Human mesothelin chimeric antigen receptor and its use

Abstract

The present invention provides compositions and methods for treating diseases associated with mesothelin expression. [Solution] The present invention relates to a method of providing an immune response in a patient by administering immune effector cells that have been engineered to express a chimeric antigen receptor (CAR) containing, for example, an antibody (e.g., scFv) that specifically targets mesothelin. In particular, the present invention relates to the use of immune effector cells, such as T cells or NK cells, engineered to express an antibody, for example, a CAR containing an antigen-binding fragment thereof, for treating cancers associated with mesothelin (or MSLN) expression.

Description

[Technical Field]

[0001] This application claims priority under International Application PCT / CN2013 / 089979 filed on 19 December 2013, International Application PCT / CN2014 / 082610 filed on 21 July 2014, and International Application PCT / CN2014 / 090509 filed on 6 November 2014, and incorporates the entire contents of each of these applications herein by reference.

[0002] Field of Invention The present invention relates to T cells engineered to express chimeric antigen receptors (CARs) for treating diseases generally associated with mesothelin expression. [Background technology]

[0003] Background of the Invention Mesothelin was originally identified by Pastan et al. as a tumor-associated antigen because its expression is limited in normal tissues but overexpressed in tumors. (Chang K, et al., Cancer Res. 1992;52(1):181-186 and Chang K, et al. Proc Natl Acad Sci USA. 1996;93(1):136-140). The mesothelin gene encodes a precursor 71kDa protein, which, when processed, produces mesothelin, a 40kDa protein fixed to the cell membrane via glycosylphosphatidylinositol (GPI) binding, and a 31kDa amino-terminal fragment called megakaryocyte-enhancing factor (MPF). Both fragments contain an N-glycosylation site. A soluble splice variant of the 40kDa carboxyl-terminal fragment, called "soluble mesothelin / MPF-associated," has been found in the serum of pancreatic ductal adenocarcinoma (PDA) patients. Johnston, F, et al. Clinical Cancer Research. 2009;15(21):6511. Mesothelin is currently being studied as both a therapeutic target and a biomarker for disease activity and treatment response. Argani P, et al. Clin Cancer Res. 2001;7(12):3862-3868.

[0004] Mesothelin is a differentiation antigen that is also present in normal tissues. Using the mouse anti-human mesothelin antibody K1 developed by Pastan's group, strong K1 reactivity, though at lower levels than typically seen in malignant tissues, has been detected in mesothelial cells lining the peritoneal cavity, pleural cavity, and pericardial cavity. Chang K, et al., Cancer Res. 1992;52(1):181-186. Weak K1 reactivity has been detected in the fallopian canal epithelium, tracheal basal epithelium, and tonsil epithelium. Mesothelin has also been found in all layers of the cornea. Jirsova K, et al. Experimental eye research. 2010;91(5):623-629. However, K1 responsiveness has not been detected in most normal tissues, including the liver, kidneys, spleen, bone marrow, lymph nodes, thymus, myocardium, tongue, skeletal muscle, skin, cerebral cortex, cerebellum, spinal cord, peripheral nerves, pituitary gland, adrenal gland, salivary gland, mammary gland, thyroid gland, parathyroid gland, testis, prostate gland, epididymis, cervical epithelium, lung parenchyma, esophagus, small intestinal epithelium, colonic epithelium, bladder epithelium, and gallbladder epithelium. Chang K, et al., Cancer Res. 1992;52(1):181-186.

[0005] Mesothelin is overexpressed in most primary pancreatic adenocarcinomas, while its expression in benign pancreatic tissue is rare and weak. (Argani P, et al. Clin Cancer Res. 2001;7(12):3862-3868). Epithelial malignant pleural mesothelioma (MPM) invariably expresses mesothelin, but sarcomatoid MPM does not. Most serous epithelial ovarian cancers and associated primary peritoneal cancers express mesothelin.

[0006] Mesothelin is a target of the innate immune response in ovarian cancer and has been proposed as a target for cancer immunotherapy. Bracci L, et al. Clin Cancer Res. 2007;13(2 Pt 1):644-653; Moschella F, et al. Cancer Res. 2011;71(10):3528-3539; Gross G, et al. FASEB J. 1992;6(15):3370-3378; Sadelain M, et al. NatRevCancer. 2003;3(1):35-45; Muul LM, et al. Blood. 2003;101(7):2563-2569; Yee C, et al. Proc Natl Acad Sci US A. 2002;99(25):16168-16173. The presence of mesothelin-specific CTLs in patients with pancreatic cancer correlates with overall survival. Thomas AM, et al. J Exp Med. 2004;200:297-306. Furthermore, Pastan and collaborators used soluble antibody fragments of anti-mesothelin antibodies conjugated to an immunotoxin for the treatment of cancer patients with mesothelin-positive tumors. This attempt demonstrated sufficient safety and some clinical activity in pancreatic cancer. Hassan R, et al. Cancer Immun. 2007;7:20 and Hassan R, et al. Clin Cancer Res. 2007;13(17):5144-5149. In ovarian cancer, this treatment strategy resulted in a minor response in one patient and stable disease in a second patient according to RECIST criteria, as well as complete resolution of ascites. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Chang K, et al., Cancer Res. 1992;52(1):181-186 [Non-Patent Document 2] Chang K, et al. Proc Natl Acad Sci USA. 1996;93(1):136-140 [Non-licensed Document 3] Johnston, F, et al. Clinical Cancer Research. 2009;15(21):6511 [Non-licensed Document 4] Argani P, et al. Clin Cancer Res. 2001;7(12):3862-3868 [Non-licensed Document 5] Jirsova K, et al. Experimental eye research. 2010;91(5):623-629 [Non-licensed Document 6] Bracci L, et al. Clin Cancer Res. 2007;13(2 Pt 1):644-653 [Non-licensed Document 7] Moschella F, et al. Cancer Res. 2011;71(10):3528-3539 [Non-licensed Document 8] Gross G, et al. FASEB J. 1992;6(15):3370-3378 [Non-licensed Document 9] Sadelain M, et al. NatRevCancer. 2003;3(1):35-45 [Non-licensed Document 10] Muul LM, et al. Blood. 2003;101(7):2563-2569 [Non-licensed Document 11] Yee C, et al. Proc Natl Acad Sci US A. 2002;99(25):16168-16173 [Non-licensed Document 12] Thomas AM, et al. J Exp Med. 2004;200:297-306 [Non-licensed Document 13] Hassan R, et al. Cancer Immun. 2007;7:20 [Non-Patent Document 14] Hassan R, et al. Clin Cancer Res. 2007;13(17):5144-5149 [Overview of the project]

[0008] Summary of the Invention The present invention relates to a method for providing an immune response in a patient by administering immune effector cells engineered to express a chimeric antigen receptor (CAR) containing, for example, an antibody (e.g., scFv) that specifically targets mesothelin. In particular, the present invention relates to the use of immune effector cells, such as T cells or NK cells, engineered to express an antibody, such as a CAR containing an antigen-binding fragment thereof, for treating cancers associated with mesothelin (or MSLN) expression. In particular, the present invention relates to adoptive cell transfer that may be particularly suitable for patients with mesothelioma (e.g., malignant pleural mesothelioma), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, or large cell lung cancer), pancreatic cancer (e.g., ductal adenocarcinoma, metastatic pancreatic cancer), ovarian cancer, colorectal cancer, and bladder cancer, or any combination thereof.

[0009] Therefore, in one aspect, the present invention relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising an anti-mesothelin binding domain (e.g., a human anti-mesothelin binding domain), a transmembrane domain, and an intracellular signaling domain comprising a stimulating domain. In one embodiment, the encoded anti-mesothelin binding domain comprises one or more (e.g., three in total) light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of the human anti-mesothelin binding domain described herein, and one or more (e.g., three in total) heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the human anti-mesothelin binding domain described herein. In one embodiment, the encoded human anti-mesothelin-binding domain includes or comprises the light chain variable region and / or the heavy chain variable region described herein (e.g., in Tables 2, 4, or 5). In one embodiment, the encoded anti-mesothelin-binding domain is an scFv comprising or comprising the light chain and heavy chain of the amino acid sequence in Table 2. In one embodiment, the anti-mesothelin binding domain (e.g., scFV) includes a light chain variable region having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the light chain variable region shown in Table 2, but with no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of Table 2; and / or includes or comprises a heavy chain variable region having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the heavy chain variable region shown in Table 2, but with no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of Table 2.In one embodiment, the human anti-mesoterin binding domain includes or comprises a sequence selected from the group consisting of SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62, or sequences having 95-99% identity with these. In one embodiment, the nucleic acid sequence encoding the human anti-mesothelin binding domain includes or comprises a sequence selected from the group consisting of SEQ ID NOs: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110, or sequences having 95-99% identity with these.

[0010] In one embodiment, the isolated nucleic acid further comprises a transmembrane domain, for example, a sequence encoding the transmembrane domain described herein. In one embodiment, the encoded transmembrane domain comprises or comprises a transmembrane domain of a protein selected from the alpha, beta, or zeta chains of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the encoded transmembrane domain comprises or comprises the sequence of Sequence ID No. 12. In one embodiment, the transmembrane domain comprises or comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of Sequence ID No. 12, but not exceeding 20, 10, or 5 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of Sequence ID No. 12.

[0011] In one embodiment, the encoded CAR includes a hinge region, for example, an anti-mesothelin-binding domain connected to a transmembrane domain by the hinge region described herein, for example, the anti-mesothelin-binding domain described herein. In one embodiment, the hinge region includes or comprises SEQ ID NO: 6 or SEQ ID NO: 8.

[0012] In one embodiment, the isolated nucleic acid molecule further comprises a co-stimulatory domain, for example, a sequence encoding the co-stimulatory domain described herein. In one embodiment, the co-stimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). In one embodiment, the encoded co-stimulatory domain comprises or comprises the sequence of SEQ ID NO: 14. In one embodiment, the co-stimulatory domain comprises or comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of SEQ ID NO: 14, but not exceeding 20, 10, or 5 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 14.

[0013] In one embodiment, the isolated nucleic acid comprises an intracellular signaling domain, for example, a sequence encoding the intracellular signaling domain described herein. In one embodiment, the isolated nucleic acid encodes a 4-1BB functional signaling domain and / or a CD3 zeta functional signaling domain. In one embodiment, the encoded intracellular signaling domain comprises the sequence of SEQ ID NO: 7 and / or the sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, the intracellular signaling domain comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of SEQ ID NO: 7 and / or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, but not exceeding 20, 10, or 5 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 7 and / or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, the encoded intracellular signaling domain comprises or consists of the sequence of SEQ ID NO: 7 and the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, wherein the sequence comprising the intracellular signaling domain is expressed in the same frame and as a single polypeptide chain.

[0014] In other aspects, the present invention relates to an isolated nucleic acid molecule encoding a CAR construct, comprising, for example, the leader sequence of SEQ ID NO: 1; an anti-mesothelin-binding domain described herein having, for example, the amino acid sequence of Table 2 or a sequence having 95-99% identity thereto; for example, the hinge region of SEQ ID NO: 2; for example, a transmembrane domain having the sequence of SEQ ID NO: 6; a co-stimulatory domain, for example, a 4-1BB co-stimulatory domain having the sequence of SEQ ID NO: 7; and a primary signaling domain, for example, a CD3 zeta-stimulatory domain having the sequence of SEQ ID NO: 9 or 10. In one embodiment, the isolated nucleic acid molecule comprises (for example, comprising) a nucleic acid sequence encoding a polypeptide having the amino acid sequence of Table 2. In one embodiment, the isolated nucleic acid molecule comprises (for example, comprising) a nucleic acid encoding a polypeptide having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of Table 2, but not exceeding 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of Table 2.

[0015] In other respects, the present invention relates to nucleic acid sequences, for example, isolated polypeptide molecules encoded by the nucleic acids described herein.

[0016] In other aspects, the present invention relates to an isolated polypeptide molecule comprising, or having, a sequence selected from the group consisting of Table 2, an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the heavy chain variable region shown in Table 2, but with no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of Table 2. In one embodiment, the isolated polypeptide comprises one or more (e.g., three in total) of the light chain complementarity determination region 1 (LC CDR1), light chain complementarity determination region 2 (LC CDR2), and light chain complementarity determination region 3 (LC CDR3) of the human anti-mesothelin binding domain described herein, and one or more (e.g., three in total) of the 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 human anti-mesothelin binding domain described herein.

[0017] In other respects, the present invention relates to an isolated chimeric antigen receptor (CAR) molecule comprising an intracellular signaling domain including the anti-mesothelin-binding domain described herein, for example, the human anti-mesothelin-binding domain described herein, a transmembrane domain, and a stimulating domain.

[0018] In one embodiment, the anti-mesothelin binding domain does not compete for binding to human mesothelin with the antigen-binding domain containing the sequence containing SEQ ID NO: 279.

[0019] In one embodiment, the anti-mesothelin binding domain competes for binding to human mesothelin with an antigen-binding domain comprising LC CDR1, LC CDR2, and LC CDR3 of anti-mesothelin light chain amino acid sequences selected from SEQ ID NO: 43 or SEQ ID NO: 49, and HC CDR1, HC CDR2, and HC CDR3 of anti-mesothelin heavy chain amino acid sequences selected from SEQ ID NO: 43 or SEQ ID NO: 49.

[0020] In one embodiment, the anti-mesothelin binding domain binds to a human mesothelin epitope different from the human mesothelin epitope targeted by the antigen-binding domain containing the sequence containing SEQ ID NO: 279. In one embodiment, the epitope comprises a sequence of amino acids selected from amino acids 314-315, 317-318, 346-349 and 369-375 of SEQ ID NO: 278, or any combination thereof. In another embodiment, the epitope comprises one or more amino acids selected from amino acids 314-315, 317-318, 346-349 and 369-375 or any combination thereof of SEQ ID NO: 278.

[0021] In one embodiment, the anti-mesotheline binding domain described herein does not bind to the N-terminus of mesotheline shown in SEQ ID NO: 278. In one embodiment, the anti-mesotheline binding domain binds to the C-terminus of human mesotheline. In one embodiment, the anti-mesotheline binding domain binds to an epitope within amino acids 450-588 of SEQ ID NO: 278. In one embodiment, the epitope bound by the anti-mesotheline binding domain includes a sequence selected from amino acids 485-490, 498-507, 532-537, and 545-572 of SEQ ID NO: 278, or a combination thereof. In one embodiment, the epitope bound by the anti-mesotheline binding domain includes one or more amino acids selected from amino acids 485-490, 498-507, 532-537, and 545-572 or any combination thereof of SEQ ID NO: 278. In these embodiments, SEQ ID NO: 278 represents amino acids 296-588 of human mesothelin, for example, the first amino acid of SEQ ID NO: 278 is amino acid 296, and the last amino acid of SEQ ID NO: 278 is amino acid 588.

[0022] In one embodiment, the anti-mesothelin binding domain includes one or more (e.g., three in total) of the light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of the human anti-mesothelin binding domain described herein, as well as one or more (e.g., three in total) of the heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of the human anti-mesothelin binding domain described herein. In one embodiment, the human anti-mesothelin binding domain includes or comprises the light chain variable region and / or the heavy chain variable region described herein (e.g., Table 2). In one embodiment, the anti-mesothelin binding domain is an scFv that includes or comprises the light chain variable region and heavy chain variable region of the amino acid sequence in Table 2. In one embodiment, the anti-mesothelin binding domain (e.g., scFV) comprises a light chain variable region having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the light chain variable region shown in Table 2, but with no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of Table 2; and / or comprising a heavy chain variable region having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the heavy chain variable region shown in Table 2, but with no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of Table 2. In one embodiment, the human anti-mesoterin binding domain includes or comprises a sequence selected from the group consisting of SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62, or a sequence having 95-99% identity with these.

[0023] In one embodiment, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain includes, for example, the sequence of SEQ ID NO: 6, an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of SEQ ID NO: 6, but with no more than 20, 10, or 5 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 6, as described herein.

[0024] In one embodiment, the anti-mesothelin binding domain is connected to a transmembrane domain by a hinge region. In one embodiment, the hinge region includes the hinge regions described herein, for example, the hinge region of Sequence ID No. 2.

[0025] In one embodiment, the isolated CAR molecule further comprises a co-stimulatory domain, e.g., the co-stimulatory domain described herein. In one embodiment, the co-stimulatory domain is a functional signaling domain or a functional variant obtained from a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). In one embodiment, the co-stimulatory domain comprises or comprises the sequence of SEQ ID NO: 7. In one embodiment, the co-stimulatory domain comprises or comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of SEQ ID NO: 7, but not exceeding 20, 10, or 5 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 7.

[0026] In one embodiment, the isolated CAR molecule includes an intracellular signaling domain, e.g., the intracellular signaling domain described herein. In one embodiment, the intracellular signaling domain includes a 4-1BB functional signaling domain and / or a CD3 zeta functional signaling domain. In one embodiment, the intracellular signaling domain includes or comprises the sequence of SEQ ID NO: 7 and / or the sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, the intracellular signaling domain includes or comprises an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of SEQ ID NO: 7 and / or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, but not exceeding 20, 10, or 5 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 7 and / or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, the intracellular signaling domain includes or comprises the sequence of SEQ ID NO: 9 and the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, where the sequence containing the intracellular signaling domain is expressed in the same frame and as a single polypeptide chain.

[0027] In other aspects, the present invention relates to an isolated CAR molecule comprising, for example, the leader sequence of SEQ ID NO: 1; an anti-mesothelin-binding domain described herein having, for example, the amino acid sequence of Table 2 or a sequence having 95-99% identity thereto; for example, the hinge region of SEQ ID NO: 2; for example, a transmembrane domain having the sequence of SEQ ID NO: 6; a co-stimulatory domain, for example, a 4-1BB co-stimulatory domain having the sequence of SEQ ID NO: 7; and a primary signaling domain, for example, a CD3 zeta-stimulating domain having the sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, the isolated CAR molecule comprises (for example, consisting of) a polypeptide having the amino acid sequence of Table 2. In one embodiment, the isolated CAR molecule comprises (for example, consisting of) a polypeptide having an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of Table 2, but not exceeding 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of Table 2. In one embodiment, the isolated CAR molecule contains or comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, and 86.

[0028] In other aspects, the present invention relates to a vector comprising a nucleic acid sequence described herein. In one embodiment, the nucleic acid sequence encodes a CAR molecule, for example, the CAR molecule described herein. In one embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenovirus vector, or retroviral vector.

[0029] In one embodiment, the vector is a lentiviral vector, for example, the lentiviral vector described herein. In one embodiment, the vector further includes a promoter. In one embodiment, the promoter is an EF-1α promoter. In one embodiment, the EF-1α promoter includes the sequence of SEQ ID NO: 11.

[0030] 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, RNA is transcribed from the in vitro transcription vector, where the vector is pD-A.anti-mesoBD OF.2bg.150A, where anti-mesoBD is the anti-mesothelin binding domain described herein. In one embodiment, the nucleic acid sequence in the vector further includes a poly(A) tail, for example, a polyA tail containing about 150 adenosine bases (SEQ ID NO: 271), for example, the polyA tail described herein. In one embodiment, the nucleic acid sequence in the vector further includes a 3'UTR, for example, at least one repeat of a 3'UTR derived from human beta-globulin, for example, the 3'UTR described herein.

[0031] In other respects, the present invention relates to cells containing vectors. The cells may be, for example, the cells described herein. In one embodiment, the cells are human T cells, for example, the T cells described herein, or human NK cells, for example, the human NK cells described herein. In one embodiment, the human T cells are CD8 + It is a 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 is diacylglycerol kinase (DGK) deficient. In one embodiment, the cell is a T cell and is Ikaros deficient. In one embodiment, the cell is a T cell and is deficient in both DGK and Ikaros.

[0032] In one respect, the CAR-expressing cells described herein may further contain a second CAR, for example, a second CAR containing a different antigen-binding domain to the same target (mesothelin) or a different target (e.g., a target other than mesothelin on stromal cells, e.g., FAP; a target other than mesothelin on prostate cancer cells, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1 or MAD-CT-2; a target other than mesothelin on ovarian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor α, claudin 6, GloboH or sperm protein 17; a target other than mesothelin on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4 or Trop-2). In one embodiment, a CAR-expressing cell comprises a first CAR that targets a first antigen and includes an intracellular signaling domain having a co-stimulatory signaling domain but lacking a primary signaling domain, and a second CAR that targets a second different antigen and includes an intracellular signaling domain having a primary signaling domain but lacking a co-stimulatory signaling domain. In one embodiment, a CAR-expressing cell comprises a first mesothelin CAR including a mesothelin-binding domain, a transmembrane domain and a co-stimulatory domain, and a second CAR that targets a non-mesothelin antigen (e.g., an antigen expressed on stromal cells, lung cancer cells, prostate cancer cells or ovarian cancer cells) and includes an antigen-binding domain, a transmembrane domain and a primary signaling domain. In another embodiment, a CAR-expressing cell comprises a first mesothelin CAR including a mesothelin-binding domain, a transmembrane domain and a primary signaling domain, and a second CAR that targets a non-mesothelin antigen (e.g., an antigen expressed on stromal cells, lung cancer cells, prostate cancer cells or ovarian cancer cells) and includes an antigen-binding domain for said antigen, a transmembrane domain and a co-stimulatory signaling domain.

[0033] In one embodiment, CAR-expressing cells include the mesothelin CAR and inhibitory CAR described herein. In one embodiment, the inhibitory CAR includes an antigen-binding domain that binds to an antigen, which is also found on normal cells, e.g., normal cells that also express mesothelin, but not on cancer cells. In one embodiment, the inhibitory CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular domain of the inhibitory molecule. For example, the intracellular domain of an inhibitory CAR may be the intracellular domain of PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta.

[0034] In other embodiments, the CAR-expressing cells described herein may express other agents, such as agents that enhance the activity or fitness of CAR-expressing cells, such as those described herein. For example, in one embodiment, the agent may be an agent that inhibits molecules that modulate or control T cell function, such as inhibitory molecules. In one embodiment, the molecules that modulate or control T cell function are inhibitory molecules. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In some embodiments, the expression of molecules that modulate or control, for example, T cell function in CAR-expressing cells can be inhibited using a drug, for example, an inhibitory nucleic acid, for example, dsRNA, for example, siRNA or shRNA, or an inhibitory protein or inhibitory system, for example, clustered and regularly arranged short palindromic sequence repeats (CRISPR), transcriptional activator-like effector nuclease (TALEN), or zinc finger endonuclease (ZFN). In some embodiments, the drug is shRNA, for example, an shRNA, for example. In some embodiments, a drug that modulates or controls, for example, T cell function, is inhibited in CAR-expressing cells. For example, a dsRNA molecule that inhibits the expression of a molecule that modulates or controls, for example, T cell function, is bound to a component of CAR, for example, a nucleic acid encoding all components.

[0035] In one embodiment, a drug that inhibits an inhibitory molecule comprises a primary polypeptide, e.g., the inhibitory molecule, conjugated to a secondary polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain as described herein. In one embodiment, the drug comprises a secondary polypeptide, e.g., a primary polypeptide or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these) and an intracellular signaling domain as described herein (e.g., comprising a co-stimulatory domain (e.g., e.g., 41BB, CD27, or CD28) and / or a primary signaling domain (e.g., the CD3 zeta signaling domain as described herein). In one embodiment, the drug comprises a first polypeptide which is PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a second polypeptide which is an intracellular signaling domain described herein (e.g., the CD28 signaling domain and / or the CD3 zeta signaling domain described herein).

[0036] In other words, the present invention provides a method for producing cells, comprising transducing cells described herein, for example, T cells or NK cells, with a vector containing a CAR molecule, for example, a nucleic acid encoding a CAR molecule described herein. In one embodiment, the vector is a lentiviral vector described herein.

[0037] The present invention also provides a method for producing RNA-modified cells that transiently express foreign RNA, such as the cells described herein, such as T cells or NK cells. The method comprises introducing in vitro transcribed RNA or synthetic RNA into cells, wherein the RNA comprises nucleic acids encoding the CAR molecules described herein.

[0038] In other words, the present invention relates to a method for providing antitumor immunity to a subject, comprising administering an effective amount of cells expressing the CAR molecule, such as the cells described herein, to the subject. In one embodiment, the cells are autologous T cells or NK cells. In one embodiment, the cells are allogeneic T cells or NK cells. In one embodiment, the subject is human.

[0039] In other words, the present invention relates to a method for treating subjects having a disease associated with mesothelin expression (e.g., proliferative disorders associated with mesothelin expression, precancerous conditions and non-cancer-related indications), which includes, for example, administering an effective amount of cells containing the CAR molecule described herein.

[0040] In one embodiment, the disease associated with mesothelin is cancer, e.g., the cancers described herein. In one embodiment, the disease associated with mesothelin is selected from the group consisting of mesothelioma (e.g., malignant pleural mesothelioma), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, or large cell lung cancer), pancreatic cancer (e.g., ductal adenocarcinoma), ovarian cancer, colorectal cancer, and bladder cancer, or any combination thereof. In one embodiment, the disease is, for example, pancreatic cancer, e.g., metastatic ductal adenocarcinoma (PDA) in a subject that has progressed with at least one prior standard treatment. In one embodiment, the disease is, for example, mesothelioma (e.g., malignant pleural mesothelioma) in a subject that has progressed with at least one prior standard treatment. In one embodiment, the disease is, for example, ovarian cancer, e.g., serous epithelial ovarian cancer, in a subject that has progressed after at least one prior standard treatment regimen.

[0041] In one embodiment, mesothelin CAR-expressing cells, such as T cells or NK cells, are administered to subjects who have previously been treated with melphalan.

[0042] In one embodiment, a CAR molecule, for example, cells expressing the CAR molecule described herein, is administered in combination with a drug that enhances the activity or fitness of the cells expressing the CAR molecule, for example, a drug described herein.

[0043] In one embodiment, CAR molecules, for example, cells expressing the CAR molecules described herein, are administered in combination with a low, immunoenhancing dose of an mTOR inhibitor. While we do not wish to be bound by theory, treatment with low, immunoenhancing doses (e.g., doses insufficient to completely suppress the immune system but sufficient to improve immune function) is thought to result in a decrease in PD-1-positive T cells or an increase in PD-1-negative cells. PD-1-positive T cells, rather than PD-1-negative T cells, may be depleted by binding to cells expressing PD-1 ligands, such as PD-L1 or PD-L2.

[0044] In one embodiment, this method can be used to optimize the performance of the CAR cells described herein in a subject. While we do not wish to be bound by theory, in one embodiment, it is thought that the performance of endogenous, unmodified immune effector cells, such as T cells, may be improved. While we do not wish to be bound by theory, in one embodiment, it is thought that the performance of mesothelin CAR-expressing cells may be improved. In another embodiment, cells that have been engineered to express or will be engineered to express CARs, such as T cells or NK cells, can be treated ex vivo by contacting them with an amount of mTOR inhibitor that increases the number of PD1-negative immune effector cells, such as T cells, or increases the ratio of PD1-negative immune effector cells, such as T cells, or PD1-positive immune effector cells, such as T cells, such as T cells.

[0045] In one embodiment, administration of a low, immunoenhancing dose of an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or a catalytic inhibitor, is initiated before administration of the CAR-expressing cells described herein, e.g., T cells or NK cells. In one embodiment, the CAR cells are administered after a sufficient time or dose of the mTOR inhibitor so that the levels of PD1-negative immunoeffector cells, e.g., T cells, or PD1-negative immunoeffector cells, e.g., the T cell / PD1-positive immunoeffector cell ratio, e.g., T cells, are increased at least transiently.

[0046] In one embodiment, cells manipulated to express CARs, e.g., T cells or NK cells, are harvested after a sufficient time or sufficient dose of a low, immunoenhancing dose of an mTOR inhibitor such that the levels of PD1-negative immunoeffector cells in or harvested from the subject, e.g., T cells or PD1-negative immunoeffector cells, e.g., the T cell / PD1-positive immunoeffector cell ratio, e.g., T cells, are increased at least transiently.

[0047] In one embodiment, a CAR molecule, for example, cells expressing the CAR molecule described herein, is administered in combination with a drug, for example, a drug described herein, for reducing one or more side effects associated with the administration of cells expressing the CAR molecule.

[0048] In one embodiment, a CAR molecule, for example, cells expressing the CAR molecule described herein, is administered in combination with a drug that treats a disease associated with mesothelin expression, for example, the drug described herein.

[0049] In one embodiment, a CAR molecule, for example, cells expressing the CAR molecule described herein, is administered in the dose and / or dosing schedule described herein.

[0050] In one embodiment, a CAR molecule, for example, cells expressing the CAR molecule described herein, is administered as a first-line treatment for a disease, for example, cancer, for example, the cancer described herein. In another embodiment, a CAR molecule, for example, cells expressing the CAR molecule described herein, is administered as a second, third, or fourth-line treatment for a disease, for example, cancer, for example, the cancer described herein.

[0051] In one embodiment, the population of cells described herein is administered.

