Treatment of cancer using anti-CD19 chimeric antigen receptors

Abstract

The present invention provides compositions and methods for treating diseases associated with expression of CD19, for example by administering a recombinant T cell comprising a CD19 CAR as described herein in combination with a kinase inhibitor (e.g., a kinase inhibitor described herein). The invention also provides kits and compositions described herein.

Description

[0001] This application is a divisional application of Chinese Patent Application 202010128384.0 (which is a divisional application of 201580018102.9). The original application was filed on April 7, 2015, and its title was "Therapeutic treatment of cancer using anti-CD19 chimeric antigen receptor".

[0002] This application claims priority to U.S. Serial No. 61 / 976,396, filed April 7, 2014; U.S. Serial No. 62 / 007,309, filed June 3, 2014; U.S. Serial No. 62 / 036,493, filed August 12, 2014; U.S. Serial No. 62 / 076,238, filed November 6, 2014; U.S. Serial No. 62 / 087,888, filed December 5, 2014; and U.S. Serial No. 62 / 097,278, filed December 29, 2014, the contents of which are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application contains a sequence list that has been submitted electronically in ASCII format and is therefore fully incorporated by reference. The ASCII copy created on April 6, 2015, is named N2067-7051WO_SL.txt and has a size of 252,236 bits. Technical Field

[0005] The present invention generally relates to the use of T cells engineered to express chimeric antigen receptor (CAR), for example, in combination with another agent such as (e.g., kinase inhibitors and / or cytokines), for the treatment of diseases associated with the expression of differentiation antigen cluster 19 protein (CD19). Background of the Invention

[0006] Many patients with B-cell malignancies are incurable with standard treatments. Furthermore, conventional treatment options often have serious side effects. Immunotherapy has been explored in cancer; however, several obstacles make achieving clinical efficacy a very difficult goal. Although hundreds of so-called tumor antigens have been identified, these antigens are often derived from the tumor itself and are therefore poorly immunogenic. Additionally, tumors employ several mechanisms to counteract and amplify their own immune response.

[0007] Recent developments of chimeric antigen receptor (CAR)-modified autologous T-cell (CART) therapy, which relies on redirecting T cells to appropriate cell surface molecules on cancer cells such as B-cell malignancies, have shown promising results in utilizing the immune system's capabilities to treat B-cell malignancies and other cancers (see, for example, Sadelain et al., Cancer Discovery 3:388-398 (2013)). Clinical results for mouse-derived CART19 (i.e., "CTL019") have shown promise in establishing complete remission in CLL patients and in children with ALL (see, for example, Kalos et al., SciTransl 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 chimeric antigen receptors on genetically modified T cells to recognize and destroy target cells, successful therapeutic T-cell therapies require the ability to proliferate and persist over time, as well as the ability to further monitor leukemia cell escape. The variable properties of T cells, whether due to dysfunction, suppression, or fatigue, will affect the performance of CAR-converted T cells, but skilled practitioners have only limited control over this at this point. For effectiveness, CAR-converted patient T cells need to persist and maintain the ability to proliferate in response to the CAR antigen. It has been shown that ALL patient T cells can do this with CART19 containing mouse scFv (see, e.g., Grupp et al., NEJM 368:1509-1518 (2013)). Invention Overview

[0008] This disclosure is characterized at least in part by compositions and methods for treating diseases such as cancer (e.g., hematologic malignancies or other B-cell malignancies) using immune effector cells (e.g., T cells or NK cells), said immune effector cells expressing chimeric antigen receptor (CAR) molecules, such as CARs that bind to B-cell antigens (e.g., differentiation antigen cluster 19 protein (CD19) (e.g., OMIM accession number 107265, SwissProt accession number P15391)). The compositions comprise immune effector cells (e.g., T cells or NK cells) expressing a B-cell-targeting CAR, and the methods include administering these immune effector cells in combination with a kinase inhibitor (e.g., one or more of CDK4 / 6 inhibitors, BTK inhibitors, mTOR inhibitors, MNK inhibitors, dual PI3K / mTOR inhibitors, or combinations thereof). In some embodiments, the combination maintains or has better clinical efficacy compared to either therapy alone. The present invention also relates to the use of engineered cells (e.g., immune effector cells (e.g., T cells or NK cells)) expressing CAR molecules that bind to B cell antigens (e.g., CD19) to treat diseases (e.g., cancers, such as blood cancers) associated with the expression of B cell antigens (e.g., CD19), wherein said engineered cells are combined with kinase inhibitors (e.g., kinase inhibitors selected from one or more of the following: cyclin-dependent kinase 4 (CDK4) inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, mTOR inhibitors, mitogen-activated protein kinase-interacting kinase (MNK) inhibitors, phosphatidylinositol 3-kinase (PI3K) / mTOR dual inhibitors, or combinations thereof).

[0009] Therefore, in one aspect, the present invention relates to a method for treating a subject (e.g., a mammal) suffering from a disease associated with the expression of a B-cell antigen (e.g., CD19). The method includes administering to the mammal an effective amount of cells (e.g., immune effector cells (e.g., T cells or NK cells) expressing a CAR molecule that binds to the B-cell antigen, said cells in combination with a kinase inhibitor (e.g., the kinase inhibitor described herein). In one embodiment, the CAR molecule binds to CD19, for example, a CD19-binding CAR molecule described herein. In other embodiments, the CAR molecule binds to one or more of CD20, CD22, or ROR1.

[0010] In one embodiment, the disease associated with the expression of B-cell antigens (e.g., expression of one or more of CD19, CD20, CD22, or ROR1) is selected from proliferative disorders such as cancer, malignant tumors, or precancerous conditions such as spinal dysplasia, myelodysplastic syndromes, or preleukemia, or non-cancer-related indications associated with the expression of B-cell antigens (e.g., expression of one or more of CD19, CD20, CD22, or ROR1). In one embodiment, the disease is a solid tumor or a liquid tumor. In one embodiment, the cancer is pancreatic cancer. In one embodiment, the disease is a hematologic malignancy. In one embodiment, the hematologic malignancy is leukemia. In one embodiment, the cancer is selected from one or more acute leukemias, including but not limited to B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), and acute lymphoblastic leukemia (ALL); and one or more chronic leukemias, including but not limited to chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). Additional hematologic malignancies or hematologic disorders include, but are not limited to, mantle cell lymphoma (MCL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, and Waldenstrom's macroglobulinemia. In some embodiments, diseases associated with the expression of B-cell antigens (e.g., one or more of CD19, CD20, CD22, or ROR1) are termed "preleukemia," which is a diverse set of hematologic disorders unified by ineffective production (or dysplasia) of myeloid hematologic cells. In some embodiments, diseases associated with the expression of B-cell antigens (e.g., one or more of CD19, CD20, CD22, or ROR1) include, but are not limited to, atypical and / or nonclassical cancers, malignancies, precancerous lesions, or proliferative disorders expressing B-cell antigens (e.g., one or more of CD19, CD20, CD22, or ROR1). Any combination of diseases associated with the expression of B-cell antigens (e.g., one or more of CD19, CD20, CD22, or ROR1) described herein can be treated with the methods and compositions described herein.

[0011] In one embodiment, the disease associated with the expression of B-cell antigens (e.g., one or more of CD19, CD20, CD22, or ROR1) is lymphoma, such as MCL, Hodgkin's lymphoma, or DLBCL. In one embodiment, the disease associated with the expression of B-cell antigens (e.g., one or more of CD19, CD20, CD22, or ROR1) is leukemia, such as SLL, CLL, and / or ALL. In one embodiment, the disease associated with the expression of B-cell antigens is multiple myeloma (e.g., CD19-negative multiple myeloma (e.g., the vast majority (99.95%) of neoplastic plasma cells have a CD19-negative phenotype), detected, for example, by both flow cytometry and RT-PCR).

[0012] In one embodiment, the kinase inhibitor is a CDK4 inhibitor (e.g., the CDK4 inhibitor described herein, e.g., 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]pyrimidine-7-one hydrochloride (also known as palbociclib or PD0332991). In one embodiment, the kinase inhibitor is a BTK inhibitor, such as the BTK inhibitor described herein, e.g., ibrutinib. In one embodiment, the kinase inhibitor is an mTOR inhibitor, such as the mTOR inhibitor described herein, e.g., ibrutinib. Rapamycin, rapamycin analogs, OSI-027. The mTOR inhibitor may be, for example, an mTORC1 inhibitor and / or an mTORC2 inhibitor, such as the mTORC1 inhibitor and / or mTORC2 inhibitor described herein. In one embodiment, the kinase inhibitor is an MNK inhibitor, such as the MNK inhibitor described herein, e.g., 4-amino-5-(4-fluoroaniline)-pyrazolo[3,4-d]pyrimidine. The MNK inhibitor may be, for example, an MNK1a, MNK1b, MNK2a, and / or MNK2b inhibitor. In one embodiment, the inhibitor may be a PI3K / mTOR dual inhibitor, such as PF-04695102.

[0013] In one embodiment, the kinase inhibitor is selected from the following CDK4 inhibitors: aloisine A; flavopiridol or HMR-1275; 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromone; crizotinib (PF-02341066); 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one, hydrochloride (P27) 6-00); 1-Methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazole-2-amine (RAF265); indisulam (E7070); roscovitine (CYC202); pabuxilib (PD0332991); dinaciclib (SCH727965); N-[5-[[(5-tert-butyl) [[Azol-2-yl]methyl]thio]thiazol-2-yl]piperidin-4-carboxamide (BMS 387032); 4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzo[acrylonitrile-2-yl]amino]benzoic acid (MLN8054); 5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethylamine (AG-024322) ; 4-(2,6-dichlorobenzoamide)-1H-pyrazole-3-carboxylic acid N-(piperidin-4-yl)amide (AT7519); 4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methanesulfonyl)phenyl]-2-pyrimidinylamine (AZD5438); XL281 (BMS908662); and ribociclib.

[0014] In one embodiment, the kinase inhibitor is a CDK4 inhibitor, such as prabuxipar (PD0332991), and prabuxipar is administered daily for a period of time 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, or 135 mg (e.g., 75 mg, 100 mg, or 125 mg), for example, for 14-21 days in a 28-day cycle, or for 7-12 days in a 21-day cycle. In one embodiment, prabuxipar is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more cycles.

[0015] In one embodiment, the kinase inhibitor is a BTK inhibitor selected from the following: 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.

[0016] In one embodiment, the kinase inhibitor is a BTK inhibitor, such as ibrutinib (PCI-32765), and ibrutinib is administered daily for a period of time at doses 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, or 600 mg (e.g., 250 mg, 420 mg, or 560 mg), for example, daily administration for a 21-day cycle or daily administration for a 28-day cycle. In one embodiment, ibrutinib is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more cycles.

[0017] In one embodiment, the kinase inhibitor is selected from the following mTOR inhibitors: tamsuloxim; desfolimex ((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-azotricyclo[30.3.1.0]). 4,9[37-carbon-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphonate, 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 N 2 -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinon-4-yl]]methoxy]butyl]-L-arginylglycyl-L-α-aspartic-L-serine-, inner salt (SF1126); and XL765.

[0018] In one embodiment, the kinase inhibitor is an mTOR inhibitor, such as rapamycin, and rapamycin is administered daily for a period of time at a dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg (e.g., 6 mg), for example, a daily administration for a 21-day cycle or a daily administration for a 28-day cycle. In one embodiment, rapamycin is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more cycles. In one embodiment, the kinase inhibitor is an mTOR inhibitor, such as everolimus, and everolimus is administered daily for a period of time 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, or 15 mg (e.g., 10 mg), for example, a daily administration for a 28-day cycle. In one implementation, everolimus is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles.

[0019] In one embodiment, the kinase inhibitor is selected from the following MNK inhibitors: CGP052088; 4-amino-3-(p-fluorophenylamino)-pyrazolo[3,4-d]pyrimidine (CGP57380); causticin; ETC-1780445-2; and 4-amino-5-(4-fluoroaniline)-pyrazolo[3,4-d]pyrimidine.

[0020] In one embodiment, the kinase inhibitor is a dual inhibitor of phosphatidylinositol 3-kinase (PI3K) and mTOR selected from the following: 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF-04691502); N-[4-[[4-(dimethylamino)-1-piperidine [4-(4,6-di-4-morpholino-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}propionitrile (BEZ-235); apitol isib (GDC-0980, RG7422); 2,4-difluoro-N-{2-(methoxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridyl}benzenesulfonamide (GSK2126458); 8-(6-methoxy-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinoline-2(3 H)-ketomaleic acid (NVP-BGT226); 3-[4-(4-morpholinylpyridino[3',2':4,5]furano[3,2-d]pyrimidin-2-yl]phenol (PI-103); 5-(9-isopropyl-8-methyl-2-morpholino-9H-purine-6-yl)pyrimidin-2-amine (VS-5584, SB2343); and N-[2-[(3,5-dimethoxyphenyl)amino]quinoline [Lin-3-yl]-4-[(4-methyl-3-methoxyphenyl)carbonyl]aminobenzenesulfonamide (XL765).

[0021] In one embodiment, a cell expresses a CAR molecule comprising an anti-CD19 binding domain (e.g., a mouse or humanized 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 comprising the anti-CD19 binding domain described herein (e.g., a mouse or humanized antibody or antibody fragment that specifically binds to CD19 as described herein), the transmembrane domain described herein, and the intracellular signaling domain described herein (e.g., an intracellular signaling domain comprising a co-stimulatory domain and / or a primary signaling domain described herein).

[0022] In one embodiment, the CAR molecule is capable of binding to CD19 (e.g., wild-type or mutant human CD19). In one embodiment, the CAR molecule includes an anti-CD19 binding domain comprising one or more (e.g., all three) light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of the anti-CD19 binding domain described herein, and one or more (e.g., all three) heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the anti-CD19 binding domain described herein, for example, an 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 anti-CD19 binding domain comprises one or more (e.g., all three) heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the anti-CD19 binding domain described herein. For example, the anti-CD19 binding domain has two variable heavy chain regions, each comprising HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the anti-CD19 binding domain comprises a mouse light chain variable region and / or a mouse heavy chain variable region as described herein (e.g., in Table 7). In one embodiment, the anti-CD19 binding domain is an scFv comprising the mouse light chain and mouse heavy chain containing the amino acid sequences of Table 7. In one embodiment, the anti-CD19 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequences of the light chain variable regions provided in Table 7, but no more than 30, 20, or 10 modifications (e.g., substitutions), or comprising a sequence having 95-99% identity with the amino acid sequences 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) to the amino acid sequences of the heavy chain variable regions provided in Table 7, but no more than 30, 20, or 10 modifications (e.g., substitutions), or comprising a sequence having 95-99% identity with the amino acid sequences of Table 7. In one embodiment, the anti-CD19 binding domain comprises the sequence of SEQ ID NO:59, or a sequence having 95-99% identity with it.In one embodiment, the anti-CD19 binding domain is scFv, and a light chain variable region comprising the amino acid sequence described herein (e.g., in Table 7) is linked via a linker (e.g., the linker described herein) to a heavy chain variable region comprising the amino acid sequence described herein (e.g., in Table 7). In one embodiment, the anti-CD19 binding domain comprises 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 the heavy chain variable region of scFv can be in any orientation, for example: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0023] In one embodiment, the CAR molecule comprises a humanized anti-CD19 binding domain comprising one or more (e.g., all three) light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of the humanized anti-CD19 binding domain described herein, and one or more (e.g., all three) heavy chain complementarity-determining regions 1 (HCCDR1), 2 (HC CDR2), and 3 (HC CDR3) of the humanized anti-CD19 binding domain described herein, for example, 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 includes one or more (e.g., all three) heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HCCDR3) of the humanized anti-CD19 binding domain described herein. For example, the humanized anti-CD19 binding domain has two variable heavy chain regions, each containing HCCDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the humanized anti-CD19 binding domain includes at least HC CDR2. In one embodiment, the light chain variable region includes one, two, three, or all four framework regions of the VK3_L25 germline sequence. In one embodiment, the light chain variable region has modifications (e.g., substitutions, such as one or more amino acid substitutions present at the corresponding positions of the mouse light chain variable region of SEQ ID NO:58 (e.g., substitutions at one or more positions 71 and 87). In one embodiment, the heavy chain variable region comprises one, two, three, or all four framework regions of the VH4_4-59 germline sequence. In one embodiment, the heavy chain variable region has modifications (e.g., substitutions, such as one or more amino acid substitutions present at the corresponding positions of the mouse heavy chain variable region of SEQ ID NO:58 (e.g., substitutions at one or more positions 71, 73, and 78). In one embodiment, the humanized anti-CD19 binding domain comprises the light chain variable region described herein (e.g., in Table 3) and / or the heavy chain variable region described herein (e.g., in Table 3). In one embodiment, the humanized anti-CD19 binding domain is an scFv of the light and heavy chains comprising the amino acid sequences of Table 3.In one embodiment, the humanized anti-CD19 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequences of the light chain variable region provided in Table 3, but no more than 30, 20, or 10 modifications (e.g., substitutions), or comprising a sequence having 95-99% identity with the amino acid sequences 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) to the amino acid sequences of the heavy chain variable region provided in Table 3, but no more than 30, 20, or 10 modifications (e.g., substitutions), or comprising a sequence having 95-99% identity with the amino acid sequences of Table 3. In one embodiment, 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 having 95-99% identity with such sequences. In one embodiment, the humanized anti-CD19 binding domain is scFv, and a light chain variable region comprising the amino acid sequence described herein (e.g., in Table 3) is linked via a linker (e.g., the linker described herein) to a heavy chain variable region comprising the amino acid sequence described herein (e.g., in Table 3). In one embodiment, the humanized anti-CD19 binding domain comprises a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO:53). The light chain variable region and the heavy chain variable region of scFv can be in any of the following orientations, for example: light chain variable region-joint-heavy chain variable region or heavy chain variable region-joint-light chain variable region.

[0024] In one embodiment, the CAR molecule comprises a transmembrane domain selected from the following proteins: the α, β, or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO:15. In one embodiment, the transmembrane domain comprises at least one, two, or three modifications (e.g., substitutions), but no more than 20, 10, or 5 modifications (e.g., substitutions) of the amino acid sequence of SEQ ID NO:15, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO:15.

[0025] In one embodiment, the anti-CD19 binding domain is connected to the transmembrane domain via a hinge region (e.g., the hinge region described herein). In one embodiment, the encoded hinge region comprises SEQ ID NO:14 or SEQ ID NO:45 or a sequence having 95-99% identity with it.

[0026] In one embodiment, the CAR molecule further comprises a sequence encoding a co-stimulatory domain (e.g., the co-stimulatory domain described herein). In one embodiment, the co-stimulatory domain comprises a functional signaling domain selected from proteins including OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137). In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO:16. In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO:51. In one embodiment, the co-stimulatory domain comprises at least one, two, or three modified (e.g., substitutions), but no more than 20, 10, or 5 modified (e.g., substitutions) amino acid sequences having the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:51, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:51.

[0027] In one embodiment, the CAR molecule further comprises a sequence encoding an intracellular signal transduction domain (e.g., the intracellular signal transduction domain described herein). In one embodiment, the intracellular signal transduction domain comprises a functional signal transduction domain of 4-1BB and / or a functional signal transduction domain of CD3ζ. In one embodiment, the intracellular signal transduction domain comprises the sequence of SEQ ID NO:16 and / or the sequence of SEQ ID NO:17. In one embodiment, the intracellular signal transduction domain comprises the sequence of SEQ ID NO:16 and / or the sequence of SEQ ID NO:43. In one embodiment, the intracellular signal transduction domain comprises a functional signal transduction domain of CD27 and / or a functional signal transduction domain of CD3ζ. In one embodiment, the intracellular signal transduction domain comprises the sequence of SEQ ID NO:51 and / or the sequence of SEQ ID NO:17. In one embodiment, the intracellular signal transduction domain comprises the sequence of SEQ ID NO:51 and / or the sequence of SEQ ID NO:43. In one embodiment, the intracellular signal transduction domain comprises at least one, two, or three modified (e.g., substitutions), but no more than 20, 10, or 5 modified (e.g., substitutions) amino acid sequences having the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:51 and / or the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:43, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:51 and / or the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:43. In one embodiment, the intracellular signal transduction 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 sequence comprising the intracellular signal transduction domain is expressed in the same reading frame and as a single polypeptide chain.

[0028] In one embodiment, the CAR molecule further comprises a leader sequence (e.g., the leader sequence described herein). In one embodiment, the leader sequence comprises the amino acid sequence of SEQ ID NO:13 or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO:13.

[0029] In one embodiment, the CAR molecule comprises a leader sequence (e.g., the leader sequence described herein, such as SEQ ID NO:13 or a leader sequence having 95-99% identity with it); an anti-CD19 binding domain described herein (e.g., an anti-CD19 binding domain comprising LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3 described herein, such as the mouse anti-CD19 binding domain described in Table 7, the humanized anti-CD19 binding domain described in Table 3, or a sequence having 95-99% identity with it); a hinge region (e.g., a hinge region described herein, such as SEQ ID NO:14 or a hinge region having 95-99% identity with it); and a transmembrane domain (e.g., a transmembrane domain described herein, such as a leader sequence having SEQ ID NO:14 or a leader sequence having 95-99% identity with it). A transmembrane domain of the sequence NO:15 or a sequence having 95-99% identity with it; an intracellular signal transduction domain (e.g., the intracellular signal transduction domain described herein, e.g., an intracellular signal transduction domain comprising a costimulatory domain and / or a primary signal transduction domain). In one embodiment, the intracellular signal transduction domain comprises a costimulatory domain, e.g., the costimulatory domain described herein, e.g., a 4-1BB costimulatory domain having the sequence SEQ ID NO:16 or SEQ ID NO:51 or having 95-99% identity with it, and / or a primary signal transduction domain (e.g., the primary signal transduction domain described herein, e.g., a CD3ζ stimulatory domain having the sequence SEQ ID NO:17 or SEQ ID NO:43 or having 95-99% identity with it).

[0030] In one embodiment, the CAR molecule comprises (e.g., consists of) the following amino acid sequences: 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 the following amino acid sequences: 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. The amino acid sequence of NO:42 has at least one, two, three, four, five, 10, 15, 20, or 30 modifications (e.g., substitutions), but no more than 60, 50, or 40 modifications (e.g., substitutions); or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences 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.

[0031] In one embodiment, the cell expressing the CAR molecule comprises a vector containing a nucleic acid sequence encoding the CAR molecule. In one embodiment, the vector is selected from DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, or retroviral vectors. In one embodiment, the vector is a lentiviral vector. In one embodiment, the vector further comprises a promoter. In one embodiment, the promoter is the EF-1 promoter. In one embodiment, the EF-1 promoter comprises the sequence of SEQ ID NO:100. In one embodiment, the vector is an in vitro transcription vector, for example, a vector for transcribing RNA of the nucleic acid molecule described herein. In one embodiment, the nucleic acid sequence in the in vitro vector further comprises a polyadenylated tail, for example, a polyadenylated tail described herein, for example, a polyadenylated tail comprising about 150 adenosine bases (SEQ ID NO:104). In one embodiment, the nucleic acid sequence in the in vitro vector further comprises a 3'UTR, for example, a 3'UTR described herein, for example, a 3'UTR comprising at least one repeat sequence of a 3'UTR derived from human β-globulin. In one embodiment, the nucleic acid sequence in the in vitro vector further comprises a promoter, for example, the T2A promoter.

[0032] In some embodiments of the compositions and methods disclosed herein, the cells expressing the CAR molecule (also referred to herein as "CAR-expressing cells") are cells or cell populations as described herein, such as human immune effector cells or cell populations (e.g., human T cells or human NK cells (e.g., human T cells or human NK cells as described herein). In one embodiment, the 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 allogeneic T cell. In one embodiment, the cell is a T cell and the T cell is diacylglycerol 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 simultaneously DGK and Ikaros deficient. It should be understood that the term "cell" as used in the compositions and methods disclosed herein encompasses compositions and methods comprising one or more cells (e.g., a cell population).

[0033] In another embodiment, (e.g., as described herein) cells expressing CAR molecules may also express another substance, such as a substance that enhances the activity of CAR-expressing cells.

[0034] In one embodiment, the method further includes administering cells expressing a CAR molecule as described herein, said cells optionally in combination with a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) and a substance that enhances the activity of CAR-expressing cells. In some embodiments, the substance is a cytokine, such as IL-7, IL-15, IL-21, or a combination thereof. In one embodiment, the method includes administering IL-7 to a subject. Cytokines may be delivered in combination with CAR-expressing cells (e.g., concurrently or shortly after administration of CAR-expressing cells). Alternatively, cytokines may be delivered some time after administration of CAR-expressing cells (e.g., after assessment of the subject's response to CAR-expressing cells).

[0035] In other embodiments, the substance that enhances the activity of CAR-expressing cells can be a substance that inhibits immunosuppressive molecules. Examples of immunosuppressive 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 TGFRβ. In one embodiment, the substance that inhibits the immunosuppressive molecule comprises a first polypeptide (e.g., an immunosuppressive molecule) that binds to a second polypeptide (e.g., an intracellular signaling domain described herein) that provides a positive signal to the cell. In one embodiment, the substance comprises, for example, a first polypeptide of 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 TGFRβ, or a fragment of any of these molecules (e.g., at least a portion of the extracellular domain of any of these molecules) and a second polypeptide as an intracellular signaling domain (e.g., comprising a co-stimulatory domain (e.g., 41BB, CD27 or CD28 as described herein) and / or a primary signaling domain (e.g., the CD3ζ signaling domain as described herein). In one embodiment, the substance 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 (e.g., the CD28 signaling domain as described herein and / or the CD3ζ signaling domain as described herein).

[0036] In one embodiment, a lymphocyte infusion, such as an allogeneic lymphocyte infusion, is used to treat cancer, wherein the lymphocyte infusion comprises at least one CAR-expressing cell (also referred to herein as a CD19 CAR-expressing cell) that binds to a B-cell antigen (e.g., CD19) as described herein. In one embodiment, an autologous lymphocyte infusion is used to treat cancer, wherein the autologous lymphocyte infusion includes at least one CD19-expressing cell.

[0037] In one implementation, cells expressing CD19 CAR (e.g., T cells) are administered to a subject who has previously received a stem cell transplant (e.g., autologous stem cell transplant).

[0038] In one implementation, cells expressing CD19 CAR (e.g., T cells) are administered to a subject who has already received a dose of melphalan.

[0039] In one embodiment, cells expressing a CAR molecule (e.g., the CAR molecule described herein) are administered in combination with a substance that improves one or more side effects (e.g., the substance described herein), wherein the one or more side effects are one or more side effects associated with the administration of cells expressing the CAR molecule.

[0040] In one embodiment, the kinase inhibitor is administered in combination with a substance that improves one or more side effects (e.g., the substance described herein), wherein the one or more side effects are one or more side effects associated with the administration of the kinase inhibitor.

[0041] In one implementation, a cell and kinase inhibitor expressing a CAR molecule (e.g., the CAR molecule described herein) is administered in combination with other substances for treating CD19-related diseases (e.g., other substances described herein).

[0042] In one implementation, cells expressing a CAR molecule (e.g., the CAR molecule described herein) are administered at the dosage and / or administration regimen described herein.

