Human mesothelin chimeric antigen receptor and uses thereof

Abstract

To provide an isolated nucleic acid molecule encoding a chimeric antigen receptor specific to mesothelin.SOLUTION: The present invention provides an isolated nucleic acid molecule encoding a chimeric antigen receptor. The chimeric antigen receptor comprises (i) a human anti-mesothelin binding domain comprising light chain complementary determining regions 1, 2, 3, comprising specific amino acid sequences, and heavy chain complementary determining regions 1, 2, 3, comprising specific amino acid sequences; (ii) a transmembrane domain; and (iii) an intracellular signaling domain comprising a stimulatory domain.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to International Application No. PCT / CN2013 / 089979, filed December 19, 2013, International Application No. PCT / CN2014 / 082610, filed July 21, 2014, and International Application No. PCT / CN2014 / 090509, filed November 6, 2014, the entire contents of each of which are incorporated herein by reference.

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

[0003] Background of the Invention Mesothelin was originally identified by Pastan et al. as a tumor-associated antigen due to its limited expression in normal tissues and overexpression 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 71 kDa protein, which is processed to produce mesothelin, a 40 kDa protein anchored to the cell membrane by glycosylphosphatidylinositol (GPI) linkages, and an amino-terminal 31 kDa truncated fragment termed megakaryocyte potentiating factor (MPF). Both fragments contain N-glycosylation sites. A soluble splice variant of the 40 kDa carboxyl-terminal fragment, termed "soluble mesothelin / MPF-related," has been found in the serum of patients with pancreatic ductal adenocarcinoma (PDA). Johnston, F, et al. Clinical Cancer Research. 2009;15(21):6511. Mesothelin is currently being investigated as both a therapeutic target and a biomarker of 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 on normal tissues. Using the mouse anti-human mesothelin antibody K1, developed by Pastan's group, strong K1 reactivity, although at lower levels than typically seen in malignant tissue, has been detected in mesothelial cells lining the peritoneal, pleural, and pericardial cavities. Chang K, et al., Cancer Res. 1992;52(1):181-186. Weak K1 reactivity has also been detected in the epithelium of the fallopian tubes, the basal epithelium of the trachea, and the epithelium of the tonsils. 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 reactivity has not been detected in the majority of normal tissues, including the liver, kidney, spleen, bone marrow, lymph nodes, thymus, cardiac muscle, 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, epididymis, cervical epithelium, lung parenchyma, esophagus, small intestinal epithelium, colonic epithelium, urinary bladder epithelium, and gallbladder epithelium. Chang K, et al., Cancer Res. 1992;52(1):181-186.

[0005] Mesothelin is overexpressed in the majority of primary pancreatic adenocarcinomas, with rare and weak expression in benign pancreatic tissue. Argani P, et al. Clin Cancer Res. 2001;7(12):3862-3868. Epithelial malignant pleural mesothelioma (MPM) universally expresses mesothelin, whereas sarcomatoid MPM does not. Most serous epithelial ovarian carcinomas and related primary peritoneal carcinomas 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 colleagues used soluble antibody fragments of anti-mesothelin antibodies conjugated to immunotoxins to treat cancer patients with mesothelin-positive tumors. This trial demonstrated adequate 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 according to RECIST criteria in one patient and stable disease in a second patient, with 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 Summary of the Invention

[0008] Summary of the Invention The present invention relates to methods of providing an immune response in a patient, for example, by administering immune effector cells engineered to express a chimeric antigen receptor (CAR) comprising 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 a CAR comprising an antibody, e.g., an antigen-binding fragment thereof, to treat cancers associated with mesothelin (or MSLN) expression. In particular, the present invention relates to adoptive cell transfer, which may be particularly suitable for patients with mesothelin-expressing cancers, such as 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., pancreatic ductal adenocarcinoma, metastatic pancreatic cancer), ovarian cancer, colorectal cancer, and bladder cancer, or any combination thereof.

[0009] Thus, in one aspect, the invention features 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 stimulatory domain. In one embodiment, the encoded anti-mesothelin binding domain comprises one or more (e.g., all three) of light chain complementarity-determining region 1 (LC CDR1), light chain complementarity-determining region 2 (LC CDR2), and light chain complementarity-determining region 3 (LC CDR3) of a human anti-mesothelin binding domain described herein, and one or more (e.g., all three) of heavy chain complementarity-determining region 1 (HC CDR1), heavy chain complementarity-determining region 2 (HC CDR2), and heavy chain complementarity-determining region 3 (HC CDR3) of a human anti-mesothelin binding domain described herein. In one embodiment, the encoded human anti-mesothelin binding domain comprises or consists of a light chain variable region described herein (e.g., in Table 2, 4, or 5) and / or a heavy chain variable region described herein (e.g., in Table 2, 4, or 5). In one embodiment, the encoded anti-mesothelin binding domain is an scFv comprising or consisting of the light and heavy chain amino acid sequences of Table 2. In certain embodiments, the anti-mesothelin binding domain (e.g., scFV) comprises or consists of a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) in the amino acid sequence of a light chain variable region shown in Table 2, or a sequence having 95-99% identity to an amino acid sequence of Table 2; and / or a heavy chain variable region comprising or consisting of an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) in the amino acid sequence of a heavy chain variable region shown in Table 2, but not more than 30, 20, or 10 modifications (e.g., substitutions) in the amino acid sequence of a heavy chain variable region shown in Table 2, or a sequence having 95-99% identity to an amino acid sequence of Table 2.In one embodiment, the human anti-mesothelin binding domain comprises or consists of 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, 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 a sequence having 95-99% identity thereto. In one embodiment, the nucleic acid sequence encoding the human anti-mesothelin binding domain comprises or consists of 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 a sequence having 95-99% identity thereto.

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

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

[0012] In one embodiment, the isolated nucleic acid molecule further comprises a sequence encoding a costimulatory domain, e.g., a costimulatory domain described herein. In one embodiment, the costimulatory 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 costimulatory domain comprises or consists of the sequence of SEQ ID NO: 14. In one embodiment, the costimulatory domain comprises or consists of an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 20, 10, or 5 modifications (e.g., substitutions) to the amino acid sequence of SEQ ID NO: 14, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 14.