[0052] In one embodiment, the CAR molecule is introduced into T cells or NK cells, for example, using in vitro transcription, and the subject (e.g., human) receives an initial dose of cells containing the CAR molecule, followed by one or more subsequent doses of cells containing the CAR molecule, where the subsequent doses are administered within 15 days after the previous dose, for example, on days 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. In one embodiment, the subject (e.g., human) receives more than one dose of cells containing the CAR molecule per week, for example, two, three, or four doses of cells containing the CAR molecule per week. In one embodiment, a subject (e.g., a human subject) receives more than one dose of cells containing CAR molecules per week (e.g., two, three, or four doses per week) (also referred to here as a cycle), followed by a week without administration of cells containing CAR molecules, and then the subject receives one or more doses of cells containing CAR molecules (e.g., more than one dose of cells containing CAR molecules per week). In another embodiment, a subject (e.g., a human subject) receives more than one cycle of cells containing CAR molecules, with intervals between cycles shorter than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, cells containing CAR molecules are administered three times per week on alternate days. In one embodiment, cells containing CAR molecules are administered for at least two, three, four, five, six, seven, eight weeks, or longer.

[0053] In one aspect, the present invention includes a population of self or allogeneic cells that have been transfected or transfected with a vector comprising a nucleic acid molecule encoding the mesothelin-CAR molecule, as described herein. In one embodiment, the vector is a retroviral vector. In one embodiment, the vector is a self-inactivating lentiviral vector as described elsewhere herein. In one embodiment, the vector is delivered to cells, for example, T cells or NK cells (e.g., by transfection or electroporation), wherein the vector comprises a nucleic acid molecule encoding the mesothelin-CAR molecule as described herein, which is transcribed as an mRNA molecule, and the mesothelin-CAR molecule is translated from the RNA molecule and expressed on the cell surface.

[0054] In other aspects, the present invention provides a population of CAR-expressing cells, for example, CAR T cells. In one embodiment, the population of CAR-expressing cells comprises a mixture of cells expressing different CARs. For example, in one embodiment, the population of CAR T cells may comprise a first cell expressing a CAR having the anti-mesothelin binding domain described herein and a second cell expressing a CAR having a different anti-mesothelin binding domain, for example, the anti-mesothelin binding domain described herein that is different from the anti-mesothelin binding domain in the CAR expressed by the first cell. As another example, a population of CAR-expressing cells may include, for example, first cells expressing a CAR containing an anti-mesothelin binding domain as described herein, and second cells expressing a CAR containing an antigen-binding domain against non-mesothelin targets (e.g., non-mesothelin targets on stromal cells, e.g., FAP; non-mesothelin targets on prostate cancer cells, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1 or MAD-CT-2; non-mesothelin targets on ovarian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor α, claudin 6, GloboH or sperm protein 17; e.g., non-mesothelin targets on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4 or Trop-2). In one embodiment, the population of CAR-expressing cells includes, for example, first cells expressing a CAR that includes a primary intracellular signaling domain and second cells expressing a CAR that includes a secondary signaling domain.

[0055] In other words, the present invention provides a population of cells in which at least one cell expresses a CAR having an anti-mesothelin binding domain described herein, and a second cell expresses another agent, for example, an agent that enhances the activity or function of the CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits a molecule that modulates or controls T cell function, for example, an inhibitory molecule. In one embodiment, the molecule that modulates or controls T cell function is an inhibitory molecule, for example, an agent described herein. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In some embodiments, the expression of molecules that modulate or control, for example, T cell function in CAR-expressing cells can be inhibited using a drug, for example, an inhibitory nucleic acid, for example, dsRNA, for example, siRNA or shRNA; or an inhibitory protein or inhibitory system, for example, clustered and regularly arranged short palindromic sequence repeats (CRISPR), transcriptional activator-like effector nuclease (TALEN) or zinc finger endonuclease (ZFN). In some embodiments, the drug is shRNA, for example, an shRNA, for example. In some embodiments, a drug that modulates or controls, for example, T cell function, is inhibited in CAR-expressing cells. For example, a dsRNA molecule that inhibits the expression of a molecule that modulates or controls, for example, T cell function, is bound to a component of CAR, for example, a nucleic acid encoding all components.

[0056] In one embodiment, a drug that inhibits an inhibitory molecule includes a primary polypeptide, e.g., an inhibitory molecule, conjugated to a secondary polypeptide that provides a positive signal to cells, e.g., an intracellular signaling domain as described herein. In one embodiment, the agent comprises, for example, a primary polypeptide of an inhibitory molecule such as PD1, PD-L1, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, CTLA4, VISTA, CD160, BTLA, LAIR1, TIM3, 2B4, TGFR beta and TIGIT, or a secondary polypeptide which is the primary polypeptide or any fragment thereof (e.g., at least a portion of the extracellular domain of any of these) and an intracellular signaling domain described herein (e.g., a co-stimulatory domain (e.g., 41BB, CD27, or CD28 as described herein) and / or a primary signaling domain (e.g., the CD3 zeta signaling domain as described herein). In one embodiment, the agent comprises a primary polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a secondary polypeptide which is the intracellular signaling domain described herein (e.g., the CD28 signaling domain and / or the CD3 zeta signaling domain as described herein).

[0057] In one embodiment, for example, a nucleic acid molecule encoding the mesothelin CAR molecule described herein is expressed as an mRNA molecule. In one embodiment, genetically modified mesothelin CAR-expressing cells, such as T cells or NK cells, can be produced by introducing or electroporating an RNA molecule encoding a desired CAR (e.g., without a vector sequence) into the cells. In one embodiment, once taken up, the mesothelin CAR molecule is translated from the RNA molecule and expressed on the surface of recombinant cells.

[0058] In other respects, the present invention relates to CAR molecules for use as pharmaceuticals, for example, the CAR molecules described herein, isolated nucleic acid molecules encoding the CAR molecules described herein, vectors containing the CAR molecules described herein, and / or cells containing the CAR molecules described herein.

[0059] In other respects, the present invention relates, for example, to the CAR molecule described herein, an isolated nucleic acid molecule encoding the CAR molecule described herein, a vector containing the CAR molecule described herein, and / or a cell containing the CAR molecule described herein, for use in treating diseases expressing the mesothelin described herein. [Brief explanation of the drawing]

[0060] [Figure 1] Figure 1 is a schematic diagram of the pD-A anti-mesoBD.OF.BBZ.2bg.150A plasmid. In this figure, sequence number 271 is referred to as "150A".

[0061] [Figure 2]Figure 2 shows the usable cell production and treatment schedules. (A) Autologous cells are obtained by leukocyte apheresis, and T cells are enriched by proliferation using anti-CD3 / CD28 mAb coated magnetic beads. Cells are grown for 8-12 days. On the last day of culture, the beads are removed using a magnetic field, the cells are washed, electroporated with human meso-CAR mRNA constructs, and cryopreserved in an insoluble medium. (B) Three treatment infusion schedules are described. In Schedule 1, the patient is administered 1 × 10⁸ human meso-carrying CAR T cells by intravenous (iv) infusion on day 0, and 1 × 10⁹ human meso-carrying CAR T cells one week later. Safety is monitored for at least one month, after which the patient may be eligible for Schedule 2. In Schedule 2, the patient is administered 1 × 10⁸ human meso-carrying CAR T cells by iv infusion three times a week for one week, followed by a one-week rest period, and then 1 × 10⁹ human meso-carrying CAR T cells three times a week for one week. In Schedule 3, patients receive 3 × 10⁸ / m² of human meso-carrying CAR T cells via IV infusion three times a week for three weeks, followed by intratumoral injection of 2 × 10⁸ human meso-carrying CAR T cells into the primary lesion on days +35 and +57.

[0062] [Figure 3] Figures 3A and 3B are representative graphs of cytotoxicity assays performed on donor 2 (healthy donor) T cells cultured with control K562 cells that do not express MSLN (as shown in Figure 3A) or K562 cells that have been transduced to express MSLN (as shown in Figure 3B) by mouse SS1 CAR or the anti-MSLN CARs M1-M12 of the present invention.

[0063] [Figure 4]Figures 4A and 4B are graphs showing IFN-γ secretion by mouse SS1 and CD19 CART and anti-MSLN CART after stimulation of MSLN+ cells. Figure 4A shows the reactivity to the transduced cell line K562-meso and its MSLN-negative parent cell line K562. Figure 4B shows the reactivity to naturally MSLN-expressing cancer cells, the ovarian cancer line Ovcar8 and the pancreatic cancer lines SW1990 and Panc0203.

[0064] [Figure 5] Figure 5 shows the clinical trial design for mesothelin CART, which was produced by transducing a CAR construct using a lentiviral vector.

[0065] [Figure 6] Figures 6A, 6B, 6C, and 6D show the antitumor activity of CART mesocellular cells.

[0066] [Figure 7] Figures 7A, 7B, and 7C show the in vivo persistence and transport of CART mesocellular cells to primary and metastatic tumor sites.

[0067] [Figure 8] Figure 8 shows cytokines and chemokines in serum after CART mesotherapy cell injection.

[0068] [Figure 9] Figures 9A and 9B show CART mesocellular induction by antitumor antibodies. Serum was obtained from MPM patients (Figure 9A) and pancreatic cancer patients (Figure 9B).

[0069] [Figure 10] Figure 10 shows tumor growth in NSG mice injected with EMMESO tumor cells. After the tumors grew to approximately 200 mm³ in size, mesoCART cells were injected via the tail vein, and measurements were taken 39 days after injection.

[0070] [Figure 11]Figures 11A and 11B show mesoCAR expression as determined by flow cytometry analysis at the time of injection (Figure 11A) or 40 days after collection from xenograft tumors.

[0071] [Figure 12] Figure 12 shows the in vitro lethal functional capacity of mesoCAR T cells isolated from the flank of NSG mice after 39 days, or cryopreserved after transduction.

[0072] [Figure 13] Figure 13 shows the inhibitory enzymes DGK and SHP1 in TILs isolated from EMMESO flank tumors compared with TILs that were allowed to rest overnight.

[0073] [Figure 14] Figures 14A, 14B, 14C, 14D, 14E, and 14F show the effects of treatment with inhibitors of mechanisms that downregulate mesoCART function (anti-PDL1, DGK inhibitors, and SSG) on tumor cell lethality (Figures 14A, 14C, and 14E) and IFN-γ cytokine secretion (Figures 14B, 14C, and 14F).

[0074] [Figure 15-1] Figures 15A, 15B, 15C, and 15D show cytokine secretion from small panels of human CART-MSLN after stimulation of various tumor cell lines. Figure 15A shows IFN-γ secretion. Figure 15B shows TNF. Figure 15C shows IL-2. Figure 15D shows IL-4. [Figure 15-2] Figures 15A, 15B, 15C, and 15D show cytokine secretion from small panels of human CART-MSLN after stimulation of various tumor cell lines. Figure 15A shows IFN-γ secretion. Figure 15B shows TNF. Figure 15C shows IL-2. Figure 15D shows IL-4.

[0075] [Figure 16]Figures 16A and 16B show the results of lethal assays of CART-MSLN-5, CART-MSLN-11, CART-MSLN-17, and mouse CART-MSLN-SS1 against Ovcar3 (Figure 16A) and U87mg (Figure 16B) tumor cells.

[0076] [Figure 17] Figures 17A and 17B show the results of a panel lethal assay of CART-MSLN against Ovcar3 tumor cells.

[0077] [Figure 18] Figure 18 shows the antitumor activity of the first set of CART-MSLN (including M5, M11, M17, and M21) in an Ovcar8 xenograft model.

[0078] [Figure 19] Figure 19 shows the antitumor activity of the second set of CART-MSLN (including M12, M14, M16, and M23) in an Ovcar8 xenograft model.

[0079] [Figure 20-1] Figures 20A, 20B, and 20C show the time-dependent loss of function of mesoCAR T cells in the tumor microenvironment (TIL) compared to fresh or thawed mesoCAR T cells. A) Cytotoxicity assay; B) IFN-γ release assay; and C) Western blot analysis of ERK signaling (via phosphorylation). [Figure 20-2] Figures 20A, 20B, and 20C show the time-dependent loss of function of mesoCAR T cells in the tumor microenvironment (TIL) compared to fresh or thawed mesoCAR T cells. A) Cytotoxicity assay; B) IFN-γ release assay; and C) Western blot analysis of ERK signaling (via phosphorylation).

[0080] [Figure 21] Figure 21 shows the effect of DGK deficiency on the cytotoxicity of mesoCAR T cells. Target cell lethality percentages were evaluated using various effector-to-target ratios.

[0081] [Figure 22] Figure 22 shows the effect of DGK deletion on IFN-γ production and release from mesoCAR T cells. IFN-γ concentrations were evaluated using various effector-to-target ratios.

[0082] [Figure 23] Figure 23 shows the effects of DGK deficiency on ERK signaling or T cell activation, and on mesoCAR T cells. B: albumin, M: mesothelin, 3 / 28: CD3 / CD28 stimulated cells.

[0083] [Figure 24] Figure 24 describes the effect of DGK deficiency on the TGF-β sensitivity of mesoCAR T cells in terms of cytotoxic activity.

[0084] [Figure 25] Figures 25A and 25B show the effect of DGK deletion on the therapeutic efficacy of mesoCAR T cells in a tumor mouse model. A) Effect on antitumor activity is shown by tumor volume over time. B) Sustained tumor-infiltrating cells and proliferation.

[0085] [Figure 26-1] Figures 26A, 26B, 26C, 26D, 26E, and 26F show cytokine production and cytotoxic mediator release in CAR-expressing T cells with reduced Ikaros levels. Figure 26A shows Ikaros expression in wild-type and Ikzf1±CAR T cells as measured by flow cytometry (left panel) and Western blotting (right panel). After stimulation with mesothelin-coated beads, PMA / ionomycin (PMA / I), or BSA-coated beads (control), the percentage of cells producing IFN-γ (Figure 26B), TNF-α (Figure 26C), IL-2 (Figure 26D), cytotoxic mediator granzyme B (Figure 26E), and CD107a expression (Figure 26F) was determined. [Figure 26-2]Figures 26A, 26B, 26C, 26D, 26E, and 26F show cytokine production and cytotoxic mediator release in CAR-expressing T cells with reduced Ikaros levels. Figure 26A shows Ikaros expression in wild-type and Ikzf1±CAR T cells as measured by flow cytometry (left panel) and Western blotting (right panel). After stimulation with mesothelin-coated beads, PMA / ionomycin (PMA / I), or BSA-coated beads (control), the percentage of cells producing IFN-γ (Figure 26B), TNF-α (Figure 26C), IL-2 (Figure 26D), cytotoxic mediator granzyme B (Figure 26E), and CD107a expression (Figure 26F) was determined.

[0086] [Figure 27] Figures 27A, 27B, and 27C show cytokine production and cytotoxic mediator release in CAR-expressing T cells with the dominant-negative allele (IkDN) of Ikaros. After stimulation with mesothelin-coated beads, PMA / ionomycin (PMA / I), or BSA-coated beads (control), the percentage of cells producing IFN-γ (Figure 27A), IL-2 (Figure 27B), and CD107a expression (Figure 27C) was determined.

[0087] [Figure 28-1]Figures 28A, 28B, 28C, 28D, and 28E show that Ikaros deletion did not enhance CAR T cell activation and signaling after antigen stimulation. Levels of CD69 (Figure 28A), CD25 (Figure 28B), and 4-1BB (Figure 28C) were determined by flow cytometry in Ikzf1±CAR T cells at the time of writing. In Figure 28D, the RAS / ERK signaling pathway was tested in wild-type (WT) and Ikaros-dominant-negative (IkDN) cells after TCR stimulation with CD3 / CD28 antibody. Levels of phosphorylated TCR signaling proteins such as phosphorylated PLCγ, phosphorylated Lck, phosphorylated JNK, phosphorylated Akt, phosphorylated ERK, phosphorylated IKKα, and IκBα were assessed by Western blotting. In Figure 28E, WT and IkDN cells transduced with mesoCAR were stimulated with BSA or mesothelin-coated beads, and downstream signaling pathways were examined by Western blotting by evaluating the levels of phosphorylated ERK and phosphorylated PLCγ. [Figure 28-2] Figures 28A, 28B, 28C, 28D, and 28E show that Ikaros deletion did not enhance CAR T cell activation and signaling after antigen stimulation. Levels of CD69 (Figure 28A), CD25 (Figure 28B), and 4-1BB (Figure 28C) were determined by flow cytometry in Ikzf1±CAR T cells at the time of writing. In Figure 28D, the RAS / ERK signaling pathway was tested in wild-type (WT) and Ikaros-dominant-negative (IkDN) cells after TCR stimulation with CD3 / CD28 antibody. Levels of phosphorylated TCR signaling proteins such as phosphorylated PLCγ, phosphorylated Lck, phosphorylated JNK, phosphorylated Akt, phosphorylated ERK, phosphorylated IKKα, and IκBα were assessed by Western blotting. In Figure 28E, WT and IkDN cells transduced with mesoCAR were stimulated with BSA or mesothelin-coated beads, and downstream signaling pathways were examined by Western blotting by evaluating the levels of phosphorylated ERK and phosphorylated PLCγ.

[0088] [Figure 29-1]Figures 29A, 29B, 29C, 29D, and 29E show that a reduction in Ikaros in CAR T cells enhances the response to target cells AE17 or mesothelin-expressing AE17 (AE17 meso) in vitro. Figure 29A shows IFN-γ production in WT and Ikzf1± mesoCAR T cells at the described effector:target cell ratio. Cell lysis of mesoCAR-expressing WT and Ikzf1± (Figure 29B) and IkDN (Figure 29C) was measured at the described effector:target cell ratio. IFN-γ production (Figure 29D) and cell lysis (Figure 29E) of transduced FAP-CAR WT and Ikzf1± cells were measured at the described effector:target cell ratio, where the target cells were FAP-expressing 3T3 cells. [Figure 29-2] Figures 29A, 29B, 29C, 29D, and 29E show that a reduction in Ikaros in CAR T cells enhances the response to target cells AE17 or mesothelin-expressing AE17 (AE17 meso) in vitro. Figure 29A shows IFN-γ production in WT and Ikzf1± mesoCAR T cells at the described effector:target cell ratio. Cell lysis of mesoCAR-expressing WT and Ikzf1± (Figure 29B) and IkDN (Figure 29C) was measured at the described effector:target cell ratio. IFN-γ production (Figure 29D) and cell lysis (Figure 29E) of transduced FAP-CAR WT and Ikzf1± cells were measured at the described effector:target cell ratio, where the target cells were FAP-expressing 3T3 cells.

[0089] [Figure 30] Figures 30A, 30B, and 30C demonstrate the efficacy of Ikaros-depleted CAR T cells against established tumors in vivo. CAR T cells were administered to mice carrying established mesothelin-expressing AE17 tumors. Tumor volume was measured after administration of mesoCAR-expressing WT mice and Ikzf1± (Figure 30A) or IkDN (Figure 30B). Tumor volume was also measured after administration of FAP-CAR-expressing WT mice and Ikzf1± (Figure 30C).

[0090] [Figure 31-1]Figures 31A, 31B, 31C, 31D, 31E, and 31F show increased persistence and resistance of Ikzf1±CAR T cells in the immunosuppressive tumor microenvironment compared to WT CAR T cells. The percentage of CAR-expressing WT or Ikzf1± cells (GFP-positive) was determined based on flow cytometry of cells collected from the spleen (Figure 31A) and tumor (Figure 31B). The functional capacity of CAR T cells collected from the spleen or tumor 3 days after infusion was evaluated by measuring IFN-γ production after stimulation with CD3 / CD28 antibody (Figure 31C) or PMA / ionomycin (PMA / I) (Figure 31D). Regulatory T cells (CD4+FoxP3+ expressing) and macrophages (CD206 expressing) were evaluated by measuring the expression of Treg or macrophage markers on CAR T cells collected from the spleen or tumor 9 days after infusion. [Figure 31-2] Figures 31A, 31B, 31C, 31D, 31E, and 31F show increased persistence and resistance of Ikzf1±CAR T cells in the immunosuppressive tumor microenvironment compared to WT CAR T cells. The percentage of CAR-expressing WT or Ikzf1± cells (GFP-positive) was determined based on flow cytometry of cells collected from the spleen (Figure 31A) and tumor (Figure 31B). The functional capacity of CAR T cells collected from the spleen or tumor 3 days after infusion was evaluated by measuring IFN-γ production after stimulation with CD3 / CD28 antibody (Figure 31C) or PMA / ionomycin (PMA / I) (Figure 31D). Regulatory T cells (CD4+FoxP3+ expressing) and macrophages (CD206 expressing) were evaluated by measuring the expression of Treg or macrophage markers on CAR T cells collected from the spleen or tumor 9 days after infusion.

[0091] [Figure 32] Figures 32A and 32B show that T cells with reduced Ikaros levels are less sensitive to the soluble inhibitors TGF-β and adenosine. MesoCAR-expressing WT, Ikzf1±, and IkDN cells were tested for their ability to produce IFN-γ (Figure 32A) and cytotoxicity (Figure 32B) in response to TGF-β or adenosine.

[0092] [Figure 33] Figures 33A and 33B are graphs showing the increase in titer against influenza vaccine strains compared to placebo. In Figure 33A, for each of the RAD001-administered cohorts in the intended treatment population, the increase in baseline geometric mean titer for each of the three influenza vaccine strains (H1N1 A / California / 07 / 2009, H3N2 A / Victoria / 210 / 2009, B / Brisbane / 60 / 2008) is shown compared to the increase in the placebo cohort 4 weeks after vaccination. The thick black line indicates a 1.2-fold increase in titer compared to placebo, which is the criterion that must be met for two of the three influenza vaccine strains to meet the primary endpoint of this trial. An asterisk "*" indicates that the GMT titer compared to placebo is greater than 1 and has at least an 80% posterior probability. Figure 33B is a graph of the same data as in Figure 33A for a subset of subjects with baseline influenza titer ≤ 1:40.

[0093] [Figure 34] Figure 34 shows a scatter plot of the ratio of the increase in geometric mean titer of RAD001 concentration against each influenza vaccine strain four weeks after vaccination. RAD001 concentration (one hour after administration) was measured after subjects received four weeks of administration. All subjects who underwent pharmacokinetic measurements were included in the analysis population. The ratio of the increase in geometric mean titer four weeks after vaccination compared to baseline is shown on the y-axis.

[0094] [Figure 35]Figure 35 is a graph showing the increase in titer against heterologous influenza strains compared to placebo. Four weeks after vaccination, each of the RAD001-administered cohorts in the intended treatment population shows a baseline increase in the geometric mean titer of influenza 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) compared to the increase in the placebo cohort. * indicates that the titer compared to placebo is greater than 1 and has a posterior probability of at least 80%.

[0095] [Figure 36] Figures 36A and 36B show graphs of IgG and IgM levels before and after influenza vaccination. Anti-A / H1N1 / California / 07 / 2009 influenza IgG and IgM levels were measured in serum obtained from subjects before and 4 weeks after influenza vaccination. No significant differences were detected in the change from baseline to 4 weeks after vaccination between the RAD001 and placebo cohorts (all p-values ​​> 0.05 by Kruskal-Wallis rank-sum test).

[0096] [Figure 37]Figures 37A, 37B, and 37C are graphs showing the decrease in PD-1-positive CD4 and CD8 cells and the increase in PD-1-negative CD4 T cells after RAD001 treatment. The percentages of PD-1-positive CD4, CD8, and PD-1-negative CD4 T cells were determined by FACS analysis of PBMC samples at baseline, 6 weeks after investigational drug treatment (week 6), and 6 weeks after discontinuation of the investigational drug and 4 weeks after influenza vaccination (week 12). Figure 37A shows that, compared to the placebo cohort (n=25), the cohorts administered RAD001 at dose levels of 0.5 mg / day (n=25), 5 mg / week (n=29), and 20 mg / week (n=30) showed a significant decrease in PD-1-positive CD4 T cells (-37.1 to -28.5%) at week 12, with p=0.002 (0.02), p=0.003 (q=0.03), and p=0.01 (q=0.05), respectively. Figure 37B shows that, compared to the placebo cohort (n=25), the cohorts administered RAD001 (n=109) at dose levels of 0.5 mg / day (n=25), 5 mg / week (n=29), and 20 mg / week (n=30) showed a significant decrease in PD-1-positive CD8 T cells (-43.3 to -38.5%) at week 12, with p=0.01 (q=0.05), p=0.007 (q=0.04), and p=0.01 (q=0.05), respectively. Figure 37C shows that, compared to the placebo cohort (n=25), the cohorts administered RAD001 (n=109) at dose levels of 0.5 mg / day (n=25), 5 mg / week (n=29), and 20 mg / week (n=30) showed a significant increase in PD-1 negative CD4 T cells (3.0–4.9%) at week 12, with p=0.0007 (q=0.02), p=0.03 (q=0.07), and p=0.03 (q=0.08), respectively.

[0097] [Figure 38]Figures 38A and 38B are graphs showing 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, adjusted for differences in baseline PD-1 expression. The percentages of PD-1-positive CD4, CD8, and PD-1-negative CD4 T cells were determined by FACS analysis of PBMC samples at baseline, 6 weeks after investigational drug treatment (week 6), and 6 weeks after investigational drug discontinuation and 4 weeks after influenza vaccination (week 12). Figure 38A shows a significant 30.2% decrease in PD-1+CD4 T cells at week 6 in the pooled RAD cohort (n=84) compared to the placebo cohort (n=25), with p=0.03 (q=0.13). The decrease in PD-1-positive CD4 T cells at week 12 in the pooled RAD compared to the placebo cohort was 32.7%, with p=0.05 (q=0.19). Figure 38B shows that the pooled RAD001 cohort (n=84) showed a significant 37.4% reduction in PD-1-positive CD8 T cells at week 6 compared to the placebo cohort (n=25), with p=0.008 (q=0.07). The reduction in PD-1-positive CD8 T cells at week 12 in the pooled RAD001 group compared to the placebo cohort was 41.4%, with p=0.066 (q=0.21). Figures 38A and 38B show the data from Figures 37A, 37B, and 37C, but the different RAD001 dose groups from Figures 37A, 37B, and 37C were pooled into a single RAD001 treatment group in Figures 38A and 38B.

[0098] [Figure 39] Figure 39 shows the increase in exercise and energy in elderly subjects in response to RAD001.

[0099] [Figure 40] Figures 40A and 40B show the predictive effects of RAD001 on P70 S6K activity in cells. Figure 40A shows P70 S6 kinase inhibited with high weekly and daily doses of RAD001, and Figure 40B shows P70 S6 kinase inhibited with low weekly doses of RAD001.

[0100] [Figure 41] Figures 41A, 41B, and 41C show Biacore T200 SPR sensorgrams for scFv SS1 (Figure 41A), M5 (Figure 41B), and M11 (Figure 41C).

[0101] [Figure 42] Figures 42A, 42B, and 42C show epitope-binning SPR sensorgrams of anti-human mesothelin scFv compared to mouse SS1 scFv. Competitive binding was observed in scFv M12, M14, M16, M17, M21, and M23 (Figure 42A). ScFv M5 (Figure 42B) and M11 (Figure 42C) bind to different epitopes than SS1.

[0102] [Figure 43] Figure 43 shows tumor growth after various mesothelin CART treatments in an OVCAR8 tumor model. Mean tumor volume ± SEM up to 62 days after tumor transplantation. T cells were administered on days 14 and 19. Small circles: Mice treated with 100 μl of PBS via the lateral tail vein; black squares: Mice treated with isotype control T cells; gray triangles: Mice treated with a single dose of SS1 CAR T cells; inverted triangles: Mice treated with two doses of SS1 CAR T cells; diamond shapes: Mice treated with a single dose of M5 CAR T cells; large circles: Mice treated with two doses of M5 CAR T cells; gray squares: Mice treated with a single dose of M11 CAR T cells; and black triangles: Mice treated with two doses of M11 CAR T cells.

[0103] [Figure 44] Figure 44 is a schematic diagram of the applicability range of human mesothelin peptides in hydrogen-deuterium exchange mass spectrometry analysis. Each black bar represents a peptide.

[0104] [Figure 45]Figures 45A and 45B are graphs showing the difference in deuterium uptake of human mesothelin when complexed with SS1 (black bars) and M5 (gray bars). The difference in deuterium uptake due to antibody binding (shown on the y-axis) is shown for each detected peptide fragment (shown on the x-axis), with Figure 45A showing peptides from amino acids 297 to 464 and Figure 45B showing peptides from amino acids 458 to 586. All differences are relative to the deuterium uptake of unbound mesothelin (control). * indicates the region of statistical significance using Tukey's test for peptides with a difference of less than 0.75 Da.

[0105] [Figure 46] Figure 46 is a schematic diagram showing the primary sequence (amino acids 296-588) of the antigen human mesothelin and the regions protected by SS1 and M5. The black bars indicate amino acids protected when complexed with SS1 (amino acids 314-315, 317-318, 346-349, and 369-375). The gray bars indicate amino acids protected when complexed with M5 (amino acids 485-490, 498-507, 532-537, and 545-572).

[0106] [Figure 47] Figure 47 is a general map showing different configurations of constructs encoding CAR and shRNA for co-expression of CAR and shRNA. Figures 47A–47D show various configurations on a single vector, for example, where the U6 regulatory shRNA is upstream or downstream of the EF1 alpha-regulated CAR-encoding component. In the representative constructs shown in Figures 47A and 47B, transcription occurs in the same direction via the U6 and EF1 alpha promoters. In the representative constructs described in Figures 47C and 47D, transcription occurs in different directions via the U6 and EF1 alpha promoters. In Figure 47E, the shRNA (and corresponding U6 promoter) is on the first vector, and the CAR (and corresponding EF1 alpha promoter) is on the second vector (Figure 16E).

[0107] [Figure 48]Figure 48 shows the structures of two representative RCAR configurations. The antigen-binding member includes an antigen-binding domain, a transmembrane domain, and a switch domain. The intracellular binding member includes a switch domain, a costimulatory signaling domain, and a primary signaling domain. The two configurations demonstrate that the first and second switch domains described herein can be oriented differently relative to the antigen-binding and intracellular binding members. Other RCAR configurations are further described here. [Modes for carrying out the invention]

[0108] Detailed description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains.

[0109] The singular form signifies one or more than one (i.e., at least one), which is the grammatical purpose of the indefinite article. For example, "component" means one component or more than one component.

[0110] The term “approximately” is intended to encompass variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1%, from a specific value when referring to measurable values ​​such as quantity or duration, insofar as such variations are appropriate to the method of disclosure.

[0111] The term “chimeric antigen receptor” or “CAR” generally refers to a set of two polypeptides, in its simplest embodiment, which, when present in an immune effector cell, provides the cell with specificity to target cells, generally cancer cells, and intracellular signaling. In some embodiments, a CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to here as the “intracellular signaling domain”) containing a functional signaling domain derived from a stimulating and / or co-stimulating molecule, such as those described below. In some aspects, the polypeptide sets are adjacent to one another. In some embodiments, the polypeptide set includes a dimerization switch that, in the presence of a dimerizing molecule, can link the polypeptides to each other, for example, linking the antigen-binding domain to the intracellular signaling domain. In one aspect, the stimulating molecule is a zeta chain associated with the T cell receptor complex. In another aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulating molecule, such as those described below. In one aspect, the co-stimulatory molecule is selected from the co-stimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27 and / or CD28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from 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 comprising 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 two functional signaling domains derived from one or more co-stimulatory molecules 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 at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from the stimulatory molecule.In one respect, the CAR includes an optional leader sequence at the amino terminus (N-ter) of the CAR fusion protein. In another respect, the CAR further includes a leader sequence at the N-terminus of the extracellular antigen-binding domain, which may optionally be cleaved from the antigen-binding domain (e.g., scFv) during cell processing and localization of the CAR to the cell membrane.

[0112] The term “signaling domain” refers to a functional portion of a protein that acts by transmitting information within a cell to regulate cellular activity via defined signaling pathways, either by producing a second messenger or by acting as an effector in response to such a messenger.

[0113] The term “mesothelin” as used herein refers to mesothelin, a 40 kDa protein anchored to the cell membrane by glycosylphosphatidylinositol (GPI) binding and an amino-terminal 31 kDa detachment fragment called megakaryocyte-enhancing factor (MPF). Both fragments contain an N-glycosylation site. The term also refers to soluble splice variants of the 40 kDa carboxyl-terminal fragment, also known as “soluble mesothelin / MPF-related.” Preferably, the term refers to human mesothelin of GenBank accession number AAH03512.1 and its naturally cleaved portion, for example, expressed on cell membranes, such as cancer cell membranes.

[0114] The term "antibody" as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be polyclonal or monoclonal, multi-chain or single-chain or complete immunoglobulins, and may originate from natural or recombinant sources. Antibodies may be tetramers of immunoglobulin molecules.

[0115] The term “antibody fragment” refers to at least one portion of an antibody that retains the ability to specifically interact with an antigen's epitope (e.g., by binding, steric hindrance, stabilization / destabilization, or spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fv(sdFv), Fd fragments consisting of a VH domain and a CH1 domain, linear antibodies, single-domain antibodies such as sdAb(either VL or VH), camel VHH domains, and bivalent fragments containing two Fab fragments linked by disulfide crosslinking at a hinge region. These include polyspecific antibodies formed from antibody fragments and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intracellular antibodies, bispecific antibodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be transplanted into polypeptide-based scaffolds such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199 describing fibronectin polypeptide minibodies).

[0116] 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 and heavy chain variable regions are sequentially linked, for example, via a synthetic linker, for example, a short-mobility polypeptide linker, and the scFv can be expressed as a single-chain polypeptide, and the scFv retains the complete specificity of the antibody from which it is derived. Unless otherwise specified, the scFv as used herein may comprise, for example, the VL and VH variable regions in either order with respect to the N-terminus and C-terminus of the polypeptide, and the scFv may comprise a VL-linker-VH or a VH-linker-VL.

[0117] The portion of the CAR of the present invention, which includes an antibody or an antibody fragment, can exist in a variety of forms, in which the antigen-binding domain is expressed as part of an adjacent polypeptide chain, for example, containing a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen-binding domain of the CAR composition of the present invention includes an antibody fragment. In another aspect, the CAR includes an antibody fragment containing an scFv.

[0118] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules as a naturally occurring higher-order structure, and this usually determines the class to which the antibody belongs.

[0119] The term "antibody light chain" refers to the smaller of two types of polypeptide chains present in antibody molecules as a naturally occurring higher-order structure. Kappa (κ) and lambda (λ) light chains are the two main antibody light chain isotypes.

[0120] The term “recombinant antibody” refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage or yeast expression system. This term should also be interpreted to mean an antibody produced by the synthesis of an antibody-encoding DNA molecule, which expresses an antibody protein or an amino acid sequence that identifies the antibody, and which is obtained using recombinant DNA or amino acid sequence technologies available and well-known in this field.

[0121] The terms “antigen” or “Ag” refer to molecules that elicit an immune response. This immune response may involve either antibody production or activation of specific immune-qualified cells, or both. Those skilled in the art will understand that virtually any macromolecule, including virtually all proteins or peptides, can act as an antigen. Furthermore, antigens may be recombinant or derived from genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response, therefore, encodes an “antigen,” as the term is used herein. Furthermore, those skilled in the art will understand that antigens do not necessarily have to be encoded solely by a 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 may be arranged in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled in the art will understand that antigens do not necessarily have to be encoded by a “gene.” It will be readily apparent that antigens may be produced by synthesis, derived from biological samples, or be macromolecules other than polypeptides. Such biological samples include, but are not limited to, tissue samples, tumor samples, and bodily fluids containing cells or other biological components.

[0122] The term “competition” refers to the ability of an antigen-binding domain, e.g., an antibody or fragment, to interfere with the binding of another antigen-binding domain, e.g., an antigen-binding domain, e.g., an antibody or fragment, to a target, e.g., mesothelin, directly or indirectly. The extent to which an antigen-binding domain, e.g., an antibody or fragment, interferes with the binding of another antigen-binding domain, e.g., an antibody or fragment, to its target, and therefore whether it can be considered competition, can be determined using a competitive binding assay. In some embodiments, a competitive binding assay is a quantitative competitive assay. For example, a particularly suitable quantitative competitive assay uses a surface plasmon resonance (SPR)-based technique to measure the binding, e.g., competition, between one antibody or fragment and another antibody or fragment for binding to an immobilized target. A representative SPR-based competitive assay is described in Example 2 herein. Other suitable quantitative competitive assays use a FACS-based technique to measure the competition between a labeled (e.g., His-tagged, biotinylated, or radioactively labeled) antibody or fragment and another antibody or fragment for binding to a target.

[0123] The term “anti-cancer effect” refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an extension of life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with the cancerous state. “Anti-cancer effect” can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies to prevent the development of cancer in the first place. The term “anti-tumor effect” refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0124] The term "self" refers to some substance originating from the same individual that is later reintroduced into the individual.

[0125] The term "homogeneous" refers to any substance originating from a different animal of the same species as the individual introducing the substance. Two or more individuals should be considered homogeneous if they do not have identical genes at one or more loci. In some respects, homogeneous substances from individuals of the same species may be genetically sufficiently different to interact antigenically.

[0126] The term "heterogeneous" refers to a graft derived from an animal of a different species.

[0127] The term "cancer" refers to a disease characterized by the uncontrolled proliferation of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. A variety of cancers are described here, including, but not limited to, mesothelioma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer.

[0128] The term “diseases associated with mesothelin expression” includes, but is not limited to, diseases or conditions associated with mesothelin expression that involve cells expressing mesothelin, such as proliferative disorders like cancer or malignant tumors, or precancerous conditions like mesothelial hyperplasia; or non-cancer-related indications associated with cells expressing mesothelin. Examples of various cancers that express mesothelin include, but are not limited to, mesothelioma, lung cancer, ovarian cancer, and pancreatic cancer.

[0129] The term “conservative 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 conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or antibody fragment of the present invention by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions involve replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CAR of the present invention may be replaced with other amino acid residues from the same side chain family, and the modified CAR can be tested for its ability to bind to mesotheline using, for example, the functional assay described herein.

[0130] The term “stimulus” refers to a primary response induced by the binding of a stimulating molecule (e.g., the TCR / CD3 complex or CAR) to its homologous ligand (or, in the case of CAR, a tumor antigen), thereby mediating a signaling event, such as, but not limited to, signaling by the TCR / CD3 complex or signaling by an appropriate NK receptor or the signaling domain of CAR. A stimulus can mediate the modification of the expression of a molecule.

[0131] The term “stimulating molecule” refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that provides a cytoplasmic signaling sequence that controls the activation of immune cells in a stimulating manner in at least one aspect of the immune cell signaling pathway. In one aspect, the signal is a primary signal that is initiated, for example, by the binding of a TCR / CD3 complex to an MHC molecule carrying a peptide, and leads to the mediation of a T cell response including, but not limited to, proliferation, activation, and differentiation. Primary cytoplasmic signaling sequences that act in a stimulating manner (also called “primary signaling domains”) may include signaling motifs known as immune receptor tyrosine 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 CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In a particular CAR of the present invention, the intracellular signaling domain in any one or more CARs of the present invention comprises an intracellular signaling sequence, for example, the primary signaling sequence of CD3 zeta. In a particular CAR of the present invention, the primary signaling sequence of CD3 zeta is the sequence provided in SEQ ID NO: 9 or equivalent residues from non-human species, such as mice, rodents, monkeys, apes, etc. In a particular CAR of the present invention, the primary signaling sequence of CD3 zeta is the sequence provided in SEQ ID NO: 10 or equivalent residues from non-human species, such as mice, rodents, monkeys, apes, etc.

[0132] The term “antigen-presenting cell” or “APC” refers to immune system cells, such as accessory cells (e.g., B cells, dendritic cells, etc.), that display exogenous antigens complexed with major histocompatibility complexes (MHCs) on their surface. T cells can recognize these complexes using T cell receptors (TCRs). APCs process the antigens and present them to T cells.

[0133] As used here, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. Intracellular signaling domains produce signals that enhance the immune effector function of CAR-containing cells, such as CAR T cells. Examples of immune effector function in CAR T cells include cytokine secretion, cytolytic activity, and helper activity.

[0134] In one embodiment, the intracellular signaling domain includes a primary intracellular signaling domain. A representative primary intracellular signaling domain may include one derived from a molecule responsible for the primary stimulus or antigen-dependent stimulus. In another embodiment, the intracellular signaling domain may include a co-stimulatory intracellular domain. A representative co-stimulatory intracellular signaling domain may include one derived from a molecule responsible for the co-stimulatory signal or antigen-independent stimulus. For example, in the case of CART, the primary intracellular signaling domain may include the cytoplasmic sequence of the T cell receptor, and the co-stimulatory intracellular signaling domain may include the cytoplasmic sequence from the co-receptor or co-stimulatory molecule.

[0135] The primary intracellular signaling domain may contain signaling motifs known as immune receptor tyrosine activation motifs or ITAMs. Examples of ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

[0136] The terms “zeta,” or also “zeta chain,” “CD3 zeta,” or “TCR-zeta” are defined as the protein provided under GenBan accession number BAG36664.1 or equivalent residues from non-human species, such as mice, rodents, monkeys, and apes, and the “zeta-stimulating domain,” or also “CD3 zeta-stimulating domain,” or “TCR-zeta-stimulating domain” are defined as amino acid residues or functional derivatives of the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit the initial signals necessary for T cell activation. In one aspect, the cytoplasmic domain of zeta contains residues 52-164 of GenBank accession number BAG36664.1 or equivalent residues from non-human species, such as mice, rodents, monkeys, and apes, that are functional orthologues. In another aspect, the “zeta-stimulating domain” or “CD3 zeta-stimulating domain” is the sequence provided under Sequence ID No. 9. In one respect, the “zeta-stimulating domain” or “CD3 zeta-stimulating domain” is the sequence provided as Sequence ID No. 10.

[0137] The term “costimulatory molecule” refers to a congenital binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules include, but are not limited to, MHCI-type 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). Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, C This product contains ligands that specifically bind to D18, 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, and CD83.

[0138] The co-stimulatory intracellular signaling domain can be the intracellular portion of a co-stimulatory molecule. Co-stimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphoid activators (SLAM proteins), and activated NK cell receptors. Examples of such molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDDS, CD7, CD287, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and CD83.

[0139] The intracellular signaling domain may include the entire intracellular portion of the derived molecule or the entire innate intracellular signaling domain or its functional fragment or derivative.

[0140] The term “4-1BB” refers to a member of the TNFR superfamily that has an amino acid sequence provided as GenBank accession number AAA62478.2 or equivalent residues from non-human species, such as mice, rodents, monkeys, and apes. In one respect, the “4-1BB costimulatory domain” is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2 or equivalent residues from non-human species, such as mice, rodents, monkeys, and apes. In another respect, the “4-1BB costimulatory domain” is the sequence provided as Sequence ID No. 7 or equivalent residues from non-human species, such as mice, rodents, monkeys, and apes.

[0141] The term "antigen-presenting cells" used here refers to immune system cells, such as accessory cells (e.g., B cells, dendritic cells, etc.), that present exogenous antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize these complexes using T cell receptors (TCRs). APCs process the antigen and present it to T cells.

[0142] The term “coding” refers to the inherent characteristic of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, that acts as a template for the synthesis of either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, as well as other polymers and macromolecules having biological properties derived therefrom, in biological processes. Therefore, a gene, cDNA, or RNA codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to that of the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand, used as a transcription template for the gene or cDNA, can be said to code for a protein or other product of that gene or cDNA.

[0143] Unless otherwise specified, “nucleotide sequences encoding an amino acid sequence” include all nucleotide sequences encoding the same amino acid sequence, including denatured versions of each other. The term “nucleotide sequences encoding a protein or RNA” also includes introns, insofar as the nucleotide sequence encoding the protein may contain introns in some version.

[0144] The term “effective dose” or “therapeutic dose” here refers to the amount of any compound, formulation, substance, or composition described herein that is interchangeable and effective in achieving a particular biological outcome. The term “endogenous” refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0145] The term “exogenous” refers to any substance introduced from or produced outside of an organism, cell, tissue, or system.

[0146] The term “expression” refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0147] The term “introduction vector” refers to a composition containing isolated nucleic acid that can be used to deliver the isolated nucleic acid into the interior of a cell. Numerous vectors are known in this field and include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “introduction vector” includes self-replicating plasmids or viruses. The term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the introduction of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral introduction vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and lentiviral vectors.

[0148] The term “expression vector” refers to a vector containing recombinant polynucleotides that includes an expression control sequence manipulatively bound to the nucleotide sequence to be expressed. An expression vector contains a sufficient cis-acting region for expression, with other expression regions supplied by a host cell or in vitro expression system. Expression vectors include all known in the art, including cosmids, plasmids (e.g., naked or liposome-encapsulated), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0149] The term “lentivirus” refers to a genus within the retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells and deliver a substantial amount of genetic information to the host cell's DNA, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. The term “lentiviral vector” refers to a vector derived from at least a portion of the lentiviral genome, including particularly self-inactivating lentiviral vectors, such as those provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors available for clinical use include, for example, Oxford BioMedica’s LENTIVECTOR® gene delivery technology and Lentigen’s LENTIMAX. TM This includes, but is not limited to, vector systems. Non-clinical lentiviral vectors are also available and are known to those skilled in the art.

[0150] The term “homologous” or “identical” refers to the subunit sequence identity between two polymer molecules, such as two DNA molecules or two RNA molecules, or two nucleic acid molecules or two polypeptide molecules. If the subunit positions of both molecules are occupied by the same monomer subunits, for example, if the positions of each of the two DNA molecules are occupied by adenine, then they are homologous or identical in that respect. Homologousness between two sequences is a function of matching or homologous positions; for example, if half the positions of the two sequences (e.g., 5 positions in a 10-subunit length polymer) are homologous, then the two sequences are 50% homologous, and if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

[0151] The term "humanized" refers to a form of non-human (e.g., mouse) antibody that is a chimeric immunoglobulin, immunoglobulin chain, or fragment (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of an antibody) containing minimal sequences derived from non-human immunoglobulins. For the most part, humanized antibodies and antibody fragments are human immunoglobulins (recipient antibodies or antibody fragments) in which residues from the recipient's complementarity-determining region (CDR) are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, that possess the desired specificity, affinity, and capability. In some cases, Fv framework region (FR) residues of human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments may contain residues not found in either the recipient antibody or the transferred CDR or framework sequence. These modifications can further refine and optimize the performance of the antibody or antibody fragment. Generally, humanized antibodies or antibody fragments contain at least one, generally two, substantial portions of variable domains, where all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin, and all or substantially all of the FR region corresponds to a human immunoglobulin sequence. Humanized antibodies or antibody fragments may also contain at least a portion of the immunoglobulin constant region (Fc), which is generally that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

[0152] The term "fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, whose entire molecule is of human origin or consists of an amino acid sequence identical to that of the human form of the antibody or immunoglobulin.

[0153] The term “isolated” means altered or removed from its natural state. For example, nucleic acids or peptides that are naturally present in a living animal are not “isolated,” but the same nucleic acids or peptides that have been partially or completely separated from their naturally occurring coexisting substances are “isolated.” Isolated nucleic acids or proteins can exist in a substantially pure form or in a non-natural environment, such as a host cell.

[0154] In the context of this invention, the following abbreviations are used for commonly occurring nucleic acid bases: "A" refers to adenosine, "C" to cytosine, "G" to guanosine, "T" to thymidine, and "U" to uridine.

[0155] The terms “manipulative binding” or “transcriptional regulation” refer to a functional binding between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. For example, when a primary nucleic acid sequence is functionally correlated with a secondary nucleic acid sequence, the primary nucleic acid sequence is manipulatively bound to the secondary nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is manipulatively bound to that coding sequence. Manipulatively bound DNA sequences may be adjacent to each other and, if it is necessary to align two protein-coding regions, they may be within the same reading frame.

[0156] The term “non-enteral” administration of immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), intratumoral or intrasternal injection or infusion.

[0157] The terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-stranded or double-stranded form, and their polymers. Unless otherwise specified, the terms include nucleic acids, including known analogs of native nucleotides, that have similar binding properties to control nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence implicitly includes its conserved modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitution can be achieved by producing sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0158] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds containing amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, these terms refer to both short chains, which are commonly called peptides, oligopeptides, and oligomers in this field, and long chains, of which there are many types commonly called proteins in this field. A “polypeptide” includes, for example, bioactive fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, recombinant peptides, or combinations thereof.

[0159] The term "promoter" refers to a DNA sequence that is recognized by or introduced into a cell's synthetic mechanism, and is necessary for the specific transcription of a polynucleotide sequence.

[0160] The term “promoter / control sequence” refers to a nucleic acid sequence necessary for the expression of a gene product that is operably bound to the promoter / control sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other regulatory elements necessary for the expression of the gene product. Promoter / control sequences, for example, express gene products in a tissue-specific manner.

[0161] The term “constitutive” promoter is a nucleotide sequence that, when operably bound to a polynucleotide encoding or identifying a gene product, causes the cell to produce that gene product under most or all physiological conditions.

[0162] The term “inducible” promoter is a nucleotide sequence that, when manipulably bound to a polynucleotide encoding or identifying a gene product, causes the cell to produce the gene product only when a substantially promoter-corresponding inducer is present within the cell.

[0163] The term “tissue-specific” promoter is a nucleotide sequence that, when manipulatively bound to a polynucleotide encoded or identified by a gene, causes a cell to produce a gene product only if the cell is substantially of the tissue type corresponding to the promoter.

[0164] In the context of scFv, the term “mobile polypeptide linker” refers to a peptide linker consisting of amino acids, such as glycine and / or serine residues, used alone or in combination to link variable heavy and variable light chain regions together. In one embodiment, the mobile polypeptide linker is a Gly / Ser linker with an amino acid sequence (Gly-Gly-Gly-Ser). nIncludes (SEQ ID NO: 38) (where n is a positive integer greater than or equal to 1). For example, n=1, n=2, n=3; n=4, n=5 and n=6, n=7, n=8, n=9 and n=10. In one embodiment, the mobile polypeptide linker includes, but is not limited to, (Gly4Ser)4 (SEQ ID NO: 27) or (Gly4Ser)3 (SEQ ID NO: 28). In other embodiments, the linker includes multiple repetitions of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO: 29). Also included in the scope of the present invention are the linkers described in WO2012 / 138475 (which is incorporated herein by reference).

[0165] The 5' cap used here (RNA cap, RNA 7-methylguanosine cap or RNA m) 7 A 5' cap (also called a G-cap) is a modified guanine nucleotide attached to the “front” or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of a terminal group bound to the first transcription nucleotide. Its presence is important for ribosome recognition and protection from RNases. Capping occurs cotranscribeally, linked to transcription and influencing each other. Immediately after transcription initiation, the 5' end of the synthesized mRNA is fused by a cap-synthesis complex that binds to RNA polymerase. This enzyme complex catalyzes the chemical reactions necessary for mRNA cap formation. Synthesis proceeds as a multi-step biochemical reaction. Modifying the cap-forming region can regulate mRNA functionality, such as stability or translation efficiency.

[0166] The term "in vitro transcribed RNA" used here refers to RNA synthesized in vitro, preferably mRNA. Generally, in vitro transcribed RNA is produced from an in vitro transcription vector. An in vitro transcription vector contains a template used for the production of in vitro transcribed RNA.

[0167] The “poly(A)” used here refers to a series of adenosines bound to mRNA by polyadenylation. In a preferred embodiment of the construct for transient expression, polyA is 50 to 5000 (SEQ ID NO: 30), preferably more than 64, more preferably more than 100, and most preferably more than 300 or 400. The poly(A) sequence can be chemically or enzymatically modified to regulate mRNA functionality such as localization, stability, or translation efficiency.

[0168] The term "polyadenylation" used here refers to the covalent bonding of a polyadenylyle portion or a modified variant thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are adenylated at their 3' end. The 3' poly(A) tail is a long sequence (often hundreds) of adenine nucleotides added to premRNA by the action of the enzyme polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to transcripts containing a specific sequence, the polyadenylation signal. The poly(A) tail and the proteins that bind to it help protect mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, mRNA efflux from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA to RNA, but can also occur later in the cytoplasm. After transcription is complete, the mRNA strand is cleaved by the action of an endonuclease complex accompanied by RNA polymerase. The cleavage site is usually characterized by the presence of the nucleotide sequence AAUAAA near the cleavage site. Once the mRNA is cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

[0169] The term "transient" as used here refers to the expression of a non-integrated transgene for a period of several hours, several days, or several weeks, where the expression period is shorter than that of a gene that is integrated into the genome or contained within a stable plasmid replicon in a host cell.

[0170] As used herein, the terms "treatment" and "treating" refer to a decrease or improvement in the progression, severity, and / or duration of a proliferative disorder or an improvement in one or more (preferably one or more recognizable symptoms) of the symptoms of a proliferative disorder, brought about by the administration of one or more therapeutic agents (e.g., one or more therapeutic agents such as the CARs of the present invention). In certain embodiments, the terms "treatment" and "treating" refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which may not necessarily be recognizable to the patient. In other embodiments, the terms "treatment" and "treating" refer to arresting the progression of a proliferative disorder physically, e.g., by stabilization of a physical parameter, physiologically, or both, e.g., by stabilization of recognizable symptoms. In other embodiments, the terms "treatment" and "treating" refer to a decrease or stabilization of tumor size or cancerous cell number.

[0171] The term "signaling pathway" refers to the biochemical relatedness among diverse signaling molecules that play a role in the transmission of signals from one part of a cell to another part of the cell. The term "cell surface receptor" refers to molecules and molecular complexes that can receive signals through the cell membrane and transmit the signals.

[0172] The term "subject" is intended to include living organisms (e.g., mammals, humans) capable of mounting an immune response.

[0173] The term "substantially purified" cells refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that are separated from other cell types that are normally associated in their natural state. In one example, a population of substantially purified cells refers to a homogeneous population of cells. In other examples, the term simply refers to cells that are separated from cells that are necessarily associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[0174] As used herein, the term "therapy" means treatment. A therapeutic effect can be obtained by alleviation, suppression, remission, or eradication of a disease state.

[0175] As used herein, the term "prevention" means a preventive or protective measure against a disease or a disease state.

[0176] The term "cancer-associated antigen" or "tumor antigen" is interchangeably used to refer to a molecule (generally a protein, carbohydrate or lipid) that is expressed, either fully or as a fragment (e.g., MHC / peptide), on the surface of cancer cells and is useful for the preferential targeting of drugs to cancer cells. In certain embodiments, the tumor antigen is a marker expressed by both normal and cancer cells, such as a cell lineage marker, such as CD19 on B cells. In certain embodiments, the tumor antigen is a cell surface molecule that is overexpressed in cancer cells as compared to normal cells, e.g., 1-fold overexpressed, 2-fold overexpressed, 3-fold or more overexpressed as compared to normal cells. In certain embodiments, the tumor antigen is a cell surface molecule that is not properly synthesized in cancer cells, e.g., a molecule that contains deletions, additions or mutations as compared to a molecule expressed in normal cells. In certain embodiments, the tumor antigen is synthesized exclusively, either fully or as a fragment (e.g., MHC / peptide), on the cell surface of cancer cells and is not synthesized or expressed on the surface of normal cells. In certain embodiments, the CARs of the present invention include CARs that include an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presented peptide. Typically, an endogenous protein-derived peptide fills the pocket of a major histocompatibility antigen complex (MHC) class I molecule and CD8 +They are recognized by T cell receptors (TCRs) on T lymphocytes. MHC complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy. TCR-like antibody targeting peptides derived from viruses or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, for example, Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified by screening libraries such as human scFv phage display libraries.

[0177] The terms "gene transfer," "transformation," or "transduction" refer to the process of transferring or introducing foreign nucleic acids into host cells. "Genetically transferred," "transformed," or "transduced" cells are those that have undergone gene transfer, transformation, or transduction of foreign nucleic acids. Cells include primary target cells and their offspring.

[0178] The term “specifically binding” refers to an antibody or ligand that recognizes and binds to a binding partner protein (e.g., a tumor antigen) present in the sample, and that antibody or ligand substantially does not recognize or bind to other molecules in the sample.

[0179] To the extent that the term is used herein, “controllable chimeric antigen receptor (RCAR)” refers to a set of polypeptides, generally two in its simplest embodiment, that, when present in RCARX cells, provide RCARX cells that are specific to target cells, generally cancer cells, and that have controllable intracellular signaling production or proliferation, thereby optimizing the immunoeffector properties of RCARX cells. RCARX cells provide specificity to target cells containing the antigen to which the antigen-binding domain is bound, at least in part, depending on the antigen-binding domain. In some embodiments, the RCAR comprises a dimerization switch, which, in the presence of a dimerization molecule, can link the intracellular signaling domain to the antigen-binding domain.

[0180] To the extent that the term is used herein, “membrane anchor” or “membrane anchoring domain” means a polypeptide or portion sufficient to fix an extracellular or intracellular domain to the plasma membrane, such as a myristoyl group.

[0181] To the extent that the term is used herein, a “switch domain” refers to a molecule that binds to another switch domain in the presence of a dimerizing molecule, generally a polypeptide-based molecule, for example, when referring to RCAR. This binding results in the functional coupling of a molecule bound to the first switch domain, e.g., a fused first, and a molecule bound to the second switch domain, e.g., a fused second. The first and second switch domains are collectively called a dimerizing switch. In some embodiments, the first and second switch domains are identical to each other, e.g., polypeptides having the same primary amino acid sequence, and are collectively called a homodimerizing switch. In some embodiments, the first and second switch domains are different to each other, e.g., polypeptides having different primary amino acid sequences, and are collectively called a heterodimerizing switch. In some embodiments, the switch is intracellular. In some embodiments, the switch is extracellular. In some embodiments, the switch domain is polypeptide-based, e.g., FKBP or FRB-based, and the dimerizing molecule is a small molecule, e.g., a rapalog. In some embodiments, the switch domain is polypeptide-based, for example, an scFv that binds to a myc peptide, and the dimerizing molecule is a polypeptide, a fragment thereof, or a polypeptide polymer, for example, a myc ligand, or a polymer of myc ligands that bind to one or more myc scFvs. In some embodiments, the switch domain is polypeptide-based, for example, a myc receptor, and the dimerizing molecule is an antibody or a fragment thereof, for example, a myc antibody.

[0182] To the extent that the term is used herein, “dimerizing molecule” refers to a molecule that facilitates the binding of the first and second switch domains, for example, when referring to RCAR. In some embodiments, the dimerizing molecule is not naturally present in the subject or is not present in concentrations that result in significant dimerization. In some embodiments, the dimerizing molecule is a small molecule, e.g., rapamycin or rapalog, e.g., RAD001.

[0183] The term “bioequivalence” refers to the amount of a drug other than the control compound (e.g., RAD001) required to produce an effect equivalent to that produced by the control dose or control amount of the control compound (e.g., RAD001). In some embodiments, the effect is the level of mTOR inhibition or the measured level of phosphorylated S6 by Western blotting, for example, measured by the assay described herein, for example, the Boulay assay, or by P70 S6 kinase inhibition, evaluated in vivo or in vitro assays. In some embodiments, the effect is a change in the PD-1-positive / PD-1-negative T cell ratio, measured by cell sorting. In some embodiments, the bioequivalence amount or dose of the mTOR inhibitor is the amount or dose that achieves the same level of P70 S6 kinase inhibition as the control dose or control amount of the control compound. In some embodiments, the bioequivalence 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 control dose or control amount of the control compound.

[0184] The term “low, immunoenhancing dose” refers to a dose of an mTOR inhibitor, such as an allosteric mTOR inhibitor, such as RAD001 or rapamycin, or a catalytic mTOR inhibitor, that partially, but not completely, inhibits mTOR activity, as measured, for example, by inhibition of P70 S6 kinase activity. For example, a method for evaluating mTOR activity by inhibition of P70 S6 kinase is described here. This dose is insufficient to produce complete immunosuppression but sufficient to enhance the immune response. In some embodiments, a low, immunoenhancing dose of an 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 PD-1-negative T cell / PD-1-positive T cell ratio. In some embodiments, a low, immunoenhancing dose of an mTOR inhibitor results in an increase in the number of naive T cells. In some embodiments, a low, immunoenhancing dose of an mTOR inhibitor results in one or more of the following: For example, in memory T cells, for example in memory T cell precursors, the following marker, CD62L 高 CD127 高 CD27+ and an increase in the expression of one or more of BCL2; For example, a decrease in KLRG1 expression in memory T cells, such as memory T cell precursors; and an increase in the number of cells having one or a combination of the following characteristics, such as memory T cell precursors: CD62L 高 increase, CD127 高 increase, CD27 + increase, KLRG1 decrease and BCL2 increase; where any of the above changes occur, for example, at least transiently, as compared to a non-treated subject.

[0185] Range: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a definitive limitation on the scope of the invention. Thus, a range description should be interpreted to specifically disclose all possible sub-ranges as well as the individual numerical values within that range. For example, a range description such as 1-6 should be interpreted to specifically disclose sub-ranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as the individual numerical values within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes anything having 95%, 96%, 97%, 98%, or 99% identity, and includes sub-ranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the width of the range.

[0186] Description Provided herein are compositions and methods for use in treating diseases such as cancer using an anti-methotrexate chimeric antigen receptor (CAR), such as a human methotrexate CAR.

[0187] In one aspect, the present invention provides a number of chimeric antigen receptors comprising antibodies or antibody fragments engineered for specific binding to mesothelin proteins. In another aspect, the present invention provides cells (e.g., T cells or NK cells) engineered to express CAR, for example, herein, CAR T cells ("CART") exhibiting anti-cancer properties. In one aspect, cells are transformed with CAR and express CAR on their cell surface. In one embodiment, cells (e.g., T cells or NK cells) are transduced with a viral vector encoding CAR. In one embodiment, the viral vector is a retroviral vector. In one embodiment, the viral vector is a lentiviral vector. In one such embodiment, cells can stably express CAR. In another embodiment, cells (e.g., T cells or NK cells) are transduced with nucleic acids encoding CAR, such as mRNA, cDNA, or DNA. In one such embodiment, cells can transiently express CAR.

[0188] In one respect, the mesothelin protein-binding portion of the CAR is an scFv antibody fragment. In another respect, such an antibody fragment is functional in that it maintains equivalent binding affinity to the IgG antibody from which it originates, i.e., it binds to the same antigen with equivalent affinity. In another respect, such an antibody fragment is functional in that it provides biological responses that include, but are not limited to, activation of the immune response, inhibition of signaling originating from its target antigen, and inhibition of kinase activity, as understood by those skilled in the art. In another respect, the mesothelin antigen-binding domain of the CAR is an scFv antibody fragment, which is human or humanized compared to the mouse sequence of the scFv from which it originates. In one embodiment, the human anti-mesothelin scFv antibody fragment includes a light chain variable region and / or a heavy chain variable region or a sequence having equivalent identity, e.g., 95–99% identity, as provided in Table 2.

[0189] In some respects, the antibodies of the present invention are incorporated into chimeric antigen receptors (CARs). In one respect, the CARs contain polypeptide sequences provided herein as SEQ ID NOs. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62, or sequences having 95-99% identity with these.

[0190] In one respect, the human scFv portion of the CAR is encoded by a transgene whose sequence is codon-optimized for expression in mammalian cells. In another respect, the entire CAR construct of the present invention is encoded by a transgene whose entire sequence is codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of synonymous codons (i.e., codons encoding the same amino acid) in coding DNA is biased across different species. Such codon degeneracy allows the same polypeptide to be encoded by diverse nucleotide sequences. Various codon optimization methods are known in the art, including, for example, the methods disclosed in at least U.S. Patents 5,786,464 and 6,114,148.

[0191] On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 39. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 40. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 41. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 42. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 43. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 44. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 45. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 46. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 47. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 48. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 49. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 50. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 51. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 52. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 53. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 54. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 55. On one side, the human mesothelin CAR molecule includes the scFv portion provided in SEQ ID NO: 56. On one side, the human mesothelin CAR molecule contains the scFv moiety provided in SEQ ID NO: 57. On one side, the human mesothelin CAR molecule contains the scFv moiety provided in SEQ ID NO: 58. On one side, the human mesothelin CAR molecule contains the scFv moiety provided in SEQ ID NO: 59. On one side, the human mesothelin CAR molecule contains the scFv moiety provided in SEQ ID NO: 60.On one hand, the human mesothelin CAR molecule contains the scFv moiety provided in SEQ ID NO: 61. On the other hand, the human mesothelin CAR molecule contains the scFv moiety provided in SEQ ID NO: 62.

[0192] In one respect, the CARs disclosed herein combine the antigen-binding domain of a specific antibody with an intracellular signaling molecule. For example, in several respects, the intracellular signaling molecule includes, but is not limited to, the CD3 zeta chain, 4-1BB and CD28 signaling modules, and combinations thereof. In one respect, the antigen-binding domain binds to mesothelin. In one respect, the mesothelin CAR includes the sequence provided in Table 2.

[0193] In one aspect, the mesothelin CAR is selected from the sequences provided in one or more of SEQ ID NOs: 63 to 86. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 63. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 64. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 65. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 66. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 67. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 68. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 69. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 70. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 71. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 72. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 73. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 74. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 75. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 76. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 77. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 78. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 79. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 80. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 81. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 82. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 83. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 84. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 85. In one aspect, the mesothelin CAR comprises the sequence provided in SEQ ID NO: 86.

[0194] Furthermore, the present invention provides mesothelin CAR compositions and their use in pharmaceuticals or methods for the treatment of cancer or any malignant tumor or autoimmune disease involving cells or tissues expressing mesothelin.

[0195] In one aspect, the present invention provides cells (e.g., T cells or NK cells) engineered to express a chimeric antigen receptor (CAR), where the CAR T cells ("CART") exhibit antitumor properties. A preferred antigen is mesothelin. In one aspect, the antigen-binding domain of the CAR contains a human anti-mesothelin antibody fragment. In another aspect, the antigen-binding domain of the CAR contains a human anti-mesothelin antibody fragment comprising scFv. Thus, the present invention provides a mesothelin CAR comprising a human anti-mesothelin-binding domain, designed to be induced in T cells or NK cells, and a method of using it for adoptive therapy.

[0196] In one respect, mesothelin CAR includes at least one intracellular signaling domain selected from the group consisting of a CD137(4-1BB) signaling domain, a CD28 signaling domain, a CD3 zeta-signaling domain, and any combination thereof. In another respect, mesothelin CAR includes at least one intracellular signaling domain of one or more co-stimulatory molecules other than a CD137(4-1BB) or CD28, a CD3 zeta-signaling domain, and any combination thereof.

[0197] Furthermore, the present invention provides mesothelin CAR compositions and their use in pharmaceuticals or methods for treating cancer or any malignant tumor or autoimmune disease involving cells or tissues expressing mesothelin.

[0198] Chimeric antigen receptor (CAR) The present invention encompasses a recombinant nucleic acid construct comprising a sequence encoding a CAR, wherein the CAR comprises an antibody that specifically binds to mesothelin, e.g., a human antibody fragment that specifically binds to mesothelin. In one respect, the mesothelin is human mesothelin, and the sequence of the antibody fragment is adjacent to and within the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. The intracellular signaling domain may include a co-stimulatory signaling domain and / or a primary signaling domain, e.g., a zeta chain. The co-stimulatory signaling domain refers to a portion of the CAR that includes at least a portion of the intracellular domain of a co-stimulatory molecule.

[0199] In a particular aspect, the CAR construct of the present invention comprises an scFv domain selected from the group consisting of SEQ ID NOs: 39-62, wherein the scFv may be preceded by an optional leader sequence such as provided in SEQ ID NO: 1, followed by an optional hinge sequence such as provided in SEQ ID NO: 2, 3, 4, or 5, a transmembrane region such as provided in SEQ ID NO: 6, an intracellular signaling domain including SEQ ID NO: 7 or 8, and a CD3 zeta sequence including SEQ ID NO: 9 or 10, wherein these domains are adjacent to each other and within the same reading frame to form a single fusion protein. Also included in the present invention are nucleotide sequences encoding polypeptides selected from the group consisting of SEQ ID NOs: 87; SEQ ID NOs: 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, and 110, or sequences having 95-99% identity with these. Also included in the present invention are each of the scFv fragments selected from the group consisting of SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62 or sequences having 95-99% identity thereto, and each of the polypeptides of the domains of SEQ ID NOs: 1, 2, and 6-9, in addition to the nucleotide sequences encoding the mesothelin CAR fusion protein of the present invention. In one aspect, a typical mesothelin CAR construct includes an optional leader sequence, an extracellular mesothelin-binding domain, a hinge, a transmembrane domain, and an intracellular stimulating domain.In one respect, a mesothelin CAR construct includes an optional leader sequence, a mesothelin-binding domain, a hinge, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular stimulatory domain. Specific mesothelin CAR constructs containing a human scFv domain are provided as SEQ ID NOs: 87-110.

[0200] The full-length CAR sequence is also provided here as 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: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86. The representative leader sequence is provided as SEQ ID NO: 1. The representative hinge / spacer sequence is provided as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. The representative transmembrane domain sequence is provided as SEQ ID NO: 6. The representative sequence of the intracellular signaling domain of the 4-1BB protein is provided as SEQ ID NO: 7. The representative sequence of the intracellular signaling domain of CD27 is provided as SEQ ID NO: 8. The representative CD3 zeta domain sequence is provided as SEQ ID NO: 9, or SEQ ID NO: 10.

[0201] In one aspect, the present invention provides a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an anti-mesothelin-binding domain, for example, described herein, which is adjacent to and within the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. In one aspect, the anti-mesothelin-binding domain is selected from one or more sequence numbers 87 to 110. In one aspect, the anti-mesothelin-binding domain includes sequence number 87. In one aspect, the anti-mesothelin-binding domain includes sequence number 88. In one aspect, the anti-mesothelin-binding domain includes sequence number 89. In one aspect, the anti-mesothelin-binding domain includes sequence number 90. In one aspect, the anti-mesothelin-binding domain includes sequence number 91. In one aspect, the anti-mesothelin-binding domain includes sequence number 92. In one aspect, the anti-mesothelin-binding domain includes sequence number 93. In one aspect, the anti-mesothelin-binding domain includes sequence number 94. On one side, the anti-mesothelin binding domain includes SEQ ID NO: 95. On one side, the anti-mesothelin binding domain includes SEQ ID NO: 96. On one side, the anti-mesothelin binding domain includes SEQ ID NO: 97. On one side, the anti-mesothelin binding domain includes SEQ ID NO: 98. On one side, the anti-mesothelin binding domain includes SEQ ID NO: 99. On one side, the anti-mesothelin binding domain includes SEQ ID NO: 100. On one side, the anti-mesothelin binding domain includes SEQ ID NO: 101. On one side, the anti-mesothelin binding domain includes SEQ ID NO: 102. On one side, the anti-mesothelin binding domain includes SEQ ID NO: 103. On one side, the anti-mesothelin binding domain includes SEQ ID NO: 104. On one side, the anti-mesothelin binding domain includes SEQ ID NO: 105. On one side, the anti-mesothelin binding domain includes SEQ ID NO: 106. On one side, the anti-mesothelin binding domain includes SEQ ID NO: 107. On the other side, the anti-mesothelin binding domain includes SEQ ID NO: 108. On the other side, the anti-mesothelin binding domain includes SEQ ID NO: 109. On the other side, the anti-mesothelin binding domain includes SEQ ID NO: 110.In one respect, the present invention provides a recombinant DNA construct comprising a transgene encoding a CAR, wherein the transgene comprises a nucleic acid sequence encoding a human anti-mesoterin-binding domain selected from one or more of the following anti-mesoterin-binding domains, e.g., one or more of sequence numbers 87-110, wherein the sequence is adjacent to and within the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. Representative intracellular signaling domains that can be used in a CAR include, but are not limited to, one or more intracellular signaling domains, e.g., CD3 zeta, CD28, 4-1BB, etc. In one respect, the nucleic acid sequence of the CAR construct of the present invention is selected from one or more of sequence numbers 111-134. In one respect, the nucleic acid sequence of the CAR construct is sequence number 111. In one respect, the nucleic acid sequence of the CAR construct is sequence number 112. In one respect, the nucleic acid sequence of the CAR construct is sequence number 113. On one plane, the nucleic acid sequence of the CAR construct is sequence number 114. On one plane, the nucleic acid sequence of the CAR construct is sequence number 115. On one plane, the nucleic acid sequence of the CAR construct is sequence number 116. On one plane, the nucleic acid sequence of the CAR construct is sequence number 117. On one plane, the nucleic acid sequence of the CAR construct is sequence number 118. On one plane, the nucleic acid sequence of the CAR construct is sequence number 119. On one plane, the nucleic acid sequence of the CAR construct is sequence number 120. On one plane, the nucleic acid sequence of the CAR construct is sequence number 121. On one plane, the nucleic acid sequence of the CAR construct is sequence number 122. On one plane, the nucleic acid sequence of the CAR construct is sequence number 123. On one plane, the nucleic acid sequence of the CAR construct is sequence number 124. On one plane, the nucleic acid sequence of the CAR construct is sequence number 125. On one plane, the nucleic acid sequence of the CAR construct is sequence number 126. On one plane, the nucleic acid sequence of the CAR construct is sequence number 127. On one side, the nucleic acid sequence of the CAR construct is sequence number 128. On the other side, the nucleic acid sequence of the CAR construct is sequence number 129.On one plane, the nucleic acid sequence of the CAR construct is sequence number 130. On another plane, the nucleic acid sequence of the CAR construct is sequence number 131. On another plane, the nucleic acid sequence of the CAR construct is sequence number 132. On another plane, the nucleic acid sequence of the CAR construct is sequence number 133. On another plane, the nucleic acid sequence of the CAR construct is sequence number 134.

[0202] Nucleic acid sequences encoding a desired molecule can be obtained using recombinant methods known in the art, such as screening libraries from cells expressing the gene, extracting the gene from a vector known to contain it, or direct isolation from cells and tissues containing it, using standard methods. Alternatively, the desired nucleic acid can be produced by synthesis rather than cloning.

[0203] The present invention includes retroviral constructs and lentiviral vector constructs that express CARs that can be directly transduced into cells. The present invention also includes RNA constructs that can be directly transduced into cells. A method for producing mRNA for use in gene transduction includes in vitro transcription (IVT) of a template using specially designed primers, followed by poly-A addition, to produce a construct generally 50–2000 nucleotides long, containing 3' and 5' untranslated sequences ("UTR"), a 5' cap and / or intra-sequence ribosome entry site (IRES), the nucleic acid to be expressed, and a poly-A tail (SEQ ID NO: 35). The RNA thus produced efficiently transduces genes into different types of cells. In one embodiment, the template contains a sequence for the CAR. In one embodiment, the RNA CAR vector is transduced into T cells by electroporation.

[0204] antigen-binding domain In one respect, the CAR of the present invention includes a target-specific binding element, also referred to as an antigen-binding domain. The selection of the antigen-binding domain depends on the type and number of antigens that define the surface of the target cell. For example, the antigen-binding domain may be selected to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease condition.

[0205] In one respect, the CAR-mediated immune effector cell response can be directed to cells expressing a desired antigen, where the CAR includes an antigen-binding domain that specifically binds to the desired antigen. In another respect, the portion of the CAR containing the antigen-binding domain includes an antigen-binding domain that targets mesothelin. In another respect, the antigen-binding domain targets human mesothelin.

[0206] The antigen-binding domain may include, but is not limited to, any domain that binds to an antigen, including, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies and their functional fragments, and alternative scaffolds known in the art to function as antigen-binding domains, such as recombinant fibronectin domains. In some cases, it is beneficial that the antigen-binding domain originates from the same species in which the CAR will ultimately be used. For example, for use in humans, it may be beneficial that the antigen-binding domain of the CAR contains human or humanized residues for the antigen-binding domain of the antibody or antibody fragment. Therefore, in one respect, the antigen-binding domain may include a human antibody or antibody fragment.

[0207] In one embodiment, the anti-mesothelin binding domain does not compete, or competes poorly, with the antigen-binding domain containing the amino acid sequence containing SEQ ID NO: 279, for example, mouse SS1 scFv, for binding to human mesothelin in the competitive assay described herein.

[0208] The amino acid sequence of mouse SS1 scFv is shown below (SEQ ID NO: 279). [ka]

[0209] In one embodiment, the anti-mesothelin binding domain competes for binding to human mesothelin with an antigen-binding domain comprising, for example, LC CDR1, LC CDR2, and LC CDR3 of anti-mesothelin light chain amino acid sequences selected from SEQ ID NO: 43 or SEQ ID NO: 49, and HC CDR1, HC CDR2, and HC CDR3 of anti-mesothelin heavy chain amino acid sequences selected from SEQ ID NO: 43 or SEQ ID NO: 49, in the competitive assay described herein. In one embodiment, the anti-mesothelin binding domain competes for binding to human mesothelin with an antigen-binding domain comprising, for example, LC CDR1 selected from SEQ ID NO: 203 or SEQ ID NO: 209, LC CDR2 selected from SEQ ID NO: 227 or SEQ ID NO: 233, and LC CDR3 selected from SEQ ID NO: 251 or SEQ ID NO: 257; and HC CDR1 selected from SEQ ID NO: 138 or SEQ ID NO: 144, HC CDR2 selected from SEQ ID NO: 156 or SEQ ID NO: 162, and HC CDR3 selected from SEQ ID NO: 179 or SEQ ID NO: 185, in the competitive assay described herein.

[0210] In one embodiment, the anti-mesothelin binding domain competes for binding to human mesothelin with an antigen-binding domain containing a sequence selected from SEQ ID NO: 43 or SEQ ID NO: 49 in the competitive assay described herein, for example.

[0211] In some embodiments, the competitive assay is an SPR-based assay. In summary, an antigen, e.g., human mesothelin, is immobilized on a surface. A control antibody is injected from above the antigen layer via a microfluidic system. Binding of the control antibody to the antigen is detected as an increase in the signal, generally expressed in response units (RUs), e.g., the control signal. After a desired time, the test antibody is injected from above the antigen layer. If the test antibody binds to a different region or epitope on the antigen, a further increase in the signal, e.g., ≥5%, ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, ≥50%, ≥55%, ≥60%, ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, or ≥95%, e.g., an increase in RUs, is detected compared to the highest signal detected by the binding of the control antibody, e.g., the control signal. If the test antibody binds to the same region or epitope of the antigen, the increase in signal is slight or nonexistent. For example, the RU increase detected is less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% compared to the highest signal detected by the binding of the control antibody. When using this SPR-based competition assay, an antibody can be said to compete with the control antibody if an increase in signal is detected less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% compared to the control signal detected by the binding of the control antibody to the antigen. When an increase in RU (rumination ratio) is detected, compared to the control signal detected by the binding of the control antibody to the antigen, the antibody can be said to be non-competitive or poorly competitive with the control antibody. This occurs when a signal of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more is detected.

[0212] The identification of epitopes to which the antigen-binding domains described herein bind can be performed by various methods known in this field. For example, the crystal structure containing the antigen-binding domain bound to or complexed with the antigen can be elucidated. In other cases, assays, such as protective assays, can be performed to identify regions contributing to the antigen epitope or to identify the epitope itself. A representative protective assay, hydrogen / deuterium exchange (HDX) mass spectrometry assay, is further described in Example 18. HDX mass spectrometry is performed to identify putative epitopes on human MSLNs, e.g., hMSLN, for mouse SS1, e.g., SEQ ID NO: 279 and M5 scFv, e.g., SEQ ID NO: 43, described herein. 296~588 For example, sequence number 278 was identified. hMSLN 296~588 For example, SEQ ID NO: 278 represents amino acids 296-588 of human mesothelin. For instance, the first amino acid in SEQ ID NO: 278 is amino acid 296, and the last amino acid in SEQ ID NO: 278 is amino acid 588. The amino acid sequence of human mesothelin, amino acids 296-588, is provided below (SEQ ID NO: 278): [ka]

[0213] The results of the HDX mass spectrometry assay were hMSLN 296~588 The results of the HDX mass spectrometry assay showed that one or more amino acids at 314-315, 317-318, 346-349, and 369-375, for example, SEQ ID NO: 278, contribute to the epitope recognized by SS1. 296~588 We have shown that one or more amino acids at 485-490, 498-507, 532-537, or 545-572, for example, SEQ ID NO: 278, contribute to the anti-mesothelin antigen-binding domain described herein, for example, M5 scFv, for example, SEQ ID NO: 43, which is recognized by this epitope.

[0214] In one embodiment, the anti-mesothelin binding domain described herein binds to an antigen-binding domain containing a sequence including sequence number 279, for example, to a human mesothelin epitope different from the human mesothelin epitope targeted by mouse SS1, for example, sequence number 278.

[0215] In one embodiment, the epitope recognized by SS1 is hMSLN 296~588 The sequence includes amino acids 314-315, 317-318, 346-349 or 369-375, for example, sequence number 278 or any combination thereof. In one embodiment, the epitope recognized by SS1 is hMSLN 296~588 It contains one or more amino acids selected from amino acids 314-315, 317-318, 346-349, or 369-375, for example, SEQ ID NO: 278.

[0216] In one embodiment, the anti-mesothelin binding domain described herein binds to the C-terminus of human mesothelin. In one embodiment, the anti-mesothelin binding domain described herein binds to an epitope in amino acids 450-588 of SEQ ID NO: 278, for example, where the epitope is found in part or in whole in amino acids 450-588, 480-580, or 485-572 of SEQ ID NO: 278. In one embodiment, the epitope recognized by the anti-mesothelin binding domain described herein is hMSLN 296~588 The sequence includes a sequence selected from amino acids 485-490, 498-507, 532-537 or 545-572, for example, SEQ ID NO: 278 or any combination thereof. In one embodiment, the epitope recognized by the anti-mesothelin binding domain described herein is hMSLN 296~588 It includes one or more amino acids selected from 485-490, 498-507, 532-537 or 545-572, for example, SEQ ID NO: 278 or any combination thereof.

[0217] In one embodiment, the anti-mesothelin binding domain includes one or more (e.g., three in total) light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of a human anti-mesothelin binding domain selected from SEQ ID NOs. 39-62, and one or more (e.g., three in total) heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of a human anti-mesothelin binding domain selected from SEQ ID NOs. 39-62. In one embodiment, the human anti-mesothelin binding domain includes the light chain variable region and / or the heavy chain variable region described herein (e.g., Table 2). In one embodiment, the anti-mesothelin binding domain includes an scFv containing the light chain variable region and heavy chain variable region of the amino acid sequence in Table 2. In one embodiment, the anti-mesothelin binding domain (e.g., scFV) includes a light chain variable region having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the light chain variable region shown in Table 2, but with no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of Table 2; and / or a heavy chain variable region having at least one, two, or three modifications (e.g., substitutions) to the amino acid sequence of the heavy chain variable region shown in Table 2, but with no more than 30, 20, or 10 modifications (e.g., substitutions), or a sequence having 95-99% identity with the amino acid sequence of Table 2.

[0218] In one embodiment, the human anti-mesothelin binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 39-62 or a sequence having 95-99% identity thereto. In one embodiment, the nucleic acid sequence encoding the human anti-mesothelin binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 87-110 or a sequence having 95-99% identity thereto. In one embodiment, the human anti-mesothelin binding domain is an scFv, wherein a light chain variable region comprising, for example, the amino acid sequence described in Table 2 or 3 is bound to, hereby, a heavy chain variable region comprising, for example, the amino acid sequence described in Table 2 or 3, via a linker, for example, the linker described herein. In one embodiment, the humanized anti-mesothelin binding domain comprises a (Gly4-Ser)n linker (SEQ ID NO: 26) (where n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4). The light chain variable region and heavy chain variable region of ScFv may be, for example, either the following orientations of light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0219] On one plane, the antigen-binding domain contains one or more sequences selected from SEQ ID NOs. 39-62. On one plane, the CAR consists of one or more sequences selected from SEQ ID NOs. 63-86.

[0220] In one respect, the antibodies of the present invention may exist in a variety of other forms, including, for example, Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, disulfide-linked Fv (sdFv), Fd fragment consisting of a VH domain and a CH1 domain, linear antibodies, single-domain antibodies such as sdAb (either VL or VH), camel VHH domain, and bivalent fragments containing two Fab fragments linked by disulfide crosslinking at a hinge region, as well as polyspecific antibodies and isolated CDRs or other epitope-binding fragments of antibodies. In one respect, the antibody fragment provided herein is scFv. In one example, human scFv may be derived from a yeast display library.

[0221] A display library is a collection that includes accessible polypeptide components and obtainable components that encode or identify said polypeptide components. Polypeptide components are varied to represent various amino acid sequences. Polypeptide components can be of any length, for example, from 3 amino acids to more than 300 amino acids. A single display library may contain more than one polypeptide component, for example, two polypeptide chains of Fab. In one representative embodiment, a display library can be used to identify anti-mesothelin binding domains. In selection, the polypeptide component of each member of the library is probed with mesothelin or a fragment thereof, and if the polypeptide component binds to mesothelin, the display library member is identified, generally by holding it on a support.

[0222] The retained display library members are obtained from the support and analyzed. The analysis includes amplification followed by selection under similar or different conditions. For example, positive and negative selection may be alternated. The analysis also includes determining the amino acid sequence of the polypeptide component, i.e., the anti-mesothelin binding domain, and purifying the polypeptide component for further characterization.

[0223] A variety of forms can be used for display libraries. An example is phage display. In phage display, protein components are generally covalently bound to bacteriophage coat proteins. This binding originates from the translation of nucleic acids encoding the protein components fused to the coat protein. The binding involves mobile peptide linkers, protease sites, or amino acids incorporated as a result of stop codon repression. Phage displays are, for example, referred to in US publications 5,223,409; Smith (1985) Science 228:1315-1317; WO92 / 18619; WO91 / 17271; WO92 / 20791; WO92 / 15679; WO93 / 01288; WO92 / 01047; WO92 / 09690; WO90 / 02809; de Haard et al. (1999) J. Biol. Chem 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8 and Hoet et al. (2005) Nat Biotechnol. As described in 23(3)344-8, bacteriophages displaying protein components can be amplified and obtained using standard phage preparation methods, such as PEG precipitation from growth medium. After selecting individual display phages, the nucleic acids encoding the selected protein components can be amplified and isolated from cells infected with the selected phages or from the phages themselves. Individual colonies or plaques can be sorted, the nucleic acids isolated, and sequenced.

[0224] Other display formats include cell-based displays (see, e.g., WO03 / 029456), protein-nucleic acid fusions (see, e.g., US No. 6,207,446), ribosome displays (e.g., Mattheakis et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol. 18:1287-92; Hanes et al. (2000) Methods Enzymol. 328:404-30; and Schaffitzel et al. (1999) J Immunol Methods. 231(1-2):119-35), and Escherichia coli periplasm displays (J Immunol Methods. 2005 Nov 22; PMID: 16337958).

[0225] In addition to using display libraries, anti-mesothelin binding domains can be obtained using other methods. For example, mesothelin or fragments thereof can be used as antigens in non-human animals, such as rodents.

[0226] In one embodiment, a non-human animal contains at least a portion of a human immunoglobulin gene. For example, it is possible to manipulate a mouse strain lacking mouse antibody production that has a large fragment of the human Ig locus. Using hybridoma technology, antigen-specific monoclonal antibodies (Mab) derived from the gene with desired specificity can be produced and selected. For example, XENOMOUSE TM See Green et al., 1994, Nat. Gen. 7:13-21; US ​​No. 2003-0070185, WO96 / 34096 (published October 31, 1996), and PCT application PCT / US96 / 05928 (filed April 29, 1996).