[0043] In one implementation, a CAR molecule is introduced into T cells, for example, using in vitro transcription, and a subject (e.g., a human) receives an initial administration of cells containing the CAR molecule and one or more subsequent administrations of cells containing the CAR molecule, wherein the one or more subsequent administrations are administered less than 15 days after the initial administration, for example, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the initial administration. In one implementation, cells containing the CAR molecule are administered to the subject (e.g., a human) more than once per week, for example, 2, 3, or 4 times per week. In one implementation, a subject (e.g., a human subject) receives more than one administration of cells containing the CAR molecule per week (e.g., 2, 3, or 4 times per week) (also referred to herein as a cycle), followed by a week without administration of cells containing the CAR molecule, and subsequently one or more additional administrations of cells containing the CAR molecule (e.g., more than once per week) to the subject. In another embodiment, the subject (e.g., a human subject) receives more than one cycle of CAR-containing cells with the time between each cycle being less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the CAR-containing cells are administered every other day, three times a week. In one embodiment, the CAR-containing cells are administered for at least two, three, four, five, six, seven, eight, or more weeks.

[0044] In one embodiment, a combination of a kinase inhibitor and cells expressing a CAR molecule (e.g., the CAR molecule described herein) is administered as a first-line treatment for a disease (e.g., cancer, e.g., the cancer described herein). In another embodiment, a combination of a kinase inhibitor and cells expressing a CAR molecule (e.g., the CAR molecule described herein) is administered as a second-line, third-line, or fourth-line treatment for a disease (e.g., cancer, e.g., the cancer described herein).

[0045] In one implementation, the cells described herein (e.g., a cell population) are administered to a subject.

[0046] In one embodiment, the method includes administering a population of cells, wherein a plurality of said cells comprise the CAR molecules described herein. In some embodiments, the population of cells expressing a CAR comprises a mixture of cells expressing different CARs. For example, in one embodiment, the population of cells expressing a CAR may comprise a first cell expressing a CAR having an anti-CD19 binding domain as described herein and a second cell expressing a CAR having a different anti-CD19 binding domain (e.g., an anti-CD19 binding domain described herein that differs from the anti-CD19 binding domain in the CAR expressed by the first cell). As another example, the population of cells expressing a CAR may comprise a first cell expressing a CAR comprising (e.g., as described herein) an anti-CD19 binding domain, and a second cell expressing a CAR comprising an antigen-binding domain against a target other than CD19 (e.g., CD123 or mesothelin). In one embodiment, the population of cells expressing a CAR comprises a first cell expressing a CAR comprising a primary intracellular signaling domain, and a second cell expressing a CAR comprising a secondary signaling domain.

[0047] In one embodiment, the method includes administering a cell population, wherein at least one cell in the population expresses a CAR having the anti-CD19 domain described herein, and administering a substance that enhances the activity of CAR-expressing cells, for example, a second cell expressing a substance that enhances the activity of CAR-expressing cells. For example, in one embodiment, the substance may be a substance that inhibits an immunosuppressive molecule. Examples of immunosuppressive 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 TGFRβ. In one embodiment, the substance that inhibits the immunosuppressive molecule comprises a first polypeptide (e.g., an inhibitory molecule) that binds to a second polypeptide (e.g., an intracellular signaling domain described herein) that provides a positive signal to the cell. In one embodiment, the substance comprises, for example, a first polypeptide of 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 TGFRβ, or a fragment of any of these molecules (e.g., at least a portion of the extracellular domain of any of these molecules) and a second polypeptide as an intracellular signaling domain (e.g., comprising a co-stimulatory domain (e.g., 41BB, CD27 or CD28 as described herein) and / or a primary signaling domain (e.g., the CD3ζ signaling domain as described herein). In one embodiment, the substance 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 (e.g., the CD28 signaling domain as described herein and / or the CD3ζ signaling domain as described herein).

[0048] In another aspect, the present invention relates to cells expressing CAR molecules as described herein, which are used as pharmaceuticals in combination with kinase inhibitors (e.g., kinase inhibitors described herein (e.g., BTK inhibitors such as ibrutinib)).

[0049] In another aspect, the present invention relates to cells expressing CAR molecules as described herein, which, in combination with kinase inhibitors (e.g., kinase inhibitors described herein (e.g., BTK inhibitors such as ibrutinib)) are used to treat diseases expressing B-cell antigens (e.g., CD19). In another aspect, the present invention relates to kinase inhibitors as described herein (e.g., BTK inhibitors such as ibrutinib), which, in combination with cells expressing CAR molecules as described herein, are used to treat diseases expressing B-cell antigens (e.g., CD19). Diseases may be, for example, cancers such as blood cancers. Cancers may be, for example, lymphoma, CLL, MCL, ALL, DLBCL, multiple myeloma, or another cancer described herein.

[0050] In another aspect, the present invention relates to cells expressing CAR molecules as described herein, which are used as drugs in combination with cytokines as described herein (e.g., IL-7, IL-15, and / or IL-21). In another aspect, the present invention relates to cytokines as described herein, which are used as drugs in combination with cells expressing CAR molecules as described herein.

[0051] In another aspect, the present invention relates to cells expressing CAR molecules as described herein, which, in combination with cytokines as described herein (e.g., IL-7, IL-15, and / or IL-21), are used to treat diseases expressing CD19. In another aspect, the present invention relates to cytokines as described herein, which, in combination with cells expressing CAR molecules as described herein, are used to treat diseases expressing CD19.

[0052] In another aspect, the present invention relates to a method for treating a mammal with Hodgkin's lymphoma, the method comprising administering to the mammal an effective amount of cells (e.g., multiple cells) expressing a CAR molecule (e.g., the CAR molecule described herein).

[0053] In one embodiment, cells expressing a CAR molecule (e.g., the CAR molecule described herein) are administered in combination with a substance that increases the efficacy of cells expressing the CAR molecule (e.g., the substance described herein).

[0054] In one embodiment, cells expressing a CAR molecule (e.g., the CAR molecule described herein) are administered in combination with a substance that improves one or more side effects (e.g., the substance described herein), wherein the one or more side effects are one or more side effects associated with the administration of cells expressing the CAR molecule.

[0055] In one implementation, cells expressing a CAR molecule (e.g., the CAR molecule described herein) are administered in combination with a substance for treating Hodgkin's lymphoma (e.g., the substance described herein).

[0056] In one implementation, cells expressing a CAR molecule (e.g., the CAR molecule described herein) are administered in combination with an immunomodulatory low-dose mTOR inhibitor (e.g., the mTOR inhibitor described herein). While not wishing to be bound by theory, it is believed that treatment with an immunomodulatory low-dose (e.g., a dose insufficient to completely suppress the immune system but sufficient to improve immune function) is associated with a decrease in PD-1 positive T cells or an increase in PD-1 negative cells. PD-1 positive T cells can be depleted, but not PD-1 negative T cells, by binding to cells expressing a PD-1 ligand (e.g., PD-L1 or PD-L2).

[0057] In one embodiment, this method can be used to optimize the performance of the CAR cells described herein in subjects. While not wishing to be bound by theory, it is believed that in one embodiment, the performance of endogenously 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 cells expressing CD19CAR is improved. In other embodiments, cells (e.g., T cells) that have been engineered or will be engineered to express CAR can be ex vivo treated with an amount of 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) to PD1-positive immune effector cells (e.g., T cells).

[0058] In one embodiment, administration of an immune-enhancing low-dose mTOR inhibitor (e.g., an allosteric inhibitor (e.g., RAD001) or a catalytic inhibitor) is initiated prior to administration of cells expressing the CAR described herein (e.g., T cells). In one embodiment, the mTOR inhibitor is RAD001 or rapamycin. In one embodiment, CAR cells are administered after a sufficient duration of mTOR inhibitor administration or after adequate administration of 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) to PD1-positive immune effector cells (e.g., T cells) has at least transiently increased.

[0059] In one implementation, cells to be engineered to express CAR, such as immune effector cells (e.g., T cells or NK cells), are harvested after sufficient time of mTOR inhibitor administration or after adequate administration of an immune-enhancing low dose of mTOR inhibitor, such that the level of PD1-negative immune effector cells (e.g., T cells) in or harvested from the subject or the ratio of PD1-negative immune effector cells (e.g., T cells) to PD1-positive immune effector cells (e.g., T cells) has increased at least transiently.

[0060] In implementation methods, any of the methods described herein also includes lymphocyte depletion of the subject, for example, prior to administration of one or more cells expressing the CAR molecule described herein (e.g., a CD19-binding CAR molecule). Lymphocyte depletion may include, for example, administration of one or more of the following: melphalan, cytoxan, cyclophosphamide, and fludarabine.

[0061] In some embodiments, the administered CAR-expressing cells comprise a regulatory CAR (RCAR), such as the RCAR described herein. The RCAR may, for example, comprise an intracellular signaling component including an intracellular signaling domain and a first switching domain; an antigen-binding component including a CD19-binding antigen-binding domain and a second switching domain; and a transmembrane domain. The method may also include administering a dimerizing molecule, for example, in an amount sufficient to induce dimerization of the first and second switching domains.

[0062] In some embodiments, CAR-expressing cells and kinase inhibitors are administered simultaneously or substantially simultaneously, for example, as a first-line therapy. In some embodiments, the method includes administering a combination of a BTK inhibitor (e.g., ibrutinib) and CAR-expressing cells (e.g., CAR19-expressing cells) to a subject as a first-line therapy.

[0063] In other embodiments, CAR-expressing cells and a kinase inhibitor are administered sequentially. For example, the kinase inhibitor is administered before CAR-expressing cells, or CAR-expressing cells are administered before the kinase inhibitor.

[0064] In some embodiments, the disease associated with CD19 expression is a blood cancer (e.g., the blood cancers described herein, such as CLL, MCL, or ALL), and the subject is or has been identified as a partial responder, non-responder, or relapsed patient to one or more blood cancer treatments (e.g., to a BTK inhibitor such as ibrutinib). In some embodiments, the subject has or has been identified as having a BTK mutation. The mutation can be, for example, a point mutation, insertion, or deletion. The mutation can be, for example, a mutation at the binding site of the BTK inhibitor (e.g., at or near the ATP-binding pocket). The mutation can cause a reduced response to the BTK inhibitor (e.g., resistance).

[0065] In some embodiments of any of the methods disclosed herein, the method includes administering a BTK inhibitor (e.g., ibrutinib) to a subject, thereby reducing the amount of the BTK inhibitor (e.g., stopping administration), and subsequently administering CAR-expressing cells (e.g., CAR19-expressing cells) to the subject.

[0066] In some implementations, the method includes administering a BTK inhibitor (e.g., ibrutinib) to a subject and subsequently administering a combination of the BTK inhibitor and CAR-expressing cells (e.g., CAR19-expressing cells) to the subject.

[0067] In some embodiments, the method includes administering a BTK inhibitor (e.g., ibrutinib) to a subject, thereby reducing the amount of the BTK inhibitor (e.g., stopping or discontinuing administration), and subsequently administering CAR-expressing cells (e.g., CAR19-expressing cells) and a second BTK inhibitor (e.g., a combination of BTK inhibitors other than the first BTK inhibitor (e.g., ibrutinib)) to the subject. In some embodiments, the second BTK inhibitor is selected from one or more of the following: GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13, or combinations thereof.

[0068] In some embodiments, the disease associated with the expression of a B-cell antigen (e.g., CD19) is a blood cancer (e.g., the blood cancer described herein, such as CLL, MCL, or ALL), and the method delays or reduces resistance to a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), CAR-expressing cells (e.g., CAR19-expressing cells) administered to the subject, or both. In some embodiments, the disease associated with the expression of CD19 is a blood cancer (e.g., the blood cancer described herein, such as CLL, MCL, or ALL), and the method prolongs remission or delays relapse of the blood cancer. For example, remission may be prolonged, relapse may be delayed, resistance may be delayed, or resistance may be reduced compared to the expected course of treatment with a kinase inhibitor or CAR-expressing cells.

[0069] Exemplary treatment options that can be used in any of the foregoing methods include one or more of the following:

[0070] In one implementation, a kinase inhibitor and CAR-expressing cells (e.g., CAR19-expressing cells) are administered to a subject (e.g., a mammal) as a first-line therapy.

[0071] In another implementation, CAR-expressing cells (e.g., CAR19-expressing cells) are administered to a subject (e.g., a mammal) after administration of a kinase inhibitor.

[0072] In other embodiments, CAR-expressing cells (e.g., CAR19-expressing cells) are administered after the kinase inhibitor is discontinued.

[0073] In other embodiments, the administration of the kinase inhibitor begins before the administration of CAR19-expressing cells, and the administration of CAR19-expressing cells is combined with the continuous administration of the kinase inhibitor.

[0074] In one implementation, a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) is administered to the subject, for example, as a first-line treatment. At predetermined time intervals (e.g., 1 or 2 months, and 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), CAR-expressing cells (e.g., CAR19-expressing cells) are administered to the subject alone or in combination with the kinase inhibitor. In some implementations, the subject's response to treatment is assessed at predetermined time intervals (e.g., before or during treatment with the kinase inhibitor and / or CAR-expressing cells). If the assessment shows the subject is a complete responder, CAR-expressing cells (e.g., CAR19-expressing cells) are not administered. If the assessment shows the subject is a partial responder to the kinase inhibitor or has a stable condition responding to the kinase inhibitor, CAR-expressing cells (e.g., CAR19-expressing cells) are administered in combination with the kinase inhibitor, for example, as described herein. If the assessment shows that the subject is a non-responder or a relapsed subject, CAR-expressing cells (e.g., CAR19-expressing cells) are administered in combination with a kinase inhibitor or a second kinase inhibitor (e.g., a second kinase inhibitor as described herein).

[0075] In other implementations, the subject (e.g., a mammal) is or is identified as a complete or partial responder to a BTK inhibitor (e.g., ibrutinib) or to cells expressing CAR19.

[0076] In some embodiments, when a subject is (or is identified as) a complete responder to a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), CAR-expressing cells (e.g., CAR19-expressing cells) are not administered to the subject during the complete response time. In some embodiments, when a subject is (or is identified as) a complete responder to a kinase inhibitor (e.g., ibrutinib), CAR-expressing cells (e.g., CAR19-expressing cells) are administered to the subject during the complete response time. In one embodiment, following the administration of CAR-expressing cells (e.g., CAR19-expressing cells), the subject experiences prolonged response or delayed relapse (e.g., compared to the expected course of disease without CAR therapy).

[0077] In some embodiments, when a subject is (or is identified as) a partial responder to a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), CAR-expressing cells (e.g., CAR19-expressing cells) are not administered to the subject during the partial response period. In other embodiments, when a subject is (or is identified as) a partial responder to a kinase inhibitor, CAR-expressing cells (e.g., CAR19-expressing cells) are administered to the subject (alone or in combination with a BTK inhibitor) during the partial response period. In one embodiment, following CAR therapy, the subject achieves a complete response and / or prolonged response or delayed relapse (e.g., compared to the expected course of disease without CAR therapy).

[0078] In some embodiments, CAR therapy is not administered to subjects who have (or are identified as having) stable disease after treatment with a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) during the stable disease period. In other embodiments, CAR therapy is administered to subjects who have (or are identified as having) stable disease after treatment with a kinase inhibitor during the stable disease period. In one embodiment, after CAR therapy, a subject exhibits a partial response, a complete response, and / or prolonged response or delayed relapse (e.g., compared to the expected course of disease without CAR therapy).

[0079] In some embodiments, when a subject has (or is identified as having) progressive disease after treatment with a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib), CAR-expressing cells (e.g., CAR19-expressing cells) are not administered to the subject during the disease progression time. In other embodiments, when a subject has (or is identified as having) progressive disease after treatment with a kinase inhibitor, CAR-expressing cells (e.g., CAR19-expressing cells) are administered to the subject during the disease progression time. In one embodiment, following CAR therapy, the subject exhibits stable disease, partial response, complete response, and / or prolonged response or delayed relapse (e.g., compared to the expected course of disease without CAR therapy).

[0080] In other embodiments, CAR-expressing cells are administered in combination with a second kinase inhibitor, wherein the second kinase inhibitor is not ibrutinib when the mammal is or has been identified as a non-responder or relapsed individual to ibrutinib. The second kinase inhibitor may be selected from one or more of the following: GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13, or combinations thereof.

[0081] In other embodiments, the subject (e.g., a mammal) is (or is identified as) a partial responder to a kinase inhibitor, and during the partial response time, the subject is given CAR-expressing cells (e.g., cells expressing CAR19) alone or in combination with a BTK inhibitor.

[0082] In other embodiments, the subject (e.g., a mammal) is (or has been identified as) a non-responder with progressive or stable disease after treatment with ibrutinib, and during the period of disease progression or stability, the subject is administered CAR-expressing cells (e.g., CAR19-expressing cells) alone or in combination with a second BTK inhibitor, wherein the second kinase inhibitor is not ibrutinib.

[0083] In another aspect, this document provides a method for treating a subject (e.g., a mammal) with a disease associated with the expression of a B-cell antigen (e.g., CD19). The method comprises administering to the subject, in combination (e.g., simultaneously (or substantially simultaneously), or sequentially), an effective amount of a kinase inhibitor as described herein (e.g., a BTK kinase inhibitor as described herein, such as ibrutinib) and CAR-expressing cells (e.g., CAR19-expressing cells).

[0084] In some implementations, the kinase inhibitor and CAR-expressing cells (e.g., CAR19 cells) are administered in combination (e.g., as a first-line therapy).

[0085] In some implementations, the kinase inhibitor is initially administered, for example, as monotherapy or first-line therapy; after the amount of the kinase inhibitor is reduced (e.g., stopped or discontinued), CAR-expressing cells (e.g., CAR19-expressing cells) are administered to the subject.

[0086] In other implementations, the kinase inhibitor is initially administered, for example, as monotherapy or first-line therapy; and subsequently, a combination of the kinase inhibitor and CAR-expressing cells (e.g., CAR19-expressing cells) is administered to the subject.

[0087] In some implementations, a kinase inhibitor is initially administered, for example, as monotherapy or first-line therapy; after the amount of the kinase inhibitor is reduced (e.g., stopped or discontinued), a combination of a second kinase inhibitor and CAR-expressing cells (e.g., cells expressing CAR19) is subsequently administered to the subject.

[0088] In some implementations, a subject's response to treatment is assessed at predetermined time intervals (e.g., before or during treatment with a kinase inhibitor and / or CAR-expressing cells). If the assessment shows the subject is a complete responder, CAR-expressing cells (e.g., CAR19-expressing cells) are not administered. If the assessment shows the subject is a partial responder to a kinase inhibitor or has a stable condition that responds to a kinase inhibitor, CAR-expressing cells (e.g., CAR19-expressing cells) are administered in combination with a kinase inhibitor, for example, as described herein. If the assessment shows the subject is a non-responder or a relapsed patient, CAR-expressing cells (e.g., CAR19-expressing cells) are administered in combination with a kinase inhibitor or a second kinase inhibitor (e.g., a second kinase inhibitor as described herein).

[0089] In some implementations, the diseases associated with the expression of B-cell antigens (e.g., CD19) are blood cancers, leukemia, lymphoma, MCL, CLL, ALL, Hodgkin's lymphoma, or multiple myeloma.

[0090] In some embodiments, the kinase inhibitor is selected from the following BTK inhibitors: ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13; and the following CDK4 inhibitors: pabuxirib, aloisine A, flapindole, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromone; crizotinib (PF-02341066, P276-00, RAF265, indisulam, roscovitine, dinaciclib, BMS). 387032, MLN8054, AG-024322, AT7519, AZD5438, BMS908662; or ribociclib; selected from the following mTOR inhibitors: rapamycin, rapamycin analogs such as everolimus, tamsulolimus, desfolimex, semapimod, AZD8055, PF04691502, SF1126, XL765, or OSI-027; or selected from the following MNK inhibitors: CGP052088, CGP57380, causticon or ETC-1780445-2, or 4-amino-5-(4-fluoroaniline)-pyrazolo[3,4-d]pyrimidine.

[0091] In some aspects, the present invention characterizes a method for treating or providing antitumor immunity to a subject suffering from Hodgkin's lymphoma (e.g., a mammal). The method comprises administering, alone or in combination with a second therapy, an effective amount of cells expressing a CD19-binding CAR molecule to the subject.

[0092] In another aspect, the present invention characterizes a method for treating or providing antitumor immunity to a subject suffering from multiple myeloma (e.g., CD19-positive or CD19-negative multiple myeloma). In one embodiment, the multiple myeloma is CD19-negative, for example, the vast majority (99.95%) of the tumor plasma cells have a CD19-negative phenotype, detected by both flow cytometry and RT-PCR, for example. The method comprises administering, alone or in combination with a second therapy (e.g., standard of care for multiple myeloma), an effective amount of cells expressing a CD19-binding CAR molecule to the subject. The method may also comprise administering a kinase inhibitor as described herein.

[0093] In embodiments of methods related to Hodgkin's lymphoma or multiple myeloma, the CAR molecule is a humanized CAR molecule, such as that described herein. 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 the following (e.g., 2, 3, 4, 5, or all): LC CDR1 of SEQ ID NO:5, LC CDR2 of SEQ ID NO:26, and LC CDR3 of SEQ ID NO:27; HC CDR1 of SEQ ID NO:19, LC CDR2 of any one of SEQ ID NO:20-23, and HC CDR3 of SEQ ID NO:24.

[0094] In some embodiments of the method associated with Hodgkin's lymphoma or multiple myeloma, a CAR molecule (e.g., CART19 or CTL019) is administered as a monotherapy. In some embodiments, the method also includes administration of a kinase inhibitor, such as a BTK inhibitor (e.g., ibrutinib), a CDK4 inhibitor, an mTOR inhibitor, or an MNK inhibitor.

[0095] In some implementations of methods related to multiple myeloma, a CAR molecule (e.g., CART19 or CTL019) is administered in combination with standard care for multiple myeloma (e.g., in combination with myeloablative chemotherapy and / or autologous stem cell transplantation salvage therapy) (e.g., after administration of melphalan (e.g., high-dose melphalan)).

[0096] In another aspect, the invention is characterized by a composition comprising cells and one or more kinase inhibitors, wherein the cells express a CAR molecule that binds to one or more B cell antigens (e.g., CD19, CD20, CD22, or ROR1), and the kinase inhibitor is selected from Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase 4 (CDK4) inhibitors, mTOR inhibitors, or mitogen-activated protein kinase-interacting kinase (MNK) inhibitors. The CAR-expressing cells and one or more kinase inhibitors may be present in a single dose form or as two or more dose forms.

[0097] In the implementation scheme, the compositions disclosed herein are used as pharmaceuticals.

[0098] In embodiments, the compositions disclosed herein are used to treat diseases associated with the expression of B-cell antigens (e.g., CD19).

[0099] Methods and compositions for producing CAR-expressing cells

[0100] In some aspects, this disclosure also provides a method for preparing a population of immune effector cells (e.g., T cells or NK cells) that can be engineered to express a CAR (e.g., the CAR described herein), the method comprising: providing the population of immune effector cells; and contacting the immune effector cells with a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib) under conditions sufficient to inhibit the target of the kinase inhibitor (e.g., BTK and / or ITK). The method may further comprise contacting the immune effector cells with a nucleic acid encoding a CAR molecule, for example, transducing the immune effector cells with a nucleic acid encoding a CAR molecule.

[0101] In some aspects, this disclosure provides a method for preparing cells expressing CAR (e.g., immune effector cells or cell populations expressing CAR), the method comprising: contacting the cells or cell populations with a kinase inhibitor (e.g., a BTK inhibitor such as ibrutinib); and introducing (e.g., transducing) a nucleic acid encoding a CAR molecule into the cells or cell populations under such conditions, thereby expressing the CAR molecule.

[0102] In some embodiments of the method for producing cells expressing CAR, the CAR molecule encoded by the nucleic acid is a CD19-binding CAR molecule. In embodiments, the method further includes culturing cells or multiple cell populations under conditions that allow cells or at least a subpopulation of cells to express the CAR molecule. In embodiments, the cells are T cells or NK cells, or the cell population includes T cells, NK cells, or both. In embodiments, the method includes contacting cells or multiple cell populations with a kinase inhibitor (e.g., for 10-20, 20-30, 30-40, 40-60, or 60-120 minutes) and subsequently removing most or all of the kinase inhibitor from the cells or multiple cell populations. In embodiments, the kinase inhibitor is added after harvesting the cells or multiple cell populations or before stimulating the cells or multiple cell populations. 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 population also includes cancer cells, such as leukemia cells or lymphoma cells. Cancer cells may be, for example, CLL cells, MCL cells, or ALL cells. In one embodiment, the kinase inhibitor inhibits a target (e.g., BTK) in cancer cells, for example, reducing its activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In another embodiment, the kinase inhibitor inhibits a target (e.g., ITK) in immune effector cells, for example, reducing its activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.

[0103] In some aspects, this disclosure 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 further comprises a population of immune effector cells.

[0104] In some embodiments, one or more immune effector cells express a CAR molecule or contain nucleic acid encoding a CAR molecule. In some embodiments, the kinase inhibitor is selected from BTK inhibitors, CDK4 inhibitors, mTOR inhibitors, or MNK inhibitors. In some embodiments, 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. In some embodiments, the reaction mixture contains cancer cells, such as hematologic malignancies. The cancer cells may be, for example, cells harvested from the subject when harvesting immune effector cells from the subject.

[0105] In some aspects, this disclosure also provides a reaction mixture comprising a population of immune effector cells and a CAR molecule or a nucleic acid encoding a CAR molecule, wherein the immune effector cells comprise covalently inactivated ITK. In embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the ITK is covalently inactivated. In some embodiments, the reaction mixture further 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 the BTK is covalently inactivated. In embodiments, BTK or ITK forms a covalent bond with a small molecule such as ibrutinib at or near its ATP-binding domain. In embodiments, BTK forms a covalent bond with a small molecule such as ibrutinib at or near its cysteine-481 residue.

[0106] In embodiments, the reaction mixture as described herein further comprises buffers or other reagents, such as a solution containing PBS. In embodiments, the reaction mixture further comprises substances that activate and / or amplify cell populations, such as substances that stimulate CD3 / TCR complex-related signaling and / or ligands that stimulate co-stimulatory molecules on the cell surface. In embodiments, this substance is a bead conjugated to an anti-CD3 antibody or a fragment thereof and / or an anti-CD28 antibody or a fragment thereof. In embodiments, the reaction mixture further comprises one or more factors for proliferation and / or viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives for cell growth. In embodiments, the reaction mixture further comprises IL-15 and / or IL-7. In embodiments, the multiple cells of the population in the reaction mixture comprise (e.g., as described herein) nucleic acid molecules containing sequences encoding CARs (e.g., sequences encoding CD19 CARs), such as the nucleic acid molecules described herein. In one embodiment, multiple cells in a population of the reaction mixture comprise a vector containing a nucleic acid sequence encoding a CAR (e.g., the CAR described herein, such as the CD19 CAR described herein). In another embodiment, the vector is a vector described herein, for example, selected from DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, or retroviral vectors. In yet another embodiment, the reaction mixture also comprises a cryoprotectant or stabilizer, such as sugars, oligosaccharides, polysaccharides and polyols (e.g., trehalose, mannitol, sorbitol, lactose, sucrose, glucose, and dextran), salts, and crown ethers. In one embodiment, the cryoprotectant is dextran.

[0107] In some embodiments, the preparation methods disclosed herein further include contacting a population of immune effector cells with a nucleic acid encoding a telomerase subunit (e.g., hTERT). The nucleic acid encoding the telomerase subunit may be DNA.

[0108] In some embodiments, the preparation methods disclosed herein also include culturing a population of immune effector cells in serum containing 2% hAB serum.

[0109] Titles, subtitles, or numbered or alphabetically numbered elements, such as (a), (b), (i), etc., are displayed solely for ease of reading. The use of titles, numbered, or alphabetically numbered elements in this document does not require steps or elements to be in alphabetical order, nor does it require that the steps or elements be independent of each other.

[0110] All publications, patent applications, patents and other references mentioned in this article are fully incorporated by way of citation.

[0111] Other features, objects, and advantages of the invention will be apparent from this description and the accompanying drawings and from the claims. Brief description of the attached diagram

[0112] Figure 1A , Figure 1B and Figure 1C This is an illustration of cytotoxicity measured in ND317 (normal donor) T cells, which were transduced with mouse anti-CD19 CAR or the humanized anti-CD19 CAR of the present invention and, as shown in the figure... Figure 1A The control K562 cells shown do not express CD19 (K562cc), such as Figure 1B The K562 cells transformed with CD19 (K562.CD19) shown in the image, or as... Figure 1C The malignant B cells (Pt14B cell isolate) isolated from a CLL patient shown are cultured together.

[0113] Figure 2A and Figure 2B This is a diagram showing the proliferative response of cells expressing humanized and mouse anti-CD19 CAR to CD19+ cells, where a higher number of live CAR+ T cells correlates with a population showing the largest CD4+ and CD8+ T cell proliferation relative to primary CLL cells.

[0114] Figure 3 This is a diagram of the deconvolution HPLC mass spectrometry of scFv of the present invention, wherein the top row depicts untreated scFv and the bottom row depicts the associated deglycosylated scFv.

[0115] Figure 4 This is a diagram illustrating conformational stability as measured by differential scanning fluidymetry. Tf of mouse scFvm It is 57℃ (thick line). Compared with the parent mouse scFv, all humanized scFv variants showed higher T values ​​at approximately 70℃. m The residues introduced through humanization have a T value that increases by more than 10°C. m .

[0116] Figure 5 This is an illustration of CD19 CAR-transduced T cell proliferation, where CAR19 cells are targeted at (a) a chronic myeloid leukemia (“CML”) cell line that is CD19 negative and therefore used as a negative control; (b) recombinant K562 cells that are CD19 positive and therefore used as a positive control; or (c) Pt14B cells isolated from CLL patients and expressing CD19 on their cell surface.

[0117] Figure 6A and Figure 6B This is a schematic diagram of a representative CAR.

[0118] Figure 7 The progression of HALLX5447 primary ALL in NSG mice after treatment with CD19-transduced CAR T cells was described. The growth of primary human ALL cells in NSG mice treated with CD19-specific CAR T cells demonstrated control of disease progression. CD19 + The mean percentage of human ALL cells was an indicator of disease burden in the peripheral blood of NSG mice up to day 65 post-tumor implantation. Black circles: mice treated with 100 μL PBS via tail vein; red squares: mice treated with simulated transduced T cells; blue triangles: mice treated with mouse CD19 CAR-transduced T cells; and purple inverted triangles: mice treated with humanized CD19 CAR-transduced T cells. Significance was calculated by ANOVA; * indicates p < 0.01.

[0119] Figure 8 The expression of CD19 in the patient's tumor cells was described. CD138 + CD45 暗 Tumor cells are stained against CD19 (x-axis) and CD38 (y-axis). Approximately 1-2% of tumor cells express the CD19 antigen.

[0120] Figure 9 shows two illustrations of cell proliferation and cell size of CART19 cells when treated with increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM).

[0121] Figure 10A and Figure 10BFigure 10A shows a series of histograms illustrating the proliferation of CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. The histograms show the proliferation of CART19 cells stimulated with tumor cell lines MOLM14, JEKO-1, and RL in the presence or absence of progressively increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). Cells were stained with CFSE and analyzed by flow cytometry to determine the percentage of proliferating cells, indicated by the scale in each histogram. Figure 10B This is a quantitative analysis of the representative histogram in Figure 10A.

[0122] Figure 11A and Figure 11B Figure 11A shows a series of flow cytometry atlases illustrating CD107a degranulation in CART19 cells stimulated with MCL cell lines in the presence or absence of ibrutinib. CD107a expression was measured on the y-axis. The images show CD107a degranulation in CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of progressively increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). Figure 11B This is a quantification of the results from Figure 11A.

[0123] Figure 12 is a series of flow cytometry atlases showing the production of intracytoplasmic IL-2 in CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of progressively increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents IL-2 expression.

[0124] Figure 13 is a series of flow cytometry atlases showing intracytoplasmic TNF-α production in CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of progressively increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents TNF-α expression.

[0125] Figure 14 is a series of flow cytometry atlases showing the production of intracytoplasmic IFN-g in CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of progressively increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM). The y-axis represents IFN-g expression.

[0126] Figure 15 is a series of illustrations showing the secretion of cytokines from CART19 cells stimulated with tumor cell lines (MOLM14, JEKO-1, and RL) in the presence or absence of progressively increasing concentrations of ibrutinib (10 nM, 100 nM, and 1000 nM).

[0127] Figure 16A , Figure 16B , Figure 16C , Figure 16D , Figure 16E and Figure 16F This demonstrates that CART19, alone or in the presence of progressively increasing concentrations of ibrutinib, kills tumor cells, as does MOLM14 ( Figure 16A and Figure 16D ), JEKO Figure 16B and Figure 16E ) and RL( Figure 16C and Figure 16F The illustration shows untransduced cells (UTD) or CART19 cells incubated with tumor cells at different ratios and the total cell throughput was assessed. Figure 16A , Figure 16B and Figure 16C ) and percentage of dead cells ( Figure 16D , Figure 16E and Figure 16F ).

[0128] Figure 17A , Figure 17B and Figure 17C This is an illustration of CART19-mediated tumor cell killing, measured by flow cytometry after 24 hours, using the total cell count. The tumor cell line MOLM14 ( Figure 17A ), JEKO Figure 17B ) and RL( Figure 17C Incubate with untransduced cells (UTD) or CART19 cells alone (ALONE) or in combination with different concentrations of ibrutinib.

[0129] Figure 18A , Figure 18B , Figure 18C and Figure 18D This is an illustration of CART19 dose exploration in an RL MCL mouse model. Tumor burden was monitored over time using bioluminescence imaging (BLI). Figure 18A and Figure 18B Monitoring overall survival over time ( ). Figure 18C ).

[0130] Figure 19A and Figure 19B This is an illustration of CART19 dosage exploration in the JEKO-1MCL mouse model. Tumor burden was monitored over time using bioluminescence imaging (BLI). Figure 19A Furthermore, the total survival time was monitored over time. Figure 19B ).

[0131] Figure 20 This is a schematic diagram showing the regimen for administering and evaluating the combination therapy of CART19 and ibrutinib in an in vivo mouse model.

[0132] Figure 21A , Figure 21B , Figure 21C and Figure 21D This is a graphic illustration showing the transduction efficiency of PBMCs used to generate CART19 T cells when cells were treated with ibrutinib before transduction. Analysis of untreated PBMCs before transduction ( Figure 21A ) and transduction ( Figure 21C CAR19 expression in PBMCs treated with ibrutinib before transduction. Figure 21B ) and transduction ( Figure 21D CAR19 expression.

[0133] Figure 22A , Figure 22B , Figure 22C , Figure 22D and Figure 22E This is a diagram illustrating the effect of ibrutinib treatment on CD3 / CD28-stimulated T cell proliferation. The effects of escalating concentrations of ibrutinib were evaluated: untreated ( Figure 22A ); 0.1 μM ibrutinib ( Figure 22B ); 0.5 μM ibrutinib ( Figure 22C ), 1μM ibrutinib ( Figure 22D ) and 5 μM ibrutinib ( Figure 22E ).

[0134] Figure 23 This is a diagram showing that ibrutinib does not affect CART19 cytotoxicity.

[0135] Figure 24 This shows that ibrutinib treatment does not promote T cells in CART19 cells. H 1 / T H 2. A series of illustrations showing cytokine skewness.

[0136] Figure 25A and Figure 25B This is a diagram showing that continuous administration of ibrutinib does not affect CART19 function when clearing tumor cells in the body. Figure 25A The number of Nalm / 6 cells detected in peripheral blood during each treatment regimen is shown. Figure 25B Kaplan-Meier survival curves show the survival of mice receiving CART19 with or without ibrutinib administration.

[0137] Figure 26A , Figure 26B , Figure 26C , Figure 26D , Figure 26E and Figure 26F This is a graph showing the CAR19 transduction efficiency in CLL patient cells at the indicated time points during ibrutinib treatment. Cells not transduced ( Figure 26A , Figure 26B and Figure 26C ) or use CAR19 transduction ( Figure 26D , Figure 26E and Figure 26F Cells stained with GAM expressed CAR19 and were present in each box in each atlas.

[0138] Figure 27 is a series of illustrations depicting the proliferation rate of untransduced cells compared to CAR19-transduced cells at the time points shown during ibrutinib treatment in a group of patients.

[0139] Figure 28A , Figure 28B and Figure 28C This is a diagram showing the lymphocytosis induced by ibrutinib treatment in a CLL patient. Cells from this patient were separated at the time points shown: baseline ( Figure 28A ); Day 1 of the second cycle ( Figure 28B ); and Day 1 of the 12th cycle ( Figure 28C ).

[0140] Figure 29A , Figure 29B and Figure 29C This is an illustration showing the reduction of CD200 expression on tumor cells in CLL patients over time with ibrutinib treatment. Each atlas contains a superposition of histograms of CD200 expression from cells separated at the indicated time points: baseline (screening); day 1 of cycle 2; and day 1 of cycle 12.

[0141] Figure 30A , Figure 30B and Figure 30C This shows that ibrutinib treatment reduces the frequency of PD1+ T cells over time in CLL patients. Cells from this patient were separated at the indicated time points: baseline ( Figure 30A ); Day 1 of the second cycle ( Figure 30B ); and Day 1 of the 12th cycle ( Figure 30C ).

[0142] Figure 31A and Figure 31B It shows the MCL cell line RL ( Figure 31A ) and JEKO-1( Figure 31B A graph illustrating the sensitivity to ibrutinib treatment.

[0143] Figure 32 This is a diagram showing the effect of ibrutinib treatment in an in vivo model of MCL.

[0144] Figure 33A and Figure 33BThese are immunohistochemical images of Hodgkin's lymphoma, showing CD19-expressing cells present in the tumor. Figure 33A 1x magnification and Figure 33B It has a magnification of 20x.

[0145] Figure 34 This is a schematic diagram of the experimental setup for a study evaluating the efficacy of CART19 therapy in patients with Hodgkin's lymphoma.

[0146] Figure 35A , Figure 35B , Figure 35C and Figure 35D Flow cytometry analysis showing PD1 and CAR19 expression on T cells. Figure 35A and Figure 35B It is a representative flow cytometry atlas showing the distribution of PD-1 and CAR19 expression on CD4+ T cells from subjects who are either complete responders (CR) or non-responders (NR) of CAR-T therapy. Figure 35C This is a graph showing the percentage of PD1 cells in a CD4+ T cell population derived from multiple groups of subjects with varying responses to CAR-T therapy. Figure 35D This is a graph showing the percentage of PD1 cells in a CD8+ T cell population derived from multiple groups of subjects with varying responses to CAR-T therapy.

[0147] Figure 36A and Figure 36B The distribution of PD1 expression in cells expressing CD4 and CAR19 is shown. Figure 36A ) or distribution in cells expressing CD8 and CAR19 ( Figure 36B The cells were derived from multiple groups of subjects who responded differently to CART therapy.

[0148] Figure 37 Flow cytometry analysis of PD1, CAR19, LAG3 and TIM3 expression on T cells from subjects who were either complete responders (CR) or non-responders (NR) of CAR-T therapy is shown.

[0149] Figure 38A and Figure 38B The expression of PD1 and LAG3 was shown in multiple groups of subjects with different responses to CART therapy. Figure 38A ) or PD1 and TIM3 expression ( Figure 38B The distribution of ).

[0150] Figure 39 This shows the plasma cell IgA immunophenotype classification from myeloma patients who received CART19 and showed a response to CART19 therapy.

[0151] Figure 40A and Figure 40B This is a graph showing the increase in influenza vaccine titers compared to a placebo. Figure 40A In the study, the increase at 4 weeks post-vaccination relative to the placebo group, for each RAD001-dosed group in the intention-to-treat population, showed an increase in the geometric mean influenza titer above baseline against each of the three influenza vaccine strains (H1N1A / California / 07 / 2009, H3N2A / Victoria / 210 / 2009, and B / Brisbane / 60 / 2008). A thick black line indicates a 1.2-fold increase in titer relative to placebo, which is required to achieve the primary endpoint of this study for two of the three influenza vaccine strains. An asterisk “*” indicates that the increase in GMT titer relative to placebo has a posterior probability greater than 1 with at least 80% confidence. Figure 40B It is the subset of subjects with a baseline influenza titer <= 1:40 and Figure 40A A diagram showing the same data.

[0152] Figure 41 A scatter plot showing the fold increase in RAD001 concentration relative to the geometric mean titer of each influenza vaccine strain at 4 weeks post-vaccination. RAD001 concentrations were measured 1 hour after administration 4 weeks post-vaccination. All subjects with pharmacokinetic values ​​were included in the analysis set. The fold increase in geometric mean titer relative to baseline at 4 weeks post-vaccination is shown on the y-axis.

[0153] Figure 42 This is a graph showing the increase in titers against heterologous influenza strains compared to placebo. The increase at 4 weeks post-vaccination compared to the placebo group, and for each RAD001-dosed group in the intention-to-treat population, showed an increase in the geometrically mean influenza titer above baseline against two heterologous influenza strains not included in the influenza vaccine (A / H1N1 strain A / New Jersey / 8 / 76 and A / H3N2 strain A / Victoria / 361 / 11). * indicates that the titer increase relative to placebo has a posterior probability greater than 1 with at least 80% probability.

[0154] Figure 43A and Figure 43B This is a graph showing IgG and IgM levels before and after influenza vaccination. Serum levels of anti-A / H1N1 / California / 07 / 2009 influenza IgG and IgM were measured from subjects before and 4 weeks after influenza vaccination. No significant differences in changes from baseline in anti-H1N1 influenza IgG and IgM levels were detected between the RAD001 group and the placebo group up to 4 weeks post-vaccination (all p-values ​​> 0.05 according to the Kruskal-Wallis rank-sum test).

[0155] Figure 44A , Figure 44B and Figure 44C This graph illustrates the percentage decrease in PD-1 positive CD4 and CD8 T cells and the increase in PD-1 negative CD4 T cells after RAD001 treatment. The percentages of PD-1 positive CD4, CD8 T cells, and PD-1 negative CD4 T cells were determined by FACS analysis of PBMC samples at baseline, 6 weeks after study drug treatment (week 6), 6 weeks after study drug discontinuation, and 4 weeks after influenza vaccination (week 12). Figure 44A The study showed a significant reduction in PD-1 positive CD4 T cells (-37.1%–-28.5%) at week 12 in the groups receiving RAD001 at dose levels of 0.5 mg / day (n=25), 5 mg / week (n=29), and 20 mg / week (n=30), respectively, at p=0.002 (0.02), p=0.003 (q=0.03), and p=0.01 (q=0.05), compared to the placebo group (n=25). Figure 44B The study showed a significant reduction (-43.3%–-38.5%) of PD-1 positive CD8 T cells at week 12 in the groups receiving RAD001 at dose levels of 0.5 mg / day (n=25), 5 mg / week (n=29), and 20 mg / week (n=30), respectively, compared to the placebo group (n=25). Figure 44C The study showed a significant increase (3.0%–4.9%) of PD-1 negative CD4 T cells at week 12 in the groups receiving RAD001 at dose levels of 0.5 mg / day (n=25), 5 mg / week (n=29), and 20 mg / week (n=30), respectively, compared to the placebo group (n=25).

[0156] Figure 45A and Figure 45B This is a graph showing, adjusted for baseline PD-1 expression differences, as a percentage of the decrease in PD-1-positive CD4 and CD8 T cells and the increase in PD-1-negative CD4 T cells after RAD001 treatment. The percentages of PD-1-positive CD4 and CD8 T cells and PD-1-negative CD4 T cells were determined by FACS analysis of PBMC samples at baseline, 6 weeks after study drug treatment (week 6), 6 weeks after study drug discontinuation, and 4 weeks after influenza vaccination (week 12). Figure 45ACompared with the placebo group (n=25), the number of PD-1+CD4 T cells in the pooled RAD group (n=84) at week 6 was significantly reduced by 30.2% at p=0.03 (q=0.13). Compared with the placebo group, the number of PD-1 positive CD4 T cells in the pooled RAD group at week 12 was reduced by 32.7% at p=0.05 (q=0.19). Figure 45B Compared with the placebo group (n=25), the pooled RAD001 group (n=84) showed a significant reduction of 37.4% in PD-1 positive CD8 T cells at week 6 (p=0.008, q=0.07). Compared with the placebo group, the pooled RAD001 group showed a 41.4% decrease in PD-1 positive CD8 T cells at week 12 (p=0.066, q=0.21). Figure 45A and Figure 45B represent Figure 44A , Figure 44B and Figure 44C The data in the middle, but Figure 44A , Figure 44B and Figure 44C The different RAD001 dosage groups were summarized into Figure 45A and Figure 45B The single RAD001 treatment group.

[0157] Figure 46 The study described increased exercise and activity in elderly subjects in response to RAD001.

[0158] Figure 47A and Figure 47B The predictive effect of RAD001 on P70 S6K activity in cells was described. Figure 47A The inhibitory effect of P70 S6 kinase was described when using higher weekly and daily doses of RAD001; Figure 47B The inhibitory effect of P70 S6 kinase when using a low weekly dose of RAD001 is described.

[0159] Figure 48A and Figure 48B The expression of the IL-7 receptor (CD127) was shown in cancer cell lines and CAR-T cells. CD127 expression in three cancer cell lines was analyzed by flow cytometry: RL (mantle cell lymphoma), JEKO (also known as Jeko-1, mantle cell lymphoma), and Nalm-6 (B-ALL). Figure 48A CD127 expression on CD3-positive (CART) cells infused and circulating in NSG mice was measured by flow cytometry. Figure 48B ).

[0160] Figure 49A , Figure 49B and Figure 49C This study demonstrates the antitumor response following CART19 treatment and subsequent IL-7 treatment. On day 6, NSG mice implanted with the mantle lymphoma cell line expressing luciferase on day 0 were treated with different doses of CART19 cells, and tumor burden was monitored. Mice were divided into four groups: those that did not receive CART19 cells and those that received 0.5 x 10^6 cells. 6 1 CART19 cell (CART19 0.5E6), 1x10 6 1 CART19 cell (CART19 1E6) or 2 x 10 6 1,000 CART19 cells (CART19 2E6). Tumor burden after CART treatment was measured by detecting bioluminescence (mean BLI). Figure 49A Accept 0.5x10 6 1 CART19 cell (CART19 0.5E6) or 1x10 6 Mice with one CART19 cell line (CART19 1E6) were randomly assigned to receive or not receive recombinant human IL-7 (rhIL-7). Figure 49A Three mice (#3827, #3829, and #3815, receiving the initial CART19 dose shown) were used to monitor tumor burden, represented by mean bioluminescence (BLI), in this study. These mice were treated with IL-7 starting on day 85. Figure 49B IL-7 was administered via intraperitoneal injection three times weekly. Tumor burden, represented by mean bioluminescence (BLI), was compared between mice that did not receive IL-7 (control) and mice that received IL-7 treatment (IL-7) before day 85 (PRE) and after day 115 (POST). Figure 49C ).

[0161] Figure 50A and Figure 50B This study visualized T cell kinetics after IL-7 treatment. The levels of human T cells in the blood of each mouse receiving IL-7 or control mouse were monitored. Figure 50A The levels of CART19 cells (CD3+ cells) detected in the blood were measured before initiating IL-7 treatment (PRE) and 14 days thereafter (Day 14). Figure 50B ).

[0162] Figure 51The structures of two exemplary RCAR layouts are described. The antigen-binding member includes an antigen-binding domain, a transmembrane domain, and a switching domain. The intracellular binding member includes a switching domain, a co-stimulatory signaling domain, and a primary signaling domain. These two layouts demonstrate that the first and second switching domains described herein can be oriented differently relative to the antigen-binding member and the intracellular binding member. Other RCAR layouts are further described herein.

[0163] Figure 52A This is an image of the RL cell line.

[0164] Figure 52B These are a set of flow cytometry scatter plots showing the expression of CD19 and CD5 in primary RL cells and RL cell lines.

[0165] Figure 52C This is an image showing the t(11;14) translocation via fluorescence in situ hybridization (FISH).

[0166] Figure 52D This demonstrates that ibrutinib inhibits IC50 in different cell lines. 50 A chart showing the percentage conversion based on MTT.

[0167] Figure 52E This is a set of images and illustrations showing the implantation of RL cells in NOD-SCID-γ chain knockout (NSG) mice and the resulting tumor burden.

[0168] Figure 52F These are a set of histological images showing MCL cells localized to multiple organs in mice.

[0169] Figure 52G These are a set of histological images of mice that have been injected with MCL-RL cells.

[0170] Figure 53A This is a set of illustrations showing the number of CD107a+CART19 cells when exposed to various MCL cell lines.

[0171] Figure 53B This is a set of illustrations showing the amounts of IL-2 and TNF-α produced by CART19 cells when exposed to various MCL cell lines.

[0172] Figure 53C This is a graph showing the percentage of CART19 cells killing various MCL cell lines at different effector:target cell ratios.

[0173] Figure 53D This is a graph showing the amount (proliferation measure) of carboxyfluorescein succinimide ester (CFSE) in CART19 cells exposed to various MCL cell lines.

[0174] Figure 53E It is a set of graphs showing the percentage of T cells before and after expansion.

[0175] Figure 53F It is a set of graphs showing the percentage of untransduced T cells or CAR-19 transduced T cells that express or produce multiple biomolecules (e.g., cytokines).

[0176] Figure 54A This is a set of images showing the activation of interleukin-2-induced T-cell kinase (ITK) in CART19 cells when specifically or non-specifically stimulated.

[0177] Figure 54B This is a set of illustrations showing CD107a surface expression (a measure of degranulation), IL-2 production, and TNF-α production in CART19 cells when using various concentrations of ibrutinib.

[0178] Figure 54C This is a set of histograms showing the amount of CFSE in CART19 cells exposed to various MCL cell lines along with ibrutinib at multiple concentrations.

[0179] Figure 54D is a set of illustrations showing the expression or production of various cytokines and biomarkers as indicators of the Th1 or Th2 state of CART19 cells when combined with different concentrations of ibrutinib.

[0180] Figure 54E This is a set of graphs showing the percentage of various MCL cell lines killed by CART19 cells when combined with different concentrations of ibrutinib.

[0181] Figure 54F This is a histogram showing the expression of multiple markers of the intrinsic cytotoxic function of CART19 cells when combined with various concentrations of ibrutinib.

[0182] Figure 55 This is a schematic diagram of an in vivo mouse model experimental design established to test the effects of CART19 and / or ibrutinib on mice injected with MCL-RL. The reading is luminescence (a measure of the number of tumor cells).

[0183] Figure 56 This is a schematic diagram of an in vivo mouse model experimental design established to test the effects of CART19 and / or ibrutinib on mice injected with MCL-RL. The reading is luminescence (a measure of the number of tumor cells).

[0184] Figure 57 This is a set of illustrations showing luminescence (a measure of the number of tumor cells) and overall survival after treatment in mice treated with different concentrations of ibrutinib.

[0185] Figure 58 This is a set of illustrations showing luminescence (a measure of the number of tumor cells) and overall survival after treatment in mice treated with ibrutinib or CART19 cells.

[0186] Figure 59 This is a diagram showing luminescence (a measure of tumor cell number) in mice treated with ibrutinib, untransduced T cells, ibrutinib combined with untransduced T cells, CART19 cells, and CART19 cells combined with ibrutinib.

[0187] Figure 60 This is a diagram showing luminescence (a measure of the number of tumor cells) in mice treated with ibrutinib alone, CART19 cells alone, or a combination of ibrutinib and CART19 cells.

[0188] Figure 61A This is a set of illustrations showing the levels of Th1 cytokines produced in mice treated with ibrutinib and / or CART19 cells. Figure 61B This is a set of illustrations showing the levels of Th2 cytokines produced in mice treated with ibrutinib and / or CART19 cells.

[0189] Figure 61C This is a graph showing the percentage of cells expressing the proliferation marker Ki67 in mice treated with CART19 cells or CART19 cells plus ibrutinib.

[0190] Figure 61D This is a graph showing the percentage of cells expressing the anti-apoptotic marker BCL-2 in mice treated with CART19 cells or CART19 cells plus ibrutinib. Invention Details

[0191] definition

[0192] Unless otherwise specified, all terms and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to this invention.

[0193] The terms “a” and “an” refer to one or more grammatical objects of the article (i.e., at least one). For example, “an element” means one or more elements.

[0194] When referring to measurable values ​​such as quantity, duration of time, etc., the term "about" is intended to cover variations of ±20% from the specified value, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1%, because such variations are suitable for carrying out the disclosed method.

[0195] The term "chimeric antigen receptor" or alternatively "CAR" refers to a group of peptides, typically two peptides in the simplest embodiment, which, in immune effector cells, provide the cell with specificity for a target cell (typically a cancer cell) and facilitate intracellular signaling. In some embodiments, the CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain"), the cytoplasmic signaling domain comprising a functional signaling domain derived from a stimulatory molecule and / or a co-stimulatory molecule as defined below. In some aspects, the group of peptides is adjacent to each other, for example, in the same polypeptide chain (e.g., including chimeric fusion proteins). In some embodiments, the group of peptides is not adjacent to each other, for example, in different polypeptide chains. In some embodiments, the group of peptides includes a dimerization switch, which, in the presence of a dimerizing molecule, can couple the peptides to each other, for example, couple the antigen-binding domain to the intracellular signaling domain. In one aspect, the stimulatory molecule is a ζ-chain associated with a T-cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In one aspect, the co-stimulatory molecule is selected from those described herein, such as 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, the intracellular signaling domain comprising a functional signaling domain derived from the 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, the intracellular signaling domain comprising both a functional signaling domain derived from the co-stimulatory molecule and a functional signaling domain derived from the stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain comprises two functional signal transduction domains derived from one or more co-stimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule. In another aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain comprises at least two functional signal transduction domains derived from one or more co-stimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule. In another aspect, the CAR includes an optional leader sequence at the N-terminus (N-ter) of the CAR fusion protein.In one aspect, the CAR also includes 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., scFv) during cellular processing and CAR localization to the cell membrane.

[0196] The term "signal transduction domain" refers to the functional portion of a protein that plays a role by transmitting information within the cell, either by generating a second messenger or by acting as an effector in response to such a messenger, thereby regulating cellular activity through defined signal transduction pathways.

[0197] As used herein, the term "CD19" refers to the differentiation antigen cluster 19 protein, which is a detectable antigenic determinant on leukemia progenitor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and SwissProt. For example, the amino acid sequence of human CD19 can be found as UniProt / SwissProt accession number P15391, and the nucleotide sequence encoding human CD19 can be found as accession number NM_001178098. As used herein, "CD19" includes proteins containing mutations (e.g., point mutations, fragments, insertions, deletions, and splicing variants of full-length wild-type CD19). CD19 is expressed in most B-cell carcinomas, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Other cell types expressing CD19 are provided below in the definition of "Diseases Associated with CD19 Expression." It is also an early marker of B-cell progenitor cells. See, for example, Nicholson et al., Mol. Immun. 34(16-17):1157-1165 (1997). In one respect, the antigen-binding portion of CAR-T recognizes and binds to antigens located within the extracellular domain of the CD19 protein. In another respect, the CD19 protein is expressed on cancer cells.

[0198] As used herein, the term "CD20" refers to an antigenic determinant known to be detectable on B cells. Human CD20 is also known as transmembrane 4-domain subfamily A member 1 (MS4A1). Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and SwissProt. For example, the amino acid sequence of human CD20 can be found under accession numbers NP_690605.1 and NP_068769.2, and the nucleotide sequences encoding transcript variants 1 and 3 of human CD20 can be found under accession numbers NM_152866.2 and NM_021950.3, respectively. In one aspect, the antigen-binding portion of the CAR recognizes and binds to antigens located within the extracellular domain of the CD20 protein. In another aspect, the CD20 protein is expressed on cancer cells.

[0199] As used herein, the term "CD22" refers to an antigenic determinant known to be detectable on leukemia precursor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and SwissProt. For example, the amino acid sequences of human CD22 isoforms 1-5 can be found under accession numbers NP001762.2, NP001172028.1, NP001172029.1, NP001172030.1, and NP001265346.1, respectively, and the nucleotide sequences encoding variants 1-5 of human CD22 can be found under accession numbers NM001771.3, NM001185099.1, NM001185100.1, NM001185101.1, and NM001278417.1, respectively. In one respect, the antigen-binding portion of the CAR recognizes and binds to antigens located within the extracellular domain of the CD22 protein. In another respect, the CD22 protein is expressed on cancer cells.

[0200] As used herein, the term "ROR1" refers to an antigenic determinant known to be detectable on leukemia precursor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and SwissProt. For example, the amino acid sequences of human ROR1 isoforms 1 and 2 precursors can be found under accession numbers NP_005003.2 and NP_001077061.1, respectively, and the mRNA sequences encoding them can be found under accession numbers NM_005012.3 and NM_001083592.1, respectively. In one aspect, the antigen-binding portion of the CAR recognizes and binds to antigens located within the extracellular domain of the ROR1 protein. In another aspect, the ROR1 protein is expressed on cancer cells.

[0201] As used herein, the term "antibody" refers to a protein that specifically binds to an antigen, or a polypeptide sequence derived from an antigen-specific immunoglobulin molecule. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[0202] The term "antibody fragment" refers to at least one portion of an antibody that retains the ability to interact specifically with an epitope of an antigen (e.g., through binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of a VH domain and a CH1 domain, linear antibodies, single-domain antibodies such as sdAb (VL or VH), camel-like VHH domains, multispecific antibodies formed from antibody fragments such as bivalent fragments containing two Fab fragments linked by disulfide bonds in a hinge region, and isolated CDR or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, macrobodies, microbodies, nanobodies, intracellular antibodies, bispecific antibodies, tripoisome antibodies, tetraspecific antibodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23:1126-1136, 2005). Antigen-binding fragments can also be grafted into peptide-based scaffolds such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin peptide microbodies).

[0203] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are sequentially linked, for example by means of a synthetic linker, such as a flexible short peptide linker, and are capable of being expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, as used herein, the scFv may have VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

[0204] The CAR of the present invention, comprising an antibody or an antibody fragment thereof, can exist in various forms, wherein the antigen-binding domain is expressed as a portion of an adjacent polypeptide chain, such as including single-domain antibody fragments (sdAbs), single-chain antibodies (scFvs), humanized antibodies, or bispecific antibodies (Harlow et al., 1999, cited in: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, cited 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 the CAR composition of the present invention comprises an antibody fragment. In yet another aspect, the CAR comprises an antibody fragment comprising scFv. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those or combinations thereof described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest”, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme).

[0205] As used herein, the term "binding domain" or "antibody molecule" refers to a protein containing at least one immunoglobulin variable domain sequence, such as an immunoglobulin chain or fragment thereof. The term "binding domain" or "antibody molecule" encompasses both antibodies and antibody fragments. In one embodiment, the antibody molecule is a multispecific antibody molecule, for example, it contains multiple immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the multiple immunoglobulin variable domain sequences has binding specificity against a first epitope and a second immunoglobulin variable domain sequence of the multiple immunoglobulin variable domain sequences has binding specificity against a second epitope. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody is specific to no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity against a first epitope and a second immunoglobulin variable domain sequence having binding specificity against a second epitope.

[0206] The CAR of the present invention, comprising an antibody or an antibody fragment thereof, can exist in various forms, wherein the antigen-binding domain is expressed as a portion of an adjacent polypeptide chain, such as including single-domain antibody fragments (sdAbs), single-chain antibodies (scFvs), humanized antibodies, or bispecific antibodies (Harlow et al., 1999, cited in: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, cited 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 the CAR composition of the present invention comprises an antibody fragment. In yet another aspect, the CAR comprises an antibody fragment comprising scFv.

[0207] The term "antibody heavy chain" refers to the larger of two types of polypeptide chains present in an antibody molecule in their natural conformation, which normally determines the class of antibody.

[0208] The term "antibody light chain" refers to the smaller of two types of polypeptide chains present in an antibody molecule in their native conformation. The κ (κ) light chain and the λ (λ) light chain refer to the two main isotypes of antibody light chains.

[0209] The term "recombinant antibody" refers to an antibody generated using recombinant DNA technology, such as an antibody expressed via a phage or yeast expression system. This term should also be interpreted as referring to an antibody produced by synthesizing a DNA molecule encoding and expressing an antibody protein, or by synthesizing a prescribed amino acid sequence for an antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequencing technologies available and well-known in the art.

[0210] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immune-active cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can act as an antigen. Additionally, antigens can be derived from recombinant DNA or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or a portion of a protein encoding a protein therefore encodes an "antigen" as used herein, wherein the protein elicits an immune response. Furthermore, those skilled in the art will understand that an antigen need not be entirely encoded by the full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Furthermore, those skilled in the art will understand that an antigen does not necessarily need to be encoded by a "gene." It will be readily apparent that antigens can be generated, synthesized, or derived from biological samples, or may be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or fluids containing other biological components.

[0211] The term "anticancer effect" refers to biological effects that can be demonstrated through a variety of means, including but not limited to, reductions in tumor volume, number of cancer cells, number of metastases, life expectancy, cancer cell proliferation, cancer cell survival, or improvement in various physiological symptoms associated with cancer. "Anticancer effect" can also be demonstrated through the ability of peptides, polynucleotides, cells, and antibodies to prevent cancer from appearing at its initial site. The term "antitumor effect" refers to biological effects that can be demonstrated through a variety of means, including but not limited to, reductions in tumor volume, number of tumor cells, tumor cell proliferation, or tumor cell survival.

[0212] The term "self" refers to any substance that is derived from the same individual to which the substance will later be reintroduced.

[0213] The term "allelic" refers to any substance derived from a different animal of the same species as the individual from which the substance was introduced. Two or more individuals are alleged to be allelopaths of each other when the genes at one or more loci are not identical. In some respects, allelopathic substances from individuals of the same species can be genetically significantly dissimilar to allow for antigenic interactions.

[0214] The term "heterogeneous" refers to a transplant derived from an animal of a different species.

[0215] The term "cancer" refers to a disease characterized by the uncontrolled growth of abnormal 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 in this article, and these 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, etc. The terms "tumor" and "cancer" are used interchangeably in this article; for example, both terms cover solid tumors and fluid-filled tumors, such as diffuse or circulating tumors. As used herein, the terms "cancer" or "tumor" include both pre-malignant and malignant cancers and tumors.

[0216] The phrase “diseases associated with CD19 expression” includes, but is not limited to, diseases associated with CD19 expression or conditions associated with cells that express or have expressed CD19 at any time, such as proliferative disorders like cancer or malignancies or precancerous conditions like spinal dysplasia, myelodysplastic syndromes, or preleukemia; or non-cancer-related indications associated with CD19-expressing cells. For the avoidance of doubt, diseases associated with CD19 expression can include conditions associated with cells that do not currently express CD19, for example, because CD19 expression has been downregulated, for example, due to treatment with a CD19-targeting molecule (e.g., CD19 CAR), but which previously expressed CD19. In one aspect, cancers associated with CD19 expression are blood cancers. In another aspect, blood cancers are leukemia or lymphoma. In one aspect, cancers associated with CD19 expression include 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), and acute lymphoblastic leukemia (ALL); and one or more chronic leukemias, including but not limited to, for example, chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). Other cancers or hematologic disorders associated with CD19 expression include, but are not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, spinal dysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom's macroglobulinemia, and "preleukemia," which is a diverse collection of hematologic disorders unified by ineffective production (or dysplasia) of myeloid hematologic cells. Other diseases associated with CD19 expression include, but are not limited to, atypical and / or nonclassical cancers, malignancies, precancerous lesions, or proliferative disorders associated with CD19 expression. Non-cancer-related indications associated with CD19 expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory diseases (allergies and asthma), and transplantation. In some embodiments, cells expressing tumor antigens express or overexpress mRNA encoding tumor antigens at any time. In one embodiment, cells expressing tumor antigens produce tumor antigen proteins (e.g., wild-type or mutant), and the tumor antigen proteins may be present at normal or reduced levels. In one embodiment, cells expressing tumor antigens produce detectable levels of tumor antigen proteins at one point in time and subsequently produce substantially no detectable tumor antigen proteins.

[0217] The phrase “diseases associated with B-cell antigen expression” includes, but is not limited to, diseases associated with one or more of CD19, CD20, CD22, or ROR1, or conditions associated with cells that express or have expressed one or more of CD19, CD20, CD22, or ROR1 at any time. Examples include proliferative disorders such as cancer or malignancies, or precancerous conditions such as myelodysplastic syndromes, myelodysplastic syndromes, or preleukemia; or non-cancer-related indications associated with cells that express one or more of CD19, CD20, CD22, or ROR1. For the avoidance of doubt, diseases associated with B-cell antigen expression can include conditions associated with cells that do not currently express B-cell antigens, for example, because the expression of that antigen has been downregulated, for example, due to treatment with a molecule targeting the B-cell antigen (e.g., a B-cell-targeting CAR), but which previously expressed that antigen. The phrase “diseases associated with B-cell antigen expression” includes diseases associated with CD19 expression as described herein.

[0218] The term "conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing an amino acid sequence. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies or antibody fragments of the present invention using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues within the CAR of this invention can be replaced with other amino acid residues from the same side chain family, and the modified CAR can be tested using the functional assays described herein.

[0219] The term "stimulation" refers to a primary response induced by the binding of a stimulating molecule (e.g., a TCR / CD3 complex or CAR) to its corresponding ligand (or, in the case of CAR, a tumor antigen), which thus mediates signal transduction events, such as, but not limited to, signal transduction via the TCR / CD3 complex or via the signal transduction domains of a suitable NK receptor or CAR. Stimulation can mediate changes in the expression of certain molecules.

[0220] The term "stimulatory molecule" refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that provide cytoplasmic signaling sequences that stimulatorily regulate immune cell activation relative to at least some aspects of immune cell signaling pathways. In one aspect, the signal is a primary signal, which is triggered, for example, by the binding of a TCR / CD3 complex to a peptide-carrying MHC molecule and leads to a T cell response, including but not limited to proliferation, activation, and differentiation. Primary cytoplasmic signaling sequences that function stimulatorily (also referred to as "primary signaling domains") may contain signaling motifs called tyrosine-based activation motifs or ITAMs. Examples of ITAMs containing cytoplasmic signaling sequences particularly useful in this invention include, but are not limited to, those derived from CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. In a specific CAR of the present invention, the intracellular signal transduction domain of any one or more CARs of the present invention comprises an intracellular signal transduction sequence, for example, a primary signal transduction sequence of CD3ζ. In a specific CAR of the present invention, the primary signal transduction sequence of CD3ζ is the sequence provided in SEQ ID NO:17 or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In a specific CAR of the present invention, the primary signal transduction sequence of CD3ζ is the sequence provided in SEQ ID NO:43 or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

[0221] The term "antigen-presenting cell" or "APC" refers to immune system cells, such as accessory cells (e.g., B cells, dendritic cells, etc.), that present foreign antigens complexed with the major histocompatibility complex (MHC) on their surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0222] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. Intracellular signaling domains generate signals that promote immune effector functions in CAR-containing cells (e.g., CAR-T cells). Examples of immune effector functions (e.g., in CAR-T cells) include cytolytic activity and cofactor activities, including the secretion of cytokines.

[0223] In one embodiment, the intracellular signaling domain may include a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may include a co-stimulatory intracellular domain. Exemplary co-stimulatory intracellular signaling domains include those derived from co-stimulatory signals or antigen-independent stimulation. For example, in the case of CAR-T, the primary intracellular signaling domain may include a cytoplasmic sequence of a T cell receptor, and the co-stimulatory intracellular signaling domain may include a cytoplasmic sequence from a co-receptor or co-stimulatory molecule.

[0224] Primary intracellular signal transduction domains may contain signal transduction motifs called immune receptor tyrosine-based activation motifs or ITAMs. Examples of primary cytoplasmic signal transduction sequences containing ITAMs include, but are not limited to, those derived from CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12.

[0225] The term “ζ” or alternatively “ζ chain,” “CD3ζ,” or “TCR-ζ” is defined as a protein or equivalent residue from a non-human species (e.g., mice, rodents, monkeys, apes, etc.) as provided in GenBank accession number BAG36664.1, and “ζ stimulatory domain” or alternatively “CD3ζ stimulatory domain” or “TCR-ζ stimulatory domain” is defined as an amino acid residue from the ζ chain cytoplasmic domain or a functional derivative thereof, said amino acid residue being sufficient to functionally propagate the initial signal necessary for T cell activation. In one aspect, the ζ cytoplasmic domain comprises residues 52 to 164 of GenBank accession number BAG36664.1 or equivalent residues from a non-human species (e.g., mice, rodents, monkeys, apes, etc.) as its functional ortholog. In one aspect, the “ζ stimulatory domain” or “CD3ζ stimulatory domain” is the sequence provided as SEQ ID NO:17. In one respect, the “ζ-stimulatory domain” or “CD3ζ-stimulatory domain” is the sequence provided as SEQ ID NO:43.

[0226] The term "co-stimulatory molecule" refers to the associated binding partner on T cells that specifically binds to co-stimulatory ligands, thus enabling T cell-mediated co-stimulatory responses (such as, but not limited to, proliferation). Co-stimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that contribute to an efficient immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Other examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, 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 ligands that specifically bind to CD83.

[0227] Co-stimulatory intracellular signal transduction domains can be the intracellular portion of co-stimulatory molecules. Co-stimulatory molecules can be found in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Examples of these 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 ligands that specifically bind to CD83.

[0228] Intracellular signal transduction domains can contain the complete intracellular portion of the molecule from which they are derived, or the complete natural intracellular signal transduction domain or its functional fragments or derivatives.

[0229] The term "4-1BB" refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank accession number AAA62478.2 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.); and "4-1BB co-stimulatory domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.). In one aspect, "4-1BB co-stimulatory domain" is the sequence provided as SEQ ID NO:16 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.).

[0230] As used herein, the term "immune effector cell" refers to cells involved in immune responses, such as those involved in promoting immune effector responses. Examples of immune effector cells include T cells, such as α / β T cells and γ / δ T cells, B cells, natural killer (NK cells, natural killer T (NKT) cells), mast cells, and myeloid-derived phagocytes.

[0231] As used herein, "immune effector function or immune effector response" refers, for example, the enhancement or promotion of immune attack on target cells by immune effector cells. For instance, immune effector function or response refers to T cell or NK cell characteristics that promote the killing of target cells or inhibit the growth or proliferation of target cells. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector function or response.

[0232] The term "encoding" refers to the intrinsic property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules having defined nucleotide sequences (e.g., rRNA, tRNA, and mRNA) or defined amino acid sequences during biological processes, and the biological properties resulting from it. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, then the gene, cDNA, or RNA encodes that protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is typically provided in the sequence listing, and the non-coding strand used as a template for gene or cDNA transcription, can be referred to as the protein or other product encoding that gene or cDNA.

[0233] Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. A phrase encoding a protein or RNA "nucleotide sequence" may also include introns to such an extent that a nucleotide sequence encoding a protein may contain introns in some form.

[0234] The terms “effective amount” or “therapeutic effective amount” are used interchangeably in this document and refer to the amount of a compound, formulation, material or composition that is effective in achieving a particular biological outcome as described herein.

[0235] The term "endogenous" refers to any substance that originates from or is produced within a living organism, cell, tissue, or system.

[0236] The term "exogenous" refers to any substance introduced from or produced outside of a living organism, cell, tissue, or system.

[0237] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0238] The term "transfer vector" refers to a composition of substances containing isolated nucleic acids and capable of delivering those isolated nucleic acids into the interior of cells. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphoteric compounds, plasmids, and viruses. Therefore, the term "transfer vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral transfer vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, etc.

[0239] The term "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence effectively linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other expression elements may be supplied by a host cell or in an in vitro expression system. Expression vectors include all those known in the art, including visceral particles, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating the recombinant polynucleotide.

[0240] The term "lentivirus" refers to a genus within the family Retroviridae. Lentivirals are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information into the host cell's DNA, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0241] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, particularly including self-inactivated lentiviral vectors as described in Milone et al., Mol. Ther. 17(8):1453–1464 (2009). Other examples of lentiviral vectors that may be used clinically include, but are not limited to, those from Oxford BioMedica. Gene delivery technology, LENTIMAX from Lentigen TM Vector systems, etc. Non-clinical lentiviral vectors are also available and are known to those skilled in the art.

[0242] The terms "homologous" or "identical" refer to the identity of secondary unit sequences between two polymer molecules (e.g., between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules). When a secondary unit position in two molecules is occupied by secondary units of the same monomer, for example, if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The identity between two sequences varies directly with the number of matching or homologous positions; for example, if half the positions in two sequences (e.g., 5 positions in a polymer of 10 secondary units in length) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10 positions) are matching or homologous, then the two sequences are 90% homologous.

[0243] A “humanized” form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as the Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of an antibody) containing a minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies and their fragments are human immunoglobulins (recipient antibodies or antibody fragments) in which residues in the complementarity-determining region (CDR) from the recipient are replaced with residues in the CDR from a non-human species (donor antibody) such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and capability. In some cases, the Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Additionally, humanized antibodies / antibody fragments may contain residues not present in either the recipient antibody or the input CDR or framework sequence. These modifications can further refine and optimize the performance of the antibody or antibody fragment. Typically, humanized antibodies or antibody fragments thereof will substantially contain all of at least one, and generally two, variable domains, wherein all or substantially all of the CDR regions correspond to those CDR regions of non-human immunoglobulins and all or a significant portion of the FR regions are those FR regions having human immunoglobulin sequences. Humanized antibodies or antibody fragments may also contain at least a portion of immunoglobulin constant regions (Fc), typically constant regions of human immunoglobulins. 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.

[0244] "Fully human" refers to immunoglobulins, such as antibodies or antibody fragments, in which the entire molecule is of human origin or consists of the same amino acid sequence as the human form of the antibody or immunoglobulin.

[0245] The term "isolated" means altered or removed from its natural state. For example, nucleic acids or peptides naturally present in living organisms are not "isolated," but the same nucleic acids or peptides that are partially or completely separated from their natural coexisting substances are "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form or may exist in non-natural environments (such as host cells).

[0246] In the context of this invention, the following abbreviations are used for commonly occurring nucleic acid bases. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0247] The term "effective linkage" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. For example, the first nucleic acid sequence is effectively linked to the second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. Similarly, if a promoter affects the transcription or expression of a coding sequence, the promoter is effectively linked to the coding sequence. Effectively linked DNA sequences can be adjacent to each other and, for example, within the same reading frame in cases where two protein-coding regions need to be linked.

[0248] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, intratumoral, or infusion techniques.

[0249] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term includes nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise stated, a particular nucleic acid sequence also inherently includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081, (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608, (985); and Rossolini et al., Mol. Cell. Probes 8:91-98, (1994)).

[0250] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limitation on the maximum number of amino acids that can constitute a protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to a short chain, such as peptides, oligopeptides, and oligomers commonly referred to in the art, and also to a longer chain, typically referred to in the art as a protein, which exists in many types. “Polypeptide” includes, for example, biologically active fragments of polypeptides, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants, modified polypeptides, derivatives, analogs, fusion proteins, and so on. Polypeptides include native peptides, recombinant peptides, or combinations thereof.

[0251] The term "promoter" refers to a DNA sequence that is recognized by the cellular synthetic apparatus or an introduced synthetic apparatus and is required to initiate the specific transcription of a polynucleotide sequence.

[0252] The term "promoter / regulatory sequence" refers to the nucleic acid sequence required to express a gene product that is effectively linked to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in others, it may also contain enhancer sequences and other regulatory elements required to express the gene product. Promoter / regulatory sequences can be, for example, those that express the gene product in a tissue-specific manner.

[0253] The term "constitutive" promoter refers to the nucleotide sequence that, when effectively linked to a polynucleotide encoding or defining a gene product, causes the gene product to be produced in the cell under most or all physiological conditions.

[0254] The term "inducible" promoter refers to a nucleotide sequence that, when effectively linked to a polynucleotide encoding or defining a gene product, causes the gene product to be produced in the cell essentially only when an inducer corresponding to that promoter is present in the cell.

[0255] The term "tissue-specific" promoter refers to a nucleotide sequence that, when effectively linked to a gene-encoded or specified polynucleotide, causes the gene product to be produced in a cell primarily only by cells of the tissue type corresponding to that promoter.

[0256] In the context of scFv, the term "flexible peptide linker" or "linker" refers to a peptide linker composed of amino acid residues such as glycine and / or serine, wherein the peptide linker is used alone or in combination to link a variable heavy chain region and a variable light chain region together. In one embodiment, the flexible peptide 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 peptide linker includes, but is not limited to, (Gly4Ser)4 (SEQ ID NO: 106) or (Gly4Ser)3 (SEQ ID NO: 107). In another embodiment, the linker comprises multiple repeating sequences of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO: 108). The scope of this invention also includes connectors described in WO 2012 / 138475, which is incorporated herein by reference.

[0257] As used in this article, the 5′ cap (also known as the RNA cap, RNA 7-methylguanosine cap, or RNAm)7 The G-cap is a modified guanine nucleotide added immediately after the transcription start point to the "front" or 5' end of eukaryotic messenger RNA. The 5' cap consists of a terminal group attached to the first nucleotide transcribed. Its presence is crucial for ribosome recognition and protection against RNase activity. Cap addition is transcription-coupled and occurs in a co-transcriptional manner, thus one influencing the other. Shortly after transcription initiation, the 5' end of the synthesizing mRNA is constrained by a cap-synthesis complex bound to RNA polymerase. This enzymatic complex catalyzes the chemical reactions required for mRNA capping. The synthesis process proceeds as a multi-step biochemical reaction. The capping portion can be modified to regulate mRNA function, such as its stability or translation efficiency.

[0258] As used herein, "in vitro transcribed RNA" refers to RNA, preferably mRNA, that has been synthesized in vitro. Typically, in vitro transcribed RNA is produced from an in vitro transcription vector. The in vitro transcription vector includes a template used to produce the in vitro transcribed RNA.

[0259] As used herein, “polyadenylation” refers to a series of adenosines linked to mRNA via polyadenylation. In a preferred embodiment of the transient expression construct, the number of polyadenylations is between 50 and 5000 (SEQ ID NO: 109), preferably greater than 64, more preferably greater than 100, and most preferably greater than 300 or 400. The polyadenylation sequence can be chemically or enzymatically modified to modulate mRNA functions such as localization, stability, or translation efficiency.

[0260] As used herein, “polyadenylation” refers to the covalent attachment of a polyadenylated moiety or its modified variant to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' polyadenylated tail is added to a long adenine nucleotide sequence (often several hundred) of the pre-mRNA by the action of an enzyme (polyadenylate polymerase). In higher eukaryotes, the polyadenylated tail is added to transcripts containing a specific sequence (polyadenylation signal). The polyadenylated tail and the proteins bound to it help protect mRNA from exonuclease degradation. Polyadenylation is also important for transcription termination, mRNA export from the nucleus, and translation. Polyadenylation occurs immediately in the nucleus after DNA is transcribed into RNA, but can also occur later in the cytoplasm. After transcription has terminated, the mRNA chain is cleaved by an endonuclease complex bound to RNA polymerase. The cleavage site is typically characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3' end at the cleavage site.

[0261] As used in this article, “transient” refers to the expression of a non-integrated transgene over a period of hours, days, or weeks, which is shorter than the expression period if the gene is integrated into the genome in the host cell or contained within a stable plasmid replicon.

[0262] The term "signal transduction pathway" refers to the biochemical relationships among various signal transduction molecules that play a role in transmitting signals from one part of the cell to another. The phrase "cell surface receptor" includes molecules and molecular complexes capable of receiving signals and transmitting signals across the cell membrane.

[0263] The term "subject" is intended to include living organisms (e.g., mammals, humans) in which an immune response can be elicited.

[0264] The term "substantially purified" cells refer to cells that are substantially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types that are normally associated with them in their natural state. In some cases, a substantially purified cell population refers to a homogeneous cell population. In other cases, the term simply refers to cells that have been separated from cells naturally associated with them in their natural state. In some respects, the cells are cultured in vitro. In other respects, the cells are not cultured in vitro.

[0265] As used in this article, the term "therapeutic" means treatment. Therapeutic effects are achieved by reducing, suppressing, alleviating, or eradicating the disease state.

[0266] As used in this article, the term "prevention" refers to the prevention or protective treatment of a disease or disease state.

[0267] In the context of this invention, "tumor antigen," "hyperproliferative disease antigen," or "antigen associated with hyperproliferative disease" refers to antigens commonly found in specific hyperproliferative diseases. In some aspects, the hyperproliferative disease antigens of this 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, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, and pancreatic cancer.

[0268] The terms "transfection," "transformation," or "transduction" refer to the process of transferring or introducing exogenous nucleic acids into host cells. "Transfected," "transformed," or "transduced" cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. Cells include primary subject cells and their progeny.

[0269] The term "specific binding" refers to an antibody or ligand that recognizes and binds to a binding partner protein (e.g., a stimulating tumor antigen) present in a sample, but which substantially does not recognize or bind to other molecules in the sample.

[0270] As used herein, a “tunable chimeric antigen receptor (RCAR)” refers to a group of peptides, typically two peptides in the simplest embodiment, that, when in RCARX cells, provide the RCARX cells with specificity against target cells (typically cancer cells) and provide tunable intracellular signaling or proliferation, thereby optimizing the immune effector properties of the RCARX cells. RCARX cells rely at least in part on an antigen-binding domain to provide specificity against target cells containing antigens bound to the antigen-binding domain. In one embodiment, the RCAR includes a dimerization switch that, in the presence of a dimerizing molecule, couples an intracellular signaling domain to an antigen-binding domain.

[0271] As used herein, “membrane anchor” or “membrane-binding domain” refers to a polypeptide or portion sufficient to anchor an extracellular or intracellular domain to the plasma membrane, such as myristoyl.

[0272] As used herein, when referring, for example, to RCAR, a “switch domain” refers to an entity that associates with another switch domain in the presence of a dimerizing molecule, typically a peptide-based entity. Association results in the functional coupling of a first switch domain linked to a first entity (e.g., fusion) and a second switch domain linked to a second entity (e.g., fusion). The first and second switch domains are collectively referred to as dimerizing switch. In embodiments, the first and second switch domains are identical to each other; for example, they are peptides having the same primary amino acid sequence and are collectively referred to as homodimerizing switch. In embodiments, the first and second switch domains are different from each other; for example, they are peptides with different primary amino acid sequences and are collectively referred to as heterodimerizing switch. In embodiments, the switch is intracellular. In embodiments, the switch is extracellular. In embodiments, the switch domain is a peptide-based entity (e.g., FKBP or FRB-based), and the dimerizing molecule is a small molecule, such as a rapalogue analog. In one embodiment, the switching domain is a peptide-based entity, such as an scFv binding to a myc peptide, and the dimer is a peptide, a fragment thereof, or a peptide multimer, such as a myc ligand or a myc ligand multimer binding to one or more myc scFvs. In another embodiment, the switching domain is a peptide-based entity, such as a myc receptor, and the dimer is an antibody or a fragment thereof, such as a myc antibody.

[0273] As used herein, for example when referring to RCAR, "dimerizing molecule" refers to an entity that promotes association between a first switching domain and a second switching domain. In embodiments, the dimerizing molecule is not naturally present in the subject or is not present at a concentration that would lead to significant dimerization. In embodiments, the dimerizing molecule is a small molecule, such as rapamycin or a rapamycin analogue, such as RAD001.

[0274] The term "bioequivalent" refers to the amount of drug other than the reference compound (e.g., RAD001) required to produce an effect equivalent to that produced by a reference dose or reference amount of the reference compound (e.g., RAD001). In one embodiment, the effect is an mTOR inhibition level, for example, as measured by P70 S6 kinase inhibition, for example, as evaluated in vivo or in vitro assays, for example, as measured by the assays described herein (e.g., Boulay assay), or by measuring phosphorylated S6 levels by Western blotting. In one embodiment, the effect is a change in the ratio of PD-1 positive / PD-1 negative T cells, as measured by cell sorting. In one embodiment, the bioequivalent amount or dose of the mTOR inhibitor is the amount or dose that achieves the same P70 S6 kinase inhibition level as the reference dose or reference amount of the reference compound. In one embodiment, the bioequivalent amount or dose of the mTOR inhibitor is the amount or dose that achieves the same level of change in the PD-1 positive / PD-1 negative T cell ratio as the reference dose or reference amount of the reference compound.

[0275] When used in conjunction with an mTOR inhibitor (e.g., an allosteric mTOR inhibitor, such as RAD001 or rapamycin, or a catalytic mTOR inhibitor), the term "immunoenhancing low dose" refers to a dose of mTOR inhibitor that partially, but not completely, inhibits mTOR activity (e.g., as measured by inhibition of P70 S6 kinase activity). Methods for evaluating mTOR activity (e.g., by inhibition of P70 S6 kinase) are discussed herein. This dose is insufficient to cause complete immunosuppression but sufficient to enhance the immune response. In one embodiment, an immunoenhancing low dose of the 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 the ratio of PD-1 negative T cells to PD-1 positive T cells. In one embodiment, an immunoenhancing low dose of the mTOR inhibitor results in an increase in the number of naive T cells. In one embodiment, an immunoenhancing low dose of the mTOR inhibitor results in one or more of the following:

[0276] Increased expression of one or more of the following markers: for example, CD62L on memory T cells (e.g., memory T cell precursors). 高 CD127高 CD27 + and BCL2;

[0277] Reduced expression of KLRG1 on memory T cells (e.g., memory T cell precursors); and

[0278] An increase in the number of memory T cell precursors, which are, for example, cells having any one or a combination of the following characteristics: CD62L 高 Add, CD127 高 Add, CD27 + Increase, KLRG1 decreases and BCL2 increases;

[0279] For example, any of the changes described above appear, for instance, at least momentarily, compared to untreated subjects.

[0280] As used herein, "refractory" refers to a disease that does not respond to treatment, such as cancer. In one implementation, refractory cancer may be resistant to treatment before or at the start of treatment. In other implementations, refractory cancer may become refractory during treatment.

[0281] As used herein, a “complete responder” refers to a subject with a disease (e.g., cancer) who demonstrates a complete response to treatment (e.g., complete remission). Complete responses can be identified, for example, using the Cheson criteria as described herein.

[0282] As used herein, "partial responder" refers to a subject with a disease (e.g., cancer) who shows a partial response to treatment (e.g., partial remission). Partial responses can be identified, for example, using the Cheson criteria.

[0283] As used herein, a “non-responder” refers to a subject with a disease (e.g., cancer) who does not show a response to treatment, for example, a patient with a stable or progressive condition. Non-responders can be identified, for example, using the Cheson criteria as described herein.

[0284] The term “recurrence,” as used herein, refers to the reappearance of a disease (e.g., cancer) after an initial responsive phase (e.g., a complete or partial response). An initial responsive phase may involve cancer cell levels falling below a certain threshold, such as below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Recurrence may involve cancer cell levels rising above a certain threshold, such as above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Recurrence can be identified, for example, using the Cheson criteria as described herein.

[0285] Scope: Throughout this disclosure, various aspects of the invention may be shown in a scope format. It should be understood that the scope format description is for convenience and brevity purposes only and should not be construed as rigidly limiting the scope of the invention. Therefore, a scope description should be considered to have all possible sub-scopes specifically disclosed and various numerical values ​​within that scope. For example, a scope such as 1 to 6 should be considered to have specifically disclosed sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and various numerical values ​​within that scope, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a scope (such as 95-99% identity) includes scopes having 95%, 96%, 97%, 98%, or 99% identity, and includes sub-scopes such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the width of the scope.

[0286] Invention Description

[0287] This document provides material compositions and methods of use for treating diseases such as cancer (e.g., hematologic malignancies or other B-cell malignancies) using immune effector cells (e.g., T cells or NK cells) expressing chimeric antigen receptors (CARs) (e.g., CARs targeting B-cell markers such as CD19). The method specifically includes administering immune effector cells (e.g., T cells or NK cells) expressing the B-cell-targeting CARs described herein in combination with another drug, such as a kinase inhibitor (e.g., the kinase inhibitors described herein).

[0288] This invention provides at least in part several experiments supporting the high efficacy of combinations of CAR therapy (e.g., B-cell-targeting CAR therapy) and kinase inhibitors (e.g., BTK inhibitors such as ibrutinib). Combinations of kinase inhibitors (e.g., BTK inhibitors such as ibrutinib) with CAR therapy can increase the efficacy of the combination therapy relative to kinase inhibitor monotherapy or a dose of CAR-expressing cells or both. These beneficial effects can, for example, allow for lower doses of kinase inhibitors or CAR-expressing cells or both while maintaining efficacy. The results described herein are applicable to a wide range of cancer types, such as hematologic malignancies and other B-cell malignancies. For example, ibrutinib inhibits BTK, which is elevated in most lymphomas. Immune effector cells expressing CAR19 (e.g., T cells or NK cells) target cancers that express CD19 on their surface, which is expressed in most B-cell malignancies. Alternatively or in combination with CAR19, any other B-cell-targeting CAR (e.g., CARs targeting one or more of the following: CD20, CD22, or ROR1) can be used in the combination therapies described herein. Therefore, the combination of CAR therapy (e.g., one or more of CD19CAR, CD20CAR, CD22CAR, or ROR1CAR) with a BTK inhibitor (e.g., ibrutinib) is suitable for treating a wide range of cancers involving excessive B-cell proliferation, including lymphoma (e.g., Hodgkin's lymphoma), MCL, CLL, DLBCL, and multiple myeloma.

[0289] According to the present invention, ibrutinib can reduce tumor masses and mobilize tumor-bearing B cells in peripheral blood (see, for example, Example 8 herein). Not wishing to be bound by theory, some lymphomas (such as MCL) are characterized by clusters of cancer cells in the proliferative centers of lymph nodes. CAR-expressing immune effector cells sometimes have difficulty penetrating these densely packed clusters. Therefore, BTK inhibitors (such as ibrutinib) can reduce tumor masses and mobilize tumor-bearing B cells in peripheral blood, thereby making lymphoma cells more susceptible to CAR-expressing cell killing.

[0290] Alternatively or in combination, BTK inhibitors (such as ibrutinib) can also affect CAR-expressing cells. This invention demonstrates that ibrutinib treatment increases the level of circulating CART19 cells (see, for example, the data shown in Example 8). It is not intended to be theoretically limited, but the increase in circulating CART19 cell levels can be, for example, the result of increased proliferation, altered T cell phenotype, or other factors. For example, ibrutinib can inhibit ITK (a kinase homologous to BTK). ITK is expressed in T cells, and inhibition of it can alter the T cell phenotype. Treatment with a kinase inhibitor (such as ibrutinib) can change the T cell phenotype from a Th2 phenotype to a Th1 phenotype and thus increase T cell proliferative capacity. Pretreatment or co-administration of a BTK inhibitor in a subject can increase T cell proliferative capacity in the subject, thus increasing the level of circulating cells expressing CAR. Furthermore, subjects pretreated with a BTK inhibitor (e.g., ibrutinib) may have a population of T cells with high proliferative capacity during their apheresis for CAR production.

[0291] In one aspect, the present invention provides a plurality of chimeric antigen receptors (CARs) comprising an antibody or antibody fragment engineered to specifically bind to a B-cell antigen (e.g., selected from one or more of CD19, CD20, CD22, or ROR1 proteins). In one aspect, the present invention provides cells (e.g., T cells) engineered to express CARs, wherein CAR T cells (“CART”) exhibit anticancer properties. In one aspect, cells are transformed with CARs and the CARs are expressed on the cell surface. In some embodiments, cells (e.g., T cells) are 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 cells can stably express CARs. In another embodiment, cells (e.g., T cells) are transfected with a nucleic acid encoding a CAR, such as mRNA, cDNA, or DNA. In some such embodiments, the cells can transiently express CARs.

[0292] In one aspect, the anti-CD19 protein-binding portion of the CAR is an scFv antibody fragment. In another aspect, such antibody fragments are functional because they retain equivalent binding affinity; for example, they bind the same antigen with comparable affinity to the IgG antibody that derived it. In another aspect, such antibody fragments are functional in that they provide a biological response, which may include, but is not limited to, activation of an immune response, inhibition of signal transduction derived from its target antigen, inhibition of kinase activity, etc., as will be understood by those skilled in the art. In one aspect, the anti-CD19 antigen-binding domain of the CAR is an scFv antibody fragment that is humanized compared to the mouse scFv sequence from which it is derived. In one aspect, the mouse scFv parental sequence is the CAR19 construct provided in PCT Publication WO 2012 / 079000 (incorporated herein by reference) and provided herein as SEQ ID NO:59. In one embodiment, the anti-CD19 binding domain is the scFv described in WO 2012 / 079000 and provided in SEQ ID NO:59.

[0293] In some aspects, the antibodies of the present invention are incorporated into a chimeric antigen receptor (CAR). In one aspect, the CAR comprises a polypeptide sequence provided as SEQ ID NO:12 in PCT Disclosure WO 2012 / 079000 and as SEQ ID NO:58 herein, wherein the scFv domain is replaced by one or more sequences selected from SEQ ID NO:1-12. In one aspect, the scFv domain of SEQ ID NO:1-12 is a humanized variant of the scFv domain of SEQ ID NO:59, which is a mouse scFv fragment that specifically binds to human CD19. Such humanization of the mouse scFv can be desired in a clinical setting where mouse-specific residues can induce a human anti-mouse antigen (HAMA) response in patients receiving CAR19 therapy (e.g., T-cell therapy transduced with a CAR19 construct).

[0294] In one aspect, the anti-CD19 binding domain (e.g., humanized scFv) portion of the CAR of the present invention is encoded by a transgene whose sequence has been codon-optimized for expression in mammalian cells. In another aspect, the complete CAR construct of the present invention is encoded by a transgene whose entire sequence has been codon-optimized for expression in mammalian cells. Codon optimization refers to the finding that the frequency of synonymous codons (i.e., codons encoding the same amino acid) in coding DNA is biased across different species. This codon degeneracy allows the same polypeptide to be encoded by multiple nucleotide sequences. Various codon optimization methods are known in the art and include, for example, methods disclosed in at least U.S. Patent Nos. 5,786,464 and 6,114,148.

[0295] In one aspect, humanized CAR19 includes the scFv portion provided in SEQ ID NO:1. In one aspect, humanized CAR19 includes the scFv portion provided in SEQ ID NO:2. In one aspect, humanized CAR19 includes the scFv portion provided in SEQ ID NO:3. In one aspect, humanized CAR19 includes the scFv portion provided in SEQ ID NO:4. In one aspect, humanized CAR19 includes the scFv portion provided in SEQ ID NO:5. In one aspect, humanized CAR19 includes the scFv portion provided in SEQ ID NO:6. In one aspect, humanized CAR19 includes the scFv portion provided in SEQ ID NO:7. In one aspect, humanized CAR19 includes the scFv portion provided in SEQ ID NO:8. In one aspect, humanized CAR19 includes the scFv portion provided in SEQ ID NO:9. In one aspect, humanized CAR19 includes the scFv portion provided in SEQ ID NO:10. In one aspect, humanized CAR19 includes the scFv portion provided in SEQ ID NO:11. In one respect, humanized CAR19 includes the scFv portion provided in SEQ ID NO:12.

[0296] In one aspect, the CAR of the present invention combines the 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 CD3ζ chain, 4-1BB, and CD28 signaling modules, and combinations thereof. In one aspect, the CD19 CAR comprises a CAR selected from the sequences provided in one or more of SEQ ID NO: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 includes the sequence provided in SEQ ID NO:39. In one aspect, the CD19 CAR includes the sequence provided in SEQ ID NO:40. In one aspect, the CD19 CAR includes the sequence provided in SEQ ID NO:41. In one aspect, the CD19 CAR includes the sequence provided in SEQ ID NO:42.

[0297] In addition, the present invention provides CD19 CAR compositions and their use in medicaments or methods for treating cancers or any malignant tumors or autoimmune diseases involving cells or tissues expressing CD19, along with other diseases.

[0298] In one aspect, the CAR of the present invention can be used to eradicate normal cells expressing CD19, and is therefore suitable for use as a cellular conditioning therapy prior to cell transplantation. In another aspect, the normal cells expressing CD19 are normal stem cells expressing CD19 and the cell transplantation is a stem cell transplantation.

[0299] In one aspect, the present invention provides engineered cells expressing a chimeric antigen receptor (CAR) (e.g., T cells), wherein the CAR-expressing cells, such as CAR T cells (“CART”), exhibit anticancer properties. A preferred antigen is CD19. In one aspect, the antigen-binding domain of the CAR comprises a partially humanized anti-CD19 antibody fragment. In another aspect, the antigen-binding domain of the CAR comprises a partially humanized anti-CD19 antibody fragment containing scFv. Therefore, the present invention provides CD19-CARs comprising a humanized anti-CD19 binding domain and engineered into immune effector cells (e.g., T cells or NK cells) and methods for using them in adoptive therapy.

[0300] In one aspect, CD19-CAR includes at least one intracellular domain selected from the CD137(4-1BB) signaling domain, the CD28 signaling domain, the CD3ζ signaling domain, and any combination thereof. In another aspect, CD19-CAR includes at least one intracellular signaling domain from one or more co-stimulatory molecules other than CD137(4-1BB) or CD28.

[0301] Chimeric antigen receptor (CAR)

[0302] This invention covers recombinant DNA constructs comprising a sequence 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 adjacent to and within the same read 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, e.g., a ζ-chain. A co-stimulatory signaling domain refers to the CAR portion comprising at least a portion of the intracellular domain of a co-stimulatory molecule. In one embodiment, the antigen-binding domain is a mouse antibody or antibody fragment as described herein. In one embodiment, the antigen-binding domain is a humanized antibody or antibody fragment.

[0303] In a specific aspect, the CAR construct of the present invention comprises an scFv domain selected from SEQ ID NO:1-12 or SEQ ID NO:59, wherein 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, SEQ ID NO:45, SEQ ID NO:47, or SEQ ID NO:49, a transmembrane region as provided in SEQ ID NO:15, an intracellular signal transduction domain comprising SEQ ID NO:16 or SEQ ID NO:51, and a CD3ζ sequence comprising SEQ ID NO:17 or SEQ ID NO:43, wherein the domains are adjacent to each other and are located in the same reading frame to form a single fusion protein. The present invention also includes nucleotide sequences encoding polypeptides selected from each scFv fragment 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. The present invention also includes nucleotide sequences encoding polypeptides encoding each scFv fragment 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 and each domain of SEQ ID NO:13-17, as well as the CD19 CAR fusion protein of the present invention. In one aspect, an exemplary CD19 CAR construct includes an optional leader sequence, an extracellular antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular stimulatory domain. In another aspect, an exemplary CD19 CAR construct includes an optional leader sequence, an extracellular antigen-binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular stimulatory domain. Specific CD19 CAR constructs containing the humanized scFv domain of the present invention are provided as SEQ ID NO:31-42 or as the mouse scFv domain of SEQ ID NO:59.

[0304] The full-length CAR sequences are also provided in this paper as SEQ ID NO:31-42 and 58, as shown in Tables 7 and 3.

[0305] An exemplary leader sequence is provided as SEQ ID NO:13. An exemplary hinge region / spacer sequence is provided as SEQ ID NO:14, SEQ ID NO:45, 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 signal transduction domain of the 4-1BB protein is provided as SEQ ID NO:16. An exemplary sequence of the intracellular signal transduction domain of CD27 is provided as SEQ ID NO:51. An exemplary CD3ζ domain sequence is provided as SEQ ID NO:17 or SEQ ID NO:43.

[0306] In one aspect, the present invention covers a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding (e.g., as described herein) an anti-CD19 binding domain, said nucleic acid sequence being adjacent to and within the same read frame as a nucleic acid sequence encoding an intracellular signal transduction domain. In one aspect, the anti-CD19 binding domain is selected from one or more of SEQ ID NO:1-12 and 58. In one aspect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of the sequences provided in one or more of SEQ ID NO: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 respect, the anti-CD19 binding domain is encoded by nucleotide residues 64 to 813 of SEQ ID NO:72.

[0307] In one aspect, the present invention covers 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 NO:61-72, the anti-CD19 binding domain being adjacent to and within the same reading frame as a nucleic acid sequence encoding an intracellular signal transduction domain. Exemplary intracellular signal transduction domains that can be used in a CAR include, but are not limited to, one or more intracellular signal transduction domains such as CD3ζ, CD28, 4-1BB, etc. In some cases, the CAR may comprise any combination of CD3ζ, CD28, 4-1BB, etc. In one aspect, the nucleic acid sequence of the CAR construct of the present invention is selected from one or more of SEQ ID NO:85-96. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:85. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:86. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:87. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:88. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:89. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:90. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:91. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:92. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:93. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:94. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:95. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:96. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:97. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:98. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO:99.

[0308] The nucleic acid sequence encoding the desired molecule can be obtained using recombination methods known in the art, such as screening a library from cells expressing the gene, deriving the gene from a vector known to contain the gene, or directly isolating the gene from cells and tissues containing the gene using standard techniques. Alternatively, the target nucleic acid can be produced synthetically rather than cloned.

[0309] This invention includes retroviral constructs and lentiviral vector constructs that express CARs that can be directly transduced into cells.

[0310] This invention also includes RNA constructs that can be directly transfected into cells. One method for generating mRNA for transfection involves using a specially designed primer for in vitro transcription (IVT) of a template, followed by the addition of polyadenylation to produce a construct containing 3' and 5' untranslated sequences (“UTR”), a 5' cap and / or an internal ribosome entry site (IRES), the nucleic acid to be expressed, and a polyadenylated tail, typically 50-2000 bases in length (SEQ ID NO: 118). The resulting RNA can be efficiently transfected into various cell types. In one embodiment, the template includes a CAR sequence. In one embodiment, the RNA CAR vector is transduced into T cells via electroporation.

[0311] Antigen-binding domain

[0312] In one aspect, the CAR of the present invention includes a target-specific binding element, also referred to as an antigen-binding domain. The choice of the structural portion depends on defining the type and number of ligands on the surface of the target cell. For example, the antigen-binding domain can be selected to recognize ligands that act as cell surface markers on the target cell associated with a specific disease state. Thus, examples of cell surface markers that can act as ligands for the antigen-binding domain in the CAR of the present invention include those associated with viral infections, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0313] In one aspect, CAR-mediated T-cell responses can be directed towards the target antigen by engineering antigen-binding domains that specifically bind to the desired antigen into the CAR.

[0314] In one aspect, the CAR portion containing the antigen-binding domain includes an antigen-binding domain targeting 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 similar 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).

[0315] The antigen-binding domain can be any domain that binds to an antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, mouse antibodies, human antibodies, humanized antibodies and their functional fragments, including but not limited to single-domain antibodies, such as the heavy chain variable domain (VH), light chain variable domain (VL) and variable domain (VHH) of camel-derived nanobody, as well as alternative scaffolds known in the art that function as antigen-binding domains, such as recombinant fibronectin domains.

[0316] In one embodiment, the CAR molecule includes an anti-CD19 binding domain comprising one or more (e.g., all three) light chain complementarity-determining regions 1 (LCCDR1), 2 (LCCDR2), and 3 (LCCDR3) of the anti-CD19 binding domain described herein, and one or more (e.g., all three) heavy chain complementarity-determining regions 1 (HCCDR1), 2 (HCCDR2), and 3 (HCCDR3) of the anti-CD19 binding domain described herein, for example, an 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 anti-CD19 binding domain comprises one or more (e.g., all three) heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the anti-CD19 binding domain described herein. For example, the anti-CD19 binding domain has two variable heavy chain regions, each comprising HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the anti-CD19 binding domain comprises a mouse light chain variable region and / or a mouse heavy chain variable region as described herein (e.g., in Table 7). In one embodiment, the anti-CD19 binding domain is an scFv comprising the mouse light chain and mouse heavy chain containing the amino acid sequences of Table 7. In one embodiment, the anti-CD19 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequences of the light chain variable regions provided in Table 7, but no more than 30, 20, or 10 modifications (e.g., substitutions), or comprising a sequence having 95-99% identity with the amino acid sequences 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) to the amino acid sequences of the heavy chain variable regions provided in Table 7, but no more than 30, 20, or 10 modifications (e.g., substitutions), or comprising a sequence having 95-99% identity with the amino acid sequences of Table 7. In one embodiment, the anti-CD19 binding domain comprises the sequence of SEQ ID NO:59, or a sequence having 95-99% identity with it. In one embodiment, the anti-CD19 binding domain is scFv, and the light chain variable region comprising the amino acid sequence described herein (e.g., in Table 7) is linked via a linker (e.g., the linker described herein) to the heavy chain variable region comprising the amino acid sequence described herein (e.g., in Table 7).In one embodiment, the anti-CD19 binding domain comprises a (Gly4-Ser)n connector, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4 (SEQ ID NO: 53). The light chain variable region and the heavy chain variable region of scFv can be in any orientation, for example: light chain variable region-connector-heavy chain variable region or heavy chain variable region-connector-light chain variable region.

[0317] In some cases, the antigen-binding domain can be derived from the same species from which the CAR will ultimately be used. For example, for human use, human residues or humanized residues containing the antigen-binding domain of an antibody or antibody fragment may be beneficial to the antigen-binding domain of the CAR.

[0318] Therefore, in one aspect, the antigen-binding domain comprises a humanized antibody or antibody fragment. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) of the mouse or humanized anti-CD19 binding domain described herein, including light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3), and / or one or more (e.g., all three) of the mouse or humanized anti-CD19 binding domain described herein, including heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HCCDR3), for example, a humanized anti-CD19 binding domain comprising one or more (e.g., all three) LC CDRs and one or more (e.g., all three) HCCDRs. In one embodiment, the humanized anti-CD19 binding domain comprises one or more (e.g., all three) heavy chain complementarity-determining regions 1 (HCCDR1), 2 (HC CDR2), and 3 (HC CDR3) of the mouse or humanized anti-CD19 binding domains described herein. For example, the humanized anti-CD19 binding domain has two variable heavy chain regions, each comprising HC CDR1, HC CDR2, and HC CDR3 as described herein. In one embodiment, the humanized anti-CD19 binding domain comprises a humanized light chain variable region and / or a humanized heavy chain variable region as described herein (e.g., in Table 3). In one embodiment, the humanized anti-CD19 binding domain comprises a humanized heavy chain variable region as described herein (e.g., in Table 3), such as at least two humanized heavy chain variable regions as described herein (e.g., in Table 3). In one embodiment, the anti-CD19 binding domain is an scFv comprising the amino acid sequence of Table 3 for both the light and heavy chains. In one embodiment, the anti-CD19 binding domain (e.g., scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequences of the light chain variable region provided in Table 3, but no more than 30, 20, or 10 modifications (e.g., substitutions), or containing a sequence having 95-99% identity with the amino acid sequences 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) to the amino acid sequences of the heavy chain variable region provided in Table 3, but no more than 30, 20, or 10 modifications (e.g., substitutions), or containing a sequence having 95-99% identity with the amino acid sequences of Table 3.In one embodiment, 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 having 95-99% identity with such sequences. In one embodiment, the nucleic acid sequence encoding the humanized anti-CD19 binding domain comprises a sequence selected from 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 having 95-99% identity with such sequences. In one embodiment, the humanized anti-CD19 binding domain is scFv, and a light chain variable region comprising the amino acid sequence described herein (e.g., in Table 3) is linked via a linker (e.g., the linker described herein) to a heavy chain variable region comprising the amino acid sequence described herein (e.g., in Table 3). In one embodiment, the humanized anti-CD19 binding domain comprises (Gly4-Ser). n The connector, wherein n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4 (SEQ ID NO: 53). The light chain variable region and the heavy chain variable region of the scFv can be in any orientation, for example: light chain variable region-connector-heavy chain variable region or heavy chain variable region-connector-light chain variable region.

[0319] In one aspect, the antigen-binding domain portion comprises one or more sequences selected from SEQ ID NO:1-12. In another aspect, the humanized CAR is selected from one or more sequences selected from SEQ ID NO:31-42. In some aspects, the non-human antibody is humanized, wherein specific sequences or regions of the antibody are modified to increase similarity to naturally occurring antibodies or fragments thereof in humans.

[0320] Humanized antibodies can be generated using a variety of techniques known in the art, including but not limited to CDR transplantation (see, for example, European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101 and 5,585,089, each of which is incorporated herein by reference in its entirety), inlay, or surface remodeling (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 by reference in its entirety), chain truncation (see, for example, U.S. Patent No. 5,565,332, which is incorporated herein by reference in its entirety) and techniques disclosed in, for example, the following: U.S. Patent Application Publication No. US2005 / 0042664, U.S. Patent Application Publication No. US2005 / 0048617, U.S. Patent No. 6,407,213, U.S. Patent 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 JS, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994). Each of these references is fully incorporated into this paper by way of citation. Often, the framework residues in the framework region are replaced with corresponding residues from the CDR donor antibody to alter, for example, improve antigen binding. These framework replacements are identified by methods well known in the art, for example, by modeling the interaction between the CDR and framework residues to identify framework residues important for antigen binding and by performing sequence comparisons to identify unusual framework residues at specific locations.(See, for example, Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entirety.)

[0321] Humanized antibodies or antibody fragments contain one or more amino acid residues of non-human origin. These non-human amino acid residues are often referred to as “input” residues, and they are generally derived from “input” variable domains. As described herein, humanized antibodies or antibody fragments contain one or more CDRs from non-human immunoglobulin molecules and framework regions, in which the amino acid residues of the framework are wholly or largely derived from human lineages. Several techniques for humanizing antibodies or antibody fragments are well known in the art and can be largely followed in the manner of Winter and collaborators (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by replacing rodent CDRs or CDR sequences with corresponding human antibody sequences, i.e., CDR transplantation (EP 239,400; PCT Publication No. WO 91 / 09967; and US 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 in their entirety). In these humanized antibodies and antibody fragments, regions substantially smaller than the intact human variable domain have been replaced by corresponding sequences from non-human species. Humanized antibodies are often human antibodies in which some CDR residues and possibly some FR residues have been replaced by residues from similar sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by matte or surface remodeling (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 tampering (US Patent No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.

[0322] Selecting the human variable domains (both heavy and light chain variable domains) to be used for generating humanized antibodies is important for reducing antigenicity. Following the so-called "best match" approach, sequences of variable domains for rodent antibodies are screened against a complete library of known human variable domain sequences. The human sequence closest to the rodent sequence is then identified and accepted as the human framework region (FR) for humanized antibodies (Sims et al., J. Immunol, 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987, the contents of which are incorporated herein by reference in their entirety). Another approach uses specific frameworks derived from common sequences of all human antibodies with specific subgroups of heavy chains or heavy chains. The same framework 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 in their entirety). In some embodiments, the framework regions of the heavy chain variable region (e.g., all four framework regions) are derived from the VH4_4-59 germline sequence. In one embodiment, the framework region may contain one, two, three, four, or five modifications, such as substitutions, for example, substitutions of amino acids from the corresponding mouse sequence (e.g., SEQ ID NO:59). In one embodiment, the framework regions of the light chain variable region (e.g., all four framework regions) are derived from the VK3_1.25 germline sequence. In one embodiment, the framework region may contain one, two, three, four, or five modifications, such as substitutions, for example, substitutions of amino acids from the corresponding mouse sequence (e.g., SEQ ID NO:59).

[0323] In some aspects, portions of the CAR compositions of the present invention containing antibody fragments are humanized, retaining high affinity for the target antigen and other advantageous biological properties. According to one aspect of the invention, the preparation of three-dimensional immunoglobulin models of humanized antibodies and antibody fragments using parental sequences and humanized sequences, through processes analyzing parental sequences and various conceptual humanized products, is generally available and familiar to those skilled in the art. Computer programs illustrating and demonstrating the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. Verification of these demonstration results allows analysis of the possible roles of residues in the functioning of the candidate immunoglobulin sequences, for example, analyzing residues affecting the ability of the candidate immunoglobulin to bind to the target antigen. In this way, FR residues can be selected and combined from the receptor sequence and the input sequence to achieve desired antibody or antibody fragment characteristics, such as increased affinity for the target antigen. Typically, CDR residues are directly and substantially involved in influencing antigen binding.

[0324] Humanized antibodies or antibody fragments can retain antigen specificity similar to (e.g., in this invention) the original antibody, and the ability to bind to human CD19. In some embodiments, humanized antibodies or antibody fragments may have improved affinity and / or specificity for binding to human CD19.

[0325] In one aspect, the anti-CD19 binding domain is characterized by a specific functional feature or property of the antibody or antibody fragment. For example, in one aspect, the CAR composition of the present invention contains a portion of the antigen-binding domain that specifically binds to human CD19. In one aspect, the antigen-binding domain has the same or similar human CD19 binding specificity as FMC63 scFv described in Nicholson et al., Mol. Immun. 34(16-17):1157-1165 (1997). In one aspect, the present 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 a fragment thereof, wherein the antibody or antibody fragment comprises a variable light chain and / or a variable heavy chain comprising the 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 selected from SEQ ID NO:1-12 or SEQ ID NO:59 of the scFv. In some aspects, the scFv is adjacent to and within the same reading frame as the leader sequence. In one respect, the leader sequence is the polypeptide sequence provided as SEQ ID NO:13.

[0326] In one aspect, the anti-CD19 binding domain is a fragment, such as a single-chain variable fragment (scFv). In another aspect, the anti-CD19 binding domain is an Fv, Fab, (Fab')2, or a 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 present invention bind to the CD19 protein with wild-type or enhanced affinity.

[0327] In some cases, scFvs can be prepared according to methods 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 generated by linking the VH and VL regions together using flexible peptide linkers. ScFv molecules contain linkers optimized for length and / or amino acid composition (e.g., Ser-Gly linkers). Linker length can significantly influence how the variable regions of the scFv fold and interact. In fact, if short peptide linkers (e.g., between 5 and 10 amino acids) are used, intrachain folding is prevented. Interchain folding is also required for the two variable regions to form a functional epitope binding site together. For examples of joint orientation and size, see, for example, Hollinger et al., 1993 Proc Natl Acad. Sci. USA 90:6444-6448, U.S. Patent Publications 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT Publications WO2006 / 020258 and WO 2007 / 024715, which are incorporated herein by reference.

[0328] The scFv may contain a linker between its VL and VH regions containing 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. The linker sequence may contain any naturally occurring amino acid. In some embodiments, the linker sequence contains the amino acids glycine and serine. In another embodiment, the linker sequence contains a set of glycine and serine repeat sequences such as (Gly4Ser). n , where n is a positive integer equal to or greater than 1 (SEQ ID NO:18). In one embodiment, the connector can be (Gly4Ser)4 (SEQ ID NO:106) or (Gly4Ser)3 (SEQ ID NO:107). Variations in connector length can preserve or enhance activity, resulting in superior efficacy in activity studies.

[0329] In some embodiments, the amino acid sequence of the antigen-binding domain (or other partial or complete CAR) can be modified, for example, by modifying the amino acid sequence described herein, for example, through conserved substitution modification. Families of amino acids with 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, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0330] In the context of two or more nucleic acid or polypeptide sequences, the percentage of identity refers to the degree to which the two or more sequences are identical. Two sequences are considered “substantially identical” if, when compared and aligned within a comparison window or a specified region for maximum correspondence, they have a specified percentage of identical amino acid residues or nucleotides (e.g., 60% identity within a specified region or, when not specified, across the entire sequence range, 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). Optionally, the identity exists in a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably in a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0331] For sequence comparisons, a sequence typically serves as a reference sequence for comparison with a test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are input into the computer, and if necessary, the coordinates of the subsequences are specified, along with the sequence algorithm program parameters. Default program parameters can be used, or alternative parameters can be specified. Based on the program parameters, the sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence. The methods used for comparing sequences are well known in the art. The best sequence alignment for comparison can be performed, 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, by the similarity search method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by the computerized execution of these algorithms (Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI in GAP, BESTFIT, FASTA and TFASTA) or by manual alignment and visual inspection (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology).

[0332] Two examples of algorithms suitable for determining sequence identity and sequence similarity percentages are the BLAST algorithm and the BLAST 2.0 algorithm, described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410 (1990), respectively. The software used for BLAST analysis is publicly available from the National Center for Biotechnology Information.

[0333] Alternatively, the identity percentage between two amino acid sequences can be determined using the E. Meyers and W. Miller algorithm (1988) Comput. Appl. Biosci. 4:11-17), which has been incorporated into the ALIGN program (version 2.0). Furthermore, the Needlema and Wunsch algorithm (1970) J. Mol. Biol. 48:444-453) (available at www.gcg.com) in the GAP program (integrated into the GCG software package) can be used, employing a Blossom 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0334] In one aspect, the present invention conceives of modifications to the amino acid sequence of an initiating antibody or fragment (e.g., scFv) to produce functionally equivalent molecules. For example, the VH or VL of the anti-CD19 binding domain (e.g., scFv) contained in the CAR can be modified to retain 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%, and 99% identity of the initiating VH or VL framework region of the anti-CD19 binding domain (e.g., scFv). The present invention conceives of modifications to complete CAR constructs, for example, modifications to one or more amino acid sequences of various domains of the CAR construct, with the aim of producing functionally equivalent molecules. CAR constructs can be modified to retain at least approximately 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%, and 99% identity with the original CAR construct.

[0335] Bispecific CAR

[0336] In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody is specific to no more than two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity against a first epitope and a second immunoglobulin variable domain sequence having binding specificity against a second epitope. In one embodiment, the first and second epitopes are on the same antigen (e.g., the same protein (or a 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, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity against the first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity against the second epitope. In one embodiment, the bispecific antibody molecule comprises a half-antibody having binding specificity against the first epitope and a half-antibody having binding specificity against the second epitope. In one embodiment, the bispecific antibody molecule comprises a half-antibody or a fragment thereof having binding specificity against a first epitope and a half-antibody or a fragment thereof having binding specificity against a second epitope. In another embodiment, the bispecific antibody molecule comprises an scFv or a fragment thereof having binding specificity against a first epitope and an scFv or a fragment thereof having binding specificity against a second epitope.

[0337] Transmembrane domain

[0338] Regarding the transmembrane domain, in several embodiments, the CAR may be designed to include a transmembrane domain connected to the extracellular domain of the CAR. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, such as one or more amino acids associated with the extracellular region of the protein from which the transmembrane region is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 extracellular region amino acids) and / or one or more amino acids associated with the intracellular region of the protein from which the transmembrane region is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 intracellular region amino acids). In one aspect, the transmembrane domain is a domain associated with one of the other domains of the CAR; for example, in one embodiment, the transmembrane domain may be derived from the same protein from which a signal transduction domain, co-stimulatory domain, or hinge domain is derived. In another aspect, the transmembrane domain is not derived from the same protein from any other domain of the derived CAR. In some cases, transmembrane domains can be selectively modified, either by amino acid substitution, to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, for example, to minimize interactions with other components of the receptor complex. In one aspect, the transmembrane domain can homodimerize with another CAR on the surface of a cell expressing the CAR. In another aspect, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interactions with binding domains of naturally occurring binding partners present in the same cells expressing the CAR.

[0339] The transmembrane domain can be derived from natural or recombinant sources. In the case of a natural source, the domain can be derived from any membrane-binding or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain whenever the CAR has bound to the target. Transmembrane domains particularly useful in this invention can comprise, for example, transmembrane domains of the following proteins: α, β, or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain may include at least, for example, transmembrane regions of the following proteins: 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, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11 a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly 9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​PAG / Cbp, NKG2D, NKG2C.

[0340] In some cases, the transmembrane domain may be connected to the extracellular region of the CAR (e.g., the antigen-binding domain of the CAR) via a hinge region (e.g., a hinge region derived from a human protein). For example, in one embodiment, the hinge region may be a human Ig (immunoglobulin) hinge region (e.g., an IgG4 hinge region, an IgD hinge region), a GS linker (e.g., the GS linker described herein), a KIR2DS2 hinge region, or a CD8a hinge region. In one embodiment, the hinge region or spacer region comprises the amino acid sequence of SEQ ID NO:14 (e.g., constitutes thereof). In one aspect, the transmembrane domain comprises the transmembrane domain of SEQ ID NO:15 (e.g., constitutes thereof).

[0341] In one aspect, the hinge region or spacer region comprises an IgG4 hinge region. For example, in one embodiment, the hinge region or spacer region comprises a hinge region having the following amino acid sequence: ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ ID NO:45). In some embodiments, the hinge region or spacer region comprises a hinge region encoded by the following nucleotide sequence:

[0342]

[0343]

[0344] In one aspect, the hinge region or spacer region comprises an IgD hinge region. For example, in one embodiment, the hinge region or spacer region comprises a hinge region having the following amino acid sequence: RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH (SEQ ID NO:47). In some embodiments, the hinge region or spacer region comprises a hinge region encoded by the following nucleotide sequence:

[0345] In one respect, the transmembrane domain can be recombined, in which case it will contain predominantly hydrophobic residues such as leucine and valine. In another respect, a phenylalanine, tryptophan, and valine triplet can be present at each end of the recombinant transmembrane domain.

[0346] Optionally, short oligomeric or polypeptide linkers between 2 and 10 amino acid lengths can form a bond between the transmembrane domain and the cytoplasmic region of the CAR. Glycine-serine duplexes provide particularly suitable linkers. For example, in one aspect, the linker comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO:49). In some embodiments, the linker is encoded by the nucleic acid sequence GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO:50).

[0347] In one respect, the hinge area or gap area includes the KIR2DS2 hinge area.

[0348] Cytoplasmic domains

[0349] The cytoplasmic domains or regions of a CAR contain intracellular signaling domains. Intracellular signaling domains are typically responsible for activating at least one of the many normal effector functions of the immune cells in which the CAR has been introduced. The term "effector function" refers to a cell's specialized function. Effector functions of T cells can be, for example, cytolytic or helper activities, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a protein portion that transduces effector function signals and directs the cell to perform its specialized function. While the entire intracellular signaling domain can often be used, in many cases it is not necessary to use the complete strand. A truncated portion of the intracellular signaling domain is used to such an extent that this truncated portion can be used in place of the complete strand, provided 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.

[0350] Examples of intracellular signal transduction domains used in the CAR of this invention include cytoplasmic sequences and co-receptors of T cell receptors (TCRs) that work synergistically to initiate signal transduction upon antigen receptor binding, as well as any derivatives or variants of these sequences and any recombinant sequences having the same functional capabilities.

[0351] It is known that signals generated by the TCR alone are insufficient to fully activate T cells and secondary and / or co-stimulatory signals are also required. Therefore, T cell activation is alleged to be mediated by two distinct classes of cytoplasmic signaling sequences: those sequences that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domains) and those sequences that function in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic domains, e.g., co-stimulatory domains).

[0352] Primary signaling domains regulate primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary intracellular signaling domains that function in a stimulatory manner may contain signaling motifs called tyrosine-based activation motifs of immune receptors or ITAMs.

[0353] Examples of ITAMs containing primary intracellular signal transduction domains that are particularly useful in this invention include those ITAMs such as CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. In one embodiment, the CAR of this invention comprises an intracellular signal transduction domain, for example, the primary signal transduction domain of CD3ζ.

[0354] In one embodiment, the primary signaling domain comprises a modified ITAM domain, such as a mutant ITAM domain having altered (e.g., increased or decreased) activity compared to the native ITAM domain. In another embodiment, the primary signaling domain comprises a primary intracellular signaling domain containing modified ITAM, such as a primary intracellular signaling domain containing optimized and / or truncated ITAM. In yet another embodiment, the primary signaling domain comprises one, two, three, four, or more ITAM motifs.

[0355] Other examples of molecules containing primary intracellular signal transduction domains that are particularly useful in this invention include those containing DAP10, DAP12, and CD32.

[0356] The intracellular signaling domain of a CAR may itself contain a CD3ζ signaling domain, or it may be combined with any other desired intracellular signaling domain useful in the context of the CAR of this invention. For example, the intracellular signaling domain of a CAR may contain a CD3ζ chain portion and a co-stimulatory signaling domain. A co-stimulatory signaling domain refers to the CAR portion containing the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, required for lymphocytes to respond effectively to antigens. 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 ligands that specifically bind to CD83. For example, CD27 co-stimulation has been shown to enhance the expansion, effector function and survival of human CAR T cells in vitro and to improve human T cell retention and antitumor activity in vivo (Song et al., Blood. 2012; 119(3):696-706). Other examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, and ITGB2. , CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), NKG2D, CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), P SGL1, 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.

[0357] Intracellular signaling sequences within the cytoplasmic portion of the CAR of the present invention can be linked together in a random or specified order. Optionally, short oligomeric or polypeptide linkers, for example, between 2 and 10 amino acid lengths (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), can form bonds between the intracellular signaling sequences. In one embodiment, a glycine-serine dinucleotide can be used as a suitable linker. In one embodiment, a single amino acid, such as alanine or glycine, can be used as a suitable linker.

[0358] In one aspect, the intracellular signal transduction domain is designed to contain two or more (e.g., 2, 3, 4, 5 or more) co-stimulatory signal transduction domains. In one embodiment, the two or more (e.g., 2, 3, 4, 5 or more) co-stimulatory signal transduction domains are separated by a linker molecule (e.g., the linker molecule described herein). In one embodiment, the intracellular signal transduction domain contains two co-stimulatory signal transduction domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0359] In one aspect, the intracellular signal transduction domain is designed to include a CD3ζ signal transduction domain and a CD28 signal transduction domain. In another aspect, the intracellular signal transduction domain is designed to include a CD3ζ signal transduction domain and a 4-1BB signal transduction domain. In another aspect, the 4-1BB signal transduction domain is the signal transduction domain of SEQ ID NO:16. In another aspect, the CD3ζ signal transduction domain is the signal transduction domain of SEQ ID NO:17.

[0360] In one aspect, the intracellular signal transduction domain is designed to include a CD3ζ signal transduction domain and a CD27 signal transduction domain. In one aspect, the CD27 signal transduction domain contains the amino acid sequence QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO:51). In one aspect, the CD27 signal transduction domain is encoded by the nucleic acid sequence AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCC CCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTA TCGCTCC (SEQ ID NO:52).

[0361] In one aspect, the CAR-expressing cells described herein may also contain a second CAR, for example, targeting the same target (CD19) or a different target (e.g., CD123 or mesothelin), such as a second CAR containing a different antigen-binding domain. In one embodiment, when the CAR-expressing cells contain two or more different CARs, the antigen-binding domains of the different CARs may be such that the antigen-binding domains do not interact with each other. For example, cells expressing first and second CARs may have the antigen-binding domain of the first CAR, for example, as a fragment that does not associate with the antigen-binding domain of the second CAR (e.g., scFv), such as the antigen-binding domain of the second CAR being VHH.

[0362] In another aspect, the CAR-expressing cells described herein may also express another substance, such as a substance that enhances the activity of CAR-expressing cells. For example, in one embodiment, this substance may be a substance that inhibits an inhibitory molecule. In some embodiments, an inhibitory molecule (e.g., PD1) may reduce the ability of CAR-expressing cells to initiate 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 TGFRβ. In one embodiment, the substance that inhibits the inhibitory molecule comprises a first polypeptide (e.g., an inhibitory molecule) that binds to a second polypeptide (e.g., an intracellular signaling domain described herein) that provides a positive signal to the cell. In one embodiment, the substance comprises, for example, 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 TGFRβ, or a fragment of any of these molecules (e.g., at least a portion of the extracellular domain of any of these molecules) and an intracellular signal transduction domain (e.g., containing a co-stimulatory domain such as 41BB, CD27 or CD28 as described herein) and / or a primary signal transduction domain. The substance 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 (e.g., the CD28 signaling domain and / or the CD3ζ signaling domain as described herein). PD1 is an inhibitory member of the CD28 receptor family, which 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). It has been shown that when two ligands of PD1, PD-L1, and PD-L2 bind to PD1, T cell activation is downregulated (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 81:281-7; 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 between PD1 and PD-L1.

[0363] In one embodiment, the substance comprises an extracellular domain (ECD) of an inhibitory molecule (e.g., programmed death 1 (PD1)) that can be fused with transmembrane domains and intracellular signaling domains such as 41BB and CD3ζ (also referred to herein as PD1CAR). In one embodiment, when used in combination with the CD19 CAR described herein, the PD1 CAR improves T cell retention. In one embodiment, the CAR is a PD1 CAR that comprises the PD1 extracellular domain as underlined in SEQ ID NO:121. In one embodiment, the PD1 CAR comprises the amino acid sequence of SEQ ID NO:121.

[0364]

[0365] In one embodiment, the PD1 CAR comprises the amino acid sequence provided below (SEQ ID NO:119).

[0366]

[0367]

[0368] In one embodiment, the substance comprises a nucleic acid sequence encoding a PD1 CAR (e.g., the PD1 CAR described herein). In one embodiment, the nucleic acid sequence of the PD1 CAR is shown below, with the PD1 ECD underlined in SEQ ID NO:120 below.

[0369]

[0370] In another aspect, the present invention provides a population of cells expressing a CAR, such as CAR-T cells. In some embodiments, the population of cells expressing a CAR comprises a mixture of cells expressing different CARs. For example, in one embodiment, the population of cells expressing a CAR may comprise a first cell expressing a CAR having an anti-CD19 binding domain as described herein and a second cell expressing a CAR having a different anti-CD19 binding domain (e.g., an anti-CD19 binding domain as described herein that differs from the anti-CD19 binding domain in the CAR expressed by the first cell). As another example, the population of cells expressing a CAR may comprise a first cell expressing a CAR containing (e.g., an anti-CD19 binding domain as described herein) and a second cell expressing a CAR containing an antigen-binding domain against a target other than CD19 (e.g., CD123). In one embodiment, the population of cells expressing a CAR comprises a first cell expressing a CAR including a primary intracellular signaling domain and a second cell expressing a CAR including a secondary signaling domain.

[0371] In another aspect, the present invention provides a cell population wherein at least one cell in the population expresses a CAR having the anti-CD19 binding domain described herein, and a second cell expresses another substance (e.g., a substance that enhances the activity of CAR-expressing cells). For example, in one embodiment, the substance may be a substance that inhibits an inhibitory molecule. In some embodiments, the inhibitory molecule may, for example, reduce the ability of CAR-expressing cells to initiate 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 TGFRβ. In one embodiment, the substance that inhibits the inhibitory molecule comprises a first polypeptide (e.g., an inhibitory molecule) that binds to a second polypeptide (e.g., an intracellular signaling domain described herein) that provides a positive signal to the cell. In one embodiment, the substance comprises, for example, 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 TGFRβ, or a fragment of any of these molecules (e.g., at least a portion of the extracellular domain of any of these molecules), and a second polypeptide as an intracellular signaling domain (e.g., comprising a co-stimulatory domain (e.g., 41BB, CD27, or CD28 as described herein) and / or a primary signaling domain (e.g., the CD3ζ signaling domain as described herein). In one embodiment, the substance 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 (e.g., the CD28 signaling domain as described herein and / or the CD3ζ signaling domain as described herein).

[0372] Regulated chimeric antigen receptor

[0373] In some embodiments, a controllable CAR (RCAR) in which CAR activity can be controlled is required to optimize the safety and efficacy of CAR therapy. Numerous ways exist to modulate CAR activity. For example, an inducible apoptosis process using, for example, caspase fused with a dimerizing 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 switching switch in CAR therapy of the present invention. In one embodiment, cells expressing the CAR of the present invention (e.g., T cells or NK cells) also contain an inducible apoptosis switching switch in which a human caspase (e.g., caspase 9) or a modified form is fused with a human FKB protein modification that allows conditional dimerization. In the presence of small molecules (e.g., rapamycin analogs (e.g., AP 1903, AP20187)), the inducible caspase (e.g., caspase 9) is activated and leads to rapid apoptosis and death of cells expressing the CAR of the present invention (e.g., T cells or NK cells). Examples of caspase-based induced apoptosis switching switches (or one or more aspects of such switching switches) have been described, for example, in US2004040047; US20110286980; US20140255360; WO 1997031899; WO2014151960; WO 2014164348; WO 2014197638; WO 2014197638; all of which are incorporated herein by reference.

[0374] In one aspect, an RCAR comprises a group of peptides, typically two peptides in the simplest embodiment, wherein the components of a standard CAR described herein (e.g., an antigen-binding domain and an intracellular signaling domain) are distributed on separate peptides or constructs. In some embodiments, the group of peptides includes a dimerization switch, which, in the presence of a dimerizing molecule, allows the peptides to couple to each other, for example, allowing the antigen-binding domain to couple to the intracellular signaling domain. In one embodiment, the CAR of the present invention utilizes dimerization switches, such as those described, for example, in WO 2014127261, which is incorporated herein by reference.

[0375] In one aspect, the RCAR comprises two polypeptides or components: 1) an intracellular signaling component comprising an intracellular signaling domain (e.g., a primary intracellular signaling domain as described herein) and a first switching domain; and 2) an antigen-binding component comprising an antigen-binding domain as described herein (e.g., targeting CD19) and a second switching domain. Optionally, the RCAR comprises a transmembrane domain as described herein. In one embodiment, the transmembrane domain may be disposed on the intracellular signaling component, on the antigen-binding component, or both. (Unless otherwise stated, the order of components or elements of the RCAR described herein may be as provided, but other orders may also be included). In other words, in one embodiment, the order is as described herein, but in other embodiments, the order may be different. For example, the order of the elements on one side of the transmembrane region may differ from the examples; for example, the placement of the switching domain relative to the intracellular signaling domain may be different, for example, inverted.

[0376] In one embodiment, the first switching domain and the second switching domain can form an intracellular or extracellular dimerizing switching switch. In one embodiment, the dimerizing switching switch can be a homodimerizing switching switch, for example, where the first switching domain and the second switching domain are identical, or a heterodimerizing switching switch, for example, where the first switching domain and the second switching domain are different from each other.

[0377] In implementations, the RCAR may include "multiple switching domains." Multiple switching domains may include heterodimerizing switching domains or homodimerizing switching domains. The multiple switching domains independently include multiple switching domains, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10, on both the first component (e.g., an antigen-binding component) and the second component (e.g., an intracellular signaling component). In one implementation, the first component may include multiple first switching domains, such as FKBP-based switching domains, and the second component may include multiple second switching domains, such as FRB-based switching domains. In another implementation, the first component may include both first and second switching domains, such as an FKBP-based switching domain and an FRB-based switching domain, and the second component may include both first and second switching domains, such as an FKBP-based switching domain and an FRB-based switching domain.

[0378] In one embodiment, the intracellular signal transduction member includes one or more intracellular signal transduction domains (e.g., primary intracellular signal transduction domains) and one or more co-stimulatory signal transduction domains.

[0379] In one embodiment, the antigen-binding member may include one or more intracellular signaling domains, such as one or more co-stimulatory signaling domains. In one embodiment, the antigen-binding member includes multiple, such as two or three co-stimulatory signaling domains described herein, such as co-stimulatory signaling domains selected from 41BB, CD28, CD27, ICOS, and OX40, and in several embodiments, does not include a primary intracellular signaling domain. In one embodiment, the antigen-binding member includes the following co-stimulatory signaling domains from 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 includes a CD3ζ domain. In such an implementation, the RCAR includes (1) an antigen-binding component comprising an antigen-binding domain, a transmembrane domain, two co-stimulatory domains, and a first switching domain; and (2) an intracellular signal transduction domain comprising a transmembrane domain or a membrane-bound domain and at least one primary intracellular signal transduction domain and a second switching domain.

[0380] One embodiment provides an RCAR in which the antigen-binding component is not tethered to the surface of the CAR cell. This allows cells with intracellular signaling components to conveniently pair with one or more antigen-binding domains without transforming cells using a sequence encoding the antigen-binding component. In such embodiments, the RCAR comprises: 1) an intracellular signaling component comprising: a first switching domain, a transmembrane domain, an intracellular signaling domain (e.g., a primary intracellular signaling domain), and a first switching domain; and 2) an antigen-binding component comprising: an antigen-binding domain and a second switching domain, wherein the antigen-binding component does not contain a transmembrane domain or a membrane-bound domain and optionally does not contain an intracellular signaling domain. In some embodiments, the RCAR may also comprise 3) a second antigen-binding component comprising a second antigen-binding domain, e.g., a second antigen-binding domain that binds to a different antigen bound by the antigen-binding domain; and a second switching domain.

[0381] This document also provides RCARs in which the antigen-binding component includes bispecific activation and targeting capabilities. In this embodiment, the antigen-binding component may include multiple, for example, 2, 3, 4, or 5 antigen-binding domains, such as scFv, wherein each antigen-binding domain binds to a target antigen, such as a different antigen or the same antigen, for example, the same or different epitopes on the same antigen. In one embodiment, multiple antigen-binding domains are tandem, and optionally, a linker or hinge region is arranged between each antigen-binding domain. Suitable linkers and hinge regions are described herein.

[0382] One embodiment provides an RCAR with a layout that allows for proliferative transformation. In this embodiment, the RCAR comprises: 1) an intracellular signaling component comprising: optionally, a transmembrane domain or a membrane-bound domain; one or more co-stimulatory signaling domains (e.g., selected from 41BB, CD28, CD27, ICOS, and OX40), and a switching domain; and 2) an antigen-binding component comprising: an antigen-binding domain, a transmembrane domain, and a primary intracellular signaling domain (e.g., a CD3ζ domain), wherein the antigen-binding component does not contain a switching domain or does not contain a switching domain dimerized with a switching domain on the intracellular signaling component. In one embodiment, the antigen-binding component does not contain a co-stimulatory signaling domain. In one embodiment, the intracellular signaling component contains a switching domain from a homodimerized switching domain. In one embodiment, the intracellular signaling member includes a first switching domain of a heterodimerization switching switch, and the RCAR includes a second intracellular signaling member that includes a second switching domain of a heterodimerization switching switch. In such embodiments, the second intracellular signaling member includes the same intracellular signaling domain as the intracellular signaling member. In one embodiment, the dimerization switching switch is intracellular. In one embodiment, the dimerization switching switch is extracellular.

[0383] In any RCAR layout described herein, the first and second transfer switch structure domains contain transfer switches based on FKBP-FRB as described herein.

[0384] This document also provides cells containing the RCAR described herein. Any cell engineered to express 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.

[0385] This document also provides nucleic acids and vectors containing sequences encoding RCARs. Sequences encoding multiple elements of the RCAR can be placed on the same nucleic acid molecule, for example, the same plasmid or vector, such as a viral vector, for example, a lentiviral vector. In one embodiment, (i) the sequence encoding an antigen-binding component and (ii) the sequence encoding an intracellular signaling component can be present on the same nucleic acid (e.g., a vector). The production of the respective proteins can be achieved, for example, by using separate promoters, or by using bicistronic transcripts (which can lead to the production of two proteins by cleaving a single translation product or by translating two separate protein products). In one embodiment, the sequence encoding a cleavable peptide (e.g., a P2A or F2A sequence) is arranged between (i) and (ii). In one embodiment, the sequence encoding an IRES (e.g., an EMCV IRES or an EV71 IRES) is arranged between (i) and (ii). In these embodiments, (i) and (ii) are transcribed into a single RNA. In one implementation, the first promoter is effectively linked to (i) and the second promoter is effectively linked to (ii), thereby transcribing (i) and (ii) into independent mRNAs.

[0386] Alternatively, sequences encoding multiple elements of the RCAR can be set on different nucleic acid molecules, such as different plasmids or vectors, such as viral vectors, for example, lentiviral vectors. For example, (i) a sequence encoding an antigen-binding component can be present on a first nucleic acid (e.g., a first vector), and (ii) a sequence encoding an intracellular signal transduction component can be present on a second nucleic acid (e.g., a second vector).

[0387] Dimerization switching

[0388] Dimerization switching can be non-covalent or covalent. In non-covalent dimerization switching, the dimerizing molecules promote non-covalent interactions between the switching domains. In covalent dimerization switching, the dimerizing molecules promote covalent interactions between the switching domains.

[0389] In one embodiment, the RCAR comprises a dimerization switching switch based on FKBP / FRAP or FKBP / FRB. FKBP12 (FKBP, or FK506 binding protein) is a rich cytoplasmic protein that serves as the initial intracellular target of the natural product (the immunosuppressive drug rapamycin). Rapamycin binds to FKBP and also to the large PI3K homolog FRAP (RAFT, mTOR). FRB is the 93-amino acid moiety of FRAP, which is sufficient to bind the FKBP-rapamycin complex (Chen, J., Zheng, XF, Brown, EJ, and Schreiber, SL (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 USA 92:4947-51).

[0390] In implementations, the FKBP / FRAP-based (e.g., FKBP / FRB-based) switching device may use dimerized molecules, such as rapamycin or rapamycin analogues.

[0391] The amino acid sequence of FKBP is as follows:

[0392]

[0393] In an implementation, the FKBP switching domain may include an FKBP fragment having the ability to bind to an FRB or fragment or its analogue in the presence of rapamycin or a rapamycin analogue, for example, the underlined portion of SEQ ID NO:122, which is:

[0394]

[0395] The amino acid sequence of FRB is as follows:

[0396]

[0397] As used herein, “FKBP / FRAP-based (e.g., FKBP / FRB-based) switching switch” refers to a dimerized switching switch comprising a first switching domain and a second switching domain, wherein the first switching domain comprises an FKBP fragment or analogue having the ability to bind to an FRB or fragment or analogue thereof in the presence of rapamycin or a rapamycin analogue (e.g., RAD001) and having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or difference of no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues with respect to the FKBP sequence of SEQ ID NO: 122 or 123; and the second switching domain comprises an FRB fragment or analogue having the ability to bind to an FRB or fragment or analogue thereof in the presence of rapamycin or a rapamycin analogue ... The FRB sequence of NO:124 has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or difference of no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues. In one embodiment, the RCAR described herein comprises a switching domain containing amino acid residues disclosed in SEQ ID NO:122 (or SEQ ID NO:123) and a switching domain containing amino acid residues disclosed in SEQ ID NO:124.

[0398] In one embodiment, the FKBP / FRB dimerization switch comprises a modified FRB switch domain exhibiting altered (e.g., enhanced) complex formation between the FRB-based switch domain (e.g., a modified FRB switch domain, an FKBP-based switch domain) and the dimerizing molecule (e.g., rapamycin or a rapamycin analog, such as 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 amino acid positions L2031, E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105, and F2108, wherein the wild-type amino acid is mutated to any other naturally occurring amino acid. In one embodiment, the mutant FRB comprises a mutation at E2032, wherein E2032 is mutated to phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I) (e.g., SEQ ID NO:125) or leucine (E2032L) (e.g., SEQ ID NO:126). In one embodiment, the mutant FRB comprises a mutation at T2098, wherein T2098 is mutated to phenylalanine (T2098F) or leucine (T2098L), for example, SEQ ID NO:127. In one embodiment, the mutant FRB comprises mutations at both E2032 and T2098, wherein E2032 is mutated to any amino acid and wherein T2098 is mutated to any amino acid, for example, SEQ ID NO:128. In one embodiment, the mutated FRB comprises E2032I and T2098L mutations, for example, SEQ ID NO:129. In another embodiment, the mutated FRB comprises E2032L and T2098L mutations, for example, SEQ ID NO:130.

[0399] Table 13. Exemplary mutant FRBs with increased affinity for dimerizing molecules.

[0400]

[0401] Other suitable dimerization switches include GyrB-GyrB-based dimerization switches, gibberellin-based dimerization switches, tag / conjugate dimerization switches, and halogen tag / snap tag dimerization switches. These types of switches and the associated dimerization molecules will be readily apparent to those skilled in the art, following the guidance provided herein.

[0402] dimer molecules

[0403] Association between switching domains is initiated by dimerizing molecules. In the presence of dimerizing molecules, interactions or associations between switching domains allow signal transduction between peptides bound to (e.g., fused to) a first switching domain and peptides bound to (e.g., fused to) a second switching domain. In the presence of unrestricted levels of dimerizing molecules, 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, and 100-fold, for example, as measured in the systems described herein.

[0404] Rapamycin and rapamycin analogues (sometimes referred to as rapamycin analogues), such as RAD001, can be used as dimerizing molecules in the FKBP / FRB-based dimerization switching devices described herein. In one embodiment, the dimerizing molecule may be selected from rapamycin (sirolimus), RAD001 (everolimus), zotarolimus, tamsulosin, AP-23573 (desfolimus), biolimus, and AP21967. Additional rapamycin analogues suitable for use with the FKBP / FRB-based dimerization switching devices are further described in the section entitled “Combination Therapies” or in the subsection entitled “Exemplary mTOR Inhibitors”.

[0405] Split CAR

[0406] In some implementations, the cells expressing CARs use split CARs. The split CAR protocol is described in more detail in publications WO 2014 / 055442 and WO 2014 / 055657. In short, a split CAR system comprises cells expressing a first CAR having a first antigen-binding domain and a co-stimulatory domain (e.g., 41BB), and the cells also expressing a second CAR having a second antigen-binding domain and an intracellular signaling domain (e.g., CD3ζ). When the cells encounter the first antigen, the co-stimulatory domain is activated and the cells proliferate. When the cells encounter the second antigen, the intracellular signaling domain is activated and cytotoxic activity is initiated. Thus, CAR-expressing cells are fully activated only in the presence of both antigens.

[0407] RNA transfection

[0408] This document discloses a method for generating in vitro transcribed RNA CARs. The invention also includes CARs encoding RNA constructs that can be directly transfected into cells. One method for generating mRNA for transfection may involve in vitro transcription (IVT) of a template with specially designed primers, followed by the addition of polyadenylation to generate a construct, typically 50-2000 bases in length (SEQ ID NO: 118), containing 3' and 5' untranslated sequences (“UTR”), a 5' cap and / or an internal ribosome entry site (IRES), the nucleic acid to be expressed, and a polyadenylated tail. The resulting RNA can be efficiently transfected into various cell types. In one aspect, the template includes the sequence of the CAR.

[0409] In one aspect, the anti-CD19 CAR is encoded by messenger RNA (mRNA). In another aspect, the mRNA encoding the anti-CD19 CAR is introduced into immune effector cells, such as T cells or NK cells, to generate CAR-expressing cells, such as CAR-T cells or CAR NK cells.

[0410] In one embodiment, an in vitro transcribed RNA CAR can be introduced into cells as a transient transfection. RNA is produced via in vitro transcription using a template generated by polymerase chain reaction (PCR). Target DNA from any source can be directly converted into a template by PCR to synthesize mRNA in vitro using suitable primers and RNA polymerase. The DNA source can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, a synthetic DNA sequence, or any other suitable DNA source. The desired in vitro transcription template is the CAR of the present invention. For example, the template of the RNA CAR comprises an extracellular region containing a single-stranded variable domain of an antitumor antibody; a hinge region; a transmembrane domain (e.g., the transmembrane domain of CD8a); and a cytoplasmic region containing intracellular signal transduction domains (e.g., signal transduction domains containing CD3ζ and 4-1BB).

[0411] In one embodiment, the DNA to be used for PCR contains a read frame. The DNA may be a naturally occurring DNA sequence derived from an organism's genome. In one embodiment, the nucleic acid may include some or all of a 5' untranslated region and / or a 3' untranslated region (UTR). The nucleic acid may 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 a 5' UTR and a 3' UTR. The DNA may alternatively be an artificial DNA sequence that is not normally expressed in naturally occurring organisms. An exemplary artificial DNA sequence is an artificial DNA sequence containing gene portions, wherein said gene portions are linked together to form a read frame encoding a fusion protein. The linked DNA portions may be from a single organism or from more than one organism.

[0412] PCR is used to generate a template for in vitro transcription of mRNA for transfection. Methods for performing PCR are well known in the art. Primers used in PCR are designed to have regions substantially complementary to a region of the DNA to be used as a PCR template. As used herein, “substantially complementary” means a nucleotide sequence in which most or all of the bases in the primer sequence are complementary or one or more bases are non-complementary or mismatched. Substantially complementary sequences are capable of annealing or hybridizing with the intended DNA target 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 a portion of nucleic acid (read frame) transcribed normally in cells, including the 5' and 3' UTRs. Primers can also be designed to amplify a portion of nucleic acid encoding a specific target domain. In one embodiment, primers are designed to amplify the coding region of human cDNA, including all or part of the 5' and 3' UTRs. Primers that can be used for PCR can be produced by synthetic methods well known in the art. A “forward primer” is a primer containing a nucleotide region substantially complementary to the nucleotides upstream of the DNA sequence to be amplified on the DNA template. "Upstream" in this document refers to the 5' position relative to the coding strand of the DNA sequence to be amplified. "Reverse primer" is a primer containing a nucleotide region substantially complementary to the double-stranded DNA template downstream of the DNA sequence to be amplified. "Downstream" in this document refers to the 3' position relative to the coding strand of the DNA sequence to be amplified.

[0413] Any DNA polymerase that can be used for PCR can be used in the methods disclosed herein. Reagents and polymerases are commercially available from numerous sources.

[0414] Chemical constructs with the ability to promote stability and / or translation efficiency can also be used. The RNA preferably has a 5'UTR and a 3'UTR. In one embodiment, the 5'UTR length is between 1 and 3000 nucleotides. The lengths of the 5'UTR and 3'UTR sequences to be added to the coding region can be varied by various methods, including but not limited to designing PCR primers that anneal to different regions of the UTR. Using this method, those skilled in the art can adjust the 5'UTR and 3'UTR lengths to the lengths required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0415] The 5'UTR and 3'UTR can be naturally occurring, endogenous 5'UTR and 3'UTR of the target nucleic acid. Alternatively, UTR sequences that are not endogenous relative to the target nucleic acid can be added by incorporating the UTR sequence into the forward and reverse primers or by any other modification to the template. The use of UTR sequences that are not endogenous relative to the target nucleic acid can be to modulate RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in the 3'UTR sequence can reduce mRNA stability. Therefore, based on UTR characteristics well known in the art, the 3'UTR can be selected or designed to increase the stability of transcribed RNA.

[0416] In one embodiment, the 5' UTR may contain a Kozak sequence of an endogenous nucleic acid. Alternatively, when a 5' UTR that is not endogenous relative to the target nucleic acid is being added via PCR as described above, a shared Kozak sequence can be redesigned by adding a 5' UTR sequence. Kozak sequences can increase the translation efficiency of some RNA transcripts, but do not appear to be necessary for the efficient translation of all RNA. Many mRNAs are known in the art to require Kozak sequences. In other embodiments, the 5' UTR may be the 5' UTR of an RNA virus whose RNA genome is stable in the cell. In other embodiments, various nucleotide analogs may be used in the 3' or 5' UTR to inhibit exonuclease degradation of the mRNA.

[0417] To enable RNA synthesis from a DNA template without gene cloning, a transcription promoter should be attached upstream of the sequence to be transcribed. When the sequence acting as an RNA polymerase promoter is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the read frame to be transcribed. In a preferred embodiment, the promoter is the T7 polymerase promoter, as described elsewhere herein. Other available promoters include, but are not limited to, the T3 RNA polymerase promoter and the SP6 RNA polymerase promoter. The common nucleotide sequences of the T7, T3, and SP6 promoters are known in the art.

[0418] In a preferred embodiment, the mRNA has caps at both the 5' end and the 3' polyadenylated tail that determine ribosome binding, translation initiation, and mRNA stability in the cell. On circular DNA templates, such as plasmid DNA, RNA polymerase produces long, cascaded products unsuitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR end produces normally sized mRNA that is not efficient in eukaryotic transfection, even if it undergoes polyadenylation post-transcriptionally.

[0419] On 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).

[0420] The conventional method for integrating polyadenylated nucleotide / T fragments into DNA templates is molecular cloning. However, the polyadenylated nucleotide / T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning methods not only cumbersome and time-consuming but also frequently unreliable. This is why there is an urgent need for methods that allow the construction of DNA templates with polyadenylated nucleotide / T 3′ fragments without cloning.

[0421] Polyadenylated / T segments of transcribed DNA templates can be generated during PCR using a reverse primer containing a polyadenylated tail (e.g., a 100T tail (SEQ ID NO: 110) (size can be 50-5000T (SEQ ID NO: 111)) or after PCR by any other method (including but not limited to DNA ligation or in vitro recombination). The polyadenylated tail also provides stability to RNA and reduces its degradation. Generally, the length of the polyadenylated tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the polyadenylated tail is between 100 and 5000 adenosine (SEQ ID NO: 112).

[0422] The polyadenylated tail of RNA can be further elongated post-transcriptionally using polyadenylated polymerases (such as E. coli polyadenylated polymerase (E-PAP)). In one embodiment, increasing the length of the polyadenylated tail from 100 nucleotides to between 300 and 400 nucleotides (SEQ ID NO: 113) results in approximately a two-fold increase in RNA translation efficiency. Additionally, conjugating different chemical groups to the 3' end can increase mRNA stability. This conjugation can contain modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the polyadenylated tail using polyadenylated polymerase. ATP analogs can also increase RNA stability.

[0423] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein contains a 5' cap. The 5' cap 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)).

[0424] The RNA produced by the methods disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates ribosome-independent binding to the mRNA and promotes translation initiation. It may contain any solute suitable for cell electroporation, and the solute may contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0425] RNA can be introduced into target cells using any of a number of different methods, including but not limited to the following commercially available methods: electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipid transfection, polymer encapsulation, peptide-mediated transfection, or biological projectile particle delivery systems such as “gene guns” (see, for example, Nishikawa et al., Hum Gene Ther., 12(8):861-70 (2001).

[0426] Non-viral delivery methods

[0427] In some respects, non-viral methods can be used to deliver nucleic acids encoding the CAR described herein into cells or tissues or subjects.

[0428] In some implementations, non-viral methods include the use of transposons (also called transposable elements). In some implementations, a transposon is a segment of DNA that can insert itself into a location in the genome; for example, it may be a segment of DNA capable of self-replication and inserting a copy into the genome, or it may be a segment of DNA spliced ​​from a longer nucleic acid and inserted into another location in the genome. For example, a transposon comprises a DNA sequence consisting of inverted repeat sequences distributed flanking the gene used for transposition.

[0429] Exemplary methods for delivering nucleic acids using transposons include the Sleeping Beauty transposon system (SBTS) and the piggyBac (PB) transposon system. 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.

[0430] SBTS comprises two components: 1) a transposon containing the transgene and 2) a transposase source. Transposases transfer the transposon from a vector plasmid (or other donor DNA) to target DNA, such as the host cell chromosome / genome. For example, a transposase binds to a vector plasmid / donor DNA, cuts the transposon (containing the transgene) from the plasmid, and inserts it into the host cell's genome. See, for example, Aronovich et al., above.

[0431] Exemplary transposons include pT2-based transposons. See, for example, Grabundzija et al., NucleicAcids 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 Tc1 / sailor-type transposases, such as SB10 or SB11 transposases (which may be, for example, overactive transposases expressed from cytomegalovirus promoters). See, for example, Aronovich et al.; Kebriaei et al.; and Grabundzija et al., all of which are incorporated herein by reference.

[0432] The use of SBTS allows for the efficient integration and expression of transgenes, such as nucleic acids encoding the CARs described herein. Methods are provided herein for generating cells (e.g., T cells or NK cells) that stably express the CARs described herein, for example, using transposon systems such as SBTS.

[0433] According to the methods described herein, in some embodiments, one or more nucleic acids (e.g., plasmids) containing SBTS components are delivered to cells (e.g., T cells or NK cells). For example, the nucleic acids are delivered using standard nucleic acid (e.g., plasmid DNA) delivery methods (e.g., the methods described herein, such as electroporation, transfection, or liposome transfection). In some embodiments, the nucleic acid contains a transposon comprising a transgene (e.g., a nucleic acid encoding the CAR described herein). In some embodiments, the nucleic acid contains a transposon comprising a transgene (e.g., a nucleic acid encoding the CAR described herein) and a nucleic acid sequence encoding a transposase. In other embodiments, two nucleic acids are provided to a system (e.g., a dual-plasmid system), for example, where the first plasmid contains a transposon comprising a transgene and the second plasmid contains a nucleic acid sequence encoding a transposase. For example, the first and second nucleic acids are co-delivered into a host cell.

[0434] In some implementations, cells expressing the CAR described herein (e.g., T cells or NK cells) are generated by using a combination of gene insertion with SBTS and genetic editing with nucleases (e.g., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR / Cas systems or engineered meganucleases, reengineered homing endonucleases).

[0435] In some implementations, the use of non-viral delivery methods allows for the reprogramming of cells (e.g., T cells or NK cells) and direct infusion of cells into the subject. Advantages of non-viral vectors include, but are not limited to, the convenience and relatively low cost of producing sufficient quantities to meet the needs of the patient population, stability during storage, and lack of immunogenicity.

[0436] Nucleic acid constructs encoding CAR

[0437] The present invention also provides nucleic acid molecules encoding one or more CAR constructs described herein. In one aspect, the nucleic acid molecules are provided as messenger RNA transcripts. In another aspect, the nucleic acid molecules are provided as DNA constructs.

[0438] Therefore, in one aspect, the present invention relates to isolated nucleic acid molecules encoding chimeric antigen receptors (CARs), wherein the CAR comprises an anti-CD19 binding domain (e.g., a humanized anti-CD19 binding domain), a transmembrane domain, and an intracellular signaling domain, said intracellular signaling domain comprising a stimulatory domain (e.g., a co-stimulatory signaling domain) and / or a primary signaling domain (e.g., a ζ-chain). In one embodiment, the anti-CD19 binding domain is the anti-CD19 binding domain described herein, for example, comprising 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, SEQ ID NO:12, and SEQ ID NO:59, or a sequence having 95-99% identity with such sequences. In one embodiment, the transmembrane domain is a transmembrane domain selected from the following proteins: the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO:15 or a sequence having 95-99% identity with it. In one embodiment, the anti-CD19 binding domain is connected to the transmembrane domain via a hinge region (e.g., the hinge region described herein). In one embodiment, the hinge region comprises a sequence of SEQ ID NO:14, SEQ ID NO:45, SEQ ID NO:47, or SEQ ID NO:49 or a sequence having 95-99% identity with it. In one embodiment, the isolated nucleic acid molecule also comprises a sequence encoding a co-stimulatory domain. In one embodiment, the co-stimulatory domain comprises a functional signal transduction domain selected from the proteins OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO:16 or a sequence having 95-99% identity with it. In one embodiment, the intracellular signal transduction domain comprises a functional signal transduction domain of 4-1BB and a functional signal transduction domain of CD3ζ.In one embodiment, the intracellular signal transduction domain comprises the sequence of SEQ ID NO:16 or SEQ ID NO:51 or a sequence having 95-99% identity with it and the sequence of SEQ ID NO:17 or SEQ ID NO:43 or a sequence having 95-99% identity with it, wherein the sequences comprising the intracellular signal transduction domain are expressed in the same reading frame and as a single polypeptide chain.

[0439] In another aspect, the present invention relates to isolated nucleic acid molecules encoding CAR constructs, said CAR constructs comprising a leader sequence of SEQ ID NO:13, an scFv domain having 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, SEQ ID NO:12 and SEQ ID NO:59 (or sequences having 95-99% identity with them), a hinge region having SEQ ID NO:14 or SEQ ID NO:45 or SEQ ID NO:47 or SEQ ID NO:49 (or sequences having 95-99% identity with them), a transmembrane domain having a sequence having SEQ ID NO:15 (or sequences having 95-99% identity with them), a 4-1BB co-stimulatory domain having a sequence having SEQ ID NO:16, or a scFv domain having a sequence having SEQ ID NO:13. The CD27 co-stimulatory domain of the sequence ID NO:51 (or a sequence having 95-99% identity with it) and the CD3ζ stimulatory domain of the sequence ID NO:17 or SEQ ID NO:43 (or a sequence having 95-99% identity with it).

[0440] In another aspect, the present invention relates to isolated polypeptide molecules encoded by the nucleic acid molecule. In one embodiment, the isolated polypeptide molecule comprises a sequence selected from 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 having 95-99% identity with such sequences.

[0441] In another aspect, the present invention relates to a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule comprising an anti-CD19 binding domain, a transmembrane domain, and an intracellular signal transduction domain comprising a stimulatory domain, wherein the 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, SEQ ID NO:12, and SEQ ID NO:59 or a sequence having 95-99% identity with such sequences.

[0442] In one embodiment, the encoded CAR molecule further comprises a sequence encoding a co-stimulatory domain. In one embodiment, the co-stimulatory domain comprises a functional signal transduction domain selected from the proteins OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137). In one embodiment, the co-stimulatory domain comprises the sequence of SEQ ID NO:16. In one embodiment, the transmembrane domain is a transmembrane domain selected from the proteins α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO:15. In one embodiment, the intracellular signal transduction domain comprises a functional signal transduction domain of 4-1BB and a functional signal transduction domain of ζ. In one embodiment, the intracellular signal transduction domain comprises the sequences of SEQ ID NO:16 and SEQ ID NO:17, wherein the sequences comprising the intracellular signal transduction domain are expressed in the same read frame and as a single polypeptide chain. In one embodiment, the anti-CD19 binding domain is connected to the transmembrane domain via 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, SEQ ID NO:47, or SEQ ID NO:49.

[0443] In another aspect, the present invention relates to an encoded CAR molecule comprising a leader sequence of SEQ ID NO:13, an scFv domain having a sequence selected from or having 95-99% identity with the following sequences: 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; 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 co-stimulatory domain having a sequence of SEQ ID NO:16 or a CD27 co-stimulatory domain having a sequence of SEQ ID NO:51, and a CD3ζ 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 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 having 95-99% identity with such sequences.

[0444] The nucleic acid sequence encoding the desired molecule can be obtained using recombination methods known in the art, such as screening a library from cells expressing the gene, deriving the gene from a vector known to contain the gene, or directly isolating the gene from cells and tissues containing the gene using standard techniques. Alternatively, the target gene can be produced synthetically rather than by cloning.

[0445] This invention also provides vectors in which the DNA of this invention is inserted. Vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow for the long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have the added advantage of being superior to vectors derived from oncoretroviruses (such as murine leukemia virus) in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. Retroviral vectors can also be, for example, gamma retroviral vectors. Gamma retroviral vectors may, for example, contain a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a target transgene, such as a gene encoding a CAR. Gamma retroviral vectors may lack viral structural genes such as gag, pol, and env. Exemplary gamma retroviral vectors include murine leukemia virus (MLV), spleen focal formation virus (SFFV), and myeloproliferative sarcoma virus (MPSV) and vectors derived from them. Other gamma retroviral vectors are described, for example, in Tobias Maetzig et al., “Gamma retroviral Vectors: Biology, Technology and Application” Viruses. 2011 Jun; 3(6): 677–713.

[0446] In another embodiment, the vector containing the nucleic acid encoding the CAR required by the present invention is an adenoviral vector (A5 / 35). In another embodiment, the expression of the nucleic acid encoding the CAR can be accomplished using transposons such as Sleeping Beauty, Crisper, CAS9, and zinc finger nucleases. See below June et al., 2009 Nature Reviews Immunology 9.10:704-716, which is incorporated herein by reference.

[0447] In short, the expression of natural or synthetic nucleic acids encoding CARs is generally achieved by efficiently linking the nucleic acid encoding the CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector is suitable for replication and integration in eukaryotes. Common cloning vectors contain transcription and translation terminators, initiation sequences, and promoters to regulate the expression of the desired nucleic acid sequence.

[0448] Using standard gene delivery protocols, the expression constructs of the present invention can also be used for nucleic acid immunotherapy and gene therapy. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0449] Nucleic acids can be cloned into a wide variety of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and entrapments. Vectors with specific applications include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0450] Alternatively, expression vectors can be delivered to cells in the form of viral vectors. 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 and molecular biology handbooks. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, suitable vectors contain a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0451] Numerous virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered in vivo or in vitro to the cells of a recipient. Numerous retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Numerous adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0452] Additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. These are typically located in a region 30–110 bp upstream of the start site, although many promoters have been shown to also contain functional elements downstream of the start site. The spacing between promoter elements tends to be flexible, allowing promoter function to be preserved when elements are reversed or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline. Depending on the promoter, the individual elements appear to function synergistically or independently to activate transcription. Exemplary promoters include the CMV IE gene promoter, the EF-1α promoter, the ubiquitous protein C promoter, or the glycerol phosphokinase (PGK) promoter.

[0453] An example of a promoter capable of expressing CAR transgenes in mammalian T cells is the EF1a promoter. The native EF1a promoter drives the expression of the α subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to the ribosome. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to effectively drive CAR expression from clone to lentiviral vector. See, for example, Milone et al., Mol. Ther. 17(8):1453–1464 (2009). In one aspect, the EF1a promoter contains the sequence provided as SEQ ID NO:100.

[0454] Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a constitutive strong promoter sequence capable of driving high-level expression of any polynucleotide sequence effectively linked to it. However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, elongation factor-1 promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also conceived as part of the present invention. The use of inducible promoters provides a molecular switching switch capable of initiating expression of the polynucleotide sequence effectively linked to it when such expression is needed or shutting down expression when such expression is not needed. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0455] The vector may also include, for example, a secretion-promoting signal sequence, a polyadenylation signal and a transcription terminator (e.g., from the bovine growth hormone (BGH) gene), elements that allow for additional body replication and replication in prokaryotes (e.g., the SV40 replication origin and ColE1 or other elements known in the art), and / or elements that allow for selection (e.g., ampicillin resistance genes and / or bleomycin markers).

[0456] To assess the expression of the CAR peptide or a portion thereof, the expression vector to be introduced into cells may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of cells expressing the CAR peptide from a population of cells seeking transfection or infection with a viral vector. In other respects, the selectable marker may be carried on a separate DNA fragment and used in co-transfection methods. The selectable marker and reporter gene may be flanked by suitable regulatory sequences capable of achieving expression in the host cell. Available selectable markers include, for example, antibiotic resistance genes, such as neo.

[0457] Reporter genes are used to identify cells that may be transfected and to evaluate the function of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and encodes a polypeptide whose expression is indicated by some easily detectable property (e.g., enzyme activity). The expression of the reporter gene is determined at an appropriate time after DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well-known and can be prepared using known techniques or are commercially available. Typically, a construct with at least a 5′ flanking region that will show the highest reporter gene expression level is identified as a promoter. Such promoter regions can be linked to reporter genes and used to evaluate the ability of various substances to regulate promoter-driven transcription.

[0458] Methods for introducing and expressing genes into cells are known in the art. In the case of expression vectors, the vectors can be readily introduced into host cells (e.g., mammalian cells, bacterial cells, yeast cells, or insect cells) by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0459] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, and electroporation. Methods for generating cells containing 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 polynucleotides into host cells is calcium phosphate transfection.

[0460] Biological methods for introducing target polynucleotides into host cells include the use of DNA vectors and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells (e.g., human cells). Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0461] Chemical approaches for introducing polynucleotides into host cells include colloidal dispersions 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 used as a delivery vehicle in vitro and in vivo is the liposome (e.g., an artificial membrane vesicle). Other methods for targeted delivery of nucleic acids are available, such as the delivery of polynucleotides using guided nanoparticles or other suitable submicron-scale delivery systems.

[0462] In the case of using non-viral delivery systems, an exemplary delivery vehicle is the liposome. The concept is to use lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, nucleic acids can bind to lipids. Lipid-bound nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, linked to liposomes by linkers that bind to both liposomes and oligonucleotides, embedded in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in micelles, or complexed with micelles, or otherwise bound to lipids. Compositions bound to lipids, lipid / DNA, or lipid / expression vectors are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or in a “collapsed” structure. They can also simply be dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances that can be naturally occurring lipids or synthetic lipids. For example, lipids include fat droplets that naturally occur in the cytoplasm, as well as compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0463] Lipids suitable for use are available from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) is available from Sigma, St. Louis, MO; diceryl phosphate (“DCP”) is available from K&K Laboratories (Plainview, NY); cholesterol (“Choi”) is available from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL.). The mother liquor of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. “Liposome” is a generic term encompassing a variety of single and multilayer lipid carriers formed by the production of closed lipid bilayers or aggregates. Liposomes can be characterized as vesicle structures with a phospholipid bilayer and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They spontaneously form liposomes when phospholipids are suspended in excess aqueous solution. Lipid components undergo self-rearrangement before forming a closed structure, entraining water and dissolved solutes between lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, this also encompasses compositions with structures in solution that differ from normal vesicle structures. For example, lipids can take the form of micelles or exist simply as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes have also been conceived.

[0464] Regardless of whether this method is used to introduce exogenous nucleic acids into host cells or otherwise expose cells to the inhibitors of the present invention, various assays can be performed to confirm the presence of recombinant DNA sequences in host cells. Such assays include, for example, "molecular biological analyses" well known to those skilled in the art, such as DNA blotting and RNA blotting, RT-PCR and PCR; and "biochemical" analyses, such as detecting the presence or absence of specific peptides, for example, identifying substances within the scope of the present invention by immunoassays (ELISA and Western blotting) or by the assays described herein.

[0465] The present invention also provides a vector comprising a nucleic acid molecule encoding a CAR. In one aspect, the CAR vector can be directly introduced into cells, such as T cells. In one aspect, the vector is a cloning vector or an expression vector, for example, the vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, microcircles, microvectors, double microchromosomes), retroviral constructs, 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.

[0466] Cell source

[0467] Prior to amplification and genetic modification or other modifications, the cell source (e.g., T cells or natural killer (NK) cells) can be obtained from the subject. The term "subject" is intended to include a living organism (e.g., a mammal) from which an immune response can be elicited. Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and their transgenic species. T cells can be obtained from a wide range of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.

[0468] In some aspects of this disclosure, immune effector cells (e.g., T cells) can be produced using any kind of technique known to those skilled in the art (such as Ficoll). TM Cells are obtained from blood components collected from a subject (separation). In a preferred aspect, cells from an individual's circulating blood are obtained via apheresis. The apheresis product generally contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, cells collected via apheresis can be washed to remove plasma fractions and, optionally, to place the cells in a suitable buffer or culture medium for subsequent processing steps. In one embodiment, the cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the washing solution is calcium-deficient and may be magnesium-deficient or may be deficient in many (if not all) divalent cations.

[0469] The initial activation step in the absence of calcium can lead to amplified activation. As will be readily apparent to those skilled in the art, the washing step can be performed by methods known to them, such as using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics CellSaver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutions with or without buffers. Alternatively, unwanted components from a single sample can be removed and the cells can be resuspended directly in a culture medium.

[0470] In one respect, by lysing red blood cells and depleting monocytes, for example, through PERCOLL TM T cells are isolated from peripheral blood lymphocytes by gradient centrifugation or by countercurrent centrifugation.

[0471] The methods described herein may include, for example, using negative selection techniques (e.g., as described herein) to select specific subsets of immune effector cells (e.g., T cells), said subsets being a population of depleted regulatory T cells or CD25+ depleted cells. Preferably, the population of depleted regulatory T cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% CD25+ cells.

[0472] In one embodiment, an anti-CD25 antibody or a fragment thereof or a CD25-binding ligand (IL-2) is used to remove regulatory T cells, such as CD25+ T cells, from a population. In one embodiment, an anti-CD25 antibody or a fragment thereof or a CD25-binding ligand is conjugated to or otherwise coated onto a substrate (e.g., beads). In one embodiment, an anti-CD25 antibody or a fragment thereof is conjugated to a substrate as described herein.

[0473] In one implementation, the material from Miltenyi is used. TM The CD25 depletion reagent removes regulatory T cells, such as CD25+ T cells, from the population. In one embodiment, the cell-to-CD25 depletion reagent ratio is 1e7 cells to 20 μL, or 1e7 cells to 15 μL, or 1e7 cells to 10 μL, or 1e7 cells to 5 μL, or 1e7 cells to 2.5 μL, or 1e7 cells to 1.25 μL. In one embodiment, for example, for depleted regulatory T cells, such as CD25+, a concentration greater than 500 million cells / ml is used. In yet another aspect, a cell concentration of 600, 700, 800, or 900 million cells / ml is used.

[0474] In one implementation, the population of immune effector cells to be depleted comprises approximately 6 x 102 9 100 CD25+ T cells. In other aspects, the depleted population of immune effector cells contains approximately 1 x 10^10 cells. 9 One to 1x10 10 100 CD25+ T cells and any integer value of CD25+ T cells between the two. In one embodiment, the resulting regulatory T cell depletion population has 2 x 100 cells. 9 One regulatory T cell, such as CD25+ cells, or fewer (e.g., 1 x 10^12 cells). 9 5x10 8 1x10 8 5x10 7 1x10 7 (one or fewer CD25+ cells).

[0475] In one embodiment, the CliniMAC system, which has a depletion-based circuitry kit, such as circuitry 162-01, is used to remove regulatory T cells, such as CD25+ cells, from a population. In one embodiment, the CliniMAC system operates on a depletion setting (e.g., DEPLETION 2.1).

[0476] We do not wish to be bound by specific theories to reduce the levels of negative regulators of immune cells in subjects before apheresis or during the production of CAR-expressing cell products (e.g., reducing unwanted immune cells, such as T cells). REG The number of cells can reduce the risk of relapse in subjects. For example, depleting T cells can reduce the risk of relapse in subjects. REG Cellular methods are known in the art. Reducing T... REG Cellular methods include, but are not limited to, cyclophosphamide, anti-GITR antibodies (the anti-GITR antibodies described herein), CD25 depletion, and combinations thereof.

[0477] In some implementations, the production method includes reducing T before (e.g., depleting) cells expressing CAR. REG The number of cells. For example, the production method includes contacting a sample (e.g., a single sample) with an anti-GITR antibody and / or an anti-CD25 antibody (or a fragment thereof, or a CD25-binding ligand), for example, to deplete T cells before producing a CAR-expressing cell (e.g., T cells, NK cells) product. REG cell.

[0478] In one implementation, before collecting cells to produce a CAR-expressing cell product, the subject is treated with reduced T cells. REG Pretreatment with one or more cell-based therapies reduces the risk of relapse in subjects compared to cell therapy expressing CARs. In one implementation, reducing T... REG Cellular methods include, but are not limited to, administering one or more of the following to a subject: 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 be performed before, during, or after the infusion of a CAR-expressing cell product.

[0479] In one embodiment, subjects are pretreated with cyclophosphamide before cell collection to produce a CAR-expressing cell product, thus reducing the risk of relapse compared to CAR-expressing cell therapy. In another embodiment, subjects are pretreated with an anti-GITR antibody before cell collection to produce a CAR-expressing cell product, thus reducing the risk of relapse compared to CAR-expressing cell therapy.

[0480] In one embodiment, the cell population to be removed is neither regulatory T cells nor tumor cells, but rather cells that otherwise adversely affect the expansion and / or function of CAR-T cells, such as cells expressing CD14, CD11b, CD33, CD15, or other markers expressed by potentially immunosuppressive cells. In one embodiment, it is conceivable to remove such cells simultaneously with regulatory T cells and / or tumor cells, or after said depletion, or in another order.

[0481] The methods described herein may include more than one selection step, such as more than one depletion step. For example, enrichment of a T cell population may be accomplished via a negative selection process using a combination of antibodies targeting cell surface markers specific to the negatively selected cells. One approach is to sort and / or select cells using a negative magnetic immunoadhesion or flow cytometry employing a mixture of monoclonal antibodies targeting ce...

Claims

1. Use of cells (e.g., cell populations) expressing CD20-binding CAR molecules in combination with one or more kinase inhibitors for the preparation of a medicament for treating diseases associated with CD20 expression, wherein the kinase inhibitor is selected from Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase 4 (CDK4) inhibitors, mammalian target of rapamycin (mTOR) inhibitors, or mitogen-activated protein kinase-interacting kinase (MNK) inhibitors.

2. Use of cells (e.g., cell populations) expressing CAR molecules that bind to CD20 for the preparation of a medicament for treating a disease associated with CD20 expression in a subject, wherein the subject has previously received a kinase inhibitor selected from BTK inhibitors, CDK4 inhibitors, mTOR inhibitors, or MNK inhibitors.

3. Use of cells (e.g., cell populations) expressing CD19-binding CAR molecules in combination with one or more kinase inhibitors for the preparation of a medicament for treating diseases associated with CD19 expression, wherein the kinase inhibitor is selected from BTK inhibitors, CDK4 inhibitors, mTOR inhibitors, or MNK inhibitors.

4. The use according to claim 1 or 3, wherein: (a) The kinase inhibitor and the cells are administered as a first-line therapy; or (b) The cells and the kinase inhibitor are formulated for simultaneous administration.

5. The use according to claim 1 or 3, wherein the cells and the kinase inhibitor are formulated for sequential delivery.

6. The use according to claim 1 or 3, wherein the cells are administered after administration of the kinase inhibitor.

7. The use according to any one of claims 1-3, wherein: (i) Prepare the cells for administration after discontinuation of kinase inhibitor administration; or (ii) Before administering the cells, begin administering the kinase inhibitor and prepare the cells for administration in combination with the continuous administration of the kinase inhibitor.

8. The use according to any one of claims 1-3, wherein the cells are formulated for administration at least 1 month, at least 2 months, at least 3 months, at least 4 months, or at least 6 months after the initiation of administration of the kinase inhibitor.

9. The use according to any one of claims 1-3, wherein: (a) The disease is identified as a complete or partial response to the kinase inhibitor, or a complete or partial response to the cells; or (b) Mammals are partial responders, refractory, or relapsed patients to the kinase inhibitor.

10. The use according to any one of claims 1-3, wherein: (a) 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; (b) The CDK4 inhibitor is selected from: pabuxirib, aloisine A, frapinol, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromone; crizotinib (PF-02341066, P276-00, RAF265, indisulam, roscovitine, dinaciclib, BMS 387032, MLN8054, AG-024322, AT7519, AZD5438, BMS908662; or ribociclib; (c) The mTOR inhibitor is selected from: rapamycin, rapamycin analogues such as everolimus, tamsuromus, desfolimex, sermamod, AZD8055, PF04691502, SF1126, XL765, or OSI-027; or (d) The MNK inhibitor is selected from: CGP052088, CGP57380, causticin, ETC-1780445-2 or 4-amino-5-(4-fluoroaniline)-pyrazolo[3,4-d]pyrimidine.

11. The use according to any one of claims 1-3, wherein the kinase inhibitor is ibrutinib.

12. The use according to claim 1 or 3, wherein the kinase inhibitor is ibrutinib, and wherein the ibrutinib is formulated for administration 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 or 600 mg daily.

13. The use according to claim 1 or 3, wherein the kinase inhibitor is ibrutinib, and wherein the ibrutinib is formulated for administration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles, optionally wherein the cycle length is 21 days or 28 days.

14. The use according to claim 1 or 2, wherein the CAR molecule comprises an anti-CD20 binding domain, a transmembrane domain, and an intracellular signal transduction domain.

15. The use according to claim 14, wherein: (a) The intracellular signal transduction domain comprises a co-stimulatory domain and a primary signal transduction domain; (b) The anti-CD20 binding domain includes light chain complementarity determination region 1 (LC CDR1), light chain complementarity determination region 2 (LC CDR2), light chain complementarity determination region 3 (LC CDR3), heavy chain complementarity determination region 1 (HC CDR1), heavy chain complementarity determination region 2 (HC CDR2) and heavy chain complementarity determination region 3 (HC CDR3) of the anti-CD20 binding domain; (c) The anti-CD20 binding domain is scFv, which includes a light chain variable region connected to the heavy chain variable region via a connector, optionally wherein the connector includes the sequence of SEQ ID NO:53; (d) The CAR molecule contains a transmembrane domain selected from the following proteins: the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154; (e) The transmembrane domain comprises the amino acid sequence of SEQ ID NO:15; (f) The anti-CD20 binding domain is connected to the transmembrane domain via a hinge region, optionally wherein the hinge region contains the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:45; or (g) The CAR molecule further comprises a leader sequence, wherein optionally the leader sequence comprises the amino acid sequence of SEQ ID NO:

13.

16. The use according to claim 15, wherein: (a) The co-stimulatory domain comprises a functional signal transduction domain selected from the following proteins: OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD11a / CD18) or 4-1BB (CD137); (b) The co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:51; (c) The intracellular signal transduction domain comprises a functional signal transduction domain of 4-1BB, a functional signal transduction domain of CD3ζ, or both, or the intracellular signal transduction domain comprises a CD27 sequence, a functional signal transduction domain of CD3ζ, or both. (d) The intracellular signal transduction domain comprises the sequence of SEQ ID NO:16, the sequence of SEQ ID NO:17, or both; (e) The intracellular signal transduction domain comprises the sequence of SEQ ID NO:16, the sequence of SEQ ID NO:43, or both; (f) The intracellular signal transduction domain comprises the sequence of SEQ ID NO:51, the sequence of SEQ ID NO:17, or both; or (g) The intracellular signal transduction domain comprises the sequence of SEQ ID NO:51, the sequence of SEQ ID NO:43, or both.

17. The use according to any one of claims 1-3, wherein the cells are formulated for use in combination with a substance that inhibits immunosuppressive molecules 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 TGFRβ.

18. The use according to any one of claims 1-3, wherein the composition comprises 1-5 x 10 7 One cell expressing CAR.

19. The use according to claim 1 or 2, wherein the disease associated with CD20 expression is cancer.

20. The use according to claim 1 or 2, wherein the disease associated with CD20 expression is a blood cancer, optionally wherein the blood cancer is selected from leukemia or lymphoma.

21. The use according to claim 19, wherein the cancer is selected from: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoblastic leukemia (ALL), Hodgkin's lymphoma, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal zone lymphoma. Spinal dysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom's macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, diffuse red pulp small B-cell lymphoma of the spleen, hairy cell leukemia, lymphoplasmacytic lymphoma, heavy chain disease, plasmacytic myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma, intranodal marginal zone lymphoma, pediatric intranodal marginal zone lymphoma, primary cutaneous follicular center lymphoma, lymphomatoid granuloma, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma in HHV8-associated multicentric Castleman's disease, primary exudative lymphoma, B-cell lymphoma or unclassifiable lymphoma.

22. The use according to claim 19, wherein the cancer is selected from MCL, CLL, ALL, Hodgkin's lymphoma or multiple myeloma.

23. The use according to any one of claims 1-3, wherein the cells are formulated for administration in combination with a cytokine, optionally wherein the cytokine is IL-7, IL-15 or IL-21.

24. The use according to claim 1 or 2, wherein the CAR is an adjustable CAR (RCAR) comprising: An intracellular signal transduction component, comprising an intracellular signal transduction domain and a first switching domain. An antigen-binding member comprising an antigen-binding domain for binding CD20 and a second switching domain; and Transmembrane domain.

25. The use according to claim 1 or 2, wherein: (a) Diseases associated with CD20 expression were identified as having BTK mutations; (b) Diseases associated with CD20 expression are blood cancers in which resistance to kinase inhibitors, cells expressing CAR molecules, or both is delayed or reduced; (c) The disease associated with CD20 expression is hematologic malignancy, in which remission to the hematologic malignancy is prolonged or recurrence of the hematologic malignancy is delayed; or (d) The cells are formulated for administration in combination with a second kinase inhibitor, wherein when a CD20-associated disease is identified as ibrutinib non-responsive or relapsed, the second kinase inhibitor is not ibrutinib, optionally, wherein the second kinase inhibitor is selected from one or more of the following: GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774 or LFM-A13, or combinations thereof.

26. The use according to claim 1 or 2, wherein: (a) A disease associated with CD20 expression is identified as partially responsive to the kinase inhibitor, and the cells are formulated to be administered alone or in combination with the kinase inhibitor during the period of partial response; or (b) CD20-associated disease that has been identified as unresponsive, progressive, or stable after treatment with ibrutinib, and said cells administered alone or in combination with a second BTK inhibitor during disease progression or stabilization, wherein the second kinase inhibitor is not ibrutinib.

27. The use according to any one of claims 1-3, wherein: (a) The use includes lymphocyte infusion with at least one of the said cells; (b) The subject has undergone lymphocyte clearance, optionally including administration of one or more of melphalan, cyclophosphamide, and fludarabine; or (c) The use also includes the formulation of an immunomodulatory low dose of an mTOR inhibitor for administration, optionally wherein the mTOR inhibitor is everolimus or rapamycin.

28. The use according to any one of claims 1-3, wherein the cell is a T cell or an NK cell, or wherein the cell population comprises T cells, NK cells, or both T cells and NK cells.

29. The use according to any one of claims 1-3, wherein the cell is a human immune effector cell, optionally wherein the cell is a human T cell or a human NK cell.

30. The use according to claim 20, wherein the hematologic malignancy is a recurrent or refractory hematologic malignancy.

31. The use according to any one of claims 1-3, which results in a reduction of T cells expressing PD1 after administration of the kinase inhibitor.

32. Use of a population of immune effector cells expressing a CAR molecule containing an anti-CD20 binding domain, a transmembrane domain, and an intracellular signal transduction domain in combination with a BTK inhibitor for the preparation of a medicament for the treatment of a CD20-related disease in a human subject with leukemia or lymphoma, optionally wherein the leukemia or lymphoma is selected from BALL, ALL, DLBCL, B-cell lymphoma, follicular lymphoma, MCL, NHL, Hodgkin's lymphoma, CLL, or SLL.

33. Methods for preparing cells expressing CAR (e.g., immune effector cells expressing CAR) or cell populations, including: (i) Expose cells or cell populations to kinase inhibitors; and (ii) Introducing (e.g., transfection) a nucleic acid encoding a CAR molecule that binds to CD20 into cells or a population of cells under conditions of CAR molecule expression.

34. Methods for preparing cells expressing CAR (e.g., immune effector cells expressing CAR) or cell populations, including: (i) Provide cells or cell populations isolated from subjects who have previously received kinase inhibitors; and (ii) Under conditions of CAR molecule expression, nucleic acid encoding a CAR molecule that binds to CD20 is introduced into a population of immune effector cells.

35. The method of claim 34, wherein the cells or cell population are isolated from the subject at least 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 after receiving the kinase inhibitor.

36. The method of claim 33 or 34, wherein the cell is a T cell or an NK cell, or wherein the cell population comprises T cells, NK cells, or both T cells and NK cells.

37. The method of claim 33, wherein: (a) The method comprises contacting cells or cell populations with the kinase inhibitor for 10-20, 20-30, 30-40, 40-60, or 60-120 minutes, and subsequently removing most or all of the kinase inhibitor from the cells or cell population; or (b) Add the kinase inhibitor after harvesting cells or cell populations or before stimulating cells or cell populations.

38. The method according to claim 33 or 34, wherein the kinase inhibitor is selected from BTK inhibitors, CDK4 inhibitors, mTOR inhibitors, or MNK inhibitors, optionally wherein: (a) 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; (b) The CDK4 inhibitor is selected from: pabuxirib, aloisine A, frapinol, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromone; crizotinib (PF-02341066, P276-00, RAF265, indisulam, roscovitine, dinaciclib, BMS 387032, MLN8054, AG-024322, AT7519, AZD5438, BMS908662; or ribociclib; (c) The mTOR inhibitor is selected from: rapamycin, rapamycin analogues such as everolimus, tamsuromus, desfolimex, sermamod, AZD8055, PF04691502, SF1126, XL765, or OSI-027; or (d) The MNK inhibitor is selected from: CGP052088, CGP57380, causticin, ETC-1780445-2 or 4-amino-5-(4-fluoroaniline)-pyrazolo[3,4-d]pyrimidine.

39. The method according to claim 33 or 34, wherein: (a) The cell population further comprises cancer cells, optionally wherein the kinase inhibitor is a BTK inhibitor, and wherein the BTK inhibitor inhibits BTK in the cancer cells; (b) The method further includes removing regulatory T cells (e.g., CD25) from the cell population. + cell); (c) The cells are human immune effector cells, or the cell population contains human immune effector cells; or (d) The cells are human T cells or human NK cells, or the cell population comprises human T cells, human NK cells, or both human T cells and human NK cells.

40. Use of cells or cell populations prepared by the method of any one of claims 33-39 for the preparation of medicaments for treating mammals suffering from diseases associated with CD20 expression.

41. A reaction mixture comprising a population of immune effector cells, a kinase inhibitor, and a CD20-binding CAR molecule or a nucleic acid encoding a CD20-binding CAR molecule.

42. The reaction mixture according to claim 41, wherein: (a) One or more immune effector cells express the CAR molecule or contain nucleic acids encoding the CAR molecule; (b) The kinase inhibitor is a BTK inhibitor selected from ibrutinib, GDC-0834, RN-486, CGI-560, CGI-1764, HM-71224, CC-292, ONO-4059, CNX-774, or LFM-A13; or (c) The reaction mixture also contains cancer cells.

43. A reaction mixture comprising a population of immune effector cells and a CD20-binding CAR molecule or a nucleic acid encoding a CD20-binding CAR molecule, wherein the immune effector cells comprise covalently inactivated ITK, optionally the reaction mixture further comprises cancer cells, and more optionally the cancer cells comprise covalently inactivated BTK.

44. A composition comprising a cell expressing a CD20-binding CAR molecule and one or more kinase inhibitors, wherein the kinase inhibitor is selected from BTK inhibitors, CDK4 inhibitors, mTOR inhibitors, or MNK inhibitors, optionally wherein the cell and the one or more kinase inhibitors are present in a single dose or as two or more doses.

45. The composition according to claim 44, for preparing a medicament for treating diseases associated with CD20 expression.

46. ​​The composition according to claim 44 or 45, wherein the cell is a human immune effector cell, optionally wherein the cell is a human T cell or a human NK cell.

47. A method for engineering immune effector cells (e.g., T cells) to express CAR molecules, comprising: Immune effector cells are exposed to a certain amount of mTOR inhibitor in vitro, wherein the certain amount of mTOR inhibitor 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) to PD1-positive immune effector cells (e.g., T cells). The nucleic acid encoding the CAR molecule is introduced (e.g., transduced) into the immune effector cells under conditions of CAR molecule expression.

48. The method of claim 47, wherein: (a) mTOR inhibitors are allosteric inhibitors (e.g., RAD001) or catalytic inhibitors; (b) The CAR molecule contains an anti-CD19 binding domain, which includes LCCDR1 of SEQ ID NO:25, LCCDR2 of SEQ ID NO:26 and LCCDR3 of SEQ ID NO:27; (c) The CAR molecule contains an anti-CD19 binding domain, wherein the anti-CD19 binding domain comprises HCCDR1 of SEQ ID NO:19, HCCDR2 of any of SEQ ID NO:20-23 and HCCDR3 of SEQ ID NO:24; (d) The CAR molecule contains an anti-CD19 binding domain, which includes the mouse light chain variable region of Table 7, the mouse heavy chain variable region of Table 7, or both. (e) The CAR molecule contains an anti-CD19 binding domain, said anti-CD19 binding domain comprising the sequence of SEQ ID NO:59 or a sequence having 95-99% identity with it; or (f) The CAR molecule contains an anti-CD19 binding domain comprising a sequence selected from the following sequences: 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 sequences having 95-99% identity with them. 49.i) use of cell populations expressing CAR molecules that bind to CD20 and ii) use of cytokines, such as IL-7, IL-15 and / or IL-21, for the preparation of drugs to treat diseases associated with CD19 expression.

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