[0013] In one embodiment, the isolated nucleic acid comprises a sequence encoding an intracellular signaling domain, e.g., an intracellular signaling domain described herein. In one embodiment, the isolated nucleic acid encodes a functional signaling domain of 4-1BB and / or a functional signaling domain of CD3 zeta. 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) but not more than 20, 10, or 5 modifications (e.g., substitutions) of 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, or a sequence having 95-99% identity 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. 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 sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

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

[0015] In another aspect, the invention pertains to isolated polypeptide molecules encoded by nucleic acid sequences, eg, the nucleic acids described herein.

[0016] In another aspect, the invention pertains to an isolated polypeptide molecule comprising or consisting of 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) but not more than 30, 20, or 10 modifications (e.g., substitutions) in an amino acid sequence of a heavy chain variable region set forth in Table 2, or a sequence having 95-99% identity to an amino acid sequence in Table 2. In one embodiment, the isolated polypeptide comprises one or more (e.g., all three) of light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of a human anti-mesothelin binding domain described herein, and one or more (e.g., all three) of heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of a human anti-mesothelin binding domain described herein.

[0017] In another aspect, the invention pertains to an isolated chimeric antigen receptor (CAR) molecule comprising an anti-mesothelin binding domain described herein, e.g., a human anti-mesothelin binding domain described herein, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain.

[0018] In one embodiment, the anti-mesothelin binding domain does not compete for binding to human mesothelin with an antigen-binding domain comprising a sequence comprising 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 an anti-mesothelin light chain amino acid sequence selected from SEQ ID NO: 43 or SEQ ID NO: 49 and HC CDR1, HC CDR2, and HC CDR3 of an anti-mesothelin heavy chain amino acid sequence selected from SEQ ID NO: 43 or SEQ ID NO: 49. In one embodiment, the anti-mesothelin binding domain competes for binding to human mesothelin with an antigen-binding domain comprising SEQ ID NO: 43 or SEQ ID NO: 49.

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

[0021] In some embodiments, the anti-mesothelin binding domain described herein does not bind to the N-terminus of mesothelin set forth in SEQ ID NO: 278. In one embodiment, the anti-mesothelin binding domain binds to the C-terminus of human mesothelin. In one embodiment, the anti-mesothelin 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-mesothelin binding domain comprises 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-mesothelin binding domain comprises one or more amino acids selected from amino acids 485-490, 498-507, 532-537, and 545-572 of SEQ ID NO: 278, or any combination thereof. 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 comprises one or more (e.g., all three) of light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of a human anti-mesothelin binding domain described herein, and one or more (e.g., all three) of heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of a human anti-mesothelin binding domain described herein. In one embodiment, the human anti-mesothelin binding domain comprises or consists of a light chain variable region described herein (e.g., in Table 2) and / or a heavy chain variable region described herein (e.g., in Table 2). In one embodiment, the anti-mesothelin binding domain is an scFv comprising or consisting of the light chain variable region and heavy chain variable region of the amino acid sequences in Table 2. In certain embodiments, the anti-mesothelin binding domain (e.g., scFV) comprises or consists of: a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) in the amino acid sequence of a light chain variable region shown in Table 2, or a sequence having 95-99% identity to an amino acid sequence of Table 2; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) in the amino acid sequence of a heavy chain variable region shown in Table 2, but not more than 30, 20, or 10 modifications (e.g., substitutions) in the amino acid sequence of a heavy chain variable region shown in Table 2, or a sequence having 95-99% identity to an amino acid sequence of Table 2. In one embodiment, the human anti-mesothelin binding domain comprises or consists of 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, 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 a sequence having 95-99% identity thereto.

[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 the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain includes a transmembrane domain described herein, e.g., having a sequence of SEQ ID NO: 6, an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 20, 10, or 5 modifications (e.g., substitutions) in the amino acid sequence of SEQ ID NO: 6, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 6.

[0024] In one embodiment, the anti-mesothelin binding domain is connected to the transmembrane domain by a hinge region, hi one embodiment, the hinge region comprises a hinge region described herein, e.g., the hinge region of SEQ ID NO:2.

[0025] In one embodiment, the isolated CAR molecule further comprises a costimulatory domain, e.g., a costimulatory domain described herein. In one embodiment, the costimulatory 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), or a functional variant thereof. In one embodiment, the costimulatory domain comprises or consists of the sequence of SEQ ID NO: 7. In one embodiment, the costimulatory domain comprises or consists of an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 20, 10, or 5 modifications (e.g., substitutions) to the amino acid sequence of SEQ ID NO: 7, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 7.

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

[0027] In another aspect, the invention pertains to an isolated CAR molecule comprising: a leader sequence, e.g., of SEQ ID NO: 1; an anti-mesothelin binding domain described herein, e.g., having an amino acid sequence of Table 2, or a sequence with 95-99% identity thereto; a hinge region, e.g., of SEQ ID NO: 2; a transmembrane domain, e.g., having a sequence of SEQ ID NO: 6; a costimulatory domain, e.g., a 4-1BB costimulatory domain having a sequence of SEQ ID NO: 7; and a primary signaling domain, e.g., a CD3 zeta stimulatory domain having a sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In one embodiment, the isolated CAR molecule comprises (e.g., consists of) a polypeptide having an amino acid sequence of Table 2. In one embodiment, the isolated CAR molecule comprises (e.g., consists of) a polypeptide having an amino acid sequence of Table 2 having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) to an amino acid sequence of Table 2, or a sequence with 95-99% identity thereto. In one embodiment, the isolated CAR molecule comprises or consists of an amino acid sequence selected from the group consisting of 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 and SEQ ID NO:86.

[0028] In another aspect, 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, e.g., a CAR molecule described herein. In one embodiment, the vector is selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

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

[0030] In one embodiment, the vector is an in vitro transcription vector, e.g., a vector that transcribes RNA of a nucleic acid molecule described herein. In one embodiment, the RNA is transcribed from an in vitro transcription vector, wherein the vector is pD-A.antimesoBD OF.2bg.150A, where antimesoBD is an antimesothelin binding domain described herein. In one embodiment, the nucleic acid sequence within the vector further comprises a poly(A) tail, e.g., a poly(A) tail comprising about 150 adenosine bases (SEQ ID NO: 271), e.g., a poly(A) tail described herein. In one embodiment, the nucleic acid sequence within the vector further comprises a 3' UTR, e.g., a 3' UTR comprising at least one repeat of the 3' UTR from human beta-globulin, e.g., a 3' UTR described herein.