[0227] In some cases, scFv can be prepared by methods known in this field (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 prepared, for example, by linking the VH and VL regions using a mobile polypeptide linker. ScFv molecules may contain linkers of optimized length and / or amino acid composition (e.g., Ser-Gly linkers). Linker length can significantly influence how the variable regions of scFv fold and interact. In fact, when short polypeptide linkers (e.g., 5-10 amino acids) are used, intrachain folding is inhibited. Interchain folding also requires the two variable regions to come together to form a functional epitope binding site. For examples of linker orientation and size, see, for example, Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, U.S. Patent Application Publications 2005 / 0100543, 2005 / 0175606, 2007 / 0014794 and PCT Publication WO2006 / 020258, and WO2007 / 024715 is incorporated herein by reference.

[0228] scFv may contain a linker between the VL and VH regions of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues. The linker sequence may contain any naturally occurring amino acids. In one embodiment, the linker sequence contains the amino acids glycine and serine. In another embodiment, the linker sequence is (Gly4Ser) nThe set includes glycine and serine repeats such as (SEQ ID NO: 135), where n is a positive integer greater than or equal to 1. In one embodiment, the linker may be (Gly4Ser)4 (SEQ ID NO: 27) or (Gly4Ser)3 (SEQ ID NO: 28). Variations in linker length may preserve or enhance activity and result in superior efficacy in activity testing.

[0229] Stability and mutation The stability of the anti-mesothelin binding domain, for example, an scFv molecule (e.g., soluble scFv), can be evaluated against the biophysical properties (e.g., thermal stability) of a conventional control scFv molecule or a full-length antibody. In one embodiment, human scFv exhibits greater thermal stability than a control binding molecule (e.g., a conventional scFv molecule) in the described assays, exceeding approximately 0.1°C, 0.25°C, 0.5°C, 0.75°C, 1°C, 1.25°C, 1.5°C, 1.75°C, 2°C, 2.5°C, 3°C, 3.5°C, 4°C, 4.5°C, 5°C, 5.5°C, 6°C, 6.5°C, 7°C, 7.5°C, 8°C, 8.5°C, 9°C, 9.5°C, 10°C, 11°C, 12°C, 13°C, 14°C, or 15°C.

[0230] The improved thermal stability of the anti-mesothelin binding domain, e.g., scFv, contributes to the subsequent overall mesothelin CAR construct, improving the therapeutic properties of the mesothelin CAR construct. The thermal stability of the anti-mesothelin binding domain, e.g., scFv, can be improved by at least about 2°C or 3°C compared to conventional antibodies. In one embodiment, the anti-mesothelin binding domain, e.g., scFv, has a thermal stability that is 1°C improved compared to conventional antibodies. In another embodiment, the anti-mesothelin binding domain, e.g., scFv, has a thermal stability that is 2°C improved compared to conventional antibodies. In yet another embodiment, the anti-mesothelin binding domain, e.g., scFv, has a thermal stability that is 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, or 15°C improved compared to conventional antibodies. For example, the scFv molecules disclosed herein can be compared with the scFv molecules or Fab fragments of the antibodies from which scFv VH and VL were derived. Thermal stability can be measured using methods known in this field. For example, in one embodiment, Tm can be measured. Methods for measuring Tm and other methods for determining protein stability are described in more detail below.

[0231] Mutations in scFv (induced by direct mutagenesis of soluble scFv) alter the stability of scFv and improve the overall stability of scFv and CART constructs. The stability of humanized scFv is compared to that of mouse scFv using measurements such as Tm, denaturation temperature, and aggregation temperature.

[0232] In one embodiment, the anti-mesothelin binding domain, e.g., scFv, has at least one mutation such that the mutated anti-mesothelin binding domain, e.g., scFv, contributes to improved stability of the anti-mesothelin construct. In another embodiment, the anti-mesothelin binding domain, e.g., scFv, has at least one, two, three, four, five, six, seven, eight, nine, or ten mutations such that the mutated anti-mesothelin binding domain, e.g., scFv, contributes to improved stability of the anti-mesothelin construct. The binding ability of the mutant scFv can be determined using the assay described in the examples.

[0233] binding affinity A wide variety of methods for determining binding affinity are known in this field. Representative methods for determining binding affinity include, for example, the use of the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ), which enables the analysis of real-time biomolecular-specific interactions by detecting changes in protein concentration within the biosensor matrix, and surface plasmon resonance. Surface plasmon resonance is an optical phenomenon. For further information, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnsson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0234] In one aspect, the portion of the CAR composition of the present invention comprising an antibody or fragment thereof comprises an amino acid sequence homologous to the amino acid sequence described herein, wherein the antibody or fragment thereof retains the desired functional properties of the anti-mesothelin antibody fragment of the present invention. In one specific aspect, the CAR composition of the present invention comprises an antibody fragment. In a further aspect, the antibody fragment comprises an scFv.

[0235] In various aspects, the portion of the CAR composition of the present invention containing an antibody or antibody fragment is manipulated by modification of one or more amino acids within one or both of the variable regions (i.e., VH and / or VL), for example, within one or more CDR regions and / or one or more framework regions. In one specific aspect, the CAR composition of the present invention contains an antibody fragment. In a further aspect, the antibody fragment contains an scFv.

[0236] Those skilled in the art will understand that the antibodies or antibody fragments of the present invention can be further modified so that their amino acid sequences are different (e.g., from the wild type), but their desired activity remains the same. For example, further nucleotide substitutions, such as amino acid substitutions at “non-essential” amino acid residues, may be made to the protein. For example, non-essential amino acid residues within a molecule may be substituted with other amino acid residues from the same side-chain family. In other embodiments, amino acid chains may be replaced with structurally similar chains that differ in the order and / or composition of side-chain family members, i.e., conservative substitutions may be made in which amino acid residues are replaced with amino acid residues having similar side chains.

[0237] The family of amino acid residues having similar side chains is defined in this art and includes 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0238] In the context of two or more nucleic acid or polypeptide sequences, the percentage of identity means two or more sequences that are the same. Two sequences are “substantially identical” if, when compared across a comparison window or specified region using one of the following sequence comparison algorithms and aligned for maximum match, or by manual alignment and visual inspection, they have a certain percentage of identical amino acid residues or nucleotides (i.e., 60% identity, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity). If desired, identity may be present over a region of at least approximately 50 nucleotides (or 10 amino acids) in length, or more preferably over a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0239] For sequence comparison, generally one sequence acts as a control sequence, and the test sequence is compared against it. When using a sequence comparison algorithm, the test sequence and control sequence are entered into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence to the control sequence based on the program parameters. Methods for aligning sequences for comparison are well known in this field. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, or the similarity search method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, either by computer-controlled implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or by manual alignment and visual inspection (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology).

[0240] Two examples of algorithms suitable for determining sequence identity percentage and sequence similarity 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, respectively. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information.

[0241] The percentage of identity between two amino acid sequences can also be determined using the algorithm from E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4:11-17, which is incorporated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4. Furthermore, the percentage of identity between two amino acid sequences can also be determined using the algorithm from Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453, which is incorporated into the GAP program in the GCG software package (available from www.gcg.com), using the Blossom 62 matrix or PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0242] In one respect, the present invention aims to modify the amino acid sequence of a starting antibody or fragment (e.g., scFv) to produce a functionally equivalent molecule. For example, the anti-mesothelin-binding domain contained in the CAR, e.g., the VH or VL of scFv, can be modified to maintain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the starting VH or VL framework region of scFv. The present invention aims to modify the entire CAR construct, for example, by modifying one or more amino acid sequences of various domains of the CAR construct, in order to produce a functionally equivalent molecule. The CAR construct 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 with the starting CAR construct.

[0243] Transmembrane domain With respect to the transmembrane domain, in various embodiments, the CAR can be designed to include a transmembrane domain that binds to the extracellular domain of the CAR. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids related to the extracellular region of the protein from which the transmembrane domain originates (e.g., one, two, three, four, five, six, seven, eight, nine, ten to up to fifteen amino acids from the extracellular region) and / or one or more additional amino acids related to the intracellular region of the protein from which the transmembrane protein originates (e.g., one, two, three, four, five, six, seven, eight, nine, ten to up to fifteen amino acids from the intracellular region). In one aspect, the transmembrane domain uses one that is related to 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 the signaling domain, co-stimulatory domain, or hinge domain originates. In other respects, the transmembrane domain does not originate from the same protein from which any of the other domains of the CAR originate. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to minimize interaction with other members of the receptor complex, for example, to avoid binding of such domain to the transmembrane domain of the same or different surface membrane proteins. In one respect, the transmembrane domain can homodimerize with other CARs on the cell surface of the CAR-expressing cell. In another respect, the amino acid sequence of the transmembrane domain may be modified or substituted to minimize interaction with the binding domain of a native binding partner present in the same CAR-expressing cell.

[0244] Transmembrane domains may be of natural origin or recombinant origin. When the origin is natural, the domain may originate from any membrane-bound or transmembrane protein. In one respect, the transmembrane domain can always signal to the intracellular domain when the CAR is bound to a target. Transmembrane domains for specific applications in the present invention may include, for example, at least the transmembrane domains of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domains are, for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITG It may include at least transmembrane regions of AM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, and NKG2C.

[0245] In one embodiment, a transmembrane domain can be bound to the extracellular region of a CAR, e.g., the antigen-binding domain of a CAR, via a hinge, e.g., a hinge from a human protein. For example, in one embodiment, the hinge may be a human Ig (immunoglobulin) hinge (e.g., IgG4 hinge, IgD hinge), a GS linker (e.g., the GS linker described herein), a KIR2DS2 hinge, or a CD8a hinge. In one embodiment, the hinge or spacer comprises (e.g., derived from) the amino acid sequence of SEQ ID NO: 2. In one aspect, the transmembrane domain comprises (e.g., derived from) the transmembrane domain of SEQ ID NO: 6.

[0246] In one aspect, the hinge or spacer includes an IgG4 hinge. For example, in one embodiment, the hinge or spacer includes a hinge with the following amino acid sequence: [ka]

[0247] In one embodiment, the hinge or spacer includes a hinge encoded by the following nucleotide sequence: [ka]

[0248] In one aspect, the hinge or spacer includes an IgD hinge. For example, in one embodiment, the hinge or spacer is an amino acid sequence [ka] Includes the hinge.

[0249] In one embodiment, the hinge or spacer is [ka] It contains a hinge encoded by the nucleotide sequence.

[0250] On one side, the transmembrane domain may be recombined, in which case it predominantly contains hydrophobic residues such as leucine and valine. On one side, a triplet of phenylalanine, tryptophan, and valine can be seen at each end of the recombinant transmembrane domain.

[0251] If desired, short oligo- or polypeptide linkers of 2 to 10 amino acids in length may form a link between the transmembrane domain and the cytoplasmic signaling region of the CAR. Glycine-serine doublets provide particularly suitable linkers. For example, in one aspect, the linker is [ka] It contains the amino acid sequence. In one embodiment, the linker is [ka] It is encoded by the nucleotide sequence.

[0252] On one surface, the hinge or spacer includes the KIR2DS2 hinge and / or a portion thereof.

[0253] Cytoplasmic domain The cytoplasmic domain or region of a CAR contains an intracellular signaling domain. This intracellular signaling domain is generally responsible for activating at least one of the normal effector functions of the immune cell into which the CAR has been introduced. The term “effector function” refers to the cellular specialization function. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including cytokine secretion. Therefore, the term “intracellular signaling domain” refers to the portion of a protein that transmits effector function signals and instructs the cell to perform its specialization function. While all intracellular signaling domains can usually be used, in many cases, the entire chain is not necessary. A cleaved portion of an intracellular signaling domain may be used in place of the complete chain, as long as it transmits effector function signals to the extent that it is used. Therefore, the term “intracellular signaling domain” is intended to include any cleaved portion of an intracellular signaling domain sufficient to transmit effector function signals.

[0254] Examples of intracellular signaling domains for use in the CAR of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act cooperatively to initiate signaling after antigen receptor binding, as well as any derivatives or variants and any recombinant sequences of these sequences having the same functional capacity.

[0255] It is known that signals produced by the TCR alone are insufficient for complete T cell activation, and that secondary and / or co-stimulatory signals are also required. Therefore, it can be said that T cell activation involves two different classes of cytoplasmic signaling sequences: one that initiates antigen-dependent primary activation via the TCR (primary intracellular signaling domain) and another that acts in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic domains, e.g., co-stimulatory domains).

[0256] Primary cytoplasmic signaling domains regulate the primary activation of the TCR complex in either a stimulating or inhibitory manner. Primary intracellular signaling domains that act in a stimulating manner may include signaling motifs known as immune receptor tyrosine activation motifs or ITAMs.

[0257] Examples of ITAM-containing primary intracellular signaling domains particularly useful in the present invention include those of CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In one embodiment, the CAR of the present invention includes an intracellular signaling domain, for example, the primary signaling domain of CD3 zeta.

[0258] In one embodiment, the primary signaling domain includes a modified ITAM domain having altered (e.g., increased or decreased) activity compared to a native ITAM domain, e.g., a mutant ITAM domain. In one embodiment, the primary signaling domain includes a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or cleaved ITAM-containing primary intracellular signaling domain. In one embodiment, the primary signaling domain includes one, two, three, four or more ITAM motifs.

[0259] Further examples of molecules containing primary intracellular signaling domains particularly useful in the present invention include those of DAP10, DAP12, and CD32.

[0260] The intracellular domain of the CAR may consist solely of a CD3 zeta signaling domain, or it may be combined with any other desired intracellular signaling domain useful in the context of the CAR of the present invention. For example, the intracellular signaling domain of the CAR may consist of a CD3 zeta chain portion and a costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient lymphocyte response to an antigen. Examples of such molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1 (also known as PD1), ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. For example, CD27 costimulation has been shown to enhance the proliferation, effector function, and survival of human CAR T cells in vitro, and to enhance human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012;119(3):696-706).Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), NKG2D, CEACAM1, CRTAM, Ly9(CD229), CD160(BY 55), including PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76 and PAG / Cbp.

[0261] The intracellular signaling domains within the cytoplasmic portion of the CAR of the present invention may be linked to each other in a random or specific order. Optionally, short oligo- or polypeptide linkers of, for example, 2 to 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) may be formed between the intracellular signaling domains. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In another embodiment, a single amino acid, such as alanine or glycine, can be used as a suitable linker.

[0262] In one aspect, the intracellular signaling domain is designed to contain two or more, for example, two, three, four, five or more, co-stimulatory signaling domains. In one embodiment, two or more, for example, two, three, four, five or more, co-stimulatory signaling domains are separated by a linker molecule, for example, the linker molecule described herein. In one embodiment, the intracellular signaling domain contains two co-stimulatory signaling domains. In one embodiment, the linker molecule is a glycine residue. In one embodiment, the linker is an alanine residue.

[0263] In one aspect, the intracellular signaling domain is designed to include the CD3 zeta signaling domain and the CD28 signaling domain. In another aspect, the intracellular signaling domain is designed to include the CD3 zeta signaling domain and the 4-1BB signaling domain. In another aspect, the 4-1BB signaling domain is the signaling domain of SEQ ID NO: 16. In another aspect, the CD3 zeta signaling domain is the signaling domain of SEQ ID NO: 17.

[0264] In one aspect, the intracellular signaling domain is designed to include the CD3 zeta signaling domain and the CD27 signaling domain. In another aspect, the CD27 signaling domain is, [ka] It contains the amino acid sequence. In one respect, the signaling domain of CD27 is [ka] It is encoded by the nucleic acid sequence.

[0265] In one respect, the CAR-expressing cells described herein may further include secondary CARs, for example, secondary CARs containing different antigen-binding domains to the same target (mesothelin) or different targets (e.g., targets other than mesothelin on stromal cells, e.g., FAP; targets other than mesothelin on prostate cancer cells, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1 or MAD-CT-2; targets other than mesothelin on ovarian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor α, claudin 6, GloboH or sperm protein 17; targets other than mesothelin on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4 or Trop-2). In one embodiment, a CAR-expressing cell contains a first CAR that targets a first antigen and includes an intracellular signaling domain having a co-stimulatory signaling domain but lacking a primary signaling domain, and a second CAR that targets a second different antigen and includes an intracellular signaling domain having a primary signaling domain but lacking a co-stimulatory signaling domain. The placement of the co-stimulatory signaling domain, e.g., 4-1BB, CD28, CD27, or OX-40, on the first CAR and the primary signaling domain, e.g., CD3 zeta, on the second CAR may limit the CAR activity for cells in which the targets of both are expressed.In one embodiment, a CAR-expressing cell contains a first mesothelin CAR comprising a mesothelin-binding domain, a transmembrane domain, and a costimulatory domain, as well as a second CAR that targets a non-mesothelin antigen (e.g., non-mesothelin targets on stromal cells, e.g., FAP; non-mesothelin targets on prostate cancer cells, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1, or MAD-CT-2; non-mesothelin targets on ovarian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor α, claudin 6, GloboH, or sperm protein 17; e.g., non-mesothelin targets on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4, or Trop-2), comprising an antigen-binding domain, a transmembrane domain, and a primary signaling domain. In another embodiment, the CAR-expressing cell includes a first mesothelin CAR comprising a mesothelin-binding domain, a transmembrane domain, and a primary signaling domain, and a second CAR that targets a non-mesothelin antigen (e.g., non-mesothelin targets on stromal cells, e.g., FAP; non-mesothelin targets on prostate cancer cells, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1, or MAD-CT-2; non-mesothelin targets on ovarian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor α, claudin 6, GloboH, or sperm protein 17; e.g., non-mesothelin targets on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4, or Trop-2), and comprises an antigen-binding domain, a transmembrane domain, and a co-stimulatory signaling domain for the antigen.

[0266] In one embodiment, CAR-expressing cells include the mesothelin CAR and inhibitory CAR described herein. In one embodiment, the inhibitory CAR includes an antigen-binding domain that binds to an antigen, which is also found on normal cells, e.g., normal cells that also express mesothelin, but not on cancer cells. In one embodiment, the inhibitory CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular domain of the inhibitory molecule. For example, the intracellular domain of an inhibitory CAR may be the intracellular domain of PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta.

[0267] In one embodiment, when a CAR-expressing cell contains two or more different CARs, the antigen-binding domains of the different CARs may be such that they do not interact with each other. For example, a cell expressing a primary CAR and a secondary CAR may have an antigen-binding domain of the primary CAR that does not associate with the antigen-binding domain of the secondary CAR, for example, VHH, but rather as a fragment, for example, scFv.

[0268] In one embodiment, the antigen-binding domain includes single-domain antigen-binding (SDAB) molecules, which include molecules in which the complementarity-determining region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain variable domains, naturally occurring light-chain-lacking binding molecules, single domains derived from conventional four-chain antibodies, manipulated domains, and non-antibody-derived single-domain scaffolds. An SDAB molecule may be any single-domain molecule in this field or of the future. SDAB molecules may originate from any species, including but not limited to, mouse, human, camel, llama, lamprey, fish, shark, goat, rabbit, and cattle. The term also includes naturally occurring single-domain antibody molecules from species other than camelids and sharks.

[0269] In one respect, SDAB molecules may be derived from the variable regions of immunoglobulins found in fish, such as those derived from immunoglobulin isotypes known as novel antigen receptors (NARs) discovered in shark serum. Methods for producing single-domain molecules derived from the variable regions of NARs ("IgNARs") are described in WO03 / 014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

[0270] In other words, SDAB molecules are naturally occurring single-domain antigen-binding molecules known as light-chain-deficient heavy-chain molecules. Such single-domain molecules are described, for example, in WO9404678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448. For clarity, this variable domain derived from naturally occurring light-chain-deficient heavy-chain molecules is referred to here as VHH or nanobody to distinguish it from conventional VH of tetra-chain immunoglobulins. Such VHH molecules may originate from camelid species, such as camels, llamas, dromedaries, alpacas, and guanacos. Non-camelid species may naturally produce light-chain-deficient heavy-chain molecules, and such VHHs are within the scope of this invention.

[0271] SDAB molecules can be recombinant, CDR transplanted, humanized, camelized, deimmunized, and / or produced in vitro (e.g., selected by phage display).

[0272] It has also been found that in cells having multiple chimeric membrane-embedded receptors containing antigen-binding domains, interactions between the multiple antigen-binding domains of the receptor can be undesirable, for example, by inhibiting the ability of one or more antigen-binding domains to bind to a congener antigen. Therefore, disclosed herein are cells having first and second non-native chimeric membrane-embedded receptors containing antigen-binding domains in which such interactions are minimized. Also disclosed herein are nucleic acids encoding first and second non-native chimeric membrane-embedded receptors containing antigen-binding domains in which such interactions are minimized, as well as methods for producing such cells and nucleic acids. In one embodiment, one antigen-binding domain of the first and second non-native chimeric membrane-embedded receptors contains an scFv, and the other contains a single VH domain, for example, a single VH domain from a camel, shark, or lamprey, or a single VH domain derived from a human or mouse sequence.

[0273] In one embodiment, the present invention comprises a first CAR and a second CAR, wherein one of the antigen-binding domains of the first CAR and the second CAR does not include a variable light chain domain and a variable heavy chain domain. In one embodiment, one of the antigen-binding domains of the first CAR and the second CAR is an scFv, and the other is not an scFv. In one embodiment, one of the antigen-binding domains of the first CAR and the second CAR comprises a single VH domain, e.g., a camel, shark, or lamprey single VH domain or a single VH domain derived from a human or mouse sequence. In one embodiment, one of the antigen-binding domains of the first CAR and the second CAR comprises a nanobody. In one embodiment, one of the antigen-binding domains of the first CAR and the second CAR comprises a camel VHH domain.

[0274] In one embodiment, one antigen-binding domain of the first CAR and the second CAR contains an scFv, and the other contains a single VH domain, e.g., a camel, shark, or lamprey single VH domain or a human or mouse sequence-derived single VH domain. In one embodiment, one antigen-binding domain of the first CAR and the second CAR contains an scFv, and the other contains a nanobody. In one embodiment, one antigen-binding domain of the first CAR and the second CAR contains an scFv, and the other contains a camel VHH domain.

[0275] In one embodiment, when presented on the cell surface, the binding of the antigen-binding domain of the first CAR to its congener antigen is not substantially reduced by the presence of the second CAR. In one embodiment, the binding of the antigen-binding domain of the first CAR to its congener antigen in the presence of the second CAR is 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the binding of the antigen-binding domain of the first CAR to its congener antigen in the absence of the second CAR.

[0276] In one embodiment, when presented on the cell surface, the antigen-binding domains of the first and second CARs bind to each other at a lower level than when they are both scFv antigen-binding domains. In one embodiment, the antigen-binding domains of the first and second CARs bind to each other at 85%, 90%, 95%, 96%, 97%, 98%, or 99% lower levels than when they are both scFv antigen-binding domains.

[0277] In other respects, the CAR-expressing cells described herein may further express other agents, such as agents that enhance the activity or fitness of the CAR-expressing cells. For example, in one embodiment, the agent may be an agent that inhibits a molecule that modulates or controls T cell function, for example, an inhibitory molecule. In one embodiment, the molecule that modulates or controls T cell function is an inhibitory molecule. An inhibitory molecule, such as PD1, in one embodiment reduces 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 TGFR beta. In some embodiments, the expression of molecules that modulate or control, for example, T cell function in CAR-expressing cells can be inhibited using a drug, for example, an inhibitory nucleic acid, for example, dsRNA, for example, siRNA or shRNA; or an inhibitory protein or inhibitory system, for example, clustered and regularly arranged short palindromic sequence repeats (CRISPR), transcriptional activator-like effector nuclease (TALEN) or zinc finger endonuclease (ZFN). In some embodiments, the drug is shRNA, for example, an shRNA, for example. In some embodiments, a drug that modulates or controls, for example, T cell function, is inhibited in CAR-expressing cells. For example, a dsRNA molecule that inhibits the expression of a molecule that modulates or controls, for example, T cell function, is bound to a component of CAR, for example, a nucleic acid encoding all components.

[0278] In one embodiment, a drug that inhibits an inhibitory molecule comprises a primary polypeptide, e.g., the inhibitory molecule, conjugated to a secondary polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the drug comprises a primary 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 and TGFR beta-T or any fragment thereof (e.g., at least a portion of the extracellular domain of any of these) and an intracellular signaling domain described herein (e.g., a co-stimulatory domain (e.g., described herein, e.g., 41BB, CD27 or CD28) and / or a primary signaling domain (e.g., In one embodiment, the drug comprises a primary polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a secondary polypeptide of an intracellular signaling domain (e.g., the CD28 signaling domain and / or the CD3 zeta signaling domain described herein). PD1 is an inhibitory member of the CD28 family of receptors, 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). Two ligands for PD1, PD-L1, and PD-L2 have been shown to downregulate T cell activation by binding to PD1 (Freeman et a. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43).PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immunosuppression can be reversed by inhibiting the local interaction between PD1 and PD-L1.

[0279] In one embodiment, the drug comprises the extracellular domain (ECD) of an inhibitory molecule, for example, programmed death 1 (PD1), which can be fused with a transmembrane domain and intracellular signaling domains such as 41BB and CD3 zeta (referred to here as a PD1 CAR). In one embodiment, the PD1 CAR, when used in combination with the mesothelin CAR described herein, improves T cell persistence. In one embodiment, the CAR is a PD1 CAR comprising the extracellular domain of PD1, underlined in SEQ ID NO: 24, and the signal sequence of amino acids 1-21 of SEQ ID NO: 24. In one embodiment, the PD1 CAR comprises the amino acid sequence of SEQ ID NO: 24. [ka]

[0280] In one embodiment, a PD1 CAR that does not contain an N-terminal signal sequence comprises the amino acid sequence (SEQ ID NO: 22) provided below. [ka]

[0281] In one embodiment, the drug comprises a PD1 CAR bound to an N-terminal signal sequence, for example, a nucleic acid sequence encoding the PD1 CAR described herein. In one embodiment, the nucleic acid sequence of the PD1 CAR is shown below, with PD1 ECD underlined in Sequence ID No. 23 below. [ka]

[0282] In other aspects, the present invention provides a population of CAR-expressing cells, for example, CAR T cells. In one embodiment, the population of CAR-expressing cells comprises a mixture of cells expressing different CARs. For example, in one embodiment, the population of CAR T cells may comprise a first cell expressing a CAR having the anti-CD19 binding domain described herein and a second cell expressing a CAR having a different anti-CD19 binding domain, for example, the anti-mesothelin binding domain described herein, which is different from the anti-mesothelin binding domain in the CAR expressed by the first cell. As another example, a population of CAR-expressing cells may include, for example, first cells expressing a CAR containing an anti-mesothelin binding domain as described herein, and second cells expressing a CAR containing an antigen-binding domain against non-mesothelin targets (e.g., non-mesothelin targets on stromal cells, e.g., FAP; non-mesothelin targets on prostate cancer cells, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1 or MAD-CT-2; non-mesothelin targets on ovarian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor α, claudin 6, GloboH or sperm protein 17; e.g., non-mesothelin targets on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4 or Trop-2). In one embodiment, the population of CAR-expressing cells includes, for example, first cells expressing a CAR that includes a primary intracellular signaling domain and second cells expressing a CAR that includes a secondary signaling domain.

[0283] In other aspects, the present invention provides a population comprising at least one cell expressing a CAR having the anti-mesothelin binding domain described herein, and a second population of cells expressing other agents, such as agents that enhance the activity or function of CAR-expressing cells. For example, in one embodiment, the agent may be an agent that modulates or controls, for example, T cell function, or inhibits it. In one embodiment, the molecule that modulates or controls T cell function is an inhibitory molecule, for example, one of the agents described herein. The inhibitory molecule can, for example, in one embodiment, 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 TGFR beta. In one embodiment, the agent that inhibits the inhibitory molecule includes a second polypeptide that provides a positive signal to the cell, for example, a first polypeptide bound to the intracellular signaling domain described herein, for example, the inhibitory molecule. In one embodiment, the agent comprises a primary 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 and TGFR beta or any of these fragments (e.g., at least a portion of the extracellular domain of any of these) and a secondary polypeptide which is an intracellular signaling domain described herein (e.g., a co-stimulatory domain (e.g., 41BB, CD27, or CD28 as described herein) and / or a primary signaling domain (e.g., the CD3 zeta signaling domain as described herein). In one embodiment, the agent comprises a primary polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a secondary polypeptide which is an intracellular signaling domain (e.g., the CD28 signaling domain and / or the CD3 zeta signaling domain as described herein).

[0284] In one aspect, the present invention provides a method comprising administering a population of CAR-expressing cells, for example, CAR T cells, or a mixture of cells expressing different CARs, in combination with another agent, for example, a kinase inhibitor such as the kinase inhibitor described herein. In the other aspect, the present invention provides a method comprising administering a population of cells in which at least one cell expresses a CAR having the anti-mesothelin-binding domain described herein, and a second cell expressing another agent, for example, an agent that enhances the activity or fitness of CAR-expressing cells, in combination with another agent, for example, a kinase inhibitor such as the kinase inhibitor described herein.

[0285] Controllable Chimeric Antigen Receptors In some embodiments, controllable carcinomas (RCARs) whose CAR activity can be controlled are desirable to optimize the safety and efficacy of CAR therapy. There are many ways to control CAR activity. For example, using a caspase fused to a dimerization domain, or, for example, inducible apoptosis (see, e.g., Di et al., N Egnl. J. Med. 2011 Nov. 3;365(18):1673-1683), can be used as a safety switch in the CAR therapy of the present invention. In some aspects, an RCAR comprises a set of polypeptides, generally two in its simplest embodiment, in which the components of the standard CAR described herein, e.g., an antigen-binding domain and an intracellular signaling domain, are delimited on another polypeptide or member. In some embodiments, the set of polypeptides includes a dimerization switch, which, in the presence of a dimerization molecule, allows the polypeptides to be linked to each other, e.g., the antigen-binding domain to be linked to the intracellular signaling domain.

[0286] In some aspects, RCAR comprises 1) an intracellular signaling domain, e.g., an intracellular signaling member comprising the primary intracellular signaling domain and the first switch domain described herein; and 2) two polypeptides or members comprising, e.g., an antigen-binding member comprising the mesothelin-targeting antigen-binding domain and the second switch domain described herein. Optionally, RCAR comprises a transmembrane domain described herein. In some embodiments, the transmembrane domain may be positioned on the intracellular signaling member, on the antigen-binding member, or on both (unless otherwise specified, when the members or components of RCAR are described herein, the order may be as described, but other orders are also included. In other words, in some embodiments, the order is as shown herein, but in other embodiments, the order may be different. For example, the order of components on one side of the transmembrane region may differ from the example, and for example, the positioning of the switch domain relative to the intracellular signaling domain may differ, for example, be reversed.

[0287] In one embodiment, the first and second switch domains can form an intracellular or extracellular dimerization switch. In one embodiment, the dimerization switch may be a homodimerization switch, for example, when the first and second switch domains are the same, or a heterodimerization switch, for example, when the first and second switch domains are different from each other.

[0288] In some embodiments, the RCAR may include a “multiswitch.” The multiswitch includes a heterodimerized switch domain or a homodimerized switch domain. The multiswitch includes a number of switch domains, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, independent of a first member, e.g., an antigen-binding member and a second member, e.g., an intracellular signaling member. In some embodiments, the first member may include a number of first switch domains, e.g., an FKBP-based switch domain, and the second member may include a number of second switch domains, e.g., an FRB-based switch domain. In some embodiments, the first member may include first and second switch domains, e.g., an FKBP-based switch domain and an FRB-based switch domain, and the second member may include first and second switch domains, e.g., an FKBP-based switch domain and an FRB-based switch domain.

[0289] In one embodiment, the intracellular signaling member includes one or more intracellular signaling domains, for example, a primary intracellular signaling domain and one or more co-stimulatory signaling domains.

[0290] In some embodiments, the antigen-binding member may comprise one or more intracellular signaling domains, e.g., one or more co-stimulatory signaling domains. In some embodiments, the antigen-binding member comprises a number of co-stimulatory signaling domains, e.g., two or three, selected from e.g., 41BB, CD28, CD27, ICOS, and OX40, as described herein, and in some embodiments, does not comprise a primary intracellular signaling domain. In some embodiments, the antigen-binding member comprises the following co-stimulatory signaling domains in an extracellular to intracellular direction: 41BB-CD27; 41BB-CD27; CD27-41BB; 41BB-CD28; CD28-41BB; OX40-CD28; CD28-OX40; CD28-41BB; or 41BB-CD28. In such embodiments, the intracellular binding member comprises a CD3 zeta domain. In one such embodiment, the RCAR comprises (1) an antigen-binding member comprising, for example, an antigen-binding domain, a transmembrane domain and two costimulatory domains and a first switch domain, as described herein; and (2) an intracellular signaling domain comprising a transmembrane domain or membrane anchoring domain and at least one primary intracellular signaling domain and a second switch domain.

[0291] In one embodiment, an RCAR is provided in which the antigen-binding member is not anchored to the surface of the CAR cell. This makes it possible to conveniently pair a cell having an intracellular signaling member with one or more antigen-binding domains without transforming the cell with a sequence encoding the antigen-binding member. In such an embodiment, the RCAR includes 1) an intracellular signaling member comprising a first switch domain, a transmembrane domain, an intracellular signaling domain, e.g., a primary intracellular signaling domain and a first switch domain; and 2) an antigen-binding member that does not include a transmembrane domain or membrane anchoring domain and optionally does not include an intracellular signaling domain, e.g., an antigen-binding domain and a second switch domain as described herein. In one embodiment, the RCAR may further include 3) a second antigen-binding member comprising a second antigen-binding domain, e.g., a second antigen-binding domain that binds to a different antigen than that bound by the antigen-binding domain; and a second switch domain.

[0292] Also provided herein is an RCAR in which the antigen-binding member has bispecific activation and targeting ability. In this embodiment, the antigen-binding member may comprise a plurality of antigen-binding domains, e.g., two, three, four, or five, such as an scFv, where each antigen-binding domain binds to a target antigen, e.g., a different antigen or the same antigen, e.g., the same or different epitopes on the same antigen. In one embodiment, the plurality of antigen-binding domains are tandem, and optionally, a linker or hinge region is placed between each of the antigen-binding domains. Appropriate linker and hinge regions are described herein.

[0293] One embodiment provides an RCAR having a configuration that enables switching of proliferation. In this embodiment, the RCAR includes 1) an intracellular signaling member optionally comprising a transmembrane domain or membrane anchoring domain; one or more costimulatory signaling domains, e.g., selected from 41BB, CD28, CD27, ICOS, and OX40, and a switch domain; and 2) an antigen-binding member comprising, e.g., an antigen-binding domain, a transmembrane domain, and a primary intracellular signaling domain, e.g., a CD3 zeta domain, which does not include a switch domain or does not include a switch domain that dimerizes with a switch domain on the intracellular signaling member. In one embodiment, the antigen-binding member does not include a costimulatory signaling domain. In one embodiment, the intracellular signaling member comprises a switch domain from a homodimerizing switch. In one embodiment, the intracellular signaling member comprises a first switch domain of a heterodimerizing switch, and the RCAR comprises a second intracellular signaling member comprising a second switch domain of a heterodimerizing switch. In such an embodiment, the second intracellular signaling member comprises the same intracellular signaling domain as the intracellular signaling member. In one embodiment, the dimerizing switch is intracellular. In one aspect, the dimerization switch is extracellular.

[0294] In any of the RCAR configurations described herein, the first and second switch domains include the FKBP / FRB-based switches described herein.

[0295] Also provided herein are cells containing the RCAR described herein. Any cells engineered to express RCAR can be used as RCARX cells. In one aspect, RCARX cells are T cells and are called RCAR T cells. In another aspect, RCARX cells are NK cells and are called RCARN cells.

[0296] Also provided herein are nucleic acids and vectors containing RCAR-coding sequences. Sequences encoding diverse components of RCAR can be placed on the same nucleic acid molecule, e.g., the same plasmid or vector, e.g., a viral vector, e.g., a lentiviral vector. In one embodiment, (i) a sequence encoding an antigen-binding member and (ii) a sequence encoding an intracellular signaling member can be present on the same nucleic acid, e.g., a vector. Production of the corresponding protein can be achieved, for example, by the use of a different promoter or by the use of a bisistronic transcript (which can produce two proteins by cleaving a single translation product or by translating two different protein products). In one embodiment, a sequence encoding a cleavable peptide, e.g., a P2A or F2A sequence, is placed between (i) and (ii). In one embodiment, a sequence encoding an IRES, e.g., an EMCV or EV71 IRES, is placed between (i) and (ii). In these embodiments, (i) and (ii) are transcribed as a single RNA. In one embodiment, a first promoter is operably bound to (i) and a second promoter is operably bound to (ii) so that (i) and (ii) are transcribed as separate mRNAs.

[0297] Alternatively, sequences encoding diverse components of RCAR can be placed on different nucleic acid molecules, such as different plasmids or vectors, such as viral vectors, such as lentiviral vectors. For example, (i) sequences encoding antigen-binding members may be placed on a first nucleic acid, such as a first vector, and (ii) sequences encoding intracellular signaling members may be located on a second nucleic acid, such as a second vector.

[0298] Dimerization switch The dimerization switch may be non-covalent or covalent. In a non-covalent dimerization switch, the dimerizing molecule promotes non-covalent interactions between switch domains. In a covalent dimerization switch, the dimerizing molecule promotes covalent interactions between switch domains.

[0299] In one embodiment, RCAR includes an FKBP / FRAP or FKBP / FRB-based dimerization switch. FKBP12 (FKBP or FK506-binding protein) is an abundant cytoplasmic protein that acts as an early intracellular target of rapamycin, a natural product immunosuppressant. Rapamycin binds to FKBP and its large PI3K homolog FRAP (RAFT, mTOR). FRB is the 93-amino acid portion of FRAP sufficient for binding to the FKBP-rapamycin complex (Chen, J., Zheng, XF, Brown, EJ & 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).

[0300] In some embodiments, FKBP / FRAP, for example, FKBP / FRB-based switches can use dimerized molecules, such as rapamycin or rapamycin analogs.

[0301] The amino acid sequence of FKBP is as follows: [ka]

[0302] In some embodiments, the FKBP switch domain may include an FRB-binding fragment of FKBP, for example, the underlined portion of Sequence ID No. 382. [ka]

[0303] The amino acid sequence of FRB is as follows: [ka]

[0304] To the extent that the term is used herein, “FKBP / FRAP, e.g., FKPP / FRB-based switch” includes an FRB-bound fragment or FKBP analogue, e.g., RAD001, and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the FKBP sequence of sequence number 382 or 383, or contains 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amix A dimerizing switch is defined as comprising a first switch domain that differs beyond no acid residues; and an FKBP-binding fragment or FRB analogue, which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the FRB sequence of SEQ ID NO: 384, or comprising a second switch domain that differs beyond 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue. In one embodiment, the RCAR described herein comprises one switch domain containing the amino acid residues disclosed in SEQ ID NO: 382 (or SEQ ID NO: 383) and one switch domain containing the amino acid residues disclosed in SEQ ID NO: 384.

[0305] In some embodiments, the FKBP / FRB dimerization switch comprises a modified FRB switch domain that exhibits altered, for example, increased affinity for a dimerizing molecule, e.g., rapamycin or rapalog, e.g., RAD001. In some embodiments, the modified FRB switch domain comprises one or more mutations selected from the amino acid positions L2031, E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105, and F2108, e.g., two, three, four, five, six, seven, eight, nine, ten, or more mutations, where the wild-type amino acid is mutated to any other naturally occurring amino acid. In one embodiment, the mutant FRB includes a mutation in E2032, where E2032 is mutated to phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I) (e.g., SEQ ID NO: 385), or leucine (E2032L) (e.g., SEQ ID NO: 386). In one embodiment, the mutant FRB includes a mutation in T2098, where T2098 is mutated to phenylalanine (T2098F) or leucine (T2098L) (e.g., SEQ ID NO: 387). In one embodiment, the mutant FRB includes mutations in both E2032 and T2098, where E2032 is mutated to any amino acid and T2098 is mutated to any amino acid (e.g., SEQ ID NO: 388). In one embodiment, the mutant FRB contains the E2032I and T2098L mutations (e.g., SEQ ID NO: 389). In another embodiment, the mutant FRB contains the E2032L and T2098L mutations (e.g., SEQ ID NO: 340).

[0306] [Table 1]

[0307] Other suitable dimerization switches include GyrB-GyrB-based dimerization switches, gibberellin-based dimerization switches, tag / binder dimerization switches, and halotag / snaptag dimerization switches. Such switches and suitable dimerization molecules will be apparent to those skilled in the art, following the guidance provided herein.

[0308] dimerization molecule The association between switch domains is facilitated by dimerizing molecules. In the presence of dimerizing molecules, the interaction or association between switch domains enables signaling between a polypeptide bound to, for example, the first switch domain (e.g., fused) and a polypeptide bound to, for example, the second switch domain (e.g., fused). In the presence of non-rate-limiting levels of dimerizing molecules, signaling increases by, for example, 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 5x, 10x, 50x, and 100x, as measured in the systems described herein.

[0309] Rapamycin and rapamycin analogs (sometimes called rapagros), such as RAD001, can be used as dimerizing molecules in the FKBP / FRB-based dimerizing switches described herein. In one embodiment, the dimerizing molecule is selected from rapamycin (sirolimus), RAD001 (everolimus), zotarolimus, temsirolimus, AP-23573 (ridafololimus), biolimus, and AP21967. Further rapamycin analogs suitable for use with FKBP / FRB-based dimerizing switches are described further in the chapter titled “Combination Therapies” or the subsection titled “Representative mTOR Inhibitors.”

[0310] RNA gene transfer Disclosed herein is a method for producing in vitro transcribed RNA CARs. The present invention also relates to CAR-coding RNA constructs that can be directly introduced into cells. The method for producing mRNA for use in gene delivery comprises in vitro transcription (IVT) of a template using specially designed primers, followed by poly-A addition, to produce a construct generally 50–2000 nucleotides long containing 3' and 5' untranslated sequences ("UTR"), a 5' cap and / or an intra-sequence ribosome entry site (IRES), the nucleic acid to be expressed, and a poly-A tail (SEQ ID NO: 35). The RNA thus produced efficiently delivers genes into different types of cells. In one aspect, the template contains the sequence for the CAR.

[0311] In one respect, mesothelin CAR is encoded by messenger RNA (mRNA). In another respect, mRNA encoding mesothelin CAR is introduced into T cells for the production of CAR T cells.

[0312] In one embodiment, an RNA CAR transcribed in vitro can be introduced into cells in the form of transient gene transfer. RNA is produced by in vitro transcription using a template produced by polymerase chain reaction (PCR). Desired DNA from any origin can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The origin of the DNA may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable origin of DNA. The desired template for in vitro transcription is the CAR of the present invention. For example, a template for an RNA CAR includes an extracellular region containing a single-chain variable domain of an antitumor antibody; a cytoplasmic region containing a hinge region, a transmembrane domain (e.g., the transmembrane domain of CD8a); and an intracellular signaling domain, for example, the signaling domain of CD3 zeta and the signaling domain of 4-1BB.

[0313] In one embodiment, the DNA used for PCR includes an open reading frame. The DNA may be derived from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the nucleic acid may include some or all of the 5' and / or 3' untranslated regions (UTRs). The nucleic acid may include exons and introns. In one embodiment, the DNA used for PCR is a human nucleic acid sequence. In another embodiment, the DNA used for PCR is a human nucleic acid sequence including the 5'UTR and 3'UTR. The DNA may be an artificial DNA sequence that is not normally expressed in naturally occurring organisms. A typical artificial DNA sequence includes portions of genes ligated together to form an open reading frame encoding a fusion protein. The ligated portions of DNA may be from a single organism or from more than one species.

[0314] PCR is used to produce templates for in vitro transcription of mRNA used for gene transfer. Methods for performing PCR are well known in this art. Primers used in PCR contain regions that are substantially complementary to the region of DNA used as the template for PCR. The term “substantially complementary” refers to 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 can anneal or hybridize with the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any part of the DNA template. For example, primers can be designed to amplify a portion of nucleic acid (open reading frame) that is normally transcribed in cells, including the 5'UTR and 3'UTR. Primers can also be designed to amplify a portion of nucleic acid that codes for a specific domain of interest. In one embodiment, primers are designed to amplify the coding region of human cDNA, including all or part of the 5'UTR and 3'UTR. Primers useful for PCR can be produced by synthetic methods well known in this art. A “forward primer” is a primer that contains a region of nucleotides substantially complementary to the nucleotides on the DNA template upstream of the DNA sequence to be amplified. The term “upstream” refers to the 5' position relative to the coding strand of the DNA sequence to be amplified. A “reverse primer” is a primer that contains a region of nucleotides substantially complementary to the double-stranded DNA template downstream of the DNA sequence to be amplified. The term “downstream” refers to the 3' position relative to the coding strand of the DNA sequence to be amplified.

[0315] Any DNA polymerase useful for PCR can be used in the method disclosed herein. Reagents and polymerases are commercially available from numerous suppliers.

[0316] Chemical structures that have the ability to promote stability and / or translation efficiency may also be used. The RNA preferably includes a 5'UTR and a 3'UTR. In one embodiment, the 5'UTR is 1 to 3000 nucleotides long. 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, the design of PCR primers that anneal to different regions of the UTR. Using this method, those skilled in the art can modify the 5'UTR and 3'UTR lengths necessary to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0317] The 5'UTR and 3'UTR may be naturally occurring, endogenous 5'UTR and 3'UTR of the desired nucleic acid. Alternatively, a non-endogenous UTR sequence can be added to the desired nucleic acid by incorporating the UTR sequence into forward and reverse primers or by some other modification of the template. The use of a non-endogenous UTR sequence in the desired nucleic acid may be useful for modifying RNA stability and / or translation efficiency. For example, AU-rich components in the 3'UTR sequence can reduce mRNA stability. Therefore, based on the UTR properties well known in this art, the 3'UTR can be selected or designed to enhance the stability of the transcribed RNA.

[0318] In one embodiment, the 5'UTR may contain a Kosack sequence of an endogenous nucleic acid. Alternatively, when a non-endogenous 5'UTR is added to a desired nucleic acid by PCR as described above, the consensus Kosack sequence can be redesigned by adding the 5'UTR sequence. While Kosack sequences can improve the translation efficiency of some RNA transcripts, they are not considered necessary for all RNA to enable efficient translation. The need for Kosack sequences for many mRNAs is known in this field. In another embodiment, the 5'UTR is the 5'UTR of an RNA virus whose RNA genome is stable in cells. In yet another embodiment, various nucleotide analogs can be used in the 3' or 5'UTR to inhibit the exonuclease degradation of mRNA.

[0319] To enable RNA synthesis from a DNA template without requiring gene cloning, a transcription promoter must be ligated upstream of the transcription sequence in the DNA template. When a sequence functioning as an RNA polymerase promoter is ligated to the 5' end of a forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame being transcribed. In one preferred embodiment, the promoter is the T7 polymerase promoter, as described elsewhere in this specification. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7, T3, and SP6 promoters are known in the art.

[0320] In a preferred embodiment, mRNA has caps above both the 5' end and the 3' poly(A) tail, which determine ribosome binding, translation initiation, and cellular stability. In circular DNA templates, such as plasmid DNA, RNA polymerase produces long, chain-like products unsuitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3'UTR end, even after polyadenylation post-transcription, results in normal-sized mRNA that is not effective for eukaryotic cell gene transfer.

[0321] 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)).

[0322] The conventional method for incorporating polyA / T stretches into DNA templates is molecular cloning. However, integrating polyA / T sequences into plasmid DNA can destabilize the plasmid, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other abnormalities. This makes the cloning procedure not only laborious and time-consuming, but also often unreliable. This is why a method that allows for the construction of DNA templates with polyA / T 3' stretches without cloning is highly desirable.

[0323] Poly(A) tails can be produced by any other method, including but not limited to DNA ligation or in vitro recombination, during or after PCR using poly(T) tail-containing reverse primers such as 100T tails (SEQ ID NO: 31) (sizes can range from 50 to 5000T (SEQ ID NO: 32)). Poly(A) tails also stabilize RNA and reduce its degradation. Generally, the length of the poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is 100 to 5000 adenosines (SEQ ID NO: 33).

[0324] The RNA poly(A) tail can be further elongated after in vitro transcription using a poly(A) polymerase such as E. coli poly(A) polymerase (E-PAP). In one embodiment, extending the poly(A) tail from 100 nucleotides to 300-400 nucleotides (SEQ ID NO: 34) approximately doubles the RNA translation efficiency. Furthermore, ligation of different chemical groups to the 3' end can increase mRNA stability. Such ligations may include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using a poly(A) polymerase. ATP analogs can further enhance RNA stability.

[0325] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the method disclosed herein includes a 5' cap. The 5' cap is provided using methods 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)).

[0326] RNA produced by the methods disclosed herein may also contain intra-sequence ribosome entry sites (IRES) sequences. IRES sequences may be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and facilitates translation initiation. They may contain any solute suitable for cell electroporation, including factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

[0327] RNA can be introduced into target cells using any of several different methods, including, but not limited to, commercially available methods such as electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated gene transfer using lipofection, polymer encapsulation, peptide-mediated gene transfer, or microparticle gun particle delivery systems such as “gene guns” (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0328] Nucleic acid constructs encoding CAR The present invention provides a CAR-transformed gene comprising nucleic acid sequences encoding one or more CAR constructs of the present invention. In one aspect, the CAR-transformed gene is provided as a messenger RNA transcript. In another aspect, the CAR-transformed gene is provided as a DNA construct.

[0329] Therefore, in one aspect, the present invention relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising an anti-mesothelin-binding domain (e.g., a human anti-mesothelin-binding domain), a transmembrane domain, and an intracellular signaling domain comprising a stimulating domain. In one embodiment, the anti-mesothelin-binding domain is an anti-mesothelin-binding domain comprising an anti-mesothelin-binding domain described herein, for example, a sequence selected from the group consisting of SEQ ID NOs: 87-111, or a sequence having 95-99% identity thereto. In one embodiment, the isolated nucleic acid molecule further comprises a sequence encoding a co-stimulating domain. In one embodiment, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO: 6 or a sequence having 95-99% identity thereto. In one embodiment, the anti-mesothelin-binding domain is connected to the transmembrane domain by a hinge region, e.g., a hinge described herein. In one embodiment, the hinge region comprises SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or a sequence having 95-99% identity thereto. In one embodiment, the isolated nucleic acid molecule further comprises a sequence encoding a costimulatory domain. In one embodiment, the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, and CD2 9, 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, and PAG / Cbp are included. In one embodiment, the co-stimulatory domain includes the sequence of Sequence ID No. 7 or a sequence having 95-99% identity thereto. In one embodiment, the intracellular signaling domain comprises a functional signaling domain 4-1BB and a functional signaling domain CD3 zeta. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 7 or SEQ ID NO: 8 or a sequence having 95-99% identity with them and the sequence of SEQ ID NO: 9 or SEQ ID NO: 10 or a sequence having 95-99% identity with them, wherein the sequences containing the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.In other aspects, the present invention relates to an scFv domain having a sequence selected from the group consisting of the leader sequence of SEQ ID NO: 1, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62 (or sequences having 95-99% identity with these), SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 The present invention relates to a nucleic acid molecular construct encoding an isolated CAR, comprising a hinge region of sequence number 5 (or a sequence 95-99% identical thereto), a transmembrane domain having the sequence of sequence number 6 (or a sequence 95-99% identical thereto), a 4-1BB costimulatory domain having the sequence of sequence number 7 (or a sequence 95-99% identical thereto), or a CD27 costimulatory domain having the sequence of sequence number 8 (or a sequence 95-99% identical thereto), and a CD3 zeta-stimulatory domain having the sequence of sequence number 9 or 10 (or a sequence 95-99% identical thereto).

[0330] In other respects, the present invention relates to an isolated polypeptide molecule encoded by a nucleic acid molecule. In one embodiment, the isolated polypeptide molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, and 86, or a sequence having 95-99% identity with these.

[0331] In other words, the present invention relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), comprising an anti-mesothelin-binding domain, a transmembrane domain, and an intracellular signaling domain including a stimulating domain, wherein the nucleic acid encoding the anti-mesothelin-binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 111; SEQ ID NOs: 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, and 134, or a sequence having 95-99% identity with these.

[0332] In one embodiment, the encoded CAR molecule further includes a sequence encoding a co-stimulatory domain. In one embodiment, the co-stimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137). In one embodiment, the co-stimulatory domain includes the sequence of SEQ ID NO: 7. In one embodiment, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain includes the sequence of SEQ ID NO: 6. In one embodiment, the intracellular signaling domain includes the functional signaling domain of 4-1BB and the functional signaling domain of CD3 zeta. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 7 and the sequence of SEQ ID NO: 9, where the sequence comprising the intracellular signaling domain is expressed in the same frame and as a single polypeptide chain. In one embodiment, the anti-mesothelin-binding domain is connected to the transmembrane domain by a hinge region. In one embodiment, the hinge region comprises SEQ ID NO: 2. In one embodiment, the hinge region comprises SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0333] In other words, the present invention relates to an isolated CAR molecule comprising a leader sequence of SEQ ID NO: 1, an scFv domain having a sequence selected from the group consisting of SEQ ID NOs: 39-62 or a sequence having 95-99% identity thereto, a hinge region of SEQ ID NO: 2 or 3 or SEQ ID NO: 4 or 5, a transmembrane domain having the sequence of SEQ ID NO: 6, a 4-1BB costimulatory domain having the sequence of SEQ ID NO: 7 or a CD27 costimulatory domain having the sequence of SEQ ID NO: 8, and a CD3 zeta-stimulating domain having the sequence of SEQ ID NO: 9 or 10. In one embodiment, the encoded CAR molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 63-86 or a sequence having 95-99% identity thereto.

[0334] The present invention further provides vectors containing CAR transgenes. In one respect, CAR vectors can be directly transduced into cells, such as T cells or NK cells. In one respect, vectors include, but are not limited to, cloning or expression vectors, such as one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double microchromosomes), retroviral constructs, and lentiviral vector constructs. In one respect, vectors can express CAR constructs in mammalian T cells or NK cells. In one respect, mammalian T cells are human T cells or human NK cells.

[0335] The present invention also includes CAR-coding RNA constructs that can be directly introduced into cells, such as T cells or NK cells. A method for producing mRNA for gene transfer involves in vitro transcription (IVT) of a template using specially designed primers, followed by poly-A addition, to produce a construct generally 50–2000 nucleotides long, containing 3' and 5' untranslated sequences ("UTR"), a 5' cap and / or an intra-sequence ribosome entry site (IRES), the gene to be expressed, and a poly-A tail. The RNA thus produced can be efficiently introduced into different types of cells. In one aspect, the template contains the sequence for the CAR.

[0336] In one respect, the mesothelin CAR transgene is encoded by messenger RNA (mRNA). In another respect, the mRNA transgene encoding mesothelin CAR is introduced into T cells or NK cells for the production of CAR T cells.

[0337] vector The present invention also provides vectors into which the DNA of the present invention is inserted. Retroviral vectors, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of the transgene and its transmission in daughter cells. Lentiviral vectors have further advantages over onchoretrovirus-derived vectors, such as mouse leukemia virus, because they can transduce non-proliferating cells such as hepatocytes. They also have the further advantage of low immunogenicity.

[0338] In one embodiment, the vector containing the nucleic acid encoding the desired CAR of the present invention is DNA, RNA, plasmid, adenovirus vector, lentiviral vector, or retrovirus vector.

[0339] In another embodiment, the vector containing the nucleic acid encoding the desired CAR of the present invention is an adenovirus vector (A5 / 35). In another embodiment, expression of the nucleic acid encoding the CAR can be achieved using transposons such as Sleeping Beauty, CRISPR, CAS9, and zinc finger nucleases. See, for example, June et al. 2009 Nature Reviews Immunology 9.10:704–716, which is incorporated herein by reference in its entirety.

[0340] In summary, the expression of CAR-encoding natural or synthetic nucleic acids is generally achieved by manipulating a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and incorporation in eukaryotes. Typical cloning vectors contain transcription and translation terminators, start sequences, and promoters useful for controlling the expression of a desired nucleic acid sequence.

[0341] The expression constructs of the present invention may also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art; see, for example, U.S. Patents 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In other embodiments, the present invention provides gene therapy vectors.

[0342] 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, phagemids, phage derivatives, animal viruses, and cosmids. Particularly desired vectors include expression vectors, replication vectors, probe-producing vectors, and sequencing vectors.

[0343] Furthermore, expression vectors can be delivered to cells in the form of viral vectors. Viral vector technology is well known in this field and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY, and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector includes a functional origin of replication, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers in at least one organism (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0344] Numerous virus-based systems have been developed for gene delivery into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Using methods known in this field, selected genes can be introduced into vectors and loaded into retroviral particles. The recombinant viruses are then isolated and delivered to target cells in vivo or ex vivo. Numerous retroviral systems are known in this field. In one embodiment, adenovirus vectors are used. Numerous adenovirus vectors are known in this field. In one embodiment, lentiviral vectors are used.

[0345] Further promoter components, such as enhancers, control the frequency of transcription initiation. Generally, these are located 30–110 bp upstream of the initiation site, although many promoters have been shown to contain similarly functional components downstream of the initiation site. The spacing between multiple promoter components is often flexible to protect promoter function when the components are in opposite positions or moved. In the thymidine kinase (TK) promoter, the spacing between promoter components may increase to 50 bp before activity begins to decline. Depending on the promoter, individual components may function cooperatively or independently in activating transcription. Representative promoters include the CMV IE gene, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK) promoters.

[0346] An example of a promoter capable of expressing CAR transgenes in mammalian T cells is the EF1 alpha promoter (EF1a or EFLα). The native EF1 promoter drives the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to ribosomes. The EF1 promoter is widely used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from transgenes cloned into lentiviral vectors. See, for example, Milone et al., Mol. Ther. 17(8):1453-1464 (2009). In one respect, the EF1 promoter contains the sequence provided in SEQ ID NO: 11.

[0347] Other examples of promoters include the very early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably bound to it. However, other constitutive promoter sequences may also be used, including but not limited to human gene promoters, such as the monkey virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) terminal repeat sequence (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus very early promoter, Roussarcoma virus promoter, as well as the actin promoter, myosin promoter, elongation factor-1α 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 intended as part of the present invention. The use of inducible promoters provides a molecular switch that can activate the expression of an operably bound polynucleotide sequence when such expression is desired and block such expression when such expression is not desired. Examples of inductive promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0348] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into cells may also include a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells being sought for gene transfer or infection by a viral vector. In other words, the selectable marker may be carried on a separate portion of DNA and used in co-gene transfer procedures. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0349] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, 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 manifested by a readily detectable characteristic, such as enzymatic activity. Reporter gene expression is assayed at an appropriate time after DNA is introduced into recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted 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 manufactured using known techniques or are commercially available. Generally, a construct with a minimum 5' flanking region exhibiting the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be ligated to the reporter gene and used to evaluate the ability of a drug to modulate promoter-driven transcription.

[0350] In one embodiment, the vector may further comprise a nucleic acid encoding a secondary CAR. In one embodiment, the secondary CAR comprises an antigen-binding domain for, for example, a non-mesothelin target on stromal cells, e.g., FAP; a non-mesothelin target on prostate cancer cells, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1, or MAD-CT-2; a non-mesothelin target on ovarian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor α, claudin 6, GloboH, or sperm protein 17; or a non-mesothelin target on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4, or Trop-2. In one embodiment, the vector comprises a nucleic acid sequence encoding a first CAR that targets a first antigen and includes an intracellular signaling domain having a co-stimulatory signaling domain but lacking a primary signaling domain, and a nucleic acid sequence encoding a second CAR that targets a second different antigen and includes an intracellular signaling domain having a primary signaling domain but lacking a co-stimulatory signaling domain. In one embodiment, the vector comprises a nucleic acid encoding a first mesothelin CAR, including a mesothelin-binding domain, a transmembrane domain, and a costimulatory domain, and a nucleic acid encoding a second CAR, including an antigen-binding domain, a transmembrane domain, and a primary signaling domain, targeting a non-mesothelin antigen (e.g., non-mesothelin targets on stromal cells, e.g., FAP; non-mesothelin targets on prostate cancer cells, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1, or MAD-CT-2; non-mesothelin targets on ovarian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor α, claudin 6, GloboH, or sperm protein 17; e.g., non-mesothelin targets on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4, or Trop-2).In another embodiment, the vector comprises a nucleic acid encoding a first mesothelin CAR, including a mesothelin-binding domain, a transmembrane domain, and a primary signaling domain, and a nucleic acid encoding a second CAR, including an antigen-binding domain, a transmembrane domain, and a co-stimulatory signaling domain, targeting a non-mesothelin antigen (e.g., non-mesothelin targets on stromal cells, e.g., FAP; non-mesothelin targets on prostate cancer cells, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1, or MAD-CT-2; non-mesothelin targets on ovarian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor α, claudin 6, GloboH, or sperm protein 17; e.g., non-mesothelin targets on lung cancer cells, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4, or Trop-2), targeting the antigen.

[0351] In one embodiment, the vector comprises a nucleic acid encoding a mesothelin CAR and a nucleic acid encoding an inhibitory CAR as described herein. In one embodiment, the inhibitory CAR includes an antigen-binding domain that binds to an antigen found on normal cells, e.g., normal cells that also express CLL, but not on cancer cells. In one embodiment, the inhibitory CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular domain of the inhibitory molecule. For example, the intracellular domain of an inhibitory CAR may be the intracellular domain of PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta.

[0352] In one embodiment, the vector comprises a nucleic acid encoding the mesothelin CAR described herein and an inhibitory nucleic acid, such as, for example, dsRNA, for example, siRNA, or shRNA, as described herein.

[0353] Methods for introducing and expressing genes in cells are known in this field. In relation to expression vectors, these vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in this field. For example, expression vectors can be introduced into host cells by physical, chemical, or biological means.

[0354] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, microparticle guns, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in this field. 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 lipofection, for example, using lipofectamine (Life Technologies).

[0355] Biological methods for introducing desired polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for gene insertion into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, for example. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0356] Chemical means 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. A typical colloidal system used as a delivery medium in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). Other state-of-the-art methods of nucleic acid targeted delivery are available, such as delivery of polynucleotides via targeted nanoparticles or other suitable submicron-sized delivery systems.

[0357] When non-viral delivery systems are used, liposomes are the typical delivery medium. Lipid formulations are intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In other respects, nucleic acids can bind to lipids. Lipid-bound nucleic acids may be encapsulated within the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, bound to liposomes by binding molecules that bind to both liposomes and oligonucleotides, encapsulated in liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained in micelles, complexed, or otherwise bound to lipids. Lipids, lipid / DNA, or lipid / expression vector binding compositions are not limited to any specific structure in solution. For example, they may exist in a bilayer structure like micelles or have a “disintegrated” structure. They may also simply be dispersed in solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that may be naturally occurring or synthetic lipids. For example, lipids include lipid droplets that occur naturally in the cytoplasm, as well as a group of compounds having long-chain aliphatic hydrocarbons such as fatty acids, alcohols, amines, amino alcohols, and aldehydes, and their derivatives.

[0358] Suitable lipids can be obtained from suppliers. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL.). Lipid storage solutions in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates much more readily than methanol. "Liposomes" is a general term encompassing a variety of single and multilayered lipid media formed by the production of encapsulated lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayered liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in excess aqueous solution, they form spontaneously. The lipid components undergo self-reorganization, subsequently forming a closed structure that traps water between lipid bilayers and dissolves the solute (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions that adopt structures different from the usual vesicle structure in solution are also included. For example, lipids can be envisioned to exist as micelle structures or simply as heterogeneous aggregates of lipid molecules. Another example to consider is lipofectamine-nucleic acid complexes.

[0359] A variety of assays can be performed to confirm the presence of recombinant DNA sequences within host cells, regardless of the method used to introduce foreign nucleic acids into host cells or otherwise expose cells to the inhibitors of the present invention. Such assays include, for example, “molecular biological” assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; and “biochemical” assays to detect the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blotting) or by assays described herein for identifying agents that fall within the scope of the present invention.

[0360] The present invention further provides a vector comprising a CAR-encoding nucleic acid molecule. In one respect, the CAR vector can be directly transduced into cells, such as T cells or NK cells. In one respect, the vector comprises, but is not limited to, one or more cloning vectors or expression vectors, such as plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double microchromosomes), retroviral constructs, and lentiviral vector constructs. In one respect, the vector can express a CAR construct in mammalian T cells. In one respect, the mammalian T cells are human T cells. In one respect, the mammalian cells are human NK cells.

[0361] Origin of Cells Prior to proliferation and genetic modification, the origin of the cells (e.g., T cells or NK cells) is obtained from a subject. The term “subject” is intended to include a viable organism (e.g., a mammal) capable of eliciting an immune response. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural fluid, splenic tissue, and tumors. In some aspects of the present invention, any number of T cell lines available in the art may be used. In some aspects of the present invention, T cells are obtained from Ficoll TMCells can be obtained from a unit of blood collected from a subject using any method known to those skilled in the art, such as separation. In one preferred aspect, cells are obtained from the circulating blood of an individual by apheresis. Apheresis products generally include lymphocytes, erythrocytes, and platelets, including T cells, monocytes, granulocytes, B cells, and other nucleated leukocytes. In one aspect, cells collected by apheresis can be washed to remove the plasma fraction, and the cells can be placed in a suitable buffer or medium for the next processing step. In one aspect of the present invention, cells are washed with phosphate-buffered saline (PBS). In another aspect, the washing solution may be calcium-deficient, magnesium-deficient, or many, if not all, divalent cations-deficient. An initial activation step in the absence of calcium may enhance activation. As will be readily apparent to those skilled in the art, the washing step can be achieved by methods known to those skilled in the art, such as using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) in accordance with the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or saline solutions with or without other buffers. Alternatively, undesirable components can be removed from the apheresis sample, and the cells can be resuspended directly in the culture medium.

[0362] In one aspect, T cells lyse red blood cells and monocytes, for example, PERCOLL TM CD3 is isolated from peripheral blood lymphocytes by depletion using gradient centrifugation or countercurrent elutriation. + CD28 + CD4 + CD8 + CD45RA + and CD45RO +Specific subpopulations of T cells, such as T cells, can be further isolated by positive or negative selection techniques. For example, in one aspect, T cells are isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3×28) conjugated beads such as DYNABEADS® M-450 CD3 / CD28 T for a period sufficient for positive selection of the desired T cells. In one aspect, the incubation period is approximately 30 minutes. Further aspects, the incubation period ranges from 30 minutes to 36 hours or longer and any integer value in between. Further aspects, the incubation period is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. Further other preferred aspects, the incubation period is 10 to 24 hours. In one aspect, the incubation period is 24 hours. For the isolation of T cells from patients with leukemia, the use of longer incubation periods, such as 24 hours, can increase cell yield. Long incubation times can be used to isolate T cells in any situation where T cells are scarce compared to other cell types, such as the isolation of tumor-infiltrating lymphocytes (TILs) from tumor tissue or isolation from immunocompromised individuals. Furthermore, the use of long incubation times can be used for CD8 + The capture efficiency of T cells can be increased. Therefore, subpopulations of T cells can be preferentially selected or eliminated at the start of culture or at any other point in the process by simply shortening or lengthening the time that T cells are bound to CD3 / CD28 beads and / or by increasing or decreasing the bead-to-T cell ratio (as further described herein). Furthermore, subpopulations of T cells can be preferentially selected or eliminated at the start of culture or at any other desired point in time by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces. Those skilled in the art will recognize that multiple selections can also be used in the context of the present invention. In some respects, it may be desirable to use a selection procedure and use “unselected” cells in the activation and proliferation processes. “Unselected” cells can also be subjected to further selection.

[0363] Enrichment of T cell populations by negative selection can be achieved by a combination of antibodies that target surface markers specific to negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies that target cell surface markers present on negatively selected cells. For example, negative selection can lead to CD4 + To enrich cells, monoclonal antibody cocktails generally contain antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some respects, generally CD4 + CD25 + CD62Lhi, GITR + and FoxP3 + It may be desirable to enrich or positively select regulatory T cells that express . Alternatively, in some respects, regulatory T cells may be depleted by anti-C25 binding beads or other similar selection methods.

[0364] In one embodiment, a population of T cells expressing IFN-γ, TNF-α, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin or other suitable molecules, such as one or more other cytokines, can be selected. A method for screening cell expression can be determined, for example, by the method described in PCT Publication WO2013 / 126712.

[0365] In one embodiment, the T cell population is diacylglycerol kinase (DGK) deficient. DGK-deficient cells are those that do not express DGK RNA or protein, or have reduced or inhibited DGK activity. DGK-deficient cells can be produced by genetic methods, for example, by administering RNA interferants, such as siRNA, shRNA, or miRNA, to reduce or inhibit DGK expression. Alternatively, DGK-deficient cells can be produced by treatment with the DGK inhibitors described herein.

[0366] In one embodiment, the T cell population is Ikaros-deficient. Ikaros-deficient cells are cells that do not express Ikaros RNA or protein, or have reduced or inhibited Ikaros activity, and Ikaros-deficient cells can be produced by genetic methods, for example, by administering RNA interferants, such as siRNA, shRNA, or miRNA, to reduce or block Ikaros expression. Alternatively, Ikaros-deficient cells can be produced by treatment with an Ikaros inhibitor, such as lenalidomide.

[0367] In some embodiments, the T cell population is DGK-deficient and Ikaros-deficient, for example, not expressing DGK and Ikaros, or having reduced or inhibited DGK and Ikaros activity. Such DGK and Ikaros-deficient cells can be produced by any of the methods described herein.

[0368] To isolate a desired cell population by positive or negative selection, the concentrations of cells and surfaces (e.g., beads or other particles) may vary. On one surface, it may be desirable to significantly reduce the volume of bead-cell mixture (e.g., increase the cell concentration) to ensure maximum contact between cells and beads. For example, on one surface, use a concentration of 2 billion cells / ml. On another surface, use 1 billion cells / ml. On yet another surface, use over 100 million cells / ml. On yet another surface, use cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million or 50 million cells / ml. On yet another surface, use cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million or 100 million cells / ml. On yet another surface, you can use concentrations of 125 million or 150 million cells / ml. The use of high concentrations can lead to increased cell yield, cell activation, and cell proliferation. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express desired target antigens, such as CD28-negative T cells, or cells from samples containing many tumor cells (e.g., leukemia blood, tumor tissue, etc.). Populations of such cells may have therapeutic value and are desirable to obtain. For example, the use of high cell concentrations can lead to the capture of CD8 cells that typically have weak CD28 expression. + This enables more efficient selection of T cells.

[0369] In related aspects, it is sometimes desirable to use low concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., bead-like particles), the interaction between particles and cells is minimized. These are selected with cells that express high levels of the desired antigen that binds to the particles. For example, CD4 + T cells express high levels of CD28, and at dilution concentrations, CD8 + It captures cells more efficiently than T cells. In one aspect, the concentration of cells used is 5 × 10 e 6 The concentration used is approximately 1 × 10⁻⁶ / ml. 5 / ml~1×10 6 / ml can be any integer value between / ml and / ml.

[0370] In other respects, cells can be incubated on a rotor at 2-10°C or room temperature for varying lengths of time and at various speeds.

[0371] Stimulating T cells can also be frozen after the washing step. While we do not wish to be bound by theory, the freezing and subsequent thawing steps provide a more homogeneous product by removing granulocytes and some monocytes from the cell population. After a washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. Many freezing solutions and parameters are known in the art and useful in this situation, but one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or PBS containing 10% dextran 40 and 5% dextrose, or 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, or 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing media, such as Hespan and PlasmaLyte A, and then freezing the cells at a rate of 1° / min at -80°C and storing them in the gas phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen, can be used.

[0372] In one respect, the cryopreserved cells are thawed and washed as described herein and allowed to rest at room temperature for 1 hour before activation using the method of the present invention.

[0373] Also intended in the context of the present invention is the collection of blood samples or apheresis products from subjects at a time prior to when the enlarged cells described herein are needed. That is, the origin of the cells to be enlarged can be collected at any time as needed, and desired cells, such as T cells, can be isolated and frozen for subsequent use in T cell therapy for various diseases or conditions that would benefit from T cell therapy as described herein. In one aspect, blood samples or apheresis are collected from generally healthy subjects. In another aspect, blood samples or apheresis are collected from generally healthy subjects who are at risk of developing the disease but have not yet developed the disease, and the desired cells are isolated and frozen for subsequent use. In another aspect, T cells can be grown, frozen, and used later. In another aspect, samples are isolated from patients immediately after diagnosis of the specific disease described herein, but before treatment. In addition, cells are isolated from blood samples or apheresis from the subject before treatment with any number of appropriate treatment modalities, including, but not limited to, natalizumab, efalizumab, antivirals, chemotherapeutic agents, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other CAMPATH, anti-CD3 antibodies, cytoxane, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate, steroids, FR901228 and other immunosuppressants such as irradiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or the p70S6 kinase, which is important for growth factor-induced signaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993).In a further respect, cells can be isolated from the patient and frozen for subsequent use (e.g., pre-, concurrently, or post-) in combination with bone marrow or stem cell transplantation, chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), or T-cell depletion therapy using antibodies such as cyclophosphamide, OKT3, or CAMPATH. In one respect, cells can be isolated in advance and frozen for subsequent treatment after B-cell depletion therapy using agents that react with CD20, such as rituximab.

[0374] In a further aspect of the present invention, T cells are obtained directly from a patient after a procedure that leaves functional T cells in the subject. In this regard, it has been observed that the quality of T cells obtained immediately after certain cancer treatments, particularly treatment with drugs that damage the immune system, and during the period when the patient is in the recovery phase from the treatment, is probably optimal or improved in terms of their ability to expand ex vivo. Similarly, after ex vivo manipulation using the method described herein, these cells may be in a favorable state for enhanced engraftment and in vivo proliferation. Therefore, in the context of the present invention, it is intended to collect hematopoietic cells, including T cells, dendritic cells, or other cells of the hematopoietic cell lineage, during this recovery period. Furthermore, in some aspects, mobilization (e.g., mobilization with GM-CSF) and pre-treatment regimens can be used to create conditions favorable for the regrowth, recirculation, regeneration, and / or proliferation of specific cell types in the subject, particularly during a defined post-treatment timeframe. Explanatory cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0375] In one embodiment, NK cells are obtained from a subject. In another embodiment, the NK cells are an NK cell line, for example, the NK-92 cell line (Conkwest).

[0376] Same type CAR In the embodiments described herein, immune effector cells may be allogeneic immune effector cells, such as T cells or NK cells. For example, the cells may be allogeneic T cells, such as allogeneic T cells lacking the expression of functional T cell receptors (TCRs) and / or human leukocyte antigens (HLAs), such as HLA class I and / or HLA class II.

[0377] T cells lacking a functional TCR can be manipulated, for example, to not express any functional TCR on their surface, not express one or more subunits containing a functional TCR, or to produce only a very small amount of functional TCR on their surface. Alternatively, T cells can express a substantially impaired TCR, for example, by expressing one or more mutant or truncated subunits of the TCR. The term “substantially impaired TCR” means that this TCR does not induce a harmful immune response in the host.

[0378] The T cells described herein can be manipulated, for example, to prevent the expression of functional HLA on their surface. For example, the T cells described herein can be manipulated to downregulate cell surface HLA expression, such as HLA class I and / or HLA class II.

[0379] In one embodiment, T cells may lack a functional TCR and functional HLA, such as HLA class I and / or HLA class II.

[0380] Modified T cells lacking functional TCR and / or HLA expression can be obtained by any suitable means, including knockout or knockdown of one or more subunits of the TCR or HLA. For example, T cells may be obtained by knockdown of the TCR and / or HLA using siRNA, shRNA, clustered and regularly arranged short palindromic sequence repeats (CRISPR) transcription activator-like effector nucleases (TALENs), or zinc finger endonucleases (ZFNs).

[0381] In one embodiment, allogeneic cells are cells that do not express or express at low levels the inhibitory molecule, for example, by any of the methods described herein. For example, cells are those that do not express or express at low levels the inhibitory molecule, which can 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 TGFR beta. For example, inhibition of the inhibitory molecule by inhibition at the DNA, RNA, or protein level can optimize the performance of CAR-expressing cells. In some embodiments, for example, inhibitory nucleic acids described herein, such as dsRNA, siRNA, or shRNA, clustered and regularly arranged short palindromic sequence repeats (CRISPR), transcriptional activator-like effector nucleases (TALENs), or zinc finger endonucleases (ZFNs) can be used.

[0382] siRNAs and shRNAs for inhibiting TCR or HLA In one embodiment, TCR expression and / or HLA expression can be inhibited using siRNA or shRNA that targets nucleic acids encoding TCR and / or HLA in T cells.

[0383] siRNA and shRNA expression in T cells can be achieved using any conventional expression system, such as a lentiviral expression system.

[0384] Representative shRNAs that downregulate the expression of TCR components are described, for example, in U.S. Publication No. 2012 / 0321667. Representative siRNAs and shRNAs that downregulate the expression of HLA class I and / or HLA class II genes are described, for example, in U.S. Publication No. 2007 / 0036773.

[0385] CRISPR to inhibit TCR or HLA As used herein, “CRISPR,” “CRISPR for TCR and / or HLA,” or “CRISPR for inhibiting TCR and / or HLA” refers to a system containing a set of clustered, regularly arranged short palindromic sequence repeats or repetitions of such sets. As used herein, “Cas” refers to a CRISPR-related protein. A “CRISPR / Cas” system is a CRISPR and Cas-derived system that can be used to repress or mutate the expression of TCR and / or HLA genes.

[0386] Naturally occurring CRISPR / Cas systems are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaeal genomes. Grissa et al. (2007) BMC Bioinformatics 8:172. This system is a type of prokaryotic immune system that confers resistance to exogenous genetic components such as plasmids and phages, providing a form of adaptive immunity. Barrangou et al. (2007) Science 315:1709-1712; Marragini et al. (2008) Science 322:1843-1845.

[0387] The CRISPR / Cas system has been modified for use in gene editing (silencing, promoting, or modifying specific genes) in eukaryotes such as mice or primates. Wiedenheft et al. (2012) Nature 482:331-8. This is achieved by introducing a plasmid containing a specially designed CRISPR and one or more suitable Cass molecules into eukaryotic cells.

[0388] CRISPR sequences, sometimes called CRISPR loci, contain alternating repeats and spacers. In naturally occurring CRISPRs, spacers typically contain exogenous bacterial sequences such as plasmids or phage sequences, while in TCR and / or HLA CRISPR / Cas systems, spacers originate from TCR or HLA gene sequences.

[0389] RNA from CRISPR loci is constitutively expressed and processed into small RNAs by Cas proteins. These contain spacers flanked by repeat sequences. The RNA then guides other Cas proteins to repress the expression of exogenous genetic components at the RNA or DNA level. Horvath et al. (2010) Science 327:167-170; Makarova et al. (2006) Biology Direct 1:7. The spacers, therefore, act as templates for RNA molecules, similar to siRNA. Pennisi (2013) Science 341:833-836.

[0390] Because these are naturally present in many different types of bacteria, the exact arrangement of CRISPR, as well as the structure, function, and number of Cas genes and their products, vary somewhat from species to species. Haft et al. (2005) PLoS Comput. Biol. 1: e60; Kunin et al. (2007) Genome Biol. 8: R61; Mojica et al. (2005) J. Mol. Evol. 60: 174-182; Bolotin et al. (2005) Microbiol. 151: 2551-2561; Pourcel et al. (2005) Microbiol. 151: 653-663; and Stern et al. (2010) Trends. Genet. 28: 335-340. For example, the Cse (Cas subtype, E. coli) protein (e.g., CasA) forms a functional complex called Cascade, which processes CRISPR RNA transcripts into spacer repeat units that hold the Cascade. Brouns et al. (2008) Science 321:960-964. In other prokaryotes, Cas6 processes CRISPR transcripts. CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 or Cas2. The Cmr (Cas RAMP module) protein in Pyrococcus friosus and other prokaryotes forms a functional complex with small CRISPR RNA, which recognizes and cleaves complementary target RNA. A simpler CRISPR system relies on the protein Cas9, which is a nuclease with two active cleavage sites for each strand of the double helix. The combination of Cas9 and modified CRISPR locus RNA can be used in systems for gene editing. Pennisi (2013) Science 341:833-836.

[0391] The CRISPR / Cas system can therefore be used to edit (add or remove base pairs) or introduce immature termination of TCR and / or HLA genes, thereby reducing TCR and / or HLA expression. The CRISPR / Cas system can also be used, like RNA interference, to block TCR and / or HLA genes in a reversible manner. In mammalian cells, for example, RNA can induce Cas proteins in the TCR and / or HL promoters and sterically block RNA polymerase.

[0392] Using technologies known in this field, such as those described in U.S. Publication No. 20140068797 and Cong (2013) Science 339:819-823, artificial CRISPR / Cas systems that inhibit TCR and / or HLA can be produced. Other artificial CRISPR / Cas systems known in this field that inhibit TCR and / or HLA can also be produced, such as those described in Tsai (2014) Nature Biotechnol., 32:6 569-576, U.S. Patents No. 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359.

[0393] TALEN for inhibiting TCR and / or HLA "TALEN," "TALEN for HLA and / or TCR," or "TALEN for inhibiting HLA and / or TCR" refers to transcription activator-like effector nucleases, which are artificial nucleases that can be used to edit HLA and / or TCR genes.

[0394] TALENs are artificially produced by fusing the TAL effector DNA-binding domain to the DNA-cleaving domain. The transcriptional activator-like effect (TALE) can be manipulated to bind to any desired DNA sequence, including a portion of an HLA or TCR gene. By combining the manipulated TALE with the DNA-cleaving domain, restriction enzymes specific to any desired DNA sequence, including HLA or TCR sequences, can be produced. These can then be introduced into cells, where they can be used for genome editing. Boch (2011) Nature Biotech. 29:135-6; and Boch et al. (2009) Science 326:1509-12; Moscou et al. (2009) Science 326:3501.

[0395] TALE is a protein secreted by Xanthomonas bacteria. Its DNA-binding domain contains a highly conserved sequence of 33-34 amino acids, excluding repeating amino acids at positions 12 and 13. These two positions are highly variable and strongly correlate with the recognition of specific nucleotides. Thus, they can be manipulated to bind to desired DNA sequences.

[0396] To produce TALENs, the TALE protein is fused with nuclease (N), which is either wild-type or a mutant FokI endonuclease. Several mutations in FokI have been made for their use in TALENs, and these improve, for example, cleavage specificity or activity. Cermak et al. (2011) Nucl. Acids Res. 39: e82; Miller et al. (2011) Nature Biotech. 29: 143-8; Hockemeyer et al. (2011) Nature Biotech. 29: 731-734; Wood et al. (2011) Science 333: 307; Doyon et al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotech. 25: 786-793; and Guo et al. (2010) J. Mol. Biol. 200: 96.

[0397] The FokI domain functions as a dimer, requiring two constructs with distinct DNA-binding domains for specific sites within the target genome, each with precise orientation and spacing. Both the number of amino acid residues between the TALEN DNA-binding domain and the FokI cleavage domain, and the number of bases between the two individual TALEN-binding sites, are considered important parameters for achieving high levels of activity. Miller et al. (2011) Nature Biotech. 29:143-8.

[0398] HLA or TCR TALENs can be used intracellularly to produce double-strand breaks (DSBs). If the repair mechanism improperly repairs the breaks by non-homologous end joining, mutations can be introduced at the break site. For example, improper repair may introduce a frameshift mutation. Alternatively, foreign DNA can be introduced into the cell along with the TALEN, and depending on the sequence and chromosomal sequence of the foreign DNA, this process can be used to correct deletions in HLA or TCR genes or to introduce such deletions into wt HLA or TCR genes, thereby reducing HLA or TCR expression.

[0399] Sequence-specific TALENs in HLA or TCR can be constructed using any method known in the art, including a variety of schemes using modular components. Zhang et al. (2011) Nature Biotech. 29:149-53; Geibler et al. (2011) PLoS ONE 6:e19509.

[0400] Zinc finger nucleases for inhibiting HLA and / or TCR "ZFN," or "zinc finger nuclease," or "ZFN for HLA and / or TCR," or "ZFN for inhibiting HLA and / or TCR" is a zinc finger nuclease, which is an artificial nuclease that can be used to edit HLA and / or TCR genes.

[0401] Similar to TALENs, ZFNs contain a FokI nuclease domain (or a derivative thereof) fused to a DNA-binding domain. In the case of ZFNs, the DNA-binding domain contains one or more zinc fingers. Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160.

[0402] Zinc fingers are small protein structural motifs stabilized by one or more zinc ions. Examples of zinc fingers include Cys2His2, which can recognize sequences of approximately 3 bp. By combining a variety of zinc fingers with known specificities, multi-finger polypeptides that recognize sequences of approximately 6 bp, 9 bp, 12 bp, 15 bp, or 18 bp can be produced. A diverse range of selection and modular assembly techniques are available for producing zinc fingers (and combinations thereof) that recognize specific sequences, including phage displays, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells.

[0403] Similar to TALENs, ZFNs must dimerize to cleave DNA. Therefore, a pair of ZFNs is needed to target non-palindromic DNA sites. The two individual ZFNs should bind to the reverse strand of DNA by their nucleases at appropriate intervals. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95:10570-5.

[0404] Similar to TALENs, ZFNs can create double-strand breaks in DNA, which, if improperly repaired, create frameshift mutations that reduce the expression and levels of HLA and / or TCR in cells. ZFNs can also be used in conjunction with homologous recombination to mutate HLA or TCR genes.

[0405] ZFNs specific to the HLA and / or TCR sequences can be constructed using any method known in this art. See, for example, Provasi (2011) Nature Med. 18: 807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al. (2008) Mol. Ther. 16: 1200-7; and Guo et al. (2010) J. Mol. Biol. 400: 96; see U.S. Publication No. 2011 / 0158957; U.S. Publication No. 2012 / 0060230.

[0406] Cell activation and proliferation Cells can generally be activated and proliferated using, for example, the methods described in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Publication No. 20060121005.

[0407] In general, the T cells of the present invention can be proliferated by contacting them with a surface bound to a drug that stimulates CD3 / TCR complex-related signals and ligands, thereby stimulating co-stimulatory molecules on the surface of the T cells. In particular, a T cell population can be stimulated as described herein by contacting it with an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody immobilized on its surface, or by contacting it with a protein kinase C activator (e.g., bryostatin) combined with a calcium ionophore. For co-stimulation of accessory molecules on the surface of T cells, ligands that bind to the accessory molecules are used. For example, a population of T cells is contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. CD4 + T cells or CD8 + Anti-CD3 and anti-CD28 antibodies are used to stimulate the proliferation of either T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), and can be used as is possible by other methods commonly known in this field (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0408] In some respects, primary stimulating and co-stimulating signals for T cells may be provided by different protocols. For example, the agents providing each signal may be in solution or bound to a surface. When bound to a surface, these agents may be bound to the same surface (i.e., “cis” form) or to a different surface (i.e., “trans” form). Alternatively, one agent may be bound to a surface while the other is in solution. In one respect, the agent providing the co-stimulating signal may be bound to the cell surface, while the agent providing the primary activation signal may be in solution or bound to a surface. In another respect, both agents may be in solution. In another respect, the agents may be in a soluble form and thus crosslink to a surface such as a cell expressing an Fc receptor or antibody or other binding agent that will bind to the agent. In this regard, see, for example, U.S. Patent Publications 20040101519 and 20060034810 for artificial antigen-presenting cells (aAPCs) whose use for T cell activation and proliferation in the present invention is considered.

[0409] On one side, two drugs are immobilized on the same bead, i.e., "cis," or on a different bead, i.e., "trans." For example, the drug providing the primary activation signal is an anti-CD3 antibody or its antigen-binding fragment, and the drug providing the co-stimulatory signal is an anti-CD28 antibody or its antigen-binding fragment, and both drugs are co-immobilized on the same bead at the same molecular weight. On one side, CD4 +The invention uses a 1:1 ratio of each antibody bound to beads for T cell proliferation. In one aspect of the invention, the ratio of anti-CD3:CD28 antibodies bound to beads is used so that an increase in T cell proliferation is observed compared to the proliferation observed with a 1:1 ratio. In one specific aspect, an increase of approximately 1 to 3 times is observed compared to the proliferation observed with a 1:1 ratio. In one aspect, the ratio of CD3:CD28 antibodies bound to beads is in the range of 100:1 to 1:100 and all integer values ​​in between. In one aspect of the invention, more anti-CD28 antibodies are bound to the particles than anti-CD3 antibodies, i.e., the CD3:CD28 ratio is less than 1. In one aspect of the invention, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to beads is greater than 2:1. In one specific aspect, antibodies with a 1:100 CD3:CD28 ratio are used bound to beads. In one aspect, antibodies with a 1:75 CD3:CD28 ratio are used bound to beads. Further, use an antibody with a 1:50 CD3:CD28 ratio bound to the beads. On one side, use an antibody with a 1:30 CD3:CD28 ratio bound to the beads. On one preferred side, use an antibody with a 1:10 CD3:CD28 ratio bound to the beads. On one side, use an antibody with a 1:3 CD3:CD28 ratio bound to the beads. On further sides, use an antibody with a 3:1 CD3:CD28 ratio bound to the beads.

[0410] T cells or other target cells can be stimulated using particle-to-cell ratios of 1:500 to 500:1 and any integer values ​​in between. As those skilled in the art will readily recognize, the particle-to-cell ratio may depend on the particle size relative to the target cells. For example, small beads can bind only a few cells, while large beads can bind many. In one respect, the particle-to-cell ratio is in the range of 1:100 to 100:1 and any integer values ​​in between, and in another respect, the ratio includes 1:9 to 9:1, and any integer values ​​in between can also be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 bound particles to T cells that result in T cell stimulation can vary as described above, however, some preferred values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with one preferred ratio being at least 1:1 particles to T cells. In one aspect, a particle-to-cell ratio of 1:1 or less is used. In one specific aspect, a preferred particle:cell ratio is 1:5. Further, the particle-to-cell ratio can vary from day to day of stimulation. For example, in one scenario, the particle-to-cell ratio is 1:1 to 10:1 on day one, and further particles are added to the cells daily or every other day until the final ratio reaches 1:1 to 1:10 (based on the number of cells on the day of addition). In one specific scenario, the particle-to-cell ratio is 1:1 on day one of stimulation and adjusted to 1:5 on days three and four of stimulation. In one scenario, particles are added daily or every other day, from 1:1 on day one of stimulation to a final ratio of 1:5 on days three and four of stimulation. In one scenario, the particle-to-cell ratio is 2:1 on day one of stimulation and 1:10 on days three and four of stimulation. In one scenario, particles are added daily or every other day, from 1:1 on day one to a final ratio of 1:10 on days three and four of stimulation. Those skilled in the art will recognize that a variety of other ratios may be suitable for use in the present invention. In particular, the ratio depends on the particle size as well as the size and type of cells. On one hand, the most typical ratios to use are around 1:1, 2:1, and 3:1 for day one.

[0411] In a further aspect of the present invention, cells such as T cells are combined with drug-coated beads, the beads and cells are subsequently separated, and then the cells are cultured. In another aspect, the drug-coated beads and cells are cultured together without being separated before culturing. In yet another aspect, the beads and cells are first enriched by the application of a force such as magnetism to increase the ligation of cell surface markers, thereby inducing cell stimulation.

[0412] For example, cell surface proteins can be ligated by bringing paramagnetic beads (3 x 28 beads) bound to anti-CD3 and anti-CD28 into contact with T cells. On one surface, the cells (e.g., 10 4 ~10 9Combine the T cells and beads (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads, 1:1 ratio) in a buffer, e.g., PBS (which does not contain divalent cations such as calcium and magnesium). Again, those skilled in the art will recognize that any cell concentration may be used. For example, the target cells may be very rare in the sample, making up only 0.01% of the sample, or the entire sample (i.e., 100%) may consist of the desired target cells. Thus, any cell number is included in the context of the present invention. On one hand, it may be desirable to significantly reduce the volume in which the particles and cells are mixed (i.e., increase the cell concentration) to ensure maximum contact between cells and particles. For example, on one hand, use a concentration of about 2 billion cells / ml. On the other hand, use more than 100 million cells / ml. Further, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml are used. Further, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml are used. Further, concentrations of 125 million or 150 million cells / ml can be used. The use of high concentrations can result in increased cell yield, cell activation, and cell proliferation. In addition, the use of high cell concentrations allows for more efficient capture of cells that may weakly express the desired target antigen, such as CD28-negative T cells. Populations of such cells may have therapeutic value and are desirable in some respects. For example, high cell concentrations can be used to capture CD8 cells that normally have weak CD28 expression. + This enables more efficient selection of T cells.

[0413] In one aspect of the present invention, the mixture can be cultured for several hours (about 3 hours) to about 14 days or any integer time unit in between. In one aspect, the mixture can be cultured for 21 days. In one aspect of the present invention, the beads and T cells are co-cultured for about 8 days. In one aspect, the beads and T cells are cultured together for 2 to 3 days. Several cycles of stimulation may also be desirable to allow the T cell culture time to 60 days or more. Suitable conditions for T cell culture include a suitable medium (e.g., minimal essential medium or RPMI medium 1640 or X-vivo 15 (Lonza)), which may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-β and TNF-α or any other additives for cell proliferation known to those skilled in the art. Other additives for cell proliferation include, but are not limited to, surfactants, plasmamenates, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. The culture medium may contain RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-vivo 15 and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and may be serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines sufficient for T cell proliferation and expansion. Antibiotics, such as penicillin and streptomycin, are only added to experimental cultures and not to the culture of cells to be injected into the target. Target cells...

Claims

1. an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising i) an antibody or antibody fragment containing a human anti-mesothelin binding domain, ii) a transmembrane domain, and iii) an intracellular signal transduction domain containing a stimulating domain, wherein the anti-mesothelin binding domain comprises one or more light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) containing the amino acid sequences described in Table 5, and one or more heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) containing the amino acid sequences described in Table 4.

2. The isolated nucleic acid molecule according to claim 1, comprising LC CDR1, LC CDR2, and LC CDR3 having the amino acid sequences shown in Table 5.

3. The isolated nucleic acid molecule according to claim 1, comprising HC CDR1, HC CDR2, and HC CDR3 having the amino acid sequences shown in Table 4.

4. The isolated nucleic acid molecule according to claim 1, comprising LC CDR1, LC CDR2, and LC CDR3 having the amino acid sequences described in Table 5, and HC CDR1, HC CDR2, and HC CDR3 having the amino acid sequences described in Table 4.

5. The isolated nucleic acid molecule according to claim 1, wherein the anti-mesothelin binding domain contains one of the amino acid sequences of the light chain variable region listed in Table 2.

6. The isolated nucleic acid molecule according to claim 1, wherein the anti-mesothelin binding domain contains one of the amino acid sequences of the heavy chain variable region listed in Table 2.

7. The isolated nucleic acid molecule according to claim 1, wherein the anti-mesothelin binding domain comprises an amino acid sequence of any of the light chain variable regions listed in Table 2 and any of the heavy chain variable regions listed in Table 2.

8. An isolated nucleic acid molecule according to any one of claims 1 to 7, wherein the anti-mesothelin binding domain is scFv.

9. An isolated nucleic acid molecule according to any one of claims 1 to 8, wherein the anti-mesothelin binding domain includes an amino acid sequence having at least one, two, or three modifications to the amino acid sequence of the light chain variable region shown in Table 2, but the number of modifications does not exceed 30, 20, or 10, or a light chain variable region having 95-99% identity with the amino acid sequence shown in Table 2.

10. An isolated nucleic acid molecule according to any one of claims 1 to 9, wherein the anti-mesothelin binding domain includes an amino acid sequence having at least one, two, or three modifications to the amino acid sequence of the heavy chain variable region shown in Table 2, but the number of modifications does not exceed 30, 20, or 10, or a heavy chain variable region having 95-99% identity with the amino acid sequence shown in Table 2.

11. An isolated nucleic acid molecule according to any one of claims 1 to 10, wherein the encoded anti-mesothelin binding domain includes a sequence selected from the group consisting of SEQ ID NOs. 39, SEQ ID NOs. 40, SEQ ID NOs. 41, SEQ ID NOs. 42, SEQ ID NOs. 43, SEQ ID NOs. 44, SEQ ID NOs. 45, SEQ ID NOs. 46, SEQ ID NOs. 47, SEQ ID NOs. 48, SEQ ID NOs. 49, SEQ ID NOs. 50, SEQ ID NOs. 51, SEQ ID NOs. 52, or sequences having 95-99% identity with these.

12. An isolated nucleic acid molecule according to any one of claims 1 to 11, wherein the nucleic acid sequence encoding the anti-mesothelin binding domain includes a sequence selected from the group consisting of SEQ ID NO: 87; SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110 or sequences having 95-99% identity with these.

13. An isolated nucleic acid molecule according to any one of claims 1 to 12, wherein the encoded CAR comprises a transmembrane domain containing a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

14. An isolated nucleic acid molecule according to any one of claims 1 to 13, wherein the encoded transmembrane domain comprises the sequence of SEQ ID NO:

6.

15. An isolated nucleic acid molecule according to any one of claims 1 to 14, wherein the encoded transmembrane domain comprises an amino acid sequence having at least one, two, or three modifications to the amino acid sequence of SEQ ID NO: 6, but the number of modifications does not exceed 20, 10, or 5, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO:

6.

16. An isolated nucleic acid molecule according to any one of claims 1 to 15, wherein the nucleic acid sequence encoding the transmembrane domain includes the sequence of SEQ ID NO: 17 or a sequence having 95-99% identity therewith.

17. An isolated nucleic acid molecule according to any one of claims 1 to 16, wherein the encoded anti-mesothelin-binding domain is connected to a transmembrane domain by a hinge region.

18. The isolated nucleic acid molecule according to claim 17, wherein the encoded hinge region includes sequence number 2 or a sequence having 95-99% identity therewith.

19. The isolated nucleic acid molecule according to claim 17, wherein the nucleic acid sequence encoding the hinge region includes the sequence of SEQ ID NO: 13 or a sequence having 95-99% identity with it.

20. Furthermore, an isolated nucleic acid molecule according to any one of claims 1 to 19, comprising a sequence encoding a co-stimulatory domain.

21. The isolated nucleic acid molecule according to claim 20, wherein the co-stimulatory domain is a functional signal transduction domain obtained from a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

22. The isolated nucleic acid molecule according to claim 20 or 21, wherein the encoded co-stimulatory domain includes the sequence of SEQ ID NO:

7.

23. The isolated nucleic acid molecule according to claim 20 or 21, wherein the encoded co-stimulatory domain comprises an amino acid sequence having at least one, two, or three modifications to the amino acid sequence of SEQ ID NO: 7, but the number of modifications does not exceed 20, 10, or 5, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO:

7.

24. The isolated nucleic acid molecule according to claim 20 or 21, wherein the nucleic acid sequence encoding the co-stimulatory domain includes the sequence of SEQ ID NO: 18 or a sequence having 95-99% identity therewith.

25. An isolated nucleic acid molecule according to any one of claims 1 to 24, wherein the encoded intracellular signaling domain comprises a functional signaling domain of 4-1BB and / or a functional signaling domain of CD3 zeta.

26. An isolated nucleic acid molecule according to any one of claims 1 to 25, wherein the encoded intracellular signaling domain comprises the sequence of SEQ ID NO: 7 and / or the sequence of SEQ ID NO: 9 or SEQ ID NO:

10.

27. An isolated nucleic acid molecule according to any one of claims 1 to 26, wherein the intracellular signaling domain comprises an amino acid sequence having at least one, two, or three modifications to the amino acid sequence of SEQ ID NO: 7 and / or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, but the number of modifications does not exceed 20, 10, or 5, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 7 and / or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO:

10.

28. The isolated nucleic acid molecule according to any one of claims 1 to 27, wherein the encoded intracellular signaling domain comprises the sequence of SEQ ID NO: 7 and the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, wherein the sequence containing the intracellular signaling domain is expressed in the same frame and as a single polypeptide chain.

29. An isolated nucleic acid molecule according to any one of claims 1 to 28, wherein the nucleic acid sequence encoding an intracellular signaling domain includes the sequence of SEQ ID NO: 18 or a sequence having 95-99% identity therewith and / or the sequence of SEQ ID NO: 20 or SEQ ID NO: 21 or a sequence having 95-99% identity therewith.

30. Furthermore, an isolated nucleic acid molecule according to any one of claims 1 to 29, comprising a leader sequence.

31. The isolated nucleic acid molecule according to claim 32, wherein the leader sequence includes sequence number 1.

32. An isolated polypeptide molecule encoded by a nucleic acid molecule according to any one of claims 1 to 32.

33. The isolated polypeptide according to claim 32, comprising an array selected from the group consisting of SEQ ID NOs: 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, and 86.

34. An isolated chimeric antigen receptor (CAR) molecule containing a human anti-mesothelin-binding domain, a transmembrane domain, and an intracellular signaling domain.

35. The isolated CAR molecule according to claim 34, comprising i) an antibody or antibody fragment containing a human anti-mesothelin binding domain, ii) a transmembrane domain, and iii) an intracellular signaling domain.

36. The isolated CAR molecule according to claim 34 or 35, wherein the anti-mesothelin binding domain does not compete for binding to human mesothelin with the antigen-binding domain containing the sequence of sequence number 279.

37. The anti-mesothelin binding domain a) One or more light chain complementarity determination regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of an anti-mesothelin binding domain sequence selected from SEQ ID NO: 43 and SEQ ID NO: 49, and one or more heavy chain complementarity determination regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of an anti-mesothelin binding domain sequence selected from SEQ ID NO: 43 and SEQ ID NO: 49; b) LC CDR1, LC CDR2, and LC CDR3 of anti-mesothelin light chain amino acid sequences selected from SEQ ID NO: 43 and SEQ ID NO: 49, and HC CDR1, HC CDR2, and HC CDR3 of anti-mesothelin heavy chain amino acid sequences selected from SEQ ID NO: 43 and SEQ ID NO: 49; or c) Sequence selected from sequence numbers 43 and 49 An isolated CAR molecule according to any one of claims 34 to 36, which competes with an antigen-binding domain containing [a specific component].

38. The isolated CAR molecule according to any one of claims 34 to 37, wherein the anti-mesothelin binding domain binds to a human mesothelin epitope different from the human mesothelin epitope targeted by the antigen-binding domain containing the sequence containing SEQ ID NO:

279.

39. An isolated CAR molecule according to any one of claims 34 to 38, wherein the anti-mesothelin binding domain binds to the C-terminus of human mesothelin.

40. The isolated CAR molecule according to claim 39, wherein the anti-mesothelin binding domain binds to an epitope in amino acids 450-588 of SEQ ID NO:

278.

41. The isolated CAR molecule according to claim 40, wherein the epitope comprises amino acids 485-490, 498-507, 532-537 or 545-572 of SEQ ID NO: 278 or any subset thereof or combination thereof.

42. An isolated CAR molecule according to any one of claims 34 to 41, wherein the human anti-mesothelin binding domain comprises one or more light chain complementarity-determining regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) containing the amino acid sequences listed in Table 5, and one or more heavy chain complementarity-determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) containing the amino acid sequences listed in Table 4.

43. An isolated CAR molecule according to any one of claims 34 to 42, comprising LC CDR1, LC CDR2, and LC CDR3 having the amino acid sequences listed in Table 5.

44. An isolated CAR molecule according to any one of claims 34 to 43, comprising HC CDR1, HC CDR2, and HC CDR3 having the amino acid sequences listed in Table 4.

45. An isolated CAR molecule according to any one of claims 34 to 44, comprising LC CDR1, LC CDR2, and LC CDR3 having the amino acid sequences described in Table 5, and HC CDR1, HC CDR2, and HC CDR3 having the amino acid sequences described in Table 4.

46. An isolated CAR molecule according to any one of claims 59 to 45, wherein the anti-mesothelin binding domain is scFv.

47. An isolated CAR molecule according to any one of claims 34 to 46, wherein the anti-mesothelin binding domain comprises a light chain and a heavy chain of the amino acid sequences listed in Table 2.

48. The isolated CAR molecule according to any one of claims 34 to 47, wherein the anti-mesothelin binding domain comprises an amino acid sequence having at least one, two, or three modifications to the amino acid sequence of the light chain variable region shown in Table 2, but the number of modifications does not exceed 30, 20, or 10, or a sequence having 95-99% identity with the amino acid sequence shown in Table 2.

49. An isolated CAR molecule according to any one of claims 34 to 48, wherein the anti-mesothelin binding domain has at least one, two, or three modifications to the amino acid sequence of the heavy chain variable region shown in Table 2, but the number of modifications does not exceed 30, 20, or 10, and the heavy chain variable region includes an amino acid sequence that is 95-99% identical to the amino acid sequence shown in Table 2.

50. An isolated CAR molecule according to any one of claims 34 to 49, wherein the anti-mesothelin binding domain comprises a sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, or sequences having 95 to 99% identity with these.

51. An isolated CAR molecule according to any one of claims 34 to 50, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

52. The isolated CAR molecule according to claim 51, wherein the transmembrane domain contains the sequence of Sequence ID No.

6.

53. The isolated CAR molecule according to claim 51, wherein the transmembrane domain comprises an amino acid sequence having at least one, two, or three modifications to the amino acid sequence of SEQ ID NO: 6, but the number of modifications does not exceed 20, 10, or 5, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO:

6.

54. An isolated CAR molecule according to any one of claims 34 to 53, wherein a human anti-mesothelin binding domain is connected to a transmembrane domain by a hinge region.

55. The isolated CAR molecule according to claim 54, wherein the hinge region includes sequence number 2 or sequence number 36 or a sequence having 95-99% identity therewith.

56. An isolated CAR molecule according to any one of claims 34 to 55, wherein the intracellular signaling domain includes a sequence encoding a co-stimulatory domain.

57. The isolated CAR molecule according to claim 56, wherein the co-stimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), CD278 (also known as "ICOS"), and 4-1BB (CD137).

58. The isolated CAR molecule according to claim 56 or 57, wherein the co-stimulatory domain contains the sequence of Sequence ID No.

7.

59. The isolated CAR molecule according to claim 56 or 57, wherein the co-stimulatory domain comprises an amino acid sequence having at least one, two, or three modifications to the amino acid sequence of SEQ ID NO: 7, but the number of modifications does not exceed 20, 10, or 5, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO:

7.

60. An isolated CAR molecule according to any one of claims 34 to 59, wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and / or a functional signaling domain of CD3 zeta.

61. The isolated CAR molecule according to claim 60, wherein the intracellular signaling domain comprises the sequence of SEQ ID NO: 7 and / or the sequence of SEQ ID NO:

9.

62. The isolated CAR molecule according to claim 60, wherein the intracellular signaling domain comprises the sequence of SEQ ID NO: 7 and / or the sequence of SEQ ID NO:

10.

63. An isolated CAR molecule according to any one of claims 34 to 55, wherein the intracellular signaling domain comprises an amino acid sequence having at least one, two, or three modifications to the amino acid sequence of SEQ ID NO: 7 and / or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, but the number of modifications does not exceed 20, 10, or 5, or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 7 and / or the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO:

10.

64. The isolated CAR molecule according to any one of claims 34 to 55, wherein the intracellular signaling domain comprises the sequence of SEQ ID NO: 7 and the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, wherein the sequences containing the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

65. Furthermore, an isolated CAR molecule according to any one of claims 59 to 64, comprising a leader sequence.

66. The isolated CAR molecule according to claim 65, wherein the leader sequence includes the amino acid sequence of SEQ ID NO: 1 or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO:

1.

67. Light chain complementarity determination region 1 (LC CDR1), light chain complementarity determination region 2 (LC CDR2), and light chain complementarity determination region 3 (LC CDR2) of any of the anti-mesothelin binding domains in SEQ ID NOs. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, and 62. A human anti-mesothelin binding domain comprising one or more CDR3) and any of the human anti-mesothelin binding domains in SEQ ID NOs. 39, SEQ ID NOs. 40, SEQ ID NOs. 41, SEQ ID NOs. 42, SEQ ID NOs. 43, SEQ ID NOs. 44, SEQ ID NOs. 45, SEQ ID NOs. 46, SEQ ID NOs. 47, SEQ ID NOs. 48, SEQ ID NOs. 49, SEQ ID NOs. 50, SEQ ID NOs. 51, SEQ ID NOs. 52, and one or more heavy chain complementarity determining regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3).

68. The human anti-mesothelin binding domain according to claim 67, which is an scFv comprising the light and heavy chains of the amino acid sequences of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO:

62.

69. The amino acid sequence of the light chain variable region shown in SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62 has at least one, two, or three modifications, but the number of modifications does not exceed 30, 20, or 10. Light chain variable regions containing an amino acid sequence or a sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 or SEQ ID NO: 62; and / or SEQ ID NO: 39, SEQ ID NO: 40 , an amino acid sequence or sequence having at least one, two, or three modifications in the heavy chain variable region amino acid sequence provided in SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 or SEQ ID NO: 62, but the number of modifications does not exceed 30, 20 or 10. The human anti-mesothelin binding domain according to claim 67 or 68, comprising a heavy chain variable region containing a sequence having 95-99% identity with the amino acid sequence of number 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, or SEQ ID NO:

62.

70. A human anti-mesothelin binding domain that does not compete for binding to human mesothelin with an antigen-binding domain containing the sequence of sequence number 279.

71. a) One or more light chain complementarity determination regions 1 (LC CDR1), 2 (LC CDR2), and 3 (LC CDR3) of an anti-mesothelin binding domain sequence selected from SEQ ID NOs. 43 and SEQ ID NOs. 49, and one or more heavy chain complementarity determination regions 1 (HC CDR1), 2 (HC CDR2), and 3 (HC CDR3) of an anti-mesothelin binding domain sequence selected from SEQ ID NOs. 43 and SEQ ID NOs. 49 b) LC CDR1, LC CDR2, and LC CDR3 of anti-mesothelin light chain amino acid sequences selected from SEQ ID NO: 43 and SEQ ID NO: 49, and HC CDR1, HC CDR2, and HC CDR3 of anti-mesothelin heavy chain amino acid sequences selected from SEQ ID NO: 43 and SEQ ID NO: 49; or c) Sequence selected from sequence numbers 43 and 49 A human anti-mesothelin binding domain according to claim 70, which competes with an antigen-binding domain containing an antigen-binding domain.

72. A human anti-mesothelin binding domain according to claim 70 or 71, wherein the antigen-binding domain containing the sequence containing sequence number 279 binds to a human mesothelin epitope different from the human mesothelin epitope targeted.

73. A human anti-mesothelin binding domain according to any one of claims 70 to 72, which binds to the C-terminus of human mesothelin.

74. The human anti-mesothelin binding domain according to claim 73, which binds to an epitope in amino acids 450-588 of sequence number 278.

75. The human anti-mesothelin-binding domain according to claim 74, wherein the epitope comprises amino acids 485-490, 498-507, 532-537 or 545-572 of Sequence ID No. 278 or any subset thereof or combination thereof.

76. A vector comprising a nucleic acid molecule encoding a CAR according to any one of claims 1 to 75.

77. The vector according to claim 76, selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenovirus vector, or retroviral vector.

78. The vector according to claim 76 or 77, further comprising a promoter.

79. The vector according to claim 78, wherein the promoter is the EF-1 promoter.

80. The vector according to claim 79, wherein the EF-1 promoter includes the sequence of sequence number 11.

81. The vector according to any one of claims 76 to 78, which is an in vitro transcription vector.

82. The vector according to claim 81, wherein the nucleic acid sequence within the vector further comprises a poly(A) tail.

83. The vector according to claim 81, wherein the nucleic acid sequence in the vector further comprises a 3' UTR.

84. A cell comprising the vector according to any one of claims 76 to 83.

85. The cell according to claim 84, which is a human T cell.

86. The cell according to claim 85, wherein the T cell is a CD8+ T cell.

87. A method for producing cells, comprising transducing T cells with a vector according to any one of claims 76 to 83.

88. A method for producing a population of RNA-modified cells, comprising introducing in vitro transcribed RNA or synthetic RNA into cells, wherein the RNA comprises a nucleic acid molecule encoding a CAR as described in any one of claims 1 to 87.

89. A method for providing anti-cancer immunity in a mammal, comprising administering an effective amount of cells expressing the CAR molecule described in any one of claims 1 to 88 to a mammal.

90. The method according to claim 89, wherein the cells are autologous T cells.

91. The method according to claim 89 or 90, wherein the cells are allogeneic T cells.

92. The method according to any one of claims 89 to 91, wherein the mammal is a human.

93. A method for treating a mammal having a disease related to mesothelin expression, comprising administering to the mammal an effective amount of cells containing the CAR molecule described in any one of claims 1 to 92.

94. The method according to claim 93, wherein the disease associated with mesothelin expression is selected from proliferative disorders or precancerous conditions such as cancer or malignant tumors, or is a non-cancer-related indication associated with mesothelin expression.

95. The method according to any one of claims 93 to 94, wherein the disease is a mesothelial-related cancer selected from the group consisting of mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer or large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, metastatic pancreatic cancer, ovarian cancer, colorectal cancer and bladder cancer or any combination thereof.

96. The method according to any one of claims 93 to 95, wherein cells expressing the CAR molecule are administered in combination with a drug that enhances the efficacy of cells expressing the CAR molecule.

97. The method according to any one of claims 93 to 96, wherein cells expressing the CAR molecule are administered in combination with a drug for reducing one or more side effects associated with the administration of cells expressing the CAR molecule.

98. The method according to any one of claims 93 to 97, wherein cells expressing the CAR molecule are administered in combination with a drug that treats a disease related to mesothelin.

99. An isolated nucleic acid molecule according to any one of claims 1 to 31, an isolated polypeptide molecule according to claim 32 or 33, an isolated CAR according to any one of claims 34 to 66, an anti-mesothelin-binding domain according to any one of claims 67 to 75, a vector according to any one of claims 76 to 83, or a cell according to any one of claims 84 to 86, for use as a pharmaceutical.

100. An isolated nucleic acid molecule according to any one of claims 1 to 31, an isolated polypeptide molecule according to claim 32 or 33, an isolated CAR according to any one of claims 34 to 66, an anti-mesothelin binding domain according to any one of claims 67 to 75, a vector according to any one of claims 76 to 83, or a cell according to any one of claims 84 to 86, for use in the treatment of a disease expressing mesothelin.

101. Furthermore, the cell according to any one of claims 84 to 86 expresses an inhibitory molecule comprising a first polypeptide which further includes at least a portion of the inhibitory molecule bound to a second polypeptide containing a positive signal from an intracellular signaling domain.

102. The cell according to claim 101, wherein the first polypeptide comprises at least a portion of PD1, and the second polypeptide comprises a co-stimulatory domain and an intracellular signaling domain.

103. The method according to claim 96, wherein the drug is an mTOR inhibitor, and the subject is administered a low, immunoenhancing dose of an mTOR inhibitor, such as RAD001 or rapamycin.

104. The method according to claim 103, wherein the mTOR inhibitor is RAD001.

105. The method according to claim 103, wherein the dose comprises an allosteric and catalytic mTOR inhibitor.

106. The method according to claim 103, wherein an mTOR inhibitor is administered for a sufficient time to decrease the proportion of PD-1 positive T cells, increase the proportion of PD-1 negative T cells, or increase the PD-1 negative T cell / PD-1 positive T cell ratio in peripheral blood of a subject or a preparation of T cells isolated from a subject.

107. The method according to claim 103, wherein immune effector cells, e.g., T cells, which are manipulated to express CAR, are collected after a sufficient time or sufficient dose of a low, immunoenhancing dose of an mTOR inhibitor such that the levels of PD1-negative immune effector cells in or collected from a subject, e.g., T cells or PD1-negative immune effector cells, e.g., the T cell / PD1-positive immune effector cell, e.g., T cell ratio are increased at least transiently.

108. The method according to claim 103, wherein the dose of the mTOR inhibitor relates to mTOR inhibition of at least 5% but not exceeding 90%, as measured, for example, by P70 S6 kinase inhibition.

109. The method according to claim 103, wherein the dose of the mTOR inhibitor relates to mTOR inhibition of at least 10% but not exceeding 40%, as measured, for example, by P70 S6 kinase inhibition.

110. A method for treating a subject having a disease related to mesothelin expression, comprising administering to a subject an effective amount of cells containing nucleic acids as described in any of claims 1 to 31, wherein the nucleic acids are introduced into T cells or NK cells using in vitro transcription, and the subject receives an initial dose of cells containing nucleic acids and one or more subsequent doses of cells containing nucleic acids, wherein the one or more subsequent doses are administered within 15 days after the previous dose, for example, on 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days.

Images