will be compared to the depth and duration of response that each patient initially achieved following standard autologous stem cell transplantation.

Individual Selection and Withdrawal Inclusion criteria

[00848] Individuals must have undergone a previous ASCT for MM and progressed within 365 days of stem cell infusion. Individuals who have undergone two previous ASCTs as part of a planned tandem ASCT consolidation regimen are eligible. Progression will be defined according to IMWG criteria for progressive disease or, for patients who achieved CR or sCR after initial ASCT, criteria for CR clearance (Durie et al. Leukemia 2006;20(9):1467-1473). N.B.: There is no requirement that patients must enroll within 365 days of prior ASCT, and patients may be treated with other agents, including experimental agents, after relapse/progression after prior ASCT prior to enrollment in this study.

[00849] Individuals must have written, signed informed consent.

[00850] Subjects must have adequate vital organ function to receive high dose melphalan, as defined by the following criteria, measured within 12 weeks prior to the melphalan infusion date:. The. Serum creatinine < 2.5 or estimated creatinine clearance >30 mL/min and not dependent on dialysis. B. SGOT < 3x the upper limit of normal and total bilirubin < 2.0 mg/dl (except for patients in whom hyperbilirubinemia is attributed to Gilbert's syndrome). w. Left ventricular ejection fraction (LVEF) >45% or, if LVEF is <45%, formal evaluation by a cardiologist has not identified any clinically significant impairment of cardiovascular function. LVEF assessment must be performed within six weeks of enrollment. d. Adequate lung function with FEV1, FVC, TLC, DLCO (after appropriate adjustment for lung volume and hemoglobin concentration) >40% of predicted values. Pulmonary function testing must be performed within six weeks of enrollment.

[00851] Individuals should have an ECOG performance status of 0-2 unless a higher performance status is due to bone pain alone.

[00852] Exclusion CriteriaIndividuals must not:

[00853] Have any active and uncontrolled infection.

[00854] Have active hepatitis B, hepatitis C, or HIV infection.

[00855] Any uncontrolled medical disorder that would preclude participation as outlined.

Treatment Regimen

[00856] Therapy for relapsed/progressive multiple myeloma

[00857] Patients may receive, prior to enrollment, therapy for relapsed/progressive multiple myeloma according to the preference of their attending physicians. Therapy may continue upon enrollment.

[00858] Patients must stop all therapy for two weeks before apheresis and for two weeks before high dose melphalan. If more than two weeks are expected to elapse between apheresis and high dose melphalan, patients may resume therapy after apheresis at the discretion of their attending physicians.

[00859] High dose of Melphalan (day -2)

[00860] Patients will be admitted to the hospital on day -3 or -2 and will undergo examination by the attending physician and routine laboratory tests, which will include monitoring parameters for tumor lysis syndrome, prior to initiation of the treatment protocol . Blood for MM monitoring laboratory testing (SPEP, quantitative immunoglobulins, and serum-free light chain analysis), will be drawn prior to initiation of therapy if such tests were not performed within 7 days of admission.

[00861] High dose therapy will consist of melphalan at a dose of 200 mg/m2 administered intravenously over approximately 20 minutes on day -2. The dose of melphalan will be reduced to 140 mg/m2 for patients >70 years of age or for patients of any age who, in the discretion of the attending physician, may not tolerate a dose of 200 mg/m2. All patients will receive standard antiemetic prophylaxis, which may include dexamethasone, and standard antibiotic prophylaxis.

[00862] Stem cell reinfusion (day 0)

[00863] Stem cell infusion will occur on day 0, at least 18 hours after administration of the high dose of melphalan. Stem cells will be infused intravenously over approximately 20 to 60 minutes after premedication, in accordance with standard institutional practice. At least 2 x 106 CD34+ dams/kg body weight must be infused. Additionally, at least 1 x 106 CD34+ progenitors/kg body weight must be available as a backup stem cell product to be infused in the event of delayed engraftment or late graft failure. G-CSF should be administered SQ starting on day +5, dosed according to standard institutional practice. Other supportive care measures such as transfusion support will be provided in accordance with standard institutional guidelines.

[00864] CART19 Cell Infusion (day +12-14)

[00865] A single dose of CART-19 transduced T cells will be administered, consisting of up to 5 x 107 CART-19 cells. The minimum acceptable dose for infusion of cells transduced with the CD19 TCRZ4-1BB vector is 1 x 107. CART-19 cells will be administered as a single dose by rapid i.v. infusion on day +12-14 after stem cell infusion. If the patient does not meet any of the inclusion criteria described here in the day 12-14 window, the CART-19 infusion may be delayed beyond day +12-14 until criteria are met.

[00866] Lenalidomide Maintenance

[00867] Individuals who have received and tolerated lenalidomide maintenance after their first ASCT will restart lenalidomide maintenance therapy at approximately day + 100, assuming no contraindications exist in the treating physician's diagnosis. The starting dose will be 10 mg daily unless prior experience dictates an alternative starting dose for a particular patient. Maintenance therapy will continue until disease progression or intolerance.

[00868] Preparation and Administration of Study Drug

[00869] CART-19 T cells are prepared at the CVPF and are not released from the CVPF until FDA-approved release criteria for the infused cells (e.g., cell dose, cell purity, sterility, average vector copy number/ cell, etc.) be met. Upon release, the cells are taken to the bedside for administration.

[00870] Cell thawing. The frozen cells will be transported on dry ice to the individual's bedside. Cells will be thawed at the head of the bed using a water bath maintained at 36°C to 38°C. The bag will be gently massaged until the cells are exactly thawed. There should be no frozen pieces left in the container. If the CART-19 cell product appears to be damaged or the bag is leaking, or otherwise appears to be compromised, it should not be infused and should be returned to CVPF as specified below.

[00871] Premedication. Side effects following T cell infusions include transient fever, chills, and/or nausea; see Cruz et al. for review (Cytotherapy 2010;12(6):743-749). It is recommended that the individual be premedicated with acetaminophen and diphenhydramine hydrochloride prior to CART-19 cell infusion. These medications can be repeated every six hours when necessary. A course of nonsteroidal anti-inflammatory medication may be prescribed if the patient continues to have a fever not relieved by acetaminophen. It is recommended that patients do not receive systemic corticosteroids such as hydrocortisone, prednisone, methylprednisolone or dexamethasone at any time except in the case of a life-challenging emergency, as this may have an adverse effect on T cells.

[00872] Febrile reaction. In the unlikely event that the individual develops sepsis or systemic bacteremia following CAR T cell infusion, appropriate cultures and medical management should be initiated. If a contaminated CART-19 T cell product is suspected, the product can be retested for sterility using archived samples that are stored at the CVPF.

[00873] Administration. The infusion will take place in an isolated room at Rhoads, using precautions for immunosuppressed patients. Transduced T cells will be administered by rapid intravenous infusion at a flow rate of approximately 10 mL to 20 mL per minute through an 18 gauge latex-free type Y blood set with a 3-way stopcock. The duration of the infusion will be based on the total volume to be infused and the recommended infusion rate. Each infusion bag will have a label affixed to it containing the following: "FOR AUTOLOGOUS USE ONLY." Additionally, the label will have at least two unique identifiers, such as the individual's initials, date of birth, and study number. Prior to the infusion, two individuals will independently verify all of this information in the presence of the individual and thus confirm that the information is correctly compared to the participant.

[00874] Emergency medical equipment (i.e., emergency trolley) will be available during the infusion in case the individual has an allergic response, or severe hypotensive crisis, or any other reaction to the infusion. Vital signs (temperature, breathing rate, pulse, and blood pressure) will be checked before and after the infusion, then every 15 minutes for at least one hour and until these signs are satisfactory and stable. The individual will be asked not to leave until the doctor deems it safe for them to do so.

Packaging

[00875] The infusion will comprise a single dose of 1-5 x 107 transduced CA T19 cells, with a minimum acceptable dose of 1 x 107 CART-19 cells for infusion. Each bag will contain an aliquot (dose-dependent volume) of cryomedium containing the following infusible grades (% v/v): 31.25% plasmalyte-A, 31.25% dextrose (5%), 0.45% NaCl , up to 7.5% DMSO, 1% dextran 40, 5% human serum albumin.

Apheresis

[00876] A large volume apheresis procedure (12 to 15 liters or 4-6 volumes of blood) is performed in the apheresis center. PBMC are obtained for CART-19 during this procedure. From a single leukapheresis, the intention is to harvest at least 5 x 109 white blood cells to manufacture CART-19 T. Reference blood leukocytes for FDA look-back requirements, and for research are also obtained and cryopreserved. The cellular product is believed to be ready for release approximately 2 to 4 weeks later. Flow cytometry lymphocyte subset quantification, including determination of B cell CD19 and CD20. Reference assessment is made for human and anti-murine anti-VSV-G antibody (HAMA). If an individual has previously had an adequate apheresis collection deposited in accordance with current Good Manufacturing Practices at the Clinical Cell and Vaccine Production Facility, these cells can be used as the cell source for manufacturing CART-19. Using a deposited apheresis product would avoid the cost, time, and risk of the individual undergoing an additional apheresis collection.

Cytoreductive Chemotherapy

[00877] Lymphodepletion chemotherapy will be high dose melphalan as described here.

CART-19 Infusion

[00878] The infusion will begin on day +12-14 after stem cell reinfusion.

[00879] On day + 12-14 before the first infusion, patients will have a CBC with differential, and assessment of CD3, CD4 and CD8 counts as chemotherapy is administered in part to induce lymphopenia.

[00880] The first dose will be administered using a single dose. The cells are thawed at the patient's bedside. The thawed cells will be administered at an infusion rate as fast as tolerated so that the duration of the infusion is approximately 10 to 15 minutes. In order to facilitate mixing, cells will be administered simultaneously using a Y adapter. Subjects will be infused and premedicated as described here. Individuals' vital signs will be assessed and pulse oximetry performed before dosing, at the end of the infusion, and every 15 minutes subsequently for 1 hour and until they are stable and satisfactory. A blood sample for determination of a baseline CART-19 level is obtained some time before the first infusion and 20 minutes to 4 hours after each infusion (and sent to TCSL).

[00881] Patients experiencing toxicities related to high doses of melphalan will have their infusion schedule delayed until these toxicities have resolved. Specific toxicities that justify delay of T cell infusions include: 1) Pulmonary: Requirement for supplemental oxygen to maintain saturation greater than 95% or presence of radiographic abnormalities on chest x-ray that are progressive; 2) Cardiac: New cardiac arrhythmia not controlled with medical management; 3) Hypotension requiring vasopressor support. 4) Active Infection: Blood cultures positive for bacteria, fungi, or virus within 48 hours of T cell infusion.

Toxicity Control

[00882] Uncontrolled T cell proliferation. Toxicity associated with allogeneic or autologous T cell infusions was controlled with a course of pharmacological immunosuppression. T-body toxicity has been reported to respond to systemic corticosteroids. If uncontrolled T cell proliferation occurs (grade 3 or 4 toxicity related to CART-19 cells), individuals may be treated with corticosteroids. Subjects will be treated with pulse methylprednisolone (2 mg/kg i.v. divided q8 hr x 2 days) followed by rapid tapering.

[00883] Furthermore, based on observations of individuals treated in another protocol, there is some concept for macrophage activation syndrome (MAS), although CD 19+ tumor burden is expected to be much lower in patients with myeloma than in in patients with CLL. Treatment and length of treatment of this toxicity will be at the discretion of the patient's physician and study investigator. Suggested management may include: If the individual has a fever greater than 101°F that lasts for more than 2 consecutive days and there is no evidence of infection (negative blood cultures, CXR, or other source), tocilizumab 4 mg/kg can be considered. The addition of corticosteroids and anti-TNF therapy may be considered at the physician's discretion.

[00884] B cell depletion. It is possible for B cell depletion and hypogammaglobulinemia to occur. This is common with anti-CD20 targeted therapies. In the event of clinically significant hypogammaglobulinemia (i.e., systemic infections), individuals will be administered intravenous immunoglobulin (IVIG) per established clinical dosing guidelines to restore normal levels of serum immunoglobulin levels, as was done with Rituximab.

[00885] Primary graft failure. Primary graft (i.e., non-graft) failure may be more common after second ASCT compared to first ASCT. Eligibility criteria stipulate that sufficient stem cells must be available for salvage reinfusion at the discretion of the attending physician in the event of primary graft failure.

Results

[00886] Three treatment-refractory advanced multiple myeloma patients have now been treated with CTL019 in this ongoing trial. The results for two of these patients show that both had substantial antitumor effects from CTL019 therapy based on the primary efficacy assessment at the three-month time point. The third patient has not yet reached the three-month time point. The results for both patients are described in greater detail below.

[00887] The first myeloma patient completed his day +100 response assessment and had a very good response to CART19 therapy. The following tests were performed with the following results: - SPEP/immunofixation: negative - urine immunofixation: kappa light chain band not measurable weak in its immunofixation (also present on day 38, so not new) Otherwise, the patient attends to criteria for strict complete remission including: - serum light chain ratio: normal - bone marrow biopsy: negative - IgA immunophenotyping: IgA is below the limit of detection.

[00888] Other than weak unmeasurable kappa light chain results from urine immunofixation, the patient meets all criteria for "stringent complete remission." Summary of plasma cell immunotyping at 3 time points (day -2, day +38, day +103) is shown in Figure 39, and demonstrates that the patient's IgA is below the limit of detection. The summary shows heavy myeloma burden on day -2 and none detectable on day +38 and +103, which classifies the patient as "MRD negative" by flow analysis. On day +103, the summary shows recovery of normal, polyclonal, CD19+ plasma cells and B cells. The patient has not had any symptoms of disease or therapy and is functioning as a normal person.

[00889] The second treated patient has not yet reached the day +100 time point. However, at this point in time, it is doing very well, but it is too early to determine the effect of the CTL019 infusion.

Example 7: CAR19 T cell/kinase inhibitor combination therapy for lining cell lymphoma.

[00890] Adoptive T cell therapy holds considerable promise for the treatment of lymphoid malignancies. Promising clinical responses in small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) and acute lymphocytic leukemia (ALL) using adoptive transfer of autologous T cells transduced with chimeric antigen receptors (CAR) against the B cell-specific CD19 antigen ( CAR19 T/CART19 cells) using CTL109. We recently reported initial data on 3 chemotherapy-refractive SLL/CLL patients enrolled in a phase I trial to treat CD19-positive malignancies using CD19-specific CAR (CAR19 T cells/CART19 cells). The method used involved genetically modifying patient-derived bulk T cells using lentivirus to express a CAR targeting CD19 that contains signaling domains derived from CD137 and TcRz. For this study cells were expanded using our anti-CD3 and -CD28 bead expansion methodology, and cells were infused early after lymphodepletion without cytokine support (Kalos M, et al. (2011) Sci Transl Med. 3: 95ra73; and Porter DL, et al. (2011) N Engl J Med 365: 725-733). These early results were extremely promising: (i) following a single course of treatment 2/3 patients achieved complete remissions and remain disease free now at 15+ months after treatment, while the third patient, who was treated with corticosteroids shortly after T cell infusion demonstrated a strong partial response. (ii) These patients were able to recapitulate the elements believed to be required for ultimate efficacy of strategies based on adoptive T cell therapy, namely, robust in vivo T cell expansion, disease eradication, T cell contraction, and persistence. functional in the long term. To date, 10 SLL/CLL patients have been treated with 2 remaining in complete clinical and molecular remission, 5 experiencing partial remission, and 3 experiencing no measurable response. In another study, two patients with B-cell ALL achieved complete remission with 1 relapsing with leukemic cells lacking CD19 expression.

[00891] Lining cell lymphoma (MCL), both before and after large cell transformation, will also likely benefit from CART19-based adoptive therapy, in particular when combined with kinase inhibitors such as those that directly affect MCL cells. .

[00892] To also analyze the combination of CART19-based adoptive therapy in combination with kinase inhibitors, high-throughput assays will be used to evaluate several inhibitors targeting the kinases critical for MCL pathogenesis: CDK4/6, BTK, and mTOR in combination with CART19 cells. The most promising combinations will be evaluated in greater detail, both in vitro and in vivo, in a mouse model of MCL xenotransplantation, which may ultimately guide the development of a clinical protocol to evaluate the combination of small molecule kinase inhibitor and immunotherapy. of CART cells in MCL patients.

[00893] In this study, preclinical studies will be performed to determine potential clinical efficacy of this method in the various subtypes of MCL and to evaluate the ability to therapeutically target MCL cells using CART19 cells either alone or in combination with small molecule inhibitors of selected proteins of the expression and activity of the kinase family that is critical for survival and growth of MCL cells.

Research Planning

[00894] In a preclinical setting, the ability to therapeutically target MCL cells, both cultured and primary-like cells, using kinase inhibitors with documented pathogenic relevance in MCL and CART19 cells will be evaluated. A high-throughput MTT assay will be used to determine the effect of these agents to identify potential optimal combinations, dosage, and timing of agent application. The 2 to 3 most promising combinations will be evaluated in greater detail with respect to cellular function, phosphorylation-based cell signaling, and gene expression first in vitro and subsequently in vivo in the MCL xenotransplantation model.

In-vitro studies to characterize the ability of CART-19 cell/kinase inhibitor combinations to effectively target MCL cells

[00895] In this objective, detailed functional, phenotypic, biochemical, and molecular assays listed above to study in vitro the impact of small molecule kinase inhibitors on MCL cells, as well as to examine interactions between CART19 cells and MCL cells and the impact of inhibitors on these interactions be examined.

[00896] Benchmarks for achieving this objective will be to generate a comprehensive data set for:

[00897] i. Document that CART19 cells are activated by and lyse cultured and primary MCL cells;

[00898] ii. Demonstrate that selected kinase inhibitors enhance the ability of each other and/or CART19 cells to eliminate MCL cells without negatively impacting CART19 cell function when appropriately applied with respect to dose and, for some, time of inhibitor vs. CART19 cell administration; It is

[00899] iii. Establish a regimen for scaling and dosing for the BTK inhibitor to be used in the in vivo MCL xenotransplantation experiments using the NSG mice.

[00900] The objectives of this study are, for example, to evaluate and identify optimal therapeutic combinations of small molecule inhibitors targeting kinases critical for MCL pathology: CDK4, BTK, and mTOR together with CART19 cells, monitor CART19 activity, and characterize the functional, biochemical, and molecular effects of therapy on MCL.

[00901] These studies should establish a rational scheme for scaling the time and dose of kinase inhibitor of BTK treatment in conjunction with CART19 therapy to be evaluated in-vivo in objective 2.

In-vivo studies to evaluate the ability of CART19 cells to target follicular lymphoma, alone and in combination with BTK inhibitor

[00902] In this objective, we will test in animal models the ability of selected inhibitor/CART19 cell combinations to affect the growth of established and primary MCL cells.

[00903] The benchmarks for accomplishing this objective will be to generate a data set for:

[00904] i. demonstrate that the selected inhibitor/CART19 cell combination markedly enhances survival of MCL-grafted animals compared to controls (single-agent and sham-treated animals);

[00905] ii. establish a regimen for scaling and dosing for the selected kinase inhibitor/CART19 combination to be used as the basis for future clinical experience.

[00906] The objectives of this study include evaluating the treatment and dose escalation defined in objective 1 for the combination of identified kinase inhibitor/CART19 plus BTK inhibitor, and testing whether BTK treatment synergizes with CART19 to target MCL in mice NSG xenotransplanted with MCL cells, both cultured and primary.

[00907] The following cell types, compounds, animals and experimental methodologies will be used to achieve the proposed objectives:

MCL cells:

[00908] Four MCL cell lines (Jeko-1, Mino, SP-49, and SP-53) and viably frozen samples of 15 primary MCL (12 typical and 3 blastoids). While cell lines develop well spontaneously, primary cells will be cultured alone as well as in the presence of conditioned medium collected from HS5 bone marrow stromal cells to improve their viability.

CART19 cells:

[00909] Primary human T cells created to express CAR19 will be generated using lentivirus transduction and using established protocols ((Kalos M, et al. (2011) Sci Transl Med. 3: 95ra73; and Porter DL, et al. (2011 ) N Engl J Med. 365: 725-733) After a single transduction event T cells typically express CAR19 at frequencies exceeding 30%.

[00910] The studies will use CART 19 populations from five SLL/CLL patients (50 to 100 vials/patient with 1x107 cells/vials are already available). CART19 cells will be identified using an anti-CAR19 idiotype specific antibody (STM). CART19 activity will be controlled both in vitro and in vivo in NSG mice in a standardized manner using NALM-6 CD19+, K562 CD19-negative, and K562 CD19-transduced cell lines. Although CART19 cell function is not restricted to MHC, CART19 cell from at least 5 MCL patients will also be used.

Kinase inhibitors

[00911] Inhibitors of the following kinases will be tested: CDK4/6 (PD0332991), BTK (PCI-32765), mTORC1 (rapamycin), MNK (4-Amino-5-(4-fluoroanilino)-pyrazole [3,4-d ]pyrimidine (Marzec M, et al. PLoS One 6:e24849); and a new compound from Eli Lilly (Gupta M, et al. (2012) Blood 119:476-487); mTOR (OSI-027), and dual PI3K/mTOR (PF-04691502).

[00912] Compounds will first be evaluated across the predetermined spectrum of effective doses, including non-toxic concentrations achieved in patient sera, to ensure optimal kinase inhibition.

Animals:

[00913] In-vivo experiments will be performed using NOD-SCID-IL-2Rgc (NSG) null mice that are bred and available from Stem Cell and Xenograft Core using breeders obtained from Jackson Laboratory (Bar Harbor). Mice will be housed in sterile conditions using HEPAfiltered microisolators and fed with irradiated food and acidified water.

[00914] Transplanted mice are treated with antibiotics (neomycin and polymyxin) for the duration of the experiment. Six to eight week old animals, equal mixes of males and females, will be used for all studies in accordance with protocols approved by the Institutional Animal Care and Use Committee.

[00915] NSG animals have been used in previous adoptive T cell transfer studies specifically to evaluate the differential activity of CART19 cells (Witzig TE, et al. (2010) Hematology Am Soc Hematol Educ Program. 2010:265-270, assay MTT). The high-throughput MTT assay to assess MCL cell growth will be performed first in response to the kinase inhibitor applied alone or in various combinations. This assay is capable of simultaneously determining the rate and viability of cell proliferation, allowing efficient evaluation of many possible combinations of small molecule inhibitors in the presence or absence of CART19 cells. The key aspects of this analysis will be to characterize the drug effect with respect to the potential synergistic, additive, or antagonistic effect. Furthermore, the effect of small molecule inhibitors on CART19 cells will be evaluated. While BTK inhibition must be B cell specific, inhibition of mTOR and CDK4/6 will affect CART19 cells. Establishing the appropriate time for drug application to minimize its potential effect on CART19 cells will be one of the objectives of these experiments.

[00916] To perform the test, MCL cells will be seeded in 96-well plates at 1x104 cells/well, in triplicates, and exposed to medium or kinase inhibitors in various combinations and various concentrations of CART-19 cells. After 48 and 74 hours, the relative number of metabolically active cells will be determined using a colorimetric MTT reduction assay (Promega).

[00917] The significance of the difference between the mean values (+/- S.D.) of controls and different treatment conditions will be evaluated using Student's t test with a P value of < 0.05 considered to be statistically significant.

Cell proliferation and apoptosis assays:

[00918] The most promising drug combinations will next be evaluated in the CFSE labeling and terminal dUTP nick end (tunnel) labeling assays to determine both the cytostatic and cytotoxic components of MCL cell growth inhibition, respectively. In the first assay, MCL cells will be labeled with the addition of BTK inhibitor CFSE and/or unlabeled CART-19 cells. After 48 hours, the cultured cells will be analyzed by FACS for the CFSE labeling pattern of MCL-like cells. The tunneling assay will be done using the BD Biosciences ApoAlert DNA Fragmentation Assay Kit according to the manufacturer's protocol.

[00919] In summary, MCL cells will be cultured with the inhibitors and/or CART19 cells for 48 or 72 hours. After being washed, the cells will be stained with labeled anti-CD20 antibody and permeabilized, washed, and incubated in TdT buffer for 1 hour at 37°C. The reaction will be stopped, the cells washed, resuspended, and analyzed by flow cytometry using CellQuest PRO software.

CART 19 functional assays:

[00920] The effector activity of CART19 cells against MCL cell lines will be evaluated using CD107 degranulation, Intracellular Cytokine Secretion (ICS) assays, proliferation cytolysis assays, and multiplex cytokine detection assays (32). For degranulation and ICS assays, Effectors (T cells) and Target (tumor cells) will be co-incubated in the presence of anti-CD107 antibody for 4 hours at E:T of 0.2:1 followed by staining for surface (CAR19, CD3, CD8, CD4) and intracellular cytokine markers according to established protocols. Cytolysis of MCL cells will be assessed using flow cytometry-based cytolysis assays. For proliferation assays, effector cells will be preloaded with CFSE (Carboxyfluorescein succinimidyl ester carboxy-fluoroscein-succinyl esterase), mixed with target cells at E:T of 0.2:1, co-incubated at 37oC for 4 days, stained for markers (CAR19, CD3, CD8, CD4) and analyzed for CFSE dilution by flow cytometry.

Multiplex cytokine assays

[00921] Cytokine production by CART19 cells in response to MCL targets will be assessed using Luminex-based bead assays as described in (STM32). For these analyzes the Invitrogen 30-plex kit will be used, which simultaneously measures IL-1ß, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40p70), IL-13, IL-15, IL-17, TNF-a, IFN-a, IFN-Y, GM-CSF, MIP-1a, MIP-1ß, IP-10 , MIG, Eotaxin, RANTES, MCP-1, VEGF, G-CSF, EGF, FGF-basic, and HGF in serum, plasma, or tissue culture supernatant.

Multiparametric flow cytometry analysis of CART19T cells:

[00922] The modulation of surface markers associated with functional activation and suppression of CART19 cells following co-incubation with tumor cells will be evaluated using four-color flow cytometry and a common BD LSR II equipped with 4 lasers (blue (488 nM ), violet (405 nM), green (532 nM), and red (633 nM) available from the University of Pennsylvania Abramson Cancer Center Flow Cytometry Core. All flow cytometry data will be analyzed using FlowJo software (TreeStar, San Carlos , CA). These analyzes will be performed essentially as described in (STM42), using a discharge channel to exclude dead cells and target cells (CD19+), and a CAR19 idiotype-specific reagent to detect CART19 cells (STM). the following markers on CART19 positive and negative cells (CD3+/CD8+ and CD3+/CD4+) after co-incubation with tumor cells, on intact or permeabilized cells when necessary. We established multiparametric panels for these markers: - activation/effector function: CD25, CD154, CD134, CD137, CD69, CD57, CD28, T-bet - inhibition: CD152 (CTLA4), PD1, LAG3, CD200 - suppression (Treg) CD4+/CD25++/CD127-, Fox-P3+

[00923] Simultaneously, MCL cells identified by CD19 and CD5 staining will be examined for expression of the immunosuppressive proteins: CD174 (PD-L1) CD173 (PD-L2) and CD152.

Inhibitory impact on cell signaling:

[00924] This part of the study will focus only on the selected compounds; those that proved to be the most effective in the functional assays (cell growth, proliferation, and apoptosis) described above. The effect will be studied separately for each drug and for the selected combinations and the studies will be adjusted to the specific compounds. For example, while the combination of mTORC1 and MNK inhibitors will assess mTORC1 signaling, in particular eIF-4E phosphorylation, BTK inhibition will focus on the PI3K-AKT and MEK-ERK reaction series, and inhibition of CDK4/6 in Rb phosphorylation. These studies will be performed by Western blotting using phosphospecific antibodies as described (Marzec M, et al. (2006) Blood. 108:1744-1750; Marzec M, et al. (2008) Blood 111: 2181-2189; Zhang Q, et al. (2011) Proc Natl Acad Sci USA 108: 11977-11982). In summary, MCL cells will be lysed and protein extracts will be assayed using the Lowry method (Bio-Rad) and loaded onto the polyacrylamide gel. To examine protein phosphorylation, the stained membranes will be incubated with phospho-specific antibodies, e.g. those specific for S6rp S235/236, eIF4E S209, 4E-BP1 T37/46, 4E-BP1 T70 (Cell Signaling) to assess mTORC1 and MNK activity and their inhibition. Then, the membranes will be incubated with the appropriate secondary antibodies, conjugated to peroxidase. The stains will be developed using the ECL Plus System from Amersham.

Genome-scale gene expression analysis:

[00925] Inhibition of cell signaling typically leads to changes in gene transcription. To determine the effects of the selected inhibitor, or individual inhibitors, on gene transcription in MCL, a genome-wide gene expression analysis will be performed in the same manner as described in Marzec M, et al. (2008) Blood 111: 21812189; Zhang Q, et al. (2011) Proc Natl Acad Sci USA 108: 11977-11982. In summary, the cells will be treated in triplicate cultures with the selected inhibitor or its diluent for 0, 4, and 8 hours. Total RNA will also be purified to enrich for mRNA which will be reverse transcribed, labeled and examined by hybridization to the Affimetrix microchip against all known gene exons. Microassay data will be normalized and summarized using RMA as implemented in GeneSpring and MAS5 algorithm. The resulting p-values will be corrected for multiple testing using false discovery rate (FDR) by the Benjamini-Hochberg Step-up Method. Differential expression testing will be performed using a variety of tools including SAM and PartekPro. Emerging genes of interest will then be clustered based on expression patterns (GeneSpring or Spotfire), and the clusters will be analyzed for functional groups and reaction sets in KEGG, Ingenuity Pathway Analysis, and Gene Ontology databases using the NIH -David as the research tool. For genes identified based on the data, independent expression confirmation by quantitative RTPCR will be performed on a larger set of samples (at least 20) from various MCL types (standard vs. blastoid and SOX11-positive vs. SOX11-negative) .

Whole Exome DNA Sequence Analysis:

[00926] To better characterize cases of MCL with respect to their pathogenesis and, for the possible extent, response to the combination therapies proposed here, the exomic DNA sequence will be examined. Whole exome capture and next generation sequencing of MCL and normal peripheral blood DNA samples will be performed using the NimbleGen Sequence Capture 2.1M Human Exome Array and the HiSeq 2000/1000 Illumina instrument.

Assessment of the effect of treatment on xenotransplanted tumors:

[00927] NSG mice will carry MCL tumors (derived from both MCL cell lines: Jeko and Mino and primary cells implanted as either tissue fragments or, less preferably, cell suspensions). Tumors will be propagated by subcutaneous implantation of small tumor fragments. Therapy will be started once the tumors have reached 0.2-0.3 cm in diameter. The kinase inhibitors will be administered by gavage at a preselected dose and time in vitro (e.g., we hope to apply the BTK inhibitor simultaneously with CART19 cells, due to its B cell specificity and expected absence of any inhibitory effect on CART19 cells) . CART-19 cells will be injected into the tail vein of mice carrying tumor at a dose of 1x107/animal, the kinase inhibitor(s) will be administered by gavage at a pre-selected dose and time in vitro. We established a dose sufficient to reproducibly eradicate malignant cells and, at the same time, not induce xenograft versus host disease. A large master stock of CART19 cells (1x 1010) will be generated and frozen to minimize variability associated with effector cellular differences. The primary measure of these experiments will be survival, which will be assessed using Kaplan/Meier curves. As a secondary measure, differential expansion of CART19 cells in animals following T cell infusion will be assessed. This will be made possible by the fact that the infused T cell product will be composed of CART19 positive and negative cells in a defined ratio. For these assays, animals will be bled weekly by bleeding from the tail vein (25 microliters at a time), followed by red blood cell lysis and staining for human CD3, CD4, CD8, and CART19. Preferential expansion of CART19 cells (at least a 2-fold increase in the ratio of CART19+/CART19-) will be evidence of selective MCL-directed CART19 cell expansion. To estimate treatment results, volumes of implanted subcutaneous tumors will be measured, determined as follows, according to the formula: volume = 0.4ab2, where a and b respectively designate long and short diameters of the tumor. Differences in tumor volumes between treated and untreated groups of mice will be statistically analyzed using a standard t-test. The mice will be sacrificed either at the end of the experiments (>30 days), or if the tumors reach > 1.2 cm in diameter, or when any evidence of the animal's suffering is observed. Differences in tumor volumes between treated and untreated groups of mice will be statistically analyzed using a standard t-test. Tumors as well as internal organs will be harvested, processed and analyzed by histology and, for selected tissues, by immunohistochemistry using the battery of antibodies against B cells (CD20, CD79a, Pax-5, CD10, BCL-6) and T cells. (CD2, CD3, CD4, CD5, CD7, CD8, TIA-1), and the proliferation marker Ki-67.

Statistical analysis:

[00928] In in vitro functional studies, the significance of the difference between the mean values (+/- S.D.) of controls and different treatment conditions will be evaluated using the Student's t test with the P value of < 0.05 considered to be statistically significant. Based on our previous experience, differences between experimental mouse groups are expected to be large. Therefore, 10 NSG mice will be used for each treatment group, which will ensure at least 90% power at a type 1 error level of 0.05 with a two-sided sample/-test, given the relationship between differences in treatment. , the mean and standard deviation is at least 3, which is expected. Data will be presented as mean ± SEM. Comparison between groups will be made using the two-sample t-test. A p value < 0.05 is considered significant. For tumor-free survival studies, groups of 10 mice will be used for survival comparison, and the disease status (tumor vs. no tumor) and tumor-free time for each mouse will be recorded. The Kaplan-Meier survival curve will be plotted and the log rank test will be performed to compare survival curves. The significance level is controlled at 0.05.

Example 8: Ibrutinib/CAR19 T cell combination therapy for lining cell lymphoma

[00929] The experiments described in this example characterize the activity of CART19 in combination with ibrutinib treatment to treat lining cell lymphoma in vitro and in vivo. Ibrutinib is a small molecule BTK inhibitor frequently used to treat some hematological cancers. The in vitro experiments described here include assessment of proliferation, cytokine production, CD107a degranulation, and cytotoxicity. Xenoplant mouse models were used to investigate the efficacy and optimal dosage of CART19 with ibrutinib treatment in vivo. Although ibrutinib shows considerable activity in MCL, about 30% of patients do not respond and among responders, only 21% to about a third experience complete remission (Wang et al. NEJM 369.6(2013):507-16). Achieving complete emission is associated with survival without improved progress. Furthermore, therapy can lead to drug resistance with a mean response duration of 17.5 months. In some settings, mutations in or immediately downstream BTK binding sites have been observed following ibrutinib therapy, highlighting a mechanism of drug resistance that may become increasingly frequent. See, for example, Woyach et al. NEJM. 370.24(2014):2286-94. Furthermore, blockade of BTK function leads to inhibition of B cell receptor (BCR) signaling and is not directly cytotoxic. See, for example, Ponader et al. Blood. 119.5(2012):1182-89. Absence of cytotoxicity and failure to eradicate malignant clones predisposes to clonal evolution under selection pressure. Additionally, preliminary findings of increased transformation to aggressive disease in patients treated with ibrutinib for CLL are considered. See, for example, Byrd et al. NEJM. 369.1(2013):32-42; and Parikh et al. Blood. 123.11(2014):1647-57.

[00930] Infusion of autologous T cells transduced with chimeric antigen receptors (CAR) against the B cell specific CD19 antigen (CTL019, CART19) leads to dramatic clinical responses in the majority of patients with various B cell neoplasms, mainly lymphoblastic leukemia acute (ALL). See, for example, Maude et al. NEJM. 371.16(2014):1507-17; and Ruella et al. Expert Opinion. Biol. The R. (2015):1-6. The presence of lymph node masses or bulky disease may lead to decreased T cell infiltration and consequent reduced antitumor activity. Bulky lymphadenopathy does not appear to impair the response to ibrutinib. Wang et al. NEJM. 369.6(2013):507- 16. Furthermore, ibrutinib has shown particular efficacy in reducing tumor masses and mobilizing neoplastic B cells in peripheral blood.

Methods

[00931] Cell lines and primary samples. MCL cell lines were obtained from ATCC (Mino, Jeko-1, SP-49) at the same time as MCL-RL was generated from a progressive pleural effusion from an MCL patient. For in vitro experiments, cell lines were maintained in culture with RPMI medium supplemented with 10% fetal calf serum, penicillin, and streptomycin. For some experiments, MCL-RL and Jeko-1 cells were transduced with click calf green luciferase/eGFP and then sorted to obtain a >99% positive population. The acute leukemia cell lines MOLM-14, K562 or NALM-6 and the TALL JURKAT cell line were used as controls. These cell lines were originally obtained from ATCC. Deidentified primary human MCL bone marrow (BM) and peripheral blood (PB) specimens were obtained from the University of Pennsylvania clinical practices. For all functional studies, primary cells were thawed at least 12 hours before the experiment and rested at 37°C.

[00932] Generation of CAR constructs and CAR T cells. The murine anti-CD19 chimeric antigen receptor (containing a CD8 hinge, 41BB costimulatory domain and CD3 zeta signaling domain) was generated as previously described. See, for example, Milone et al. Molecular Therapy: the Journal of the American Society of Gene Therapy. 17.8 (2009):1453-64. Production of CAR-expressing T cells was performed as previously described. See, for example, Gill et al. Blood. 123.15 (2014): 2343-54. Normal donor CD4 and CD8 T cells or PB mononuclear cells (PBMC) were obtained from the Human Immunology Core of the University of Pennsylvania. T cells were seeded at 1x106/mL, with a CD4:CD8 ratio of 1:1 and expanded in X-vivo medium 15 (Lonza, 04-418Q), 5% human serum AB (Gemini, 100-512) , penicillin/streptomycin (Gibco, 15070063), and Glutamax (Gibco, 35050061) using anti-CD3/CD28 Dynabeads (Life Technologies, 11161D) added on day 1 of culture and removed on day 6. T cells were transduced with lentivirus on day 2 T cells were expanded in culture for 8 to 15 days and harvested when the average cell volume was below 300 fl. T cells were then cryopreserved in FBS 10% DMSO for future experiments. Before all experiments, T cells were thawed and rested overnight at 37°C.

[00933] Ibrutinib. ibrutinib (PCI-32765) was purchased from MedKoo (#202171) or Selleck Biochemicals (#S2680) as a powder or DMSO solution. For in vitro experiments, ibrutinib was diluted to concentrations of 10, 100 and 1000 nM. For in vivo experiments, ibrutinib powder was dissolved in a 10% HP-beta-cyclodextrin solution (1.6 mg/mL) and administered to mice in drinking water.

[00934] Multiparametric flow cytometry analysis. Anti-human antibodies were purchased from Biolegend, eBioscience, or Becton Dickinson. Cells were isolated from in vitro culture or animals, washed once in PBS supplemented with 2% fetal calf serum, and stained for 15 minutes at room temperature. For cell number quantification, Countbright beads (Invitrogen) were used according to the manufacturer's instructions. In all analyses, the population of interest was controlled based on forward vs. forward dispersal characteristics. Laterals followed by singlet control, and live cells were controlled using Live Dead Aqua (Invitrogen). Time tracking was included for quality control. CAR19 surface expression was previously detected as described. See, for example, Kalos et al. Science Translational Medicine. 3.95(2011):95ra73. Flow cytometry was performed on a four-laser Fortessa-LSR cytometer (Becton-Dickinson) and analyzed with FlowJo X 10.0.7r2 (Tree Star).

[00935] Degranulation test. The degranulation test was previously carried out as described. See, for example, Kalos et al. Science Translational Medicine. 3.95(2011):95ra73. T cells were incubated with target cells in a 1:5 ratio in T cell media. Antibodies anti-CD107a-PECY7 (Biolegend), anti-CD28 (BD Biosciences), anti-CD49d (BD Biosciences), and monensin (BD Biosciences) were added to the coculture. After 4 hours, cells were harvested and stained for CAR expression, CD3, CD8, and Live aqua staining (Invitrogen). Cells were fixed and permeabilized (Invitrogen Fix/Perm buffers) and intracellular staining was then performed to detect multiple cytokines (IFN, TNFa, IL-2, GM-CSF, MIP1b).

[00936] Proliferation assay. T cells were washed and resuspended at 1x107/mL in 100 ul of PBS and stained with 100 ul of 2.5 uM CFSE (Invitrogen) for 5 minutes at 37° C. The reaction was then quenched with cold media, and cells were washed three times. Targets were irradiated at a dose of 100 Gy. T cells were incubated in a 1:1 ratio with irradiated target cells for 120 hours, adding media within 24 hours. Cells were then harvested, stained for CD3, CAR, and Live Dead aqua (Invitrogen), and Countbright beads (Invitrogen) were added prior to flow cytometric analysis for absolute quantification.

[00937] Cytotoxicity assays. Luciferase/eGFP+ NALM-6 or RL cells were previously used for cytotoxicity assay as described. See, for example, Gill et al. Blood. 123.15(2014):2343-54. Targets were incubated at the indicated ratios with effector T cells for 4 or 16 hours. Killing was calculated by bioluminescence imaging on a Xenogen IVIS-200 Spectrum camera.

[00938] Cytokine measurements. Target and effector cells were co-incubated in a 1:1 ratio in T cell media for 24 h. The supernatant was collected and analyzed by Luminex 30-plex array (Luminex Corp, FLEXMAP 3D) according to the manufacturer's protocol (Invitrogen). See, for example, Kalos et al. Science Translational Medicine. 3.95(2011):95ra73.

[00939] In vivo experiments. NOD-SCID -/- Y-chain (NSG) mice originally obtained from Jackson Laboratories were purchased from Stem Cell and Xenograft Core of the University of Pennsylvania. All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee (IACUC). Schematic diagrams of the xenograft models used are discussed here. Cells (MCL cell lines or T cells) were injected in 200 μl of PBS at the indicated concentration into the tail veins of mice. Bioluminescent imaging was performed using a Xenogen IVIS-200 Spectrum camera and analyzed with LivingImage v software. 4.3.1 (Caliper LifeSciencies). Animals were euthanized at the end of the experiment or when they met the pre-specified purposes according to IACUC protocols.

[00940] Immunohistochemistry. Immunohistochemical (IHC) staining of formalin-fixed paraffin-embedded tissues was performed on a Leica Bond-III instrument using the Bond Polymer Refine Detection System. Antibodies against CD3, CD4, CD8, Pax5, and CyclinD1 were used undiluted. Heat-induced epitope retrieval was performed for 20 minutes with ER2 solution (Leica Microsystems AR9640). Images were digitally acquired using the Aperio ScanScopeTM.

[00941] Statistical analysis. All statistics were performed using GraphPad Prism 6 for windown, version 6.04.

Lining cell lymphoma cell lines

[00942] Most lining cell lymphoma (MCL) lines in existence have been immortalized and propagated for many generations in vitro, thereby losing their dependence on B cell receptor signaling. Therefore, they are insufficiently sensitive to ibrutinib . In vitro experiments were performed to determine the sensitivity of MCL cell lines to ibrutinib treatment. These cell lines were also used to evaluate the efficacy of ibrutinib/CART19 combination treatment in experiments also discussed in this example. MCL cells were harvested from the pleural effusion of a patient with multiplied relapsed MCL. Both the original cells (RLprimary) and a cell line derived from them (RL) had a blastoid morphology, typical MCL immunophenotype and were positive for the classical t(11;14) translocation by fluorescence in-situ hybridization (FISH) ( FIG. 52A, 52B, 52C).

[00943] RL and JEKO-1 cells were cultured with different doses of ibrutinib (0.1 nM, 1nM, 10nM, 100nM, 1µM, and 10 µM) and sensitivity to ibrutinib was determined by measuring the reduction in bioluminescence ( BLI). As shown in FIG. 31A, RL cells were sensitive to ibrutinib treatment in a dose-dependent manner. However, JEKO-1 cells were resistant to ibrutinib treatment (no demonstration of reduced bioluminescence as a function of increased ibrutinib dosage).

[00944] The sensitivity of RL, Jeko-1, and Mino cells to ibrutinib was also analyzed using an MTT assay. Exposure of RL to increasing concentrations of ibrutinib in vitro led to a dose-dependent inhibition of proliferation and downstream mediator phosphorylation, indicating an on-target effect of ibrutinib, with an IC50 of 10nM. In comparison, the established MCL cell lines, Jeko-1 and Mino, were relatively resistant to ibrutinib, with IC50 results up to 10 µM (FIG. 52D). To confirm RL as a viable model for in vivo experiments, immunodeficient NOD-SCID γ-chain knockout mice (NSG) were engrafted with 1x10 luciferase-expressing RL cells (FIG. 52E). Their tumor burden and survival were assessed. After intravenous injection, MCL was engrafted into all mice and localized to the spleen and liver, followed by dissemination to the bone marrow, blood, and lymph nodes (FIG. 52F). The histology of the affected spleen and liver is consistent with MCL parenchymal infiltration. (FIG. 52G).

[00945] These results showed that these different cell lines can therefore be used to equally present ibrutinib-sensitive and ibrutinib-resistant MCL.

Lining cell lymphoma cells are sensitive to killing by CART19

[00946] Most of the preclinical work showing the efficacy of CART19 has been done using B-ALL cell lines, which are not sensitive to ibrutinib. Furthermore, the best clinical responses to date have been reported in patients with B-ALL, whereas patients with indolent B-cell malignancies have reportedly inferior responses.

[00947] To show that MCL is sensitive to killing by CART19 in this model, healthy donor T cells were transduced with an anti-CD19 CAR construct that has been used in clinical trials. See, for example, Porter, NEJM 2011. A series of in vitro experiments were performed to show that the ibrutinib-sensitive cell line RL and the ibrutinib-resistant cell line Jeko-1 lead to equivalent CART-19 degranulation, cytokine production , extermination and proliferation (FIG. 53A, 53B, 53C, 53D). Furthermore, peripheral blood or bone marrow was obtained from two MCL patients in the leukemic phase, allowing the expansion and transduction of autologous T cells with anti-CD19 CAR. Autologous patient-derived CART19 cells were reactive to MCL whereas non-transduced T cells were non-reactive to their respective autologous MCL (FIG. 53E, 53F). These results indicate that MCL is sensitive to the effector functions of CART19.

Evaluation of treatment with ibrutinib/CART19 in vitro

[00948] An effect of ibrutinib on T cells has previously been discounted based on short-term activity assays, as described in Honigberg et al. Proc. Natl. Academic. Sci. USA. 107(2010):13075-80. Then, an analysis of the effect of ibrutinib on the T cell kinase ITK was reported to support an immunomodulatory role of ibrutinib on CD4 T cells by inhibiting Th2-type polarization. See Dubovsky et al. Blood 122.15(2013):2539- 49. Cytokine analysis of patients treated with CART19 by multiple groups indicates that CART19 therapy is associated with both Th1 (IL2, IFNγ, TNF), Th2 (IL-4, IL-5 , IL-10) and other cytokines (see, for example, Kalos et al. Science Translational Medicine 3.95(2011):95ra73). In this example, the effect of CART19 function was assessed with ibrutinib above and below ibrutinib concentrations that would be expected in patients (average peak concentration in serum 100-150ng/mL) See, Advani et al. J. Clin. Oncol. 2013; 31:88.

[00949] CART19 cells were found to contain ITK. Nonspecific stimulation of CART19 cells by the TCR in the presence of ibrutinib led to a reduction in phosphorylated ITK (pITK-Y180) as previously reported for CD4+ T cells. See Dubovsky et al. Blood 122.15(2013):2539-49. In comparison, specific stimulation of CART19 cells by CAR did not lead to decreased ITK activation (FIG. 54A). This observation indicated that CART19 function would likely not be adversely affected by ibrutinib exposure.

[00950] Additionally, the short- and long-term in vitro function of CART19 cells in the presence of ibrutinib was determined. Ibrutinib at clinically pertinent concentrations did not impair CART19 cell proliferation, degranulation, or cytokine production; although at supraphysiological concentrations, there was inhibition of CART19 cell functions, probably representing non-specific toxicity (FIG. 54B, 54C, 10B).

[00951] In particular, PBMCs isolated from a normal healthy donor were transduced with a lentiviral anti-CD19 CAR construct as described above. The resulting CAR19-expressing T cells (CART19) were cultured and inoculated to determine proliferation and expansion capacity. A 1:1 ratio of CD4:CD8 expressing T cells were cultured with or without different concentrations of ibrutinib (10nM, 100nM, and 1000 nM ibrutinib). Ibrutinib was added at each cell passage. The number of cells was counted on day 0, day 5, day 6, day 7, day 9, and day 10 (FIG. 9A). Cell volume was similarly monitored (FIG. 9B).

[00952] CFSE staining and flow cytometry analysis were similarly used to evaluate the proliferation of CART19 cells in the presence of ibrutinib after stimulation by MOLM14, JEKO-1, and RL tumor cell lines. MOLM14 is an AML cell line, and JEKO-1 and RL are lining cell lymphoma cell lines. Specifically, RL cells are a novel MCL cell line derived from neoplastic B cells obtained from a pleural effusion from a patient with relapsed MCL. CART19 cells and tumor cells were mixed in a 1:1 ratio and proliferation was assessed for 5 days. The percentage of cell proliferation is designated in each histogram in FIG. 10A. Quantification of proliferation as shown in FIG. 10B shows that high doses of ibrutinib could inhibit CART 19 cell proliferation during coculture with MCL cell lines.

[00953] T cell degranulation indicates the activation of cytolytic T cells and the ability to initiate antigen-specific cytotoxicity. CD107a is a functional marker of T cell degranulation transiently expressed on the cell surface after T cell stimulation. Flow cytometry analysis was used to quantify CART19 T cells expressing CD107a after stimulation with MOLM14 tumor cell lines, JEKO-1, and RL. Cells expressing CD107a were present in Q2 (quadrant 2) of the cell profiles shown in FIG. 11A. Quantifying the results obtained from the profiles in FIG. 11A is shown in FIG. 11B. These results indicate that coculture of CART19 cells with MCL-RL or JEKO-1 led to bulky CAR-specific CD107a degranulation.

[00954] Cytokine production by CART19 T cell in the presence of ibrutinib was similarly quantified after stimulation by different tumor cell lines. The production of IL-2, TNF-a, and IFN-y was evaluated by flow cytometry. The cells that produce the cytokines are present in quadrant 2 of the profiles shown in FIG. 12, FIG. 13, and FIG. 14. CART19 T cells stimulated by JEKO-1 and RL showed an increase in cytokine expression. Likewise, increasing concentrations of ibrutinib treatment did not affect the percentage of CART19 cytokine-producing cells.

[00955] Cytokine secretion of CART19 cells after stimulation by tumor cells and in the presence of varying concentration of ibrutinib was analyzed by LUMINEX 30-plex assay. Cytokines secreted by TH1 cells, such as IL-2, IFN-γ, and TNF-a, were analyzed. Cytokines secreted by TH2 cells, including IL-4, IL-5, IL-6, IL-10, IL-13, IL-15, IL-17, MIP1a, GM-CSF, MIP-1b, CP-1, IL-1Ra, IL-7, IP-10, IL-1b, VEGF, G-CSF, EGF, HGF, IFNa, IL-12, RANTES, Eotaxin, IL-2R, MIG, and IL-8 were analyzed. As shown in FIG. 15, TH1 and TH2 cytokines were secreted by CART19 T cells.

[00956] Likewise, experiments using two different techniques indicated that there was no difference in Th1/Th2 polarization between CART19 cells exposed to ibrutinib and not exposed to ibrutinib (FIG. 54D).

[00957] The killing of MCL cells by CART19 cells was increased in the presence of ibrutinib, suggesting at least an additive effect of the combination (FIG. 54E). However, the intrinsic cytotoxic function of CART19 cells was not increased in the presence of ibrutinib. (FIG. 54F).

[00958] Additional bioluminescence assays were performed to evaluate CART19 cell killing of tumor cells. CART19 cells were seeded with MOLM14, JEKO-1, and RL tumor cells carrying a luciferase reporter at varying ratios, such as 1:1; 1:0.5; 1:0.25; and 1:0 in a 96-well plate, in duplicate. After 24 hours, bioluminescence was detected and quantified. The results indicated that bioluminescence in MOLM14 samples did not decrease after incubation with CART19 cells. However, for JEKO-1 and RL cells, bioluminescence decreased in the presence of CART19 cells, indicating that CART19 cells mediated the killing of JEKO-1 and RL cells (FIG. 16A, 16B, 16C, 16D, 16E, and 16F). . There was a further decrease in bioluminescence when treated with ibrutinib, indicating that the combination of ibrutinib and CART19 caused JEKO-1 cell killing and increased RL than with CART19 treatment alone. Consistent with these results, calculation of total cells after each treatment showed that CART19 treatment of JEKO-1 and RL cells caused a reduction in cell number and a further reduction when treated with CART19 and ibrutinib (FIG. 17B and 17C). , suggesting that the combination therapy was not only effective in killing MCL cells, but led to efficient killing of MCL cell lines.

Evaluation of treatment with ibrutinib/CART19 in vivo

[00959] In these experiments, lining cell lymphoma mouse models were used to evaluate combination therapy with CART19 and ibrutinib in vivo.

[00960] Schematic diagrams such as those shown in FIGs. 20, 55, and 5A were used in this example. FIG. 20 shows a schematic diagram of testing CART19/ibrutinib combination therapy in in vivo mouse models of MCL. 1x106 cells from RL (MCL-3), JEKO-1 (MCL-4), and NALM6 (MCL-5) cell lines are injected into NSG mice (20 mice for each experiment). After 1 week to allow for engraftment, treatment is initiated on Day 0, where treatment is: 0.5- 1x106 CART19 cells (by injection), 25 mg/kg/day ibrutinib (oral gavage), or CART19 + treatment with ibrutinib. On Day 7, Day 14, Day 21, and Day 28, bioluminescence is imaged to monitor tumor size. Mice receiving ibrutinib treatment are continuously treated with ibrutinib. The survival of the mice is also monitored. FIG. 55 and 56 show additional schematic diagrams of testing CART19/ibrutinib combination therapy in in vivo mouse models of MCL. 2 x 106MCL-RL cells are injected into NSG mice. After a week to allow for engraftment (engraftment confirmed by bioluminescence imaging), treatment is initiated on Day 7 (after MCL-RL cell injection) where treatment is: vehicle control, CART19 cells (2 x 106 cells), and/or ibrutinib (125 mg/kg/day). On days 14, 21, 28, and 35 (after MCL-RL cell injection), bioluminescence is imaged to monitor tumor size.

[00961] The effect of treatment with ibrutinib alone in an in vivo mouse model of MCL was examined. RL cells transfected with the GFP/luciferase gene were intravenously injected into immunodeficient NSG mice, resulting in 100% MCL engraftment in the liver and spleen, with eventual expansion in the lymph nodes and bone marrow. The mice were treated with varying doses of ibrutinib, 25 mg/kg/day and 250 mg/kg/day. Average bioluminescence, representing tumor growth, was assessed at multiple time points. As shown in FIG. 32, RL-derived tumors demonstrated dose-related sensitivity. Additional experiments titrating ibrutinib doses in RL cell-containing mice were performed and are shown in FIG. 57. A higher dose led to better antitumor activity without increasing toxicity, in line with the highest dose of ibrutinib used in the clinic for MCL (Wang et al. NEJM 369.6(2013): 507-16).

[00962] The completion of the CART19 dose was carried out in the same way. Two MCL cell lines, RL (MCL-1) and JEKO-1 (MCL-2), carrying a GFP luciferase reporter were injected into NSG mice on day 0. CART19 T cells were injected on day 7 at dosages varied, for example, at 0.5 x e6 cells, 1 x e6 cells, or 2 x e6 cells. The mice were monitored, for example, for 100 days. At various time points, mice were monitored for tumor size (e.g., bioluminescence imaging) (FIG. 18A and 18B, and FIG. 19A), and for overall survival (e.g., Kaplan- Meier) (FIG. 18C and FIG. 19B). Different doses of CART19 cells show dose-dependent antitumor efficacy, with 2 x 106 CART19 cells/mouse being the most effective dose (FIG. 19A).

[00963] These studies provided an opportunity to conduct a head-to-head comparison of the two therapies for MCL. As shown in FIG. 58, long-term survival was only achieved in mice treated with CART-19 cells. There was no difference in antitumor effect when comparing untransduced T cells plus ibrutinib with ibrutinib alone. Therefore, in all subsequent experiments, the control groups were vehicle and ibrutinib alone (FIG. 59).

[00964] The addition of CART19 to ibrutinib was similarly tested, as detailed in the schematic diagram in FIG. 56. Assessment of the effect of ibrutinib on tumor burden indicated modestly delayed tumor growth at early time points. In comparison, CART19 cell therapy led to a clear decrease in tumor burden over several weeks. In mice receiving only CART19 cells, this was followed by an indolent relapse beginning on Day 40, whereas mice that were treated with CART19 cells as well as ibrutinib had no detectable disease until Day 80 (FIG. 60). Histopathology of organs harvested at the end of the experiment showed persistence of the disease in all mice treated with control and ibrutinib with foci of tumor necrosis in those treated with ibrutinib. Most mice treated with CART19 alone showed long-term indolent relapse that was accompanied by persistence of CART19 cells, whereas mice treated with CART19-ibrutinib showed tumor clearance and disappearance of CART19 from involved organs (data not shown).

Mechanism of the combined effect of CART19 and ibrutinib

[00965] The in vitro experiments here indicated that ibrutinib did not clearly impair or enhance CART19 effector functions in the short term. In vivo studies showed that ibrutinib monotherapy had a modest antitumor effect. The results indicated that ibrutinib could significantly enhance the antitumor function of CART19 cells (FIG. 60). Therefore, experiments were carried out to determine the mechanism for this effect.

[00966] Inhibition of ITK has been shown to inhibit Th2 polarization and tilt towards a Th1 phenotype (Dubovsky et al. Blood 122.15(2013):2539-49). In mice treated with CART19 cells and ibrutinib, an increase in Th1 cells when compared to CART19 cell monotherapy was not observed using this assay (FIG. 61A, 61B). However, exposing mice to ibrutinib led to an increase in peripheral CART19 cells. There was no difference in the proliferation marker Ki67 between the treatment group and a control group (FIG. 61C), so this assay did not detect a difference in proliferation. Similarly, there was no difference in the antiapoptotic marker Bcl2 or the apoptosis marker phosphotidyl serine, suggesting that the difference in CART cell numbers was not related to an impairment of apoptosis (FIG. 61D). Because ibrutinib has been associated with peripheral lymphocytosis in patients, experiments were performed to determine whether this was similarly seen in mice treated with ibrutinib alone. One week after starting ibrutinib, there were more circulating MCL cells and fewer nodal/organ MCL cells in ibrutinib-treated mice. Interestingly, this increase was observed equally in an ibrutinib-resistant and -sensitive in vivo model, as was also observed in NSG mice engrafted with the NALM-6 acute leukemia cell line (data not shown).

[00967] To understand the role of ibrutinib in T cell expansion in vivo, we engrafted NSG mice with MCL-RL WT cells and treated them with luciferase-positive T cells. Likewise, mice treated with CTL019 and CTL019-ibrutinib showed intense T cell expansion compared to UTD or UTD-ibrutinib (data not shown). We next investigated the frequency of different T cell subsets in vivo and saw no differences in PB T cells 1 week after T cell infusion (data not shown). Considering that CXCR4 is involved in ibrutinib-directed B cell mobilization in humans, we checked CXCR4 expression in vivo in PB T cells from mice treated with CTL019 or CTL019-ibrutinib: the level of CXCR4 was similar in the 2 groups (data no. shown). Finally, inhibitory/costimulatory receptor expression in PB of T cells from mice treated with CART19 and CART19-ibrutinib was analyzed. No differences in TIM3, LAG3, CD137 or CTLA4 expression were evident, however a trend towards reduced PD-1 expression was noted in mice treated with CTL019 and UTD combined with ibrutinib. Conclusions

[00968] Therapies for B cell malignancies include small molecule inhibitors of BCR signaling and T cell-based therapies targeting CD19. In the setting of relapsed MCL, the BTK inhibitor ibrutinib is now FDA approved and generates high initial response rates. Unfortunately, these responses tend to be transient and require higher drug doses than those used for CLL. CART-19 leads to durable responses in patients with high-risk B-ALL, and may also be effective in other B-cell malignancies. Preliminary data suggest that responses of mature B-cell malignancies to CART-19 may be lower than those of B-ALL, but the mechanism of this disparity has not yet been investigated. This example investigated the impact of adding ibrutinib to CART19 in the treatment of MCL.

[00969] Different MCL cell lines with varying sensitivities to ibrutinib (IC50 ranging from 10 nM to 10 µM) were used for in vitro experiments. These different cell lines were used to equally model ibrutinib-sensitive and ibrutinib-resistant MCL. At all but the highest doses of ibrutinib, CART19 cell function was unchanged, with intact T cell expansion kinetics, tumor death and recognition, and cytokine production. Furthermore, the results did not reveal a T helper polarization on ibrutinib exposure. This finding may be due to a combination of factors, including the use of a mixed culture of CD4 and CD8 cells, compared to the CD4-only experimental model performed by Dubovksy et al. Blood 122. 15(2013): 2539-49. Likewise, ibrutinib-sensitive and ibrutinib-resistant cell lines strongly activated CART19 cells and induced death, cytokine production, and proliferation. The combination of CART19 and ibrutinib in vitro leads to at least additive tumor killing. The results in this example show superiority of CART19 over ibrutinib when each was used as monotherapy at clinically relevant doses and administration schedules (single dose for CART19, continuous administration for ibrutinib).

[00970] A systemic xenograft MCL model was also generated in this example, using the MCL-RL cell line generated in a laboratory. Treatment of these mice with different doses of allogeneic CAR19 T cells led to a dose-dependent antitumor effect. A similar dose response to CART-19 was also observed in the ibrutinib-resistant JEKO-1 cell line. MCL- RL was treated in vivo with different doses (e.g. 0, 25 and 125 mg/kg/day) of Ibrutinib, respectively leading to a median overall survival of 70, 81 and 100 days (p < 0.001). A direct in vivo comparison of ibrutinib 125 mg/kg and CART19 showed significantly improved tumor control for CART19-treated mice. Similarly, mice engrafted with MCL-RL were treated with vehicle, ibrutinib, CART19, or the combination of CART19 and ibrutinib (iCART19). At clinically relevant doses, MCL monotherapy with CART19 was superior to ibrutinib monotherapy, and the combination of ibrutinib with CART19 led to an enhanced antitumor effect. In particular, the iCART19 combination in vivo led to initially higher circulating levels of CART19 cells, followed by profound tumor responses, and relapses were significantly delayed when ibrutinib was added to CART19. The iCART19 combination resulted in improved tumor control with 80% of mice achieving complete remission and long-term disease-free survival. Mechanistically, ibrutinib-treated mice had higher numbers of circulating CART19 cells without changes in Th1/Th2 or memory phenotype. Therefore, the results here showed that ibrutinib can be combined with CART19 in a rational manner and suggest that the properties of each of these therapies can compensate for the deficiencies of the other, thereby leading to enhanced long-term antitumor effect. The experiments and results of combining BCR signaling inhibition with anti-CD19-targeted T-cell therapy pave the way for rational combinations of non-cross-resistance therapies for B-cell malignancies.

[00971] The kinetics of tumor response and relapse suggest that ibrutinib serves to deepen the initial response achieved by CTL019 alone, or enhance the long-term immunosurveillance capacity of CTL019 cells.

Example 9: Ibrutinib/CAR19 T-cell Combination Therapy for Chronic Lymphocytic Leukemia

[00972] Ibrutinib is also used to treat chronic lymphocytic leukemia (CLL). Ibrutinib has not demonstrated cytotoxic effects on T cells or NK cells. However, in CLL cells, Ibrutinib promotes programmed cell death and inhibits tumor cell adhesion and migration. In this example, experiments were performed to examine: 1) the effect of Ibrutinib on CART19 production in patients undergoing Ibrutinib therapy; and 2) the optimal timing of treatment with Ibrutinib in combination with CART19 for optimal in vivo function.

Effect of Ibrutinib on CART19 function and production

[00973] Normal donor PBMCs were obtained using apheresis as described herein, for example, in Example 6. The PBMCs were incubated with 5 µM Ibrutinib for 30 minutes or were left untreated (for control), and then the cells were washed twice. The cells were then transduced with CAR19-containing lentivirus constructs to generate CART19 T cells using the methods described herein, for example in Example 4. The number of CART19 T cells generated from FACs analysis of Ibrutinib-treated PBMCs was performed to determine the number of CART19 T cells generated from Ibrutinib-treated PBMCs compared to untreated PBMCs. As shown in Figure 21, 15% of transduced Ibrutinib-treated PBMCs expressed CAR19 and 12% of transduced untreated PBMCs expressed CAR19. These results demonstrate that ibrutinib treatment does not affect the lentiviral transduction efficiency or T cell production of CART19. Therefore, CART19 T cells can be manufactured from CLL patients undergoing Ibrutinib treatment.

[00974] Further in vitro analyzes were performed to determine the effect of Ibrutinib on CART19 T cell proliferation, CART19 cytotoxicity, and the TH1:TH2 cytokine production ratio.

[00975] To assess cell proliferation, cells were stained with CFSE to detect proliferating cells and analyzed by FACS, as described in Example 4. CART19 T cells were incubated with varying concentrations of Ibrutinib (0.1 µM, 0 .5 µM, 1 µM, and 5 µM), and were stimulated with CD3/CD28 beads or left unstimulated. In FIG. 22, histograms were overlaid to compare unstimulated to CD3/CD28-stimulated CART19 T cells at each concentration of Ibrutinib. For each concentration of Ibrutinib, CD3/CD28-stimulated T cell proliferation was observed, thus demonstrating that Ibrutinib treatment did not affect CART19 cell proliferation.

[00976] The effect of Ibrutinib on cytotoxicity of CART19 T cells was also evaluated, using methods described in Example 4. CAR19-transduced and non-transduced T cells were treated with media, DMSO, or 1 µM Ibrutinib. A flow-based killing assay was performed using titration effector to target (E;T) ratios with effector CART19 cells (media, DMSO, or Ibrutinib treated) to determine specific cytotoxic activity against target cells expressing CD19. As shown in Figure 23, CART19 T cells treated with Ibrutinib demonstrated the same percentage of specific cytotoxic activity against cells expressing target CD19 as controls (CART19 T cells treated with media or DMSO). Therefore, Ibrutinib treatment does not affect the cytotoxicity of CART19.

[00977] Ibrutinib treatment may limit TH2 activation, and therefore may promote TH1 selective pressure on T cells and distort TH1/TH2 cytokines in human CLL patients. The assays were performed to evaluate TH1 and TH2 cytokine production in the presence or absence of Ibrutinib or DMSO (control). As shown in figure 24, Ibrutinib does not promote distortion of TH1 and TH2 cytokines in CART 19 cells.

Efficacy of ibrutinib/CART19 combination treatment in vivo

[00978] The function of CART19 was evaluated in an in vivo mouse model. Nalm/6 cells (human acute lymphoblastic leukemia cell line) were implanted into NSG mice, and the mice were monitored daily for at least 50 days. The Nalm/6 tumor model produces tumors that are not sensitive to ibrutinib, and therefore allows analysis of CART19 function in vivo and efficacy in reducing cancer treatment/tumor volume. Starting on day 7, mice were administered DMSO (control) or Ibrutinib daily by oral gavage. CART19 was administered on day 7 or day 9, or mice were left untreated (control). On days 4, 11, 18, 25, and 32, the number of Nalm/6 cells circulating in the mice was measured, for example, by FACS analysis of peripheral blood, to determine the effectiveness of CART19 to clear Nalm/6 cells in the presence or absence of Ibrutinib. For example, cells were stained with human anti-CD19 antibody to determine the percentage of human CD19+ cells (Nalm/6) in the blood of tumor-bearing NSG mice.

[00979] As shown in Figure 25A, mice that did not receive CART19 injections exhibited an increase in Nalm/6 tumor cells. However, mice that received CART19 injections in combination with daily DMSO administration showed successful clearance (reduction) of Nalm/6 cells. Mice that received injections of CART19 in combination with daily administration of Ibrutinib also showed successful clearance of Nalm/6 cells with the same kinetics and efficacy as mice that received DMSO treatment. Therefore, these results demonstrate that Ibrutinib treatment does not impair the function of CART19 in clearing Nalm/6 cells from this tumor model.

[00980] The health status of the mice was monitored for at least 50 days after injection of the Nalm/6 cells. The Kaplan-Meier survival curve of FIG. 25B shows that none of the mice that were untreated (did not receive CART19 T cells) and received DMSO or Ibrutinib survived more than 30 days after injection of Nalm/6 cells. However, treatment with CART19 T cells, DMSO or Ibrutinib increased the survival of mice. Thus, these results taken together with the tumor burden results of FIG. 13A demonstrate that Ibrutinib treatment does not impair CART19 function in clearing tumor cells of the NSG-Nalm/6 tumor model in vivo.

Optimal Timing of Ibrutinib/CART19 Combination Treatment in CLL Patients

[00981] The optimal time for Ibrutinib treatment of CLL during CART19 administration was evaluated using samples from CLL patients who underwent Ibrutinib treatment for one year. PBMC samples from 9 CLL patients (Patient 111330026, Patient 111330030, Patient 111330039, Patient 111330056, Patient 111330073, Patient 111330074, Patient 111330081, Patient 111330086, and Patient 111330111) were isolated in different cycles of Ibrutinib treatment and were used to make CART19 T cells. PBMC samples were collected before Ibrutinib treatment to stabilize a baseline, and then collected for Ibrutinib treatment on cycle 2, day 1, and cycle 12, day 1. Several different parameters of CART19 manufacturing was evaluated, such as transduction, proliferation, cytotoxicity, and cytokine production. Other assessments may include ex vivo immunophenotyping, such as assessment of memory, inhibitor molecules, and exhaustion.

[00982] FIG. 26 shows the results of flow cytometry analysis of CAR19 transduction of T cells from Patient 111330030, which was representative of the results obtained from the other 8 patients after CAR transduction. PBMCs were collected from patients at the indicated times (baseline, e.g., before treatment; cycle 2 on day 1; and cycle 12 on day 1) and were transduced with lentiviral vectors containing CAR19, using methods as described, e.g. in Example 4. The transduced PBMCs were then stained for annexin, CD3, CD4, and GAM (to detect CAR), and then analyzed by FACS analysis. The region packed into the graphs in FIG. 14 shows the percentage of cells that were GAM positive, and successfully transduced to CART19 T cells. The bottom three graphs show that CAR19 was successfully transduced in about 23% of cells at baseline, 28% of cells at cycle 2, day 1, and 44% of cells at cycle 12, day 1.

[00983] Next, the proliferation rate (or population doublings) of the CART19 cells or the non-transduced cells (control) was assessed at each time point (baseline, cycle 2 on day 1, and cycle 12 on day 1) for 12 days. FIG. 27 shows graphical representations of population doublings over 12 days for three patients, Patient 111330039 (#39), Patient 111330026 (#26), and Patient 111330030 (#30). These results indicate that with regard to proliferation rate, PBMCs isolated between or on cycle 2, day 1 and cycle 12, day 1 are preferred for CAR transduction.

Potential mechanisms for ibrutinib treatment affecting CART19 function

[00984] Various mechanisms of Ibrutinib treatment that may affect CART19 function have been evaluated from CLL patient samples.

[00985] Analysis of cells expressing CD19 (CD19+) was evaluated by FACS analysis. PBMCs from a CLL patient undergoing Ibrutinib treatment were isolated at baseline (before Ibrutinib treatment), cycle 2 day 1, and cycle 12 day 1 and were subsequently stained for CD19. FACS analysis showed that Ibrutinib causes a decrease in cells expressing CD19 (FIG. 28A, FIG. 28B, and FIG. 28C). Therefore, these results indicate that treatment with Ibrutinib induces lymphocytosis.

[00986] Additional analysis was performed to examine CD200 expression on tumor cells over time during Ibrutinib treatment. The immunosuppressive molecule CD200 is upregulated on primary B-cell CLL tumor cells. CD200 binds to its receptor, CD200R, which is expressed on cells of the monocyte/macrophage lineage and on T lymphocytes. The interaction of CD200 with its receptor releases an inhibitory signal to the macrophage lineage, altering cytokine profiles from TH1 to TH2 and results in the induction of regulatory T cells. Samples from Patients 111330030, 111330026, and 111330039 at baseline (screen), cycle 2, day 1, and cycle 12, day 1 were stained for annexin, CD19 (to classify for CD19-expressing tumor cells), and CD200 and analyzed by FACS. Histograms detecting CD200 expression in tumor cells from each time point were overlaid for each patient (FIG. 29A, 29B, and 29C). Generally, CD200 expression on tumor cells decreased over time during ibrutinib treatment.

[00987] The frequency of T cells expressing PD1 during Ibrutinib treatment was also evaluated. Patient samples were obtained at baseline, cycle 2, day 1, and cycle 12, day 1 and stained with annexin, CD3, CD8, and PD1. Cells that were negative for annexin and positive for CD3 were analyzed for expression of CD8 and PD1. Cells expressing CD8 and PD1 are designated by the box in FIG. 30A, 30B, and 30C. Comparison of FACS profiles of cells at baseline (FIG. 30A), cycle 2, day 1 (FIG. 30B), and cycle 12, day 1 (FIG. 30C), indicates that ibrutinib treatment decreases the frequency of cells expressing PD1 over time.

[00988] The data obtained from the experiments described above indicate that the ideal time to administer CART19 therapy to CLL patients receiving Ibrutinib is between cycle 2 and cycle 12, or in cycle 12.

Example 10: CAR19 T-cell Therapy for Hodgkin Lymphoma

[00989] CAR19 T cell therapy can also be used to treat Hodgkin's lymphoma (HL). Hodgkin's lymphoma is characterized by the presence of malignant Hodgkin Reed-Sternberg (HRS) cells that are derived from clonal central germline B cells. There are several factors that indicate the therapeutic efficacy of CAR19 T cell therapy for HL. CD19 staining of HL tumors shows cells expressing CD19 (CD19+) within the tumor and tumor microenvironment (FIG. 33). One study showed that a clonal B cell population (CD20+CD27+ALDH+) expressing CD19 is responsible for the generation and maintenance of Hodgkin's lymphoma cell lines, and also circulates in the blood of most HL patients (Jones et al. , Blood, 2009, 113(23):5920-5926). This clonal B cell population has also been suggested to give rise to or contribute to the generation of malignant HRS cells. Thus, CART19 therapy would deplete this B cell population that contributes to tumorigenesis or tumor cell maintenance. Another study showed that B cell elimination slows solid tumor development in multiple murine models (Kim et al., J Immunotherapy, 2008, 31(5):446-57). In support of the idea that elimination of B cells in the HL tumor microenvironment results in some antitumor effect, current therapies, such as rituxan, are being clinically tested for targeting and elimination of tumor B cells in HL (Younes et al., Blood, 2012, 119(18):4123-8). Again, carcinogenesis related to chronic inflammation has also been shown to be B cell dependent (de Visser, et al., Cancer Cell, 2005, 7(5):411-23). The results of these studies indicate that targeting the B cell population, particularly in the HL tumor microenvironment, would be useful for treating HL, reducing or inhibiting disease progression or tumor development.

[00990] Furthermore, normal B cells expressing CD19 also infiltrate the tumor microenvironment in HL. Previous studies with CART19 therapy in CLL and ALL (e.g., described in Examples 4 and 5) show that exposure of CART19 to CD19+ targets leads to cytokine production and macrophage production. Thereby, modulation of the HL tumor microenvironment from a pro-tumor microenvironment to an anti-tumor microenvironment can be activated by infusion of CART19 to interact with normal CD19+ B cells present in HL. For example, exposure of CART19 to targets expressing CD19 causes cytokine production, e.g., inflammatory cytokines, which promote antitumor activity through the expansion of cytotoxic T cells, activation of macrophages, and recruitment of other immune effector cells with various functions that inhibit development. tumor cells, such as leukocytes, macrophages, and antigen-presenting cells. Because target CD19+ B cells may not be malignant (e.g., normally circulating B cells), a transient rather than prolonged CART19 effect may be preferred for modulation of the tumor microenvironment.

[00991] A study to examine the therapeutic efficacy of CART19 therapy in HL patients can be carried out as described below (FIG. 34). The study will also evaluate the safety and tolerability of CART19 in HL subjects, and determine the effect of CART19 cells on the HL tumor microenvironment.

[00992] 8 patients with classic HL are treated in this study. Patients are of all ages, although separate protocols for drug release can be established for pediatric and adult patients. Patients in this study do not have any potentially curative treatment options available (such as autologous stem cell transplantation (ASCT) or allogeneic), or are not suitable for such curative treatment options. For example, patients can be any of the following: PET+ after salvage chemotherapy, PET+ after brentuximab treatment, or PET+ after ASCT with or without prior exposure to brentuximab. Patients will have a limited prognosis (several months less than or equal to 2 years of expected survival) with currently available therapies. And finally, patients will not have received anti-CD20 antibody therapy. Patients are excluded due to lack of viability, for example, if the patient has insufficient numbers of T cells for 6 infusions of CART19.

[00993] A CAR19 mRNA is produced by in vitro transcription. CAR19 mRNA is electroporated into donor T cells, and the resulting cells are expanded and stimulated by incubation with CD3/CD28 beads. Dosages containing 1x108 - 5x108 RNA-electroporated CAR19 T cells are delivered to the patient three times a week for two weeks (e.g., on days 0, 2, 4, 7, 9, and 11). Overall response rate will be assessed by CT, PET and clinical scan at 1st month after treatment. Response and survival will be monitored monthly for the first 6 months, then every 3 months until 2 years after the first CART19 infusion (day 0). Monitoring techniques include tumor or lymph node biopsy (e.g., for immunohistochemical analysis and/or RNA for gene expression profiling) and PET scanning before and after CART19 treatment. For example, the effect of CART19 cells on the HL tumor microenvironment is analyzed by comparing the results of gene expression profiling performed on accessible lymph node biopsies from selected patients before treatment and approximately one week after treatment (or the appropriate time after treatment to allow changes in cell phenotype). To assess the safety and tolerability of CART19 treatment, the frequency and severity of adverse events are reported, including the frequency of cytokine release syndrome (CRS) and macrophage activation syndrome (MAS).

[00994] Chemotherapy can be administered concurrently with CART19 treatment. The first dose of CART19 may be preceded by lymphodepletion chemotherapy, e.g. cytoxan.

Example 11: Non-responsive subgroup of CLL patients exhibits increased expression of immune checkpoint inhibitor molecules

[00995] In this study, clinically manufactured CART19 cells from 34 CLL patients were evaluated for expression of immune checkpoint inhibitor molecules, such as PD-1, LAG3, and TIM3. The response of this cohort to CART19 was known and consequently a correlation between response and biomarker expression patterns could be assessed.

[00996] CART19 cells manufactured from CLL patients with different responses to CART therapy were analyzed by flow cytometry to determine the expression of CAR and the immune checkpoint inhibitor molecules PD-1, LAG3, and TIM3. CART19 cells were from: healthy donors (HD) (n=2); CLL patients who responded to CART therapy (CR) (n=5); CLL patients who partially responded to CART therapy (PR) (n=8); CLL patients who did not respond to CART therapy (NR) (n=21). Cells were stained with fluorescently labeled antibodies that specifically recognize CD3, CD4, CD8, CD27, CD45RO, the CAR19 molecule, and immune checkpoint molecules PD-1, LAG3, and TIM3, according to standard methods for analysis by flow cytometry known in the art. The expression of each marker, e.g., CD4+, CD8+, etc., was determined by flow cytometry analysis software, and subpopulations (e.g., CD4+ T cells, CD8+ T cells, or CAR19-expressing T cells) were also analyzed for expression of immune checkpoint molecules PD-1, LAG3, and TIM3.

[00997] An example of the flow cytometry analysis profiles used to determine surface marker expression is shown in figures 35A and 35B. T cells expressing CD4 were determined using flow cytometry, and were also analyzed for CAR19 and PD-1 expression, so that the x-axis of the profiles indicates CAR19 expression (the top left (Q5) and bottom quadrants left (Q8) show CAR19- negative CD4+ cells, while the top right (Q6) and bottom right (Q7) quadrants show CAR19-expressing CD4+ cells) and the y axis shows PD-1 expression (bottom left (Q8) and right (Q7) quadrants show PD-1 negative CD4+ cells and top left (Q5) and right (Q6) quadrants show CD4+ cells expressing PD-1). In the CD4+ population of a CART responder, 44.7% of total cells expressed PD-1, and about 22.3% of CAR19-expressing cells were PD-1 positive, while 27.2% of CAR19-expressing cells were PD-1 negative (Figure 35A). In contrast, in the CD4+ population of a non-responder, there was a significant decrease in total CAR19-expressing cells (about 15.3% compared to 49.5% in CR), with 14.7% of CAR19-expressing cells being positive. of PD-1 while only 0.64% were PD-1 negative (Figure 35B). Comparison between the profiles in Figure 35A and Figure 35B shows a much higher percentage of CD4+ cells from a non-responder express PD-1 (about 92.9%) compared to the CART responder (about 44.7%) .

[00998] Using the methods and analysis described above, the percentage of cells expressing PD-1 (PD-1+) from the CD4+ population and the CD8+ population was determined for each patient in each response group. Non-responders were shown to have a higher percentage of PD-1+ cells in both the CD4+ (Figure 35C) and CD8+ (Figure 35D) populations compared to those who responded to CAR therapy (CR); the increase in mean PD-1 percentage was statistically significant for both CD4+ and CD8+ populations. Partial responders (PR) exhibited higher percentages of PD-1+ cells than responders (CR) in both CD4+ (Figure 35C) and CD8+ (Figure 35D) populations.

[00999] Next, the percentage of cells expressing PD-1 (PD-1+) of the CD4+ population expressing CAR19 and the CD8+ population expressing CD19 was determined for each patient in each response group. Similar analysis was performed as above, with the additional step of analyzing CD4+ and CD8+ cells for CAR19 expression, and after identifying CAR19-expressing cells, determining the percentage of PD-1-expressing cells from the CAR19-expressing cell populations. A similar trend as that observed in the global CD4+ and CD8+ populations was observed for the CD4+ and CD8+ populations expressing CAR19: non-responders were shown to have a higher percentage of PD-1+ cells in both the CD4+ (Figure 36A) and CD8+ (Figure 36B) populations. compared to those who responded to CAR therapy (CR); the increase in the percentage of PD-1 was statistically significant for both CD4+ and CD8+ populations. Partial responders (PR) exhibited higher percentages of PD-1+ cells than responders (CR), both in CD4+ (Figure 36A) and CD8+ (Figure 36B) populations.

[001000] Another analysis was performed to determine the distribution of cells expressing PD-1, LAG3, and TIM3 from patients with different responses to CAR therapy. Representative cell profile analysis for expression of PD-1, LAG3, and TIM3 in the CD4+ population is shown in Figure 37. Cell populations were first analyzed for expression of CD4+ and CD8+. The CD4+ population (or CD8+ population, not shown) was then analyzed for PD-1 and CAR19 expression (Figure 37, left profiles). As previously described, non-responders (NR) had a significantly increased percentage of cells that were total PD-1+ compared to CART responders (CR) (about 92.9% of PD-1 positive for NR compared to 44.7% PD-1 positive for CR). Furthermore, in nonresponders, CAR19-expressing cells were mostly PD-1 positive (14.7% PD-1 positive and CAR+ compared to 0.64% PD-1 negative and CAR+). Then the populations were analyzed for the coexpression of PD-1 and LAG3 (figure, central profiles). Cells that expressed both PD-1 and LAG3 are shown in the top right quadrant (Q2). Nonresponders had a significantly increased percentage of cells that expressed both immune checkpoint inhibitors, PD-1 and LAG3, compared to CART responders (67.3% compared to 7.31%). PD-1 expression was also analyzed with TIM3 expression. In figure 37, profiles on the right, the box indicates cells that express both PD-1 and TIM3. Similar to the results obtained with PD-1 and LAG3, non-responders had a significantly higher percentage of cells expressing both immune checkpoint inhibitors, PD-1 and TIM3, compared to CART responders (83.3% in compared to 28.5%). The percentage of cells expressing PD-1 (PD1+), cells expressing PD-1 and LAG3 (PD1+LAG3+), and cells expressing PD-1 and TIM3 (PD1+TIM3+) was determined for each patient in each response group using the flow cytometry analysis as described above. Non-responders were shown to have an increased percentage of PD1+ LAG3+ cells (FIG. 38A) and PD1+TIM3+ cells (Figure 38B) compared to CART-responsives which was statistically significant for both cell populations. Partial responders also showed an increased percentage of both cell populations compared to CART responders, with the averages being decreased compared to non-responders.

[001001] These results indicate that patients who do not respond to CAR therapy exhibit increased expression of immune checkpoint inhibitors (e.g., PD-1, LAG3, and TIM3) compared to patients who respond or partially respond to CAR therapy. CAR. Thus, these results show that agents that inhibit or decrease the expression of immune checkpoint inhibitors, e.g., PD-1, LAG3, or TIM3, may be useful for administration to patients receiving CAR therapy to prevent immunosuppression through series of immune checkpoint reaction (e.g., mediated by PD-1, LAG3, or TIM3), thereby increasing the efficacy of CAR-expressing cells.

Example 12: Effects of mTOR inhibition on immunosenescence in the elderly

[001002] One of the reaction series most clearly linked to aging is the mTOR reaction series. The mTOR inhibitor rapamycin has been shown to extend lifespan in mice and improve a variety of age-related conditions in aged mice (Harrison, DE et al. (2009) Nature 460:392-395; Wilkinson JE et al. (2012) Aging Cell 11:675-682; and Flynn, JM et al. (2013) Aging Cell 12:851-862). Thus, these findings indicate that mTOR inhibitors may have beneficial effects on aging and aging-related conditions in humans.

[001003] An age-related phenotype that can be studied in a short clinical testing time frame is immunosenescence. Immunosenescence is the decline in immune function that occurs in the elderly, inducing an increased susceptibility to infection and a diminished response to vaccines, including influenza vaccine. The decline in immune function with age is the accumulation of defects, including a decrease in the ability of hematopoietic stem cells (HSCs) to generate naive lymphocytes, and an increase in the number of exhausted PD-1 positive lymphocytes that have defective responses to antigenic stimulation (Boraschi, D et al. (2013) Sci. Transl. Med.5:185ps8; Lages, CS et al. (2010) Aging Cell 9:785-798; and Shimatani, K et al., (2009) Proc. Natl. Acad. Sci. USA 106:15807-15812). Studies in aged mice showed that 6 weeks of treatment with the mTOR inhibitor rapamycin rejuvenated HSC function by inducing improved production of naïve lymphocytes, improved response to influenza vaccine, and prolonged lifespan (Chen, C et al. (2009 ) Sci. Signal. 2:ra75).

[001004] To evaluate the effects of mTOR inhibition on aging-related phenotypes and whether the mTOR inhibitor RAD001 improves immunosenescence, the response to the influenza vaccine in elderly volunteers receiving RAD001 or placebo was evaluated. The findings presented here suggest that RAD001 enhanced the influenza vaccine response in elderly volunteers at doses that were well tolerated. RAD001 also reduced the percentage of Programmed Death (PD)-1 positive CD4 and CD8 T lymphocytes that accumulate with age. These results show that mTOR inhibition has beneficial effects on immunosenescence in elderly volunteers.

[001005] As described here, a 6-week treatment with the mTOR inhibitor RAD001, a rapamycin analog, improved the response to the influenza vaccine in elderly human volunteers.

Methods Study Population

[001006] Elderly Volunteers >= 65 years of age without unstable underlying medical illnesses were enrolled at 9 sites in New Zealand and Australia. Screening exclusion criteria included hemoglobin <9.0 g/dL, white blood cell count <3,500/mm3, neutrophil count <2,000/mm3, or platelet count <125,000/mm3, uncontrolled diabetes, unstable ischemic heart disease , clinically significant underlying pulmonary disease, history of an immunodeficiency or receiving immunosuppressive therapy, history of coagulopathy or medical condition requiring long-term anticoagulation, estimated glomerular filtration rate < 30 mL/minute, presence of severe uncontrolled hypercholesterolemia (>350 mg/minute dL, 9.1 mmol/L) or hypertriglyceridemia (>500 mg/dL, 5.6 mmol/L).

[001007] Baseline demographics between treatment branches were similar (Table 7). Of the 218 individuals enrolled, 211 completed the study. Seven subjects withdrawn from the study. Five subjects withdrew due to adverse events (AEs), one subject withdrew consent, and one subject left the study as a result of protocol violation.

[001008] Table 7: Demographic and reference characteristics of Study Patients *Body mass index is weight in kilograms divided by the square of height in meters

Study Design and Conduct

[001009] From December 2011 to April 2012, 218 elderly volunteers were enrolled in a randomized, observer-blind, placebo-controlled experiment. Subjects were randomized to treatment arms using a validated automated randomization system with a RAD001 to placebo ratio of 5:2 at each treatment arm. The treatment arms were: RAD001 0.5 mg daily or placebo RAD001 5 mg weekly or placebo RAD001 20 mg weekly or placebo

[001010] The experiment was observer-blind because the placebo cohorts in RAD001 0.5 mg daily and 20 mg weekly differed slightly from the RAD001 tables in those cohorts. The study team evaluating the subjects was not fully blinded. The duration of treatment for all cohorts was 6 weeks during which time subjects underwent in-clinic safety assessments every 2 weeks. After subjects were dosed for 4 weeks, steady-state levels of RAD001 were measured pre-dose and at one hour post-dose. Following completion of the 6-week course of study drug, subjects were given a 2-week drug-free break to reverse any possible RAD001-induced immunosuppression, and were then given a 2012 seasonal influenza vaccine (Agrippal®, Novartis Vaccines and Diagnostics, Siena, Italy) containing the strains H1N1 A/California/ 07/2009, H3N2 A/Victoria/210/2009, B/Brisbane/60/ 2008. Four weeks after the influenza vaccine, individuals had serum collected for influenza titer assessments. Antibody titers for the 3 influenza vaccine strains as well as for 2 heterologous strains (strain A/H1N1 A/New Jersy/8/76 and strain A/H3N2 A/Victoria/361/11) were measured by inhibition assay standard hemagglutination test (Kendal, AP et al. (1982) Concepts and procedures for laboratory-based influenza surveillance. Atlanta: Centers for Disease Control and Prevention B17-B35). A/H1N1/California/07/2009-specific IgG and IgM levels were measured in serum samples taken before and 4 weeks after the influenza vaccine as previously described (Spensieri, F. et al. (2013) Proc. Natl Acad Sci USA 110:14330-14335). The results were expressed as fluorescence intensity.

[001011] All subjects provided written informed consent. The study was conducted in accordance with the principles of Good Clinical Practice and was approved by ethics committees and regulatory agencies.

Security

[001012] Adverse event assessment and blood collection for hematological and biochemical safety assessments were performed during study visits. Adverse event information was also collected from diaries that subjects completed at home during the 6 weeks they were on study drug. Data on all adverse events were collected from the time of informed consent until 30 days after the last study visit. Events were classified by investigators as mild, moderate or severe.

Statistical analysis

[001013] The primary geometric analysis of mean titration relationships was done using a normal Bayesian regression model with non-informative priors. This model was fit for each antibody titer on the log scale. The primary outcome in each model was the day 84 assessment. The day 63 assessment was included in the outcome vector. The model fitted using SAS 9.2 proc mixed with the prior declaration. The covariance structure of the matrix was considered unstructured (option type = UN). A flat antecedent was used. For secondary analysis of seroconversion rates, logistic regression was used.

[001014] The treatment population was defined as all individuals who received at least one full dose of study drug and who did not have any deviations from main protocols impacting efficacy data. 199 out of the total 218 individuals enrolled in the study were in the treatment population.

Immunophenotyping

[001015] Peripheral blood mononuclear cells were isolated from whole blood collected at 3 time points: reference line; after 6 weeks of treatment with the study drug; and at study termination when subjects were off study drug for 6 weeks and 4 weeks after influenza vaccine. Seventy-six PBMC subgroups were analyzed by flow cytometry using 8 color immunophenotyping panels at the Human Immune Monitoring Center at Stanford University, CA, USA as previously described (Maecker, HT et al. (2012) Nat Rev Immunol. 12: 191-200). Seventy-six PBMC subgroups were analyzed by flow cytometry using 8 color lyophilized immunophenotyping panels (BD Lyoplate, BD Biosciences, San Diego, CA). PBMC samples with >80% viability and yield of 2x106cells or greater were included in the analysis.

[001016] Relative changes from baseline immunophenotypes to week 6 of study drug treatment and baseline to study termination (week 12) were calculated for each of the RAD001 dosing cohorts. Student's t-test was conducted to examine whether the relative change of baseline immunophenotypes for the two blood sampling time points was significantly different from zero, respectively, within each dosage group after adjusting for the placebo effect. Imputation of missing data in treatment effect analysis was not conducted. Therefore if a patient has a lack of phenotypic data at baseline, this patient should not be included in the analysis for this phenotype. If a patient has a lack of phenotype data at weeks 6 or 12, then this patient will not contribute to the analysis of this phenotype at the affected time point.

[001017] 608 tests on 76 phenotypes under 3 dosage groups were conducted to compare the treatment effect against the placebo effect. The stratified false discovery rate (FDR) methodology was implemented to control the occurrence of false positives associated with multiple tests and provided considerably better intensity. The cell type was considered as the stratification factor and FDR calculation (q value) corrected within each stratum respectively. All null hypotheses were rejected at a significance level of 0.05 with a corresponding q value < 0.1. The multiple test adjustment strategy with rejection at significance level 0.05 and corresponding to q<0.1 ensured that less than 10% of the findings are false.

[001018] In a second analysis, the immunophenotype changes between the pooled treatment and placebo groups, where all three RAD001 dosage groups were combined. To determine which immunophenotype changes differentiated between the treated and placebo groups, within-patient cell count ratios for each measured phenotype were calculated between baseline and Week 6 of study drug treatment and between baseline and the end of the study (Week 12). Ratios were log-transformed, and analyzed by analysis of covariance at each time point in order to detect a difference between the pooled treatment and placebo groups. 152 tests on 76 phenotypes were performed to compare the pooled treatment effect against the placebo effect. The stratified false finding rate (FDR) control methodology was implemented to control the occurrence of false positives associated with multiple testing although it provides considerably better power (Benjamini, Y. et al. (1995) J. Roy. Statist. 57: 289-300; and Sun, L. et al. (2006) Genet. Epidemiol. 30:519-530). A cell type group was taken as the stratification factor and FDR (q-value) calculation was conducted within each stratum respectively. All null hypotheses at a significance level of 0.05 and q-value less than 20% were rejected. This can be interpreted that the rejection of only those hypotheses with P values less than 0.05 and less than 20% probability of each observed significant result is due to multiple testing.

Results

[001019] In general, RAD001 was well tolerated, particularly the dosage regimens of 0.5 mg daily and 5 mg weekly. No deaths occurred during the study. Three subjects experienced four serious adverse events (SAEs) that were assessed as unrelated to RAD001. The 4 SAEs were retinal hemorrhage of the left eye with subsequent blindness in an individual with normal platelet counts who completed a 6-week course of 5 mg weekly of RAD001 6 weeks previously; severe back pain in a placebo-treated subject and severe gastroenteritis in a placebo-treated subject. A list of treatment-related adverse events (AEs) with an incidence >2% in any treatment group is provided in Table 8. The most common RAD001-related AE was mouth ulcer, which in most cases was mild. severity. In general, subjects receiving RAD001 had a similar incidence of AEs as those treated with placebo. Only one severe AE was evaluated as related to RAD001 mouth ulcers in an individual treated with 20 mg weekly of RAD001. Table 8: Incidence of treatment-related AEs >2% in any treatment group by preferred term

[001020] The ability of RAD001 to improve immune function in elderly volunteers was evaluated by measuring the serological response to the 2012 seasonal influenza vaccine. Geometric mean titrations (GMT) of hemagglutination inhibition (HI) for each of the 3 strains of influenza vaccine and 4 weeks post influenza vaccine are provided in Table 9. The primary analysis variable was the ratio of GMT to HI (4 weeks post vaccine/reference). The study was intensified to be able to demonstrate that in at least 2 out of 3 vaccine lines there was 1) an increase in GMT > 1.2-fold with respect to placebo; and 2) a posterior probability of no less than 80% that the corrected placebo-GMT ratio exceeded. This purpose was chosen because a 1.2-fold increase in the influenza GMT ratio induced by the MF-59 vaccine adjuvant was associated with a decrease in influenza illness (Iob, A et al. (2005) Epidemiol Infect 133:687-693 ).

[001021] Table 9. HI GMTs for each influenza vaccine lineage in reference and at 4 weeks after influenza vaccine Reference line indicates 2 weeks before influenza vaccine Week 4 indicates 4 weeks after influenza vaccine N is number of individuals per cohort GMT is geometric mean titer GMT ratio is the GMT in week 4 post vaccination/GMT in reference CV% indicates the coefficient of variation

[001022] In the intended to treat (ITT) population, the low dose immune boosting RAD001 cohort (0.5 mg daily or 5 mg weekly) but not the high dose cohort (20 mg weekly) meets the primary purpose of the study (Figure 40A). This demonstrates that there is a distinct immunomodulatory mechanism of RAD001 at low doses, and that at the highest dose, the known immunosuppressive effects of mTOR inhibition can be considered. Furthermore, the results suggest a trend toward improved immune function in the elderly following treatment with a low dose of immune-boosting RAD001.

[001023] In a subgroup analysis, the subgroup of subindividuals with low baseline influenza titers (< 1:40) experienced a greater increase associated with RAD001 in titers than the ITT population (Figure 40B). These data show that RAD001 is particularly effective in enhancing the influenza vaccine response of individuals who did not have protective titers (>1:40) at baseline, and therefore were at increased risk of influenza illness.

[001024] Scatterplots of RAD001 concentration versus increase in titer for each influenza vaccine strain show an inverse response/exposure relationship (Figure 41). Modeling and simulation based on mTOR-mediated phosphorylation of S6 kinase (S6K) predict that the 20 mg weekly dose regimen inhibits mTOR-mediated S6K activity almost completely, the 5 mg weekly dose regimen inhibits S6K activity more than 50 %, and the 0.5 mg daily dosing regimen inhibits S6K phosphorylation by approximately 38% over the dosing interval (Tanaka, C et al., (2008) J. Clin. Oncol 26:1596-1602). Thus, partial inhibition of mTOR, e.g., mTOR-mediated S6K phosphorylation, with a low immune-enhancing RAD001 dose, may be as, if not more effective, than nearly complete inhibition of mTOR with a high RAD001 dose in enhancing the response. immune system in the elderly.

[001025] Seroconversion rates 4 weeks after influenza vaccination were also evaluated. Seroconversion was defined as the change from a negative pre-vaccination titer (i.e., HI titer <1:10) to post-vaccination HI titer >1:40 or at least a 4-fold increase from a pre-vaccination HI titer. non-negative vaccination (> 1:10). In the intention-to-treat population, seroconversion rates for the B and H3N2 lineages were increased in RAD001 compared to the placebo cohorts, although the increases did not meet statistical significance (Table 10). In the subpopulation of individuals with baseline influenza titers <= 1:40, RAD001 treatment also increased seroconversion rates for the B and H3N2 lineages, and these results reached statistical significance for the B lineage at 0.5 mg of daily dose cohort. These data also show that RAD001 enhanced the serological response to influenza vaccination in the elderly.

[001026] Table 10. Percentage of individuals with influenza seroconversion 4 weeks after vaccination *Odds ratio for seroconversion between RAD001 and Placebo significantly different from 1 (two-sided value < 0.05 obtained by logistic regression as a fixed effect)

[001027] Current seasonal influenza vaccines often provide protection against continually emerging strains of influenza that present as variants of previously circulating viruses. However, influenza vaccine mice in the presence of the mTOR inhibitor rapamycin, compared with placebo, developed a broader serological response to influenza. The broader serological response included antibodies to conserved epitopes expressed by multiple influenza subtypes that provide protection against infection with heterologous strains of influenza not contained in the vaccine (Keating, R et al. (2013) Nat Immunology 14:2166-2178). To determine whether RAD001 enhanced the serological response to influenza in elderly volunteers, HI titers for the 2 heterologous influenza strains not contained in the influenza vaccine (A/H1N1 A/New Jersey/8/76 strain and A/H3N2 A/ Victoria/361/11) were measured. The increase in HI GMT ratios for heterologous strains was greater in RAD001 compared to placebo cohorts (Figure 42). Furthermore, seroconversion rates for heterologous strains were higher in RAD001 compared to placebo cohorts. The increase in seroconversion rates in the 5 and 20 mg weekly RAD001 dosing cohorts was statistically significant for the heterologous H3N2 strain (Table 11). The H3N2 seroconversion rate for the pooled RAD001 cohorts was 39% versus 20% for the placebo cohort (p=0.007). The results presented here suggest that inhibition of mTOR amplifies the serological response of elderly volunteers to the influenza vaccine, and increases antibody titers to heterologous strains of influenza not contained in the seasonal influenza vaccine.

[001028] Enhanced serological response to heterologous strains of influenza in mice treated with rapamycin was associated with an inhibition of class switching in B cells and an increase in anti-influenza IgM levels (Keating, R. et al. (2013) Nat Immunol 14:2166-2178). However, class switching inhibition may not be involved in the enhanced serological response in humans treated with RAD001 because anti-influenza IgM and IgG levels after vaccination did not differ between the RAD001 and placebo-treated cohorts (Figure 43).

[001029] Table 11: Percentage of individuals who seroconverted to heterologous influenza strains 4 weeks after seasonal influenza vaccine * Odds ratio for seroconversion between RAD001 and Placebo significantly different from 1 (two-sided p-value < 0.05 obtained by logistic regression with treatment as a fixed effect)

[001030] To determine the mechanism by which RAD001 enhanced immune function in elderly volunteers, immunophenotyping was performed on PBMC samples obtained from subjects at baseline, after 6 weeks of study drug treatment, and 4 weeks after influenza vaccination (6 weeks after discontinuation of study drug). Although the percentage of most PBMC subsets did not differ between the RAD001 and placebo cohorts, the percentage of PD-1 positive CD4 and CD8 cells was lower in the RAD001 compared to placebo cohorts (Figure 44). PD-1 positive CD4 and CD8 cells accumulate with age and have defective responses to antigen stimulation because PD-1 inhibits T cell receptor-induced T cell proliferation, cytokine production, and cytolytic function (Lages, C.S. and another, (2010) Aging Cell 9:785-798). There was an increase in the percentage of PD-1 positive T cells over time in the placebo cohort. At week 12 (4 weeks post vaccination), this increase may have been due to influenza vaccination, as the influenza virus has been shown to increase PD-1 positive T cells (Erikson, JJ et al. (2012) JCI 122:2967 -2982). However, the percentage of PD-1 positive CD4 T cells increased from baseline at week 6 and 12 in all RAD001 cohorts (Figure 44A). The percentage of PD-1 positive CD8 cells also decreased from baseline at both weeks 6 and 12 in the two lower dose RAD001 cohorts (Figure 44B). The percentage of PD-1 negative CD4 T cells was assessed and increased in the RAD001 cohorts compared to the placebo cohorts (Figure 44C).

[001031] Under more rigorous statistical analysis, where results from the RAD001 cohorts were pooled and adjusted for differences in baseline PD-1 expression, there was a statistically significant 30.2% decrease in PD-1 CD4 T cells positive at week 6 in the pooled RAD cohort (n=84) compared to the placebo cohort (n=25) with p=0.03 (q=0.13) (Figure 45A). The decrease in PD-1 positive CD4 T cells at week 12 in the pooled RAD compared to the placebo cohort is 32.7% with p=0.05 (q=0.19). Figure 45B shows a statistically significant 37.4% decrease in PD-1 positive CD8 T cells at week 6 in the pooled RAD001 cohort (n=84) compared to the placebo cohort (n=25) with p=0.008 (q= 0.07). The decrease in PD-1 positive CD8 T cells at week 12 in the pooled RAD001 compared to the placebo cohort is 41.4% with p=0.066 (q=0.21). Thus, the results in Figures 44 and 45 together suggest that the RAD001-associated decrease in the percentage of PD-1 positive CD4 and CD8 T cells may contribute to enhanced immune function.

Conclusion

[001032] In conclusion, the data presented here show that the mTOR inhibitor RAD001 ameliorates the age-related decline in immune function in elderly humans when evaluated in response to influenza vaccination, and that this improvement is achieved with a balance of risk/ acceptable benefit. In a study of aged mice, 6-week treatment with the mTOR inhibitor rapamycin increased not only the response to influenza vaccination but also extended lifespan, suggesting that improved immunosenescence may be a marker of a broader effect on aging-related phenotypes.

[001033] Since RAD001 dosing was discontinued 2 weeks before vaccination, the immune enhancing effects of RAD001 may be mediated by changes in a pertinent cell population that persist after discontinuation of drug treatment. The results presented here show that RAD001 decreased the percentage of PD-1-depleted CD4 and CD8 positive cells compared to placebo. PD-1 expression is induced by TCR signaling and remains high upon persistent antigen stimulation including chronic viral infection. While not intended to be linked by theory, it is possible that RAD001 reduces chronic immune activation in elderly volunteers and thereby leads to a decrease in PD-1 expression. RAD001 can similarly directly inhibit PD-1 expression as has been reported for the immunophilin cyclosporin A (Oestreich, KJ et al. (2008) J Immunol. 181:4832-4839). A RAD001-induced reduction in the percentage of PD-1 positive T cells is likely to improve the quality of T cell responses. This is consistent with previous studies showing that mTOR inhibition improved the quality of the PD-1 CD8 T cell response. memory to vaccination in mice and primates (Araki, K et al. (2009) Nature 460:108-112). In aged mice, inhibition of mTOR has similarly been shown to increase the number of hematopoietic stem cells, leading to increased production of treatment-naïve lymphocytes (Chen, C et al. (2009) Sci Signal 2:ra75). Although significant differences in the percentages of lymphocytes that did not receive treatment in the RAD001 versus placebo cohorts were not detected in this example, this possible mechanism can also be investigated.

[001034] The mechanism by which RAD001 amplified the serological response to heterologous strains of influenza can also be investigated. Rapamycin has similarly been shown to inhibit class switching in B cells after influenza vaccination. As a result, a unique repertoire of anti-influenza antibodies was generated which provided cross-lineage protection against lethal infection with influenza virus subtypes not contained in the influenza vaccine (Keating, R et al. (2013) Nat Immunol. 14 :2166-2178). The results described here did not show that RAD001 altered B cell class switching in elderly subjects who had discontinued RAD001 2 weeks before influenza vaccination. Although the underlying mechanism requires further elucidation, the enhanced serological response to heterologous influenza strains described here may confer enhanced protection from influenza disease at ages when there is a mismatch between the seasonal vaccine and circulating influenza strains in the community.

[001035] The effect of RAD001 on influenza antibody titers was comparable to the effect of the MF59 vaccine adjuvant which is approved to enhance the elderly's response to influenza vaccination (Podda, A (2001) Vaccine 19:2673-2680) . Therefore, RAD001-directed enhancement of the antibody response to influenza vaccination may translate into clinical benefit as demonstrated with MF59-adjuvanted influenza vaccine in the elderly (Iob, A et al. (2005) Epidemiol Infect. 133:687- 693). However, RAD001 is also used to suppress the immune response of organ transplant patients. These apparently paradoxical results raise the possibility that the immunomodulatory effects of mTOR inhibitors may be dose and/or antigen dependent (Ferrer, IR et al. (2010) J Immunol. 185:2004-2008). A trend toward an inverse vaccination response/RAD001 exposure relationship was seen here. It is possible that complete mTOR inhibition suppresses immune function by the normal cyclophilin-rapamycin mechanism, whereas partial mTOR inhibition, at least in the elderly, enhances immune function due to a distinct aging-related phenotype inhibition. Of interest, mTOR activity is increased in a variety of tissues including hematopoietic stem cells in aging animal models (Chen C. et al. (2009) Sci Signal 2:ra75 and Barns, M., et al. (2014 ) Int J Biochem Cell Biol. 53:174-185). Thus, decline of mTOR activity to levels seen in young tissue, rather than more complete suppression of mTOR activity, may be of clinical benefit in aging indications.

[001036] The safety profile of mTOR inhibitors such as RAD001 in the treatment of aging-related indications was of concern. The toxicity of RAD001 at doses used in oncology or organ transplant indications includes rates of stomatitis, diarrhea, nausea, cytopenias, hyperlipidemia, and hyperglycemia that would be unacceptable for many aging-related indications. However, these AEs are related to minimal levels of RAD001 in the blood. Therefore, the RAD001 dosing regimens used in this study were chosen to minimize trough levels. The mean RAD001 trough levels of the 0.5 mg per day, 5 mg per week, and 20 mg per week cohorts were 0.9 ng/mL, below 0.3 ng/mL (the lower limit of quantification). , and 0.7 ng/mL, respectively. These trough levels are significantly lower than the trough levels associated with dosing regimens used in organ transplantation and cancer patients. Furthermore, the limited 6-week course of treatment decreased the risk of adverse events. These results suggest that the dosing regimens used in this study may have an acceptable risk/benefit ratio for some conditions in the elderly. However, significant numbers of subjects in the experiments described here developed mouth ulcers even when dosed as low as 0.5 mg per day. Therefore, the low-dose safety profile of immune-enhancing RAD001 warrants another study. The development of mTOR inhibitors with clearer safety profiles than currently available rapalogues may provide better therapeutic options in the future for conditions associated with aging.

Example 13: Enhancement of Immune Response to Vaccine in Adult Individuals

[001037] Immune function declines in the elderly, leading to an increased incidence of infection and a diminished response to vaccination. As a first step in determining whether mTOR inhibition has anti-aging effects in humans, a randomized placebo-controlled trial was administered to determine whether the mTOR inhibitor RAD001 reverses the aging-related decline in immune function as assessed in response to vaccination in volunteers. elderly. In all cases, appropriate express consents were obtained and the study was approved by national health authorities.

[001038] The following 3 dosage regimens of RAD001 were used in the study: 20 mg per week (trough level: 0.7 ng/mL) 5 mg per week (trough level was below detection limits) 0.5 mg per day ( minimum level: 0.9 ng/mL)

[001039] These dosage regimens were chosen because they have lower trough levels than doses of RAD001 approved for transplant and oncology indications. The trough level is the lowest level of a drug in the body. The trough level of RAD001 associated with the 10 mg daily oncology dosing regimen is approximately 20 ng/mL. The trough level associated with the transplant dosing regimen of 0.75-1.5 mg twice daily is approximately 3 ng/mL. In comparison, the trough level associated with the dosing regimens used in our immunization study was 3-20 times lower.

[001040] Since RAD001-related AEs are associated with trough levels, the 3 dosage regimens were predicted to have adequate safety for normal volunteers. Furthermore, the 3 doses were predicted to give a range of mTOR inhibition. P70 S6 Kinase (P70 S6K) is a downstream target that is phosphorylated by mTOR. Phosphorylation levels of P70 S6K serve as a measure of mTOR activity. Based on modeling and simulation of P70 S6K phosphorylation data obtained in preclinical and clinical studies of RAD001, 20 mg per week was predicted to almost completely inhibit mTOR activity for a full week, whereas 5 mg per week and 0 .5 mg per day was predicted to partially inhibit mTOR activity.

[001041] Elderly volunteers >= 65 years of age were randomized to one of 3 treatment groups with RAD001 (50 subjects per subdivision) or placebo (20 individuals per subdivision). Subjects were treated with study drug for 6 weeks, given a 2-week break, and then received influenza (Aggrippal, Novartis) and pneumococcal (Pneumovax 23, Merck) vaccinations. Response to influenza vaccination was assessed by measuring geometric mean titers (GMTs) by hemagglutination inhibition assay to the 3 influenza strains (influenza subtypes H1N1, H3N2, and B) in the influenza vaccine 4 weeks after vaccination. The primary purposes of the study were (1) safety and tolerability and (2) a 1.2-fold increase in influenza titers compared to placebo in 2/3 of the influenza vaccine strains 4 weeks after vaccination. This purpose was chosen because a 1.2-fold increase in influenza titers is associated with a decrease in influenza illness following vaccination, and is therefore clinically pertinent. Daily doses of 5 mg per week and 0.5 mg per day were well tolerated and unlike daily doses of 20 mg per week, met the primary purpose of GMT (Figure 40A). RAD001 not only improved the response to influenza vaccination, but also improved the response to pneumococcal vaccination compared to placebo in elderly volunteers. The pneumococcal vaccine contains antigens from 23 pneumococcal serotypes. Antibody titers to 7 of the serotypes were measured in our subjects. Antibody titers to 6/7 serotypes were increased in all 3 RAD cohorts compared to placebo.

[001042] Combined influenza and pneumococcal titer data suggest that partial mTOR inhibition (less than 80-100%) is more effective in reversing the aging-related decline in immune function than more complete mTOR inhibition.

Example 14: Low-Dose mTOR Inhibition Increases Energy and Exercise

[001043] In preclinical models, inhibition of mTOR with the rapalog rapamycin increases spontaneous physical activity in elderly mice (Wilkinson et al. Rapamycin slows aging in mice. (2012) Aging Cell; 11:675-82). Of interest, subjects in the 0.5 mg daily dosing cohort described in Example 13 similarly reported increased energy and exercise capacity compared to placebo in questionnaires administered one year after dosing (Figure 46). These data suggest that partial mTOR inhibition with rapalogues may have beneficial effects on aging-related morbidity from immune function alone.

Example 15: Inhibition of P70 S6 kinase with RAD001

[001044] Modeling and simulation were performed to predict daily and weekly dose ranges of RAD001 that are predicted to partially inhibit mTOR activity. As noted above, P70 S6K is phosphorylated by mTOR and is the downstream target of mTOR that is most closely linked to aging because knockout of P70 S6K increases lifespan. Therefore, modeling was performed of doses of RAD001 that partially inhibit P70 S6K activity. Weekly dosing in the range of >= 0.1 mg and < 20 mg is predicted to achieve partial inhibition of P70 S6K activity (Figure 47).

[001045] For daily dosing, RAD001 concentrations of 30 pM to 4 nM partially inhibited P70 S6K activity in cell lines (Table 12). These serum concentrations are predicted to be obtained with RAD001 doses >= 0.005 mg to < 1.5 mg per day.

[001046] Table 12: Percentage of inhibition of P70 S6K activity in HeLa cells in vitro

Conclusion

[001047] Methods of treating morbidity related to aging, or generally enhancing an immune response, with doses of mTOR inhibitors that only partially inhibit P70 S6K. The efficacy of partial mTOR inhibition with low doses of RAD001 in aging indications is an unexpected finding. Dose of RAD001 ranging from >= 0.1 mg to < 20 mg weekly and >= 0.005 mg to < 1.5 mg daily will achieve partial mTOR inhibition and is therefore expected to have efficacy in aging-related morbidity or in enhancing the immune response.

Example 16: Exogenous IL-7 enhances CAR T cell function

[001048] Following adoptive transfer of CAR T cells, some patients experience limited persistence of CAR T cells, which may result in suboptimal levels of antitumor activity. In this example, the effects of administering exogenous human IL-7 are evaluated in mouse xenograft models where an initial suboptimal response to CAR T cells was observed.

[001049] Expression of the IL-7 receptor CD127 was first evaluated in different cancer cell lines and in CAR-expressing cells. Two lining cell lymphoma cell lines (RL and Jeko-1) and one B-ALL cell line (Nalm-6) were analyzed by flow cytometry for CD127 expression. As shown in Figure 48A, out of the three cancer cell lines tested, RL was shown to have the highest expression of CD127, followed by Jeko-1 and Nalm-6. CART19 cells were infused into NSG mice and CD127 expression was assessed on circulating CART19 cells by flow cytometry. As shown in Figure 48B, CD127 is uniformly expressed on all circulating CART19 cells.

[001050] Next, the effect of exogenous lL-7 treatment on the antitumor activity of CART19 cells was evaluated in an animal model of lymphoma. NSG mice were engrafted with a luciferase-expressing coat cell line (RL luc) on day 0 (D0), followed by CART19 cell treatment on day 6. NSG mice were divided into groups, where one group received no cells. CART19, a second group received 0.5 x106 CART19 cells, a third group received 1x106 CART19 cells, and a fourth group received 2x106 CART19 cells. Tumor size was monitored by measuring the average bioluminescence of the engrafted tumors for more than 80 days. Only mice receiving 2x106 CART19 cells demonstrated tumor rejection and inhibition of tumor growth (Figure 49A). Mice from both groups receiving 0.5x106CART19 cells or 1x106CART19 cells showed a suboptimal antitumor response. Mice from these two groups were then randomized, where three mice (mouse #3827 and #3829 which received 0.5x106 CART19 cells, and mouse #3815 which received 1x106 CART19 cells) received exogenous recombinant human IL-7 at a dosage of 200 ng/mouse by intraperitoneal injection three times weekly starting on day 85, and two mice did not. The tumor burden of mice receiving exogenous IL-7 from day 85 to 125, as detected by average bioluminescence, is shown in Figure 49B. All mice receiving IL-7 showed a dramatic response of 1-3 log reduction in volume loading. Mice that originally received a higher dose of CART19 cells (mouse #3815 that received 1x106 CART19 cells) showed a more profound response. When comparing the tumor burden of mice that received lL-7 treatment to control, before and after lL-7 treatment, the reduction in tumor burden was only observed in the mice that received lL-7 treatment ( Figure 49C).

[001051] T cell dynamics after lL-7 treatment in the animal lymphoma model were also examined. Human CART19 cells were not detectable in the blood before IL-7 treatment. Upon IL-7 treatment, there was a rapid but variable increase in the number of T cells in the treated mice (Figure 50A). The T cell expansion observed in mice receiving IL-7 also correlates with tumor response. The mouse with the highest number of T cells detected in the blood at peak expansion during lL-7 treatment (mouse #3815) had the most robust reduction in tumor burden (see 49B). Furthermore, the time of peak expansion correlated with the injected T cell dose as a baseline. The number/level of CD3-expressing cells in the blood were also measured before and after lL-7 treatment. In control mice, cells expressing very little CD3 were detected, while IL-7-treated mice showed a significant increase in CD3+ cells after lL-7 treatment (Figure 50B).

[001052] Together, the results in this example demonstrate that treatment with exogenous IL-7 increases T cell proliferation and antitumor activity in vivo, indicating that the use of IL-7 in patients with sub-optimal results after CAR therapy may improve the antitumor response in these patients.

EQUIVALENTS

[001053] The descriptions of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been described with reference to specific aspects, it is evident that other aspects and variations of this invention may be devised by others skilled in the art without departing from the actual scope and spirit of the invention. The appended claims are intended to be construed to include all equivalent aspects and variations.

Claims (41)

1. Use of a population of cells expressing a CAR molecule that binds to CD19 ("CAR-19 expressing cells") in combination with one or more kinase inhibitors, characterized by the fact that it is for preparing a composition or medicine for the treatment of a hematological cancer in a mammal, the one or more kinase inhibitors being a Bruton's tyrosine kinase (BTK) inhibitor. 2. Use according to claim 1, characterized by the fact that: (a) the kinase inhibitor and CAR19-expressing cells are for administration to the mammal as a first line of therapy; or (b) the CAR19-expressing cells are for administration to the mammal after administration of the kinase inhibitor, optionally, wherein: (i) the CAR19-expressing cells are for administration after ceasing administration of the kinase inhibitor; or (ii) CAR19-expressing cells are for administration in combination with continued administration of the kinase inhibitor. 3. Use according to claim 1 or 2, characterized by the fact that the mammal is identified as being a complete or partial responder to the BTK inhibitor (e.g., ibrutinib) or a complete or partial responder to CAR19-expressing cells . 4. Use according to any one of claims 1 to 3, characterized in that the BTK inhibitor is selected from ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC -292, ONO-4059, CNX-774, or LFM-A13. 5. Use according to any one of claims 1 to 4, characterized by the fact that the kinase inhibitor is ibrutinib and the ibrutinib has a dose of 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 or 600 mg per day. 6. Use according to any one of claims 1 to 5, characterized in that the cells express a CAR molecule comprising an anti-CD19 binding domain, a transmembrane domain and an intracellular signaling domain. 7. Use according to claim 6, characterized by the fact that: (i) the intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain; (ii) the anti-CD19 binding domain comprises a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2), a light chain complementarity determining region 3 (LC CDR3), a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementarity determining region 2 (HC CDR2), and a heavy chain complementarity determining region 3 (HC CDR3) of a binding domain anti-CD19; (iii) the anti-CD19 binding domain comprises a murine light chain variable region of Table 7, a murine heavy chain variable region of Table 7, or both; (iv) the anti-CD19 binding domain comprises an LC CDR1 of SEQ ID NO: 25, an LC CDR2 of SEQ ID NO: 26 and an LC CDR3 of SEQ ID NO: 27; (v) the anti-CD19 binding domain comprises an HC CDR1 of SEQ ID NO: 19, an HC CDR2 of any one of SEQ ID NOS: 20-23 and an HC CDR3 of SEQ ID NO: 24; (vi) the anti-CD19 binding domain comprises a sequence from SEQ ID NO: 59, or a sequence with 95 to 99% of its identification; and/or (vii) the anti-CD19 binding domain is linked to the transmembrane domain by a hinge region, for example, the hinge region comprising a sequence from SEQ ID NO: 14 or SEQ ID NO: 45. 8. Use according to claim 6 or 7, characterized by the fact that the anti-CD19 binding domain is a humanized anti-CD19 binding domain, optionally wherein: (a) the humanized anti-CD19 binding domain comprises a sequence selected from: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, or a sequence with 95 to 99% identity to it; and/or (b) the humanized anti-CD19 binding domain is an scFv comprising a light chain variable region linked to a heavy chain variable region via a linker, for example, the linker comprising a sequence of SEQ ID NO: 53. 9. Use according to any one of claims 1 to 8, characterized by the fact that: (i) the CAR molecule comprises a transmembrane domain of a protein selected from: 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 or CD154, optionally, wherein the transmembrane domain comprises a sequence from SEQ ID NO: 15; (ii) the CAR molecule comprises a costimulatory domain, for example, the costimulatory domain comprising a functional signaling domain of a protein selected from: OX40, CD2, CD27, CD28, ICAM-1, LFA- 1 (CD11a/CD18) or 4-1BB (CD137), for example, with the co-stimulatory domain comprising a sequence from SEQ ID NO: 16 or SEQ ID NO: 51; (iii) the CAR molecule comprises an intracellular signaling domain, for example, wherein: (a) the intracellular signaling domain comprises a functional 4-1BB signaling domain, a functional CD3 zeta signaling domain, or both ; or (b) the intracellular signaling domain comprises a CD27 sequence, a functional CD3 zeta signaling domain, or both, (iv) the CAR molecule further comprises a leader sequence, e.g., the leader sequence comprising a amino acid sequence of SEQ ID NO: 13; or (v) the CAR molecule comprises an amino acid sequence of SEQ ID NO: 58, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 42. 10. Use according to claim 9, characterized in that the intracellular signaling domain comprises: (a) a sequence of SEQ ID NO: 16, a sequence of SEQ ID NO: 17, or both; (b) a sequence of SEQ ID NO: 16, a sequence of SEQ ID NO: 43, or both; (c) a sequence of SEQ ID NO: 51, a sequence of SEQ ID NO: 17, or both; or (d) a sequence of SEQ ID NO: 51, a sequence of SEQ ID NO: 43, or both. 11. Use according to any one of claims 1 to 10, characterized by the fact that the composition is for use in combination with an agent that inhibits an immune inhibitory molecule selected from: PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g. CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta. 12. Use according to any one of claims 1 to 11, characterized in that the composition comprises 1-5 x 108 cells expressing CAR19. 13. Use according to any one of claims 1 to 12, characterized in that the composition comprises about 5 x 106, 1 x 107, 2 x 107 or 5 x 107 cells expressing CAR19. 14. Use according to any one of claims 1 to 12, characterized in that the CAR19-expressing cells are administered in a dosage of 104 to 109 CAR19-expressing cells/kg of body weight. 15. Use according to any one of claims 1 to 14, characterized by the fact that the hematological cancer is a hematological cancer associated with the expression of CD19. 16. Use according to any one of claims 1 to 15, characterized in that the hematological cancer is selected from leukemia or lymphoma. 17. Use according to any one of claims 1 to 16, characterized by the fact that the hematological cancer is selected from: (i) chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, leukemia acute lymphoblastic leukemia (ALL), Hodgkin's lymphoma, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia or a follicular large cell lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (marginal zone lymphoma extranodal mucosa-associated lymphoid tissue), marginal zone lymphoma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma/leukemia, diffuse pulp splenic red, small B cell lymphoma, hairy variant cell leukemia, lymphoplasmacytic lymphoma, heavy chain disease, plasma cell myeloma, solitary bone plasmacytoma, extra bone plasmacytoma, marginal zone nodal lymphoma, pediatric marginal zone nodal lymphoma, lymphoma of the primary cutaneous follicular center, lymphomatoid granulomatosis, large B-cell lymphoma of the primary mediastinum (thymus), intravascular large B-cell lymphoma, ALB lymphoma + large B-cell lymphoma, large B-cell lymphoma arising in associated multicentric Castleman's disease to HHV8, primary effusion lymphoma, B-cell lymphoma or non-classifying lymphoma; or (ii) MCL, CLL, ALL, Hodgkin lymphoma or multiple myeloma. 18. Use according to any one of claims 1 to 17, characterized in that the composition is for use in combination with a cytokine, optionally, the cytokine being IL-7, IL-15 or IL-21. 19. Use according to any one of claims 1 to 18, characterized by the fact that the CAR is an optionally adjustable CAR (RCAR), wherein the RCAR comprises: (a) an intracellular signaling member comprising a domain of intracellular signaling and a first exchange domain, (b) an antigen-binding member comprising an antigen-binding domain that binds CD19 and a second exchange domain; and (c) a transmembrane domain. 20. Use according to any one of claims 1 to 19, characterized by the fact that: (i) the mammal is identified as having a BTK mutation; (ii) the mammal is identified as a partial responder, non-responder or relapser to one or more therapies for the hematological cancer; (iii) the mammal is identified as being a partial responder to the kinase inhibitor and cells expressing CAR19, alone or in combination with the BTK inhibitor, are administered to the mammal during the period of partial response; (iv) the BTK inhibitor is ibrutinib, the mammal is identified as being a non-responder having progressive or stable disease after treatment with ibrutinib, and the CAR19-expressing cells are for administration to the mammal, alone or in combination with a second BTK inhibitor. BTK other than ibrutinib, during the period of progressive or stable disease; (v) the mammal has optionally undergone lymphocyte depletion, with lymphocyte extirpation comprising the administration of one or more of melphalan, cytoxan, cyclophosphamide and fludarabine; or (vi) the mammal suffers a reduction in PD1-expressing T cells following administration of the kinase inhibitor. 21. Use according to any one of claims 1 to 20, characterized by the fact that: (i) resistance to the kinase inhibitor, CAR19-expressing cells, or both, is delayed or decreased; or (ii) remission of hematological cancer is prolonged or relapse of hematological cancer is delayed. 22. Use according to any one of claims 1 to 21, characterized by the fact that the BTK inhibitor is ibrutinib, the mammal is identified as being a non-responder or relapser to ibrutinib and the cells expressing CAR19 are administered in combination with a second kinase inhibitor other than ibrutinib. 23. Use according to claim 20 or 22, characterized in that the second kinase inhibitor is chosen from one or more of GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774 or LFM-A13 or a combination thereof. 24. Use according to any one of claims 1 to 23, characterized by the fact that the kinase inhibitor is ibrutinib and the ibrutinib is formulated for administration during 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, or more cycles, for example, with the cycle length being 21 or 28 days. 25. Use according to any one of claims 1 to 24, characterized by the fact that: (i) administration of CAR19-expressing cells is initiated at least 1, 2, 3 or 4 weeks, or 1, 2, 3, 4, 6, 9, 12, 15, 18 or 24 months, after the start of administration of the kinase inhibitor; (ii) the cells and the kinase inhibitor are formulated for simultaneous administration; or (iii) the cells and the kinase inhibitor are formulated for sequential release. 26. Use according to any one of claims 1 to 25, characterized in that the use comprises carrying out an infusion of lymphocytes with at least one cell that expresses CAR19. 27. Use according to any one of claims 1 to 26, characterized in that it further comprises administering a low, immunity-enhancing dose of an mTOR inhibitor to the mammal, optionally, wherein the mTOR inhibitor is everolimus or rapamycin. 28. Method for producing a cell that expresses CAR19 (e.g., an immune effector cell that expresses CAR19), or a population of cells, characterized by the fact that it comprises: (a) contacting the cell or population of cells with an inhibitor of BTK; and (b) introducing (e.g., by transduction) a nucleic acid encoding a CAR molecule that binds to CD19 into the cell or population of cells under conditions such that the CAR molecule is expressed. 29. The method of claim 28, wherein the cell is a T cell or NK cell, or the cell population includes T cells, NK cells, or both. 30. Method, according to claim 28 or 29, characterized by the fact that: (i) comprises contacting the cell or population of cells with the BTK inhibitor for 10-20, 20-30, 30-40, 40-60 or 60-120 minutes and subsequently removing most or all of the BTK inhibitor from the cell or cell population; (ii) the BTK inhibitor is added after the cell or cell population is harvested or before the cell or cell population is stimulated; or (iii) the BTK inhibitor is selected from: ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774 or LFM-A13. 31. Method according to any one of claims 28 to 30, characterized by the fact that the cell population also optionally comprises cancer cells, with the BTK inhibitor inhibiting a BTK in the cancer cells. 32. Method according to any one of claims 28 to 31, characterized by the fact that it further comprises depleting regulatory T cells (e.g., CD25+ cells) from the cell population. 33. Reaction mixture, characterized by the fact that it comprises a population of immune effector cells, a BTK inhibitor and a CAR molecule that binds to CD19 or a nucleic acid that encodes a CAR molecule that binds to CD19. 34. Reaction mixture according to claim 33, characterized by the fact that: (i) one or more of the immune effector cells express the CAR molecule or comprise the nucleic acid that encodes the CAR molecule; (ii) the BTK inhibitor is selected from ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774 or LFM-A13; or (iii) the reaction mixture additionally comprises cancer cells. 35. Reaction mixture, characterized by the fact that it comprises a population of immune effector cells and a CAR molecule that binds to CD19 or a nucleic acid that encodes a CAR molecule that binds to CD19, the immune effector cells comprising Covalently inactivated ITK. 36. Reaction mixture according to claim 35, characterized by the fact that it further comprises cancer cells, optionally, the cancer cells comprising covalently inactivated BTK. 37. Composition, characterized by the fact that it comprises a cell that expresses a CAR molecule that binds to CD19 (a “CAR19-expressing cell”) and one or more kinase inhibitors, said one or more kinase inhibitors including a Bruton's tyrosine kinase (BTK) inhibitor, optionally, wherein the CAR19-expressing cell and the one or more kinase inhibitors are present in a single dose form, or as two or more dose forms. 38. Use of the composition, as defined in claim 37, characterized by the fact that it is in the manufacture of a medicine. 39. Use, according to any one of claims 1 to 27 or 38, method, according to any one of claims 28 to 32, or composition, according to claim 37, characterized by the fact that the cell expressing CAR19 is a human immune effector cell (e.g., a human T cell or human NK cell) or cell population. 40. Use of an immune effector cell that expresses a CAR molecule that binds to CD19 in combination with a Bruton's tyrosine kinase inhibitor, characterized by the fact that it is in the manufacture of a medicine. 41. Use of a composition comprising an effective amount of the immune effector cell or combination therapy, as defined in claim 40, characterized by the fact that it is in the manufacture of a medicament to provide antitumor immunity in an individual.