[0031] In another aspect, the invention pertains to a cell comprising the vector. The cell can be, e.g., a cell described herein. In one embodiment, the cell is a human T cell, e.g., a T cell described herein, or a human NK cell, e.g., a human NK cell described herein. In one embodiment, the human T cell is a CD8 + The cell 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 the T cell is diacylglycerol kinase (DGK) deficient. In one embodiment, the cell is a T cell, and the T cell is Ikaros deficient. In one embodiment, the cell is a T cell, and the T cell is both DGK and Ikaros deficient.

[0032] In one aspect, a CAR-expressing cell described herein can further comprise a second CAR, e.g., a second CAR that includes a different antigen binding domain, to the same target (mesothelin) or a different target (e.g., a target other than mesothelin on a stromal cell, e.g., FAP; a target other than mesothelin on a prostate cancer cell, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1, or MAD-CT-2; a target other than mesothelin on an ovarian cancer cell, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor alpha, claudin 6, GloboH, or sperm protein 17; e.g., a target other than mesothelin on a lung cancer cell, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4, or Trop-2). In one embodiment, the CAR-expressing cells comprise a first CAR that targets a first antigen and comprises an intracellular signaling domain with a costimulatory signaling domain but no primary signaling domain, and a second CAR that targets a second, different antigen and comprises an intracellular signaling domain with a primary signaling domain but no costimulatory signaling domain. In one embodiment, the CAR-expressing cells comprise a first mesothelin CAR that comprises a mesothelin-binding domain, a transmembrane domain, and a costimulatory domain, and a second CAR that targets an antigen other than mesothelin (e.g., an antigen expressed on stromal cells, lung cancer cells, prostate cancer cells, or ovarian cancer cells) and comprises an antigen-binding domain, a transmembrane domain, and a primary signaling domain. In another embodiment, the CAR-expressing cells comprise a first mesothelin CAR that comprises a mesothelin-binding domain, a transmembrane domain, and a primary signaling domain, and a second CAR that targets an antigen other than mesothelin (e.g., an antigen expressed on stromal cells, lung cancer cells, prostate cancer cells, or ovarian cancer cells) and comprises an antigen-binding domain for the antigen, a transmembrane domain, and a costimulatory signaling domain.

[0033] In one embodiment, the CAR-expressing cells comprise a mesothelin CAR and an inhibitory CAR described herein. In one embodiment, the inhibitory CAR comprises an antigen-binding domain that binds to an antigen found on normal cells, e.g., normal cells that also express mesothelin, but not on cancer cells. In one embodiment, the inhibitory CAR comprises the antigen-binding domain, transmembrane domain, and intracellular domain of an inhibitory molecule. For example, the intracellular domain of the inhibitory CAR can 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 can further express other agents, e.g., agents that enhance the activity or fitness of the CAR-expressing cells, e.g., agents described herein. For example, in one embodiment, the agent can be an agent that inhibits a molecule that modulates or regulates, e.g., inhibits, T cell function. In some embodiments, the molecule that modulates or regulates T cell function is an inhibitory molecule. 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, an agent, such as an inhibitory nucleic acid, such as a dsRNA, e.g., an siRNA or shRNA, as described herein, or an inhibitory protein or inhibitory system, such as a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription activator-like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), can be used to inhibit the expression of a molecule that regulates or controls, e.g., inhibits, T cell function in a CAR-expressing cell. In some embodiments, the agent is an shRNA, e.g., an shRNA described herein. In some embodiments, an agent that regulates or controls, e.g., inhibits, T cell function is inhibited in a CAR-expressing cell. For example, a dsRNA molecule that inhibits the expression of a molecule that regulates or controls, e.g., inhibits, T cell function is linked to a nucleic acid encoding a component, e.g., all components, of a CAR.

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

[0036]

[0013] In another aspect, the invention provides a method of producing a cell described herein, e.g., a T cell or an NK cell, comprising transducing a cell with a vector comprising a nucleic acid encoding a CAR molecule, e.g., 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 a population of RNA-engineered cells, e.g., cells described herein, e.g., T cells or NK cells, that transiently express a foreign RNA. The method includes introducing into the cells in vitro transcribed RNA or synthetic RNA, wherein the RNA includes a nucleic acid encoding a CAR molecule described herein.

[0038] In another aspect, the invention relates to a method of providing anti-tumor immunity to a subject, comprising administering to the subject an effective amount of cells expressing a CAR molecule described herein, e.g., a cell described herein, comprising a CAR molecule-containing cell. 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 another aspect, the invention features a method of treating a subject having a disease associated with expression of mesothelin (e.g., proliferative diseases, precancerous conditions, and non-cancer-related indications associated with expression of mesothelin), comprising administering to the subject an effective amount of cells comprising a CAR molecule, e.g., as described herein.

[0040] In one embodiment, the disease associated with mesothelin is cancer, e.g., a cancer 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., pancreatic ductal adenocarcinoma), ovarian cancer, colorectal cancer, and bladder cancer, or any combination thereof. In one embodiment, the disease is pancreatic cancer, e.g., metastatic pancreatic ductal adenocarcinoma (PDA), e.g., in a subject that has progressed on at least one prior standard of care. In one embodiment, the disease is mesothelioma (e.g., malignant pleural mesothelioma), e.g., in a subject that has progressed on at least one prior standard of care regimen. In one embodiment, the disease is ovarian cancer, e.g., serous epithelial ovarian cancer, e.g., in a subject that has progressed after at least one prior standard of care regimen.

[0041] In one embodiment, mesothelin CAR-expressing cells, e.g., T cells or NK cells, are administered to a subject who has previously received melphalan.

[0042] In one embodiment, the cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered in combination with an agent that enhances the activity or fitness of the cells expressing the CAR molecule, e.g., an agent described herein.

[0043] In one embodiment, cells expressing CAR molecules, for example, CAR molecules described herein, are administered in combination with a low, immune-enhancing dose of an mTOR inhibitor.Without wishing to be bound by theory, treatment with a low, immune-enhancing dose (for example, a dose that is insufficient to completely suppress the immune system but sufficient to improve immune function) is thought to be accompanied by a decrease in PD-1 positive T cells or an increase in PD-1 negative cells.PD-1 positive T cells, but not PD-1 negative T cells, can be depleted by binding with cells expressing PD-1 ligands, for example, PD-L1 or PD-L2.

[0044] In one embodiment, this method can be used to optimize the performance of CAR cells described herein in a subject. Without wishing to be bound by theory, it is believed that in some embodiments, the performance of endogenous, unmodified immune effector cells, e.g., T cells, is improved. Without wishing to be bound by theory, it is believed that in some embodiments, the performance of mesothelin CAR-expressing cells is improved. In other embodiments, cells, e.g., T cells or NK cells, that have been or will be engineered to express a CAR can be treated ex vivo by contacting them with an amount of an mTOR inhibitor that increases the number of PD1-negative immune effector cells, e.g., T cells, or increases the ratio of PD1-negative immune effector cells, e.g., T cells / PD1-positive immune effector cells, e.g., T cells.

[0045] In some embodiments, administration of a low, immune-enhancing dose of an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or a catalytic inhibitor, is initiated prior to administration of the CAR-expressing cells described herein, e.g., T cells or NK cells. In some embodiments, the CAR cells are administered after a sufficient time or sufficient dosing of the mTOR inhibitor such that the level of PD1-negative immune effector cells, e.g., T cells or the ratio of PD1-negative immune effector cells, e.g., T cells / PD1-positive immune effector cells, e.g., T cells, has increased, at least transiently.

[0046] In one embodiment, the cells, e.g., T cells or NK cells, to be engineered to express a CAR are harvested after a sufficient time or sufficient dosing of a low, immune enhancing dose of an mTOR inhibitor such that the level of PD1-negative immune effector cells, e.g., the ratio of T cells or PD1-negative immune effector cells, e.g., T cells / PD1-positive immune effector cells, e.g., T cells, in or harvested from the subject has been at least transiently increased.

[0047] In one embodiment, cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered in combination with an agent to mitigate one or more side effects associated with administration of cells expressing a CAR molecule, e.g., an agent described herein.

[0048] In one embodiment, cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered in combination with an agent that treats a disease associated with mesothelin expression, e.g., an agent described herein.

[0049] In one embodiment, cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered at a dose and / or dosing schedule described herein.

[0050] In one embodiment, cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered as a first line treatment for a disease, e.g., cancer, e.g., a cancer described herein. In other embodiments, cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered as a second, third, or fourth line treatment for a disease, e.g., cancer, e.g., a cancer described herein.

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

[0052] In one embodiment, the CAR molecule is introduced into T cells or NK cells using, e.g., in vitro transcription, and the subject (e.g., human) receives an initial administration of cells comprising the CAR molecule, followed by one or more subsequent administrations of cells comprising the CAR molecule, wherein the subsequent administration or administrations are administered within 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days, after the previous administration. In one embodiment, the subject (e.g., human) receives more than one administration per week of cells comprising the CAR molecule, e.g., two, three, or four administrations per week of cells comprising the CAR molecule. In one embodiment, a subject (e.g., a human subject) receives more than one administration of cells comprising a CAR molecule per week (e.g., two, three, or four administrations per week) (also referred to herein as a cycle), followed by one week without administration of cells comprising a CAR molecule, followed by one or more administrations of cells comprising a CAR molecule (e.g., more than one administration of cells comprising a CAR molecule per week). In other embodiments, a subject (e.g., a human subject) receives more than one cycle of cells comprising a CAR molecule, with the interval between each cycle being less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, cells comprising a CAR molecule are administered every other day, three times per week. In one embodiment, cells comprising a CAR molecule are administered for at least 2, 3, 4, 5, 6, 7, 8, or more weeks.

[0053] In one aspect, the invention includes a population of autologous or allogeneic cells that have been transduced or transduced with a vector comprising a nucleic acid molecule encoding a mesothelin-CAR molecule, e.g., 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 (e.g., by gene transfer or electroporation) to cells, e.g., T cells or NK cells, where the vector comprises a nucleic acid molecule encoding a mesothelin CAR molecule 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 another aspect, the present invention provides a population of CAR-expressing cells, e.g., CAR T cells. In some embodiments, 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 can include a first cell expressing a CAR with an anti-mesothelin binding domain described herein and a second cell expressing a CAR with a different anti-mesothelin binding domain, e.g., an 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 can include, e.g., a first cell expressing a CAR comprising an anti-mesothelin binding domain, as described herein, and a second cell expressing a CAR comprising an antigen binding domain for a target other than mesothelin (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 alpha, claudin 6, GloboH, or sperm protein 17; e.g., a target other than mesothelin 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, a first cell expressing a CAR comprising a primary intracellular signaling domain and a second cell expressing a CAR comprising a secondary signaling domain.

[0055] In another aspect, the invention provides a population of cells in which at least one cell in the population expresses a CAR having an anti-mesothelin binding domain described herein, and a second cell expresses another agent, e.g., an agent that enhances the activity or function of the CAR-expressing cell. For example, in one embodiment, the agent can be an agent that inhibits a molecule that regulates or controls, e.g., inhibits, T cell function. In some embodiments, the molecule that regulates or controls T cell function is an inhibitory molecule, e.g., 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, an agent, such as an inhibitory nucleic acid, for example, a dsRNA, for example, an siRNA or shRNA, as described herein; or an inhibitory protein or inhibitory system, for example, a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription activator-like effector nuclease (TALEN) or a zinc finger endonuclease (ZFN), can be used to inhibit the expression of a molecule that regulates or controls, for example, inhibits, T cell function in CAR-expressing cells. In some embodiments, the agent is an shRNA, for example, an shRNA described herein. In some embodiments, an agent that regulates or controls, for example, inhibits, T cell function is inhibited in CAR-expressing cells. For example, a dsRNA molecule that inhibits the expression of a molecule that regulates or controls, for example, inhibits, T cell function is bound to a nucleic acid encoding a component, for example, all components, of a CAR.

[0056] In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, e.g., 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, e.g., a first polypeptide or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., a costimulatory domain (e.g., 41BB, CD27, or CD28, e.g., described herein) and / or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and / or a CD3 zeta signaling domain described herein).

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

[0058] In another aspect, the invention pertains to a CAR molecule, e.g., a CAR molecule described herein, an isolated nucleic acid molecule encoding a CAR molecule described herein, a vector comprising a CAR molecule described herein, and / or a cell comprising a CAR molecule described herein, for use as a medicament.

[0059] In another aspect, the invention pertains to a CAR molecule described herein, an isolated nucleic acid molecule encoding a CAR molecule described herein, a vector comprising a CAR molecule described herein, and / or a cell comprising a CAR molecule described herein, e.g., for use in treating a disease that expresses mesothelin, as described herein. [Brief explanation of the drawings]

[0060] [Figure 1] Figure 1 is a schematic diagram of the pD-A.anti-mesoBD.OF.BBZ.2bg.150A plasmid. This diagram refers to SEQ ID NO: 271 as "150A."

[0061] [Figure 2]Figure 2 shows possible cell production and treatment schedules. (A) Autologous cells are obtained by leukapheresis, and T cells are enriched by expansion using anti-CD3 / CD28 mAb-coated magnetic beads. Cells are expanded for 8–12 days. On the final day of culture, the beads are removed using a magnetic field, and the cells are washed, electroporated with the human meso-CAR mRNA construct, and cryopreserved in a lysis-insensitive medium. (B) Three treatment infusion schedules are described. In Schedule 1, patients receive 1 × 10 human meso-loaded CAR T cells via intravenous (iv) infusion on day 0, followed one week later by 1 × 10 human meso-loaded CAR T cells. Safety is monitored for a minimum of one month, after which patients may be eligible for Schedule 2. In Schedule 2, patients receive 1 × 10 human meso-loaded CAR T cells via iv infusion three times per week for one week, followed by a one-week rest period followed by 1 × 10 human meso-loaded CAR T cells three times per week for one week. In Schedule 3, patients will receive 3x108 / m2 human meso-loaded CAR T cells by iv infusion three times a week for three weeks, followed by intratumoral injections of 2x108 human meso-loaded CAR T cells into the primary lesion on days +35 and +57.

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

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

[0064] [Figure 5] FIG. 5 shows the clinical trial design for mesothelin CART produced by transducing a CAR construct with a lentiviral vector.

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

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

[0067] [Figure 8] FIG. 8 shows cytokines and chemokines in serum after CART mesocell infusion.

[0068] [Figure 9] Figures 9A and 9B show CART mesocell induction of anti-tumor antibodies. Serum was obtained from an MPM patient (Figure 9A) and a pancreatic cancer patient (Figure 9B).

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

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

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

[0072] [Figure 13] FIG. 13 shows the inhibitory enzymes DGK and SHP1 in TILs isolated from EMMESO flank tumors compared to TILs rested overnight.

[0073] [Figure 14] Figures 14A, 14B, 14C, 14D, 14E, and 14F show the effect of treatment with inhibitors of inhibitory mechanisms that downregulate mesoCART function (anti-PDL1, DGK inhibitor, and SSG) on tumor cell killing (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 a small panel of human CART-MSLNs after stimulation with 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 a small panel of human CART-MSLNs after stimulation with 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 lethality assays of CART-MSLN-5, CART-MSLN-11, CART-MSLN17, and murine 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 killing assay of a panel of CART-MSLNs against Ovcar3 tumor cells.

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

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

[0079] [Figure 20-1] Figures 20A, 20B, and 20C show the functional loss of mesoCAR T cells over time 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 functional loss of mesoCAR T cells over time 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 deletion on the cytotoxicity of mesoCAR T cells. The percentage of target cell killing is assessed at different effector:target ratios.

[0081] [Figure 22] Figure 22 shows the effect of DGK deletion on IFN-γ production and release from mesoCAR T cells. The concentration of IFN-γ is assessed at different effector:target ratios.

[0082] [Figure 23] Figure 23 shows the effect of DGK deletion on ERK signaling or T cell activation, mesoCAR T cells. B: Albumin, M: Mesothelin, 3 / 28: CD3 / CD28 stimulator cells.

[0083] [Figure 24] Figure 24 describes the effect of DGK deletion on TGF-β sensitivity of mesoCAR T cells for 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 as shown by tumor volume over time. B) Persistence and proliferation of tumor-infiltrating cells.

[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 levels of Ikaros. Figure 26A shows Ikaros expression in wild-type and Ikzfl±CAR T cells as measured by flow cytometry (left panel) and Western blot (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), and IL-2 (Figure 26D), the cytotoxic mediator granzyme B (Figure 26E), and CD107a expression (Figure 26F) were 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 levels of Ikaros. Figure 26A shows Ikaros expression in wild-type and Ikzfl±CAR T cells as measured by flow cytometry (left panel) and Western blot (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), and IL-2 (Figure 26D), the cytotoxic mediator granzyme B (Figure 26E), and CD107a expression (Figure 26F) were determined.

[0086] [Figure 27] Figures 27A, 27B, and 27C show cytokine production and cytotoxic mediator release in CAR-expressing T cells harboring a dominant-negative allele of Ikaros (IkDN). 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 deletion of Ikaros 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 indicated time points. In Figure 28D, the RAS / ERK signaling pathway was examined in wild-type (WT) and Ikaros dominant-negative cells (IkDN) after TCR stimulation with CD3 / CD28 antibodies. 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 blot. In Figure 28E, mesoCAR-transduced WT and IkDN cells were stimulated with BSA or mesothelin-coated beads, and downstream signaling pathways were examined by Western blot by assessing the levels of phosphorylated ERK and phosphorylated PLCγ. [Figure 28-2] Figures 28A, 28B, 28C, 28D, and 28E show that deletion of Ikaros 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 indicated time points. In Figure 28D, the RAS / ERK signaling pathway was examined in wild-type (WT) and Ikaros dominant-negative cells (IkDN) after TCR stimulation with CD3 / CD28 antibodies. 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 blot. In Figure 28E, mesoCAR-transduced WT and IkDN cells were stimulated with BSA or mesothelin-coated beads, and downstream signaling pathways were examined by Western blot by assessing the levels of phosphorylated ERK and phosphorylated PLCγ.

[0088] [Figure 29-1]Figures 29A, 29B, 29C, 29D, and 29E show that reducing Ikaros in CAR T cells enhances responses to target cells AE17 or mesothelin-expressing AE17 (AE17 meso) in vitro. Figure 29A shows IFN-γ production in WT and Ikzfl± meso CAR T cells at the indicated effector:target cell ratios. Cytolysis of mesoCAR-expressing WT and Ikzfl± (Figure 29B) and IkDN (Figure 29C) was measured at the indicated effector:target cell ratios. IFN-γ production (Figure 29D) and cytolysis (Figure 29E) of WT and Ikzfl± transduced with FAP-CAR were measured at the indicated effector:target cell ratios, where target cells were FAP-expressing 3T3 cells. [Figure 29-2] Figures 29A, 29B, 29C, 29D, and 29E show that reducing Ikaros in CAR T cells enhances responses to target cells AE17 or mesothelin-expressing AE17 (AE17 meso) in vitro. Figure 29A shows IFN-γ production in WT and Ikzfl± meso CAR T cells at the indicated effector:target cell ratios. Cytolysis of mesoCAR-expressing WT and Ikzfl± (Figure 29B) and IkDN (Figure 29C) was measured at the indicated effector:target cell ratios. IFN-γ production (Figure 29D) and cytolysis (Figure 29E) of WT and Ikzfl± transduced with FAP-CAR were measured at the indicated effector:target cell ratios, where target cells were FAP-expressing 3T3 cells.

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

[0090] [Figure 31-1]Figures 31A, 31B, 31C, 31D, 31E, and 31F show the 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 from cells harvested from the spleen (Figure 31A) and tumor (Figure 31B). The functional capacity of CAR T cells harvested from the spleen or tumor 3 days after infusion was assessed 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+ expression) and macrophages (CD206 expression) were assessed by measuring the expression of Treg or macrophage markers on CAR T cells harvested from the spleen or tumor 9 days after infusion. [Figure 31-2] Figures 31A, 31B, 31C, 31D, 31E, and 31F show the 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 from cells harvested from the spleen (Figure 31A) and tumor (Figure 31B). The functional capacity of CAR T cells harvested from the spleen or tumor 3 days after infusion was assessed 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+ expression) and macrophages (CD206 expression) were assessed by measuring the expression of Treg or macrophage markers on CAR T cells harvested 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, Ikzfl±, 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 influenza geometric mean titers against influenza vaccine strains compared to placebo. In Figure 33A, the increase above baseline in influenza geometric mean titers against 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 for each of the RAD001-treated cohorts in the intention-to-treat population. The thick black line indicates a 1.2-fold increase in titer relative to placebo, 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 a GMT titer against placebo greater than 1, with at least an 80% posterior probability. Figure 33B is a graph of the same data as Figure 33A for the subset of subjects with baseline influenza titers ≦1:40.

[0093] [Figure 34] Figure 34 shows a scatter plot of RAD001 concentrations 4 weeks after vaccination versus the fold increase in geometric mean titers for each influenza vaccine strain. RAD001 concentrations (1 hour post-dose) were measured after subjects received 4 weeks of dosing. All subjects who underwent pharmacokinetic measurements were included in the analysis population. The fold increase in geometric mean titers 4 weeks after vaccination compared to baseline is shown on the y-axis.

[0094] [Figure 35]Figure 35 is a graph showing the increase in titers against heterologous influenza strains compared to placebo. Four weeks after vaccination, the increase above baseline in influenza geometric mean titers 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) is shown for each RAD001-treated cohort in the intention-to-treat population, relative to the increase in the placebo cohort. * indicates a titer against placebo greater than 1 with at least an 80% posterior probability.

[0095] [Figure 36] Figures 36A and 36B are 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 in anti-H1N1 influenza IgG and IgM levels 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 and increase in PD-1-negative CD4 T cells after treatment with RAD001. The percentage of PD-1-positive CD4, CD8, and PD-1-negative CD4 T cells was determined by FACS analysis of PBMC samples at baseline, 6 weeks after study drug treatment (Week 6), and 6 weeks after discontinuation of study drug and 4 days after influenza vaccination (Week 12). Figure 37A shows that there was a significant decrease (-37.1 to -28.5%) in PD-1 positive CD4 T cells at week 12 in cohorts receiving RAD001 at dose levels of 0.5 mg / day (n=25), 5 mg / week (n=29), and 20 mg / week (n=30) compared to the placebo cohort (n=25), with p=0.002 (0.02), p=0.003 (q=0.03), and p=0.01 (q=0.05), respectively. Figure 37B shows that there was a significant decrease (-43.3 to -38.5%) in PD-1 positive CD8 T cells at week 12 in 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) compared to the placebo cohort (n=25), 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 there was a significant increase (3.0-4.9%) in PD-1 negative CD4 T cells at week 12 in cohorts receiving RAD001 (n=109) at dose levels of 0.5 mg / day (n=25), 5 mg / week (n=29), and 20 mg / week (n=30) compared to the placebo cohort (n=25), 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 decrease in the percent of 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 percent of PD-1-positive CD4, CD8, and PD-1-negative CD4 T cells was determined by FACS analysis of PBMC samples at baseline, 6 weeks after study drug treatment (Week 6), and 6 weeks after study drug discontinuation and 4 days after influenza vaccination (Week 12). Figure 38A shows that there was 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), p=0.03 (q=0.13). The decrease in PD-1-positive CD4 T cells at Week 12 in the pooled RAD cohort compared to the placebo cohort was 32.7%, p=0.05 (q=0.19). Figure 38B shows that there was a significant 37.4% reduction in PD-1 positive CD8 T cells at week 6 in the pooled RAD001 cohort (n=84) compared to the placebo cohort (n=25), p=0.008 (q=0.07). The reduction in PD-1 positive CD8 T cells at week 12 in the pooled RAD001 compared to the placebo cohort was 41.4%, p=0.066 (q=0.21). Figures 38A and 38B show the data from Figures 37A, 37B, and 37C, except that the different RAD001 dose groups from Figures 37A, 37B, and 37C have been pooled into a single RAD001 treatment group in Figures 38A and 38B.

[0098] [Figure 39] FIG. 39 shows increased activity and energy in elderly subjects in response to RAD001.

[0099] [Figure 40] Figures 40A and 40B show the predicted effects of RAD001 on P70 S6K activity in cells. Figure 40A shows P70 S6 kinase inhibited by weekly and daily high doses of RAD001, and Figure 40B shows P70 S6 kinase inhibited by weekly low 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 for scFvs M12, M14, M16, M17, M21, and M23 (Figure 42A). ScFvs M5 (Figure 42B) and M11 (Figure 42C) bind to a different epitope than SS1.

[0102] [Figure 43] Figure 43 shows tumor growth after various mesothelin CAR T treatments in the OVCAR8 tumor model. Mean tumor volume ± SEM up to 62 days after tumor implantation. T cells were administered on days 14 and 19. Small circles: mice treated with 100 μl of PBS via the lateral tail vein; closed 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; diamonds: 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 closed triangles: mice treated with two doses of M11 CAR T cells.

[0103] [Figure 44] Figure 44 is a schematic diagram of human mesothelin peptide coverage 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 the peptide from amino acids 297 to 464 shown in Figure 45A and the peptide from amino acids 458 to 586 shown in Figure 45B. All differences are relative to the deuterium uptake of unbound mesothelin (control). * indicates the area of ​​statistical significance using Tukey's test for peptides that differ by less than 0.75 Da.

[0105] [Figure 46] Figure 46 is a schematic diagram showing the primary sequence of the antigen human mesothelin (amino acids 296-588) and the regions protected by SS1 and M5. Black bars indicate amino acids protected when complexed with SS1 (amino acids 314-315, 317-318, 346-349, and 369-375). 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 arrangements of CAR and shRNA-encoding constructs for co-expression of CAR and shRNA. Figures 47A-47D show various arrangements on a single vector, for example, where the U6-controlled shRNA is upstream or downstream of the EF1 alpha-controlled 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 a first vector, and the CAR (and corresponding EF1 alpha promoter) is on a second vector (Figure 16E).

[0107] [Figure 48]Figure 48 shows the structures of two representative RCAR configurations. The antigen binding member comprises an antigen binding domain, a transmembrane domain, and a switch domain. The intracellular binding member comprises 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 in different orientations relative to the antigen binding member and the intracellular binding member. Other RCAR configurations are further described herein. DETAILED DESCRIPTION OF THE INVENTION

[0108] Detailed Description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0109] The terms "a," "an," and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the indefinite article. By way of example, "an" or "an" means one component or more than one component.

[0110] The term "about," when referring to a measurable value such as an amount, duration, etc., is intended to encompass variations of ±20%, or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the particular value, provided such variations are appropriate for the practice of the disclosed methods.

[0111]

[0013] The term "chimeric antigen receptor" or alternatively "CAR" refers to a set of polypeptides, generally two in the simplest embodiment, which, when present in an immune effector cell, provides the cell with specificity for a target cell, generally a cancer cell, and intracellular signal production. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule, as described below. In some aspects, the set of polypeptides are adjacent to one another. In some embodiments, the set of polypeptides comprises a dimerization switch that can link the polypeptides to one another in the presence of a dimerization molecule, e.g., link the antigen binding domain to the intracellular signaling domain. In one aspect, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, as described below. In one aspect, the costimulatory molecule is selected from a costimulatory molecule 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 a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule.In one aspect, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, where the leader sequence may optionally be cleaved from the antigen-binding domain (e.g., scFv) during cellular 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 the cell to control cellular activity through a defined signaling pathway, either by producing second messengers or by functioning as an effector by responding to such messengers.

[0113] As used herein, the term "mesothelin" refers to mesothelin, a 40 kDa protein anchored to cell membranes by a glycosylphosphatidylinositol (GPI) linkage and an amino-terminal 31 kDa shed fragment called megakaryocyte potentiating factor (MPF). Both fragments contain N-glycosylation sites. The term also refers to a soluble splice variant of the 40 kDa carboxyl-terminal fragment, also called "soluble mesothelin / MPF-related." Preferably, the term refers to human mesothelin, GenBank Accession No. AAH03512.1, and its naturally cleaved portions, e.g., expressed on cell membranes, e.g., cancer cell membranes.

[0114] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins, and can be derived from natural or recombinant sources. An antibody can be a tetramer of immunoglobulin molecules.

[0115] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope on an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), 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), camelid VHH domains, multispecific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope-binding fragments of an antibody. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0116] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, an scFv as used herein can comprise, e.g., the VL and VH variable regions in either order with respect to the N- and C-termini of the polypeptide, and the scFv can comprise VL-linker-VH or VH-linker-VL.

[0117] The portion of the CAR of the present invention comprising an antibody or antibody fragment thereof can exist in a variety of forms, where the antigen-binding domain is expressed as part of a contiguous polypeptide chain, including, for example, a 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 comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment comprising an scFv.

[0118] The term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, which usually determine the class to which the antibody belongs.

[0119] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0120] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed in a bacteriophage or yeast expression system. The term should also be construed to mean an antibody that has been produced by the synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein or an amino acid sequence that specifies the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology that is available and well known in the art.

[0121] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve either antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response, therefore, encodes an "antigen," as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, with these nucleotide sequences arranged in various combinations to encode a polypeptide that elicits the desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It will be readily apparent that an antigen can be synthetically produced or derived from a biological sample, or may be a macromolecule other than a polypeptide. Such biological samples include, but are not limited to, tissue samples, tumor samples, and body 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 thereof, to prevent another antigen-binding domain, e.g., an antigen-binding domain provided herein, e.g., an antibody or fragment thereof, from binding, directly or indirectly, to a target, e.g., mesothelin. The extent to which an antigen-binding domain, e.g., an antibody or fragment thereof, prevents another antigen-binding domain, e.g., an antibody or fragment thereof, from binding to a target, and therefore can be considered competition, can be determined using a competitive binding assay. In some embodiments, the competitive binding assay is a quantitative competition assay. For example, one particularly suitable quantitative competition assay uses surface plasmon resonance (SPR)-based techniques to measure binding, e.g., competition, between one antibody or fragment thereof and another antibody or fragment thereof for binding to an immobilized target. An exemplary SPR-based competition assay is described in Example 2 herein. Another suitable quantitative competition assay uses FACS-based techniques to measure competition between a labeled (e.g., His-tagged, biotinylated, or radioactively labeled, among others) antibody or fragment thereof and another antibody or fragment thereof 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 increase in 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 condition. An "anti-cancer effect" can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies in preventing 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 tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0124] The term "autologous" refers to any material originating from the same individual that is later reintroduced into the individual.

[0125] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual to which the material is introduced. Two or more individuals are said to be allogeneic to one another if the genes at one or more loci are not identical. In some aspects, allogeneic material derived from individuals of the same species may be sufficiently genetically different to interact antigenically.

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

[0127] The term "cancer" refers to a disease characterized by the uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers are described herein and include, but are 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, lung cancer, and the like.

[0128] The term "disease associated with mesothelin expression" includes, but is not limited to, a disease or condition associated with mesothelin expression associated with cells that express mesothelin, including, for example, a proliferative disorder such as a cancer or malignant tumor, or a precancerous condition such as mesothelin hyperplasia; or a non-cancer-related indication associated with cells that express mesothelin. Examples of various cancers that express mesothelin include, but are not limited to, mesothelioma, lung cancer, ovarian cancer, pancreatic cancer, etc.

[0129] The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies or antibody fragments of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of the invention can 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 mesothelin, for example, using the functional assays described herein.

[0130] The term "stimulation" refers to a primary response elicited by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex or a CAR) to its cognate ligand (or tumor antigen in the case of a CAR), thereby mediating a signaling event, such as, but not limited to, signaling by the TCR / CD3 complex or by an appropriate NK receptor or by the signaling domain of the CAR. Stimulation can mediate altered expression of certain molecules.

[0131] The term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides cytoplasmic signaling sequences that regulate immune cell activation in a stimulatory manner for at least some aspect of the immune cell signaling pathway. In one aspect, the signal is a primary signal, initiated, for example, by binding of a peptide-loaded MHC molecule to the TCR / CD3 complex, and leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. Primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that act in a stimulatory manner can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. Examples of ITAM-containing cytoplasmic signaling sequences that are particularly useful in the present invention include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In certain CARs of the invention, the intracellular signaling domain in any one or more CARs of the invention comprises an intracellular signaling sequence, e.g., the primary signaling sequence of CD3 zeta. In certain CARs of the invention, the primary signaling sequence of CD3 zeta is the sequence provided in SEQ ID NO: 9 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc. In certain CARs of the invention, the primary signaling sequence of CD3 zeta is the sequence provided in SEQ ID NO: 10 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.

[0132] The term "antigen-presenting cell" or "APC" refers to a cell of the immune system, such as an accessory cell (e.g., B cell, dendritic cell, etc.), that displays foreign antigens on its surface complexed with major histocompatibility complexes (MHC). T cells can recognize these complexes using the T cell receptor (TCR). APCs process antigens and present them to T cells.

[0133] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain generates a signal that enhances the immune effector function of a CAR-containing cell, e.g., a CAR T cell. For example, in a CAR T cell, examples of immune effector function include cytolytic activity and helper activity, including cytokine secretion.

[0134] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain. Exemplary primary intracellular signaling domains may include those derived from molecules responsible for primary or antigen-dependent stimulation. In some embodiments, the intracellular signaling domain may comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains may include those derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain may comprise the cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain may comprise the cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0135] The primary intracellular signaling domain may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, 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," alternatively "zeta chain," "CD3 zeta," or "TCR-zeta" are defined as the protein provided as GenBank Accession No. BAG36664.1 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and a "zeta stimulatory domain" or alternatively "CD3 zeta stimulatory domain" or "TCR-zeta stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain, or a functional derivative thereof, sufficient to functionally transmit an early signal necessary for T cell activation. In one aspect, the cytoplasmic domain of zeta comprises residues 52-164 of GenBank Accession No. BAG36664.1, or the equivalent residues from a functional ortholog of a non-human species, e.g., mouse, rodent, monkey, ape, etc. In one aspect, a "zeta stimulatory domain" or "CD3 zeta stimulatory domain" is the sequence provided as SEQ ID NO:9. In one aspect, the "zeta stimulatory domain" or "CD3 zeta stimulatory domain" is the sequence provided as SEQ ID NO:10.

[0137] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response, such as, but not limited to, proliferation, by the T cell. 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 costimulatory 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 These include 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 costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and ligands that specifically bind to CD83.

[0139] The intracellular signaling domain can comprise the entire intracellular portion of the molecule from which it is derived or the entire naturally occurring intracellular signaling domain or a functional fragment or derivative thereof.

[0140] The term "4-1BB" refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc. In one aspect, a "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc. In one aspect, a "4-1BB costimulatory domain" is the sequence provided as SEQ ID NO:7, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.

[0141] As used herein, "antigen-presenting cells" refer to immune system cells, such as accessory cells (e.g., B cells, dendritic cells, etc.), that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize these complexes using T cell receptors (TCRs). APCs process antigens and present them to T cells.

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

[0143] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The term nucleotide sequence encoding a protein or RNA may also include introns, to the extent that a nucleotide sequence encoding a protein may, in some versions, contain introns.

[0144] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, substance, or composition described herein that is effective to achieve a particular biological result. The term "endogenous" refers to any substance that is derived from or produced within an organism, cell, tissue, or system.

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

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

[0147] The term "transfer vector" refers to a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphipathic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes self-replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the introduction of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0148] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or an 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 the recombinant polynucleotide.

[0149] The term "lentivirus" refers to a genus in the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells and deliver significant amounts of genetic information into the DNA of host cells, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, including, in particular, self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that can be used in the clinic include, for example, Oxford BioMedica's LENTIVECTOR® Gene Delivery Technology and Lentigen's LENTIMAX®. TM These include, but are not limited to, vector systems, etc. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.

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

[0151] The term "humanized" refers to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit that has the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments may contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications may further refine and optimize antibody or antibody fragment performance. Generally, a humanized antibody or antibody fragment thereof will comprise a substantial portion of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. A humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

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

[0153] The term "isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially pure form, or it can exist in a non-native environment such as, for example, a host cell.

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

[0155] The term "operably linked" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, where necessary to join two protein-coding regions, in the same reading frame.

[0156] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), intratumoral or intrasternal injection or infusion techniques.

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

[0158] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds comprising 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 may comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and to longer chains, of which there are many types commonly referred to in the art as proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others. A polypeptide includes a naturally occurring peptide, a recombinant peptide, a recombinant peptide, or a combination thereof.

[0159] The term "promoter" refers to a DNA sequence recognized by or introduced into the synthetic machinery of a cell necessary for the specific transcription of a polynucleotide sequence.

[0160] The term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence, and in other instances, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. A promoter / regulatory sequence is one that, for example, expresses a gene product in a tissue-specific manner.

[0161] The term "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a cell to produce the gene product under most or all physiological conditions of the cell.

[0162] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a cell to produce the gene product only when an inducer corresponding to the promoter is present in the cell.

[0163] The term "tissue-specific" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoded by or specified by a gene, causes a cell to produce a gene product substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0164] The term "flexible polypeptide linker" as used in the context of scFvs refers to a peptide linker consisting of amino acids such as glycine and / or serine residues, used alone or in combination to link the variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker, having the amino acid sequence (Gly-Gly-Gly-Ser) n(SEQ ID NO: 38) (wherein 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 flexible polypeptide linker includes, but is not limited to, (Gly4Ser)4 (SEQ ID NO: 27) or (Gly4Ser)3 (SEQ ID NO: 28). In another embodiment, the linker comprises multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO: 29). Also included within the scope of the present invention are linkers described in WO2012 / 138475 (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 added to the "front" or 5' end of eukaryotic messenger RNA shortly after the initiation of transcription. The 5' cap consists of a terminal group attached to the first transcribed nucleotide. Its presence is important for ribosome recognition and protection from RNases. Capping occurs co-transcriptionally, coupled with transcription. Shortly after the initiation of transcription, the 5' end of synthesized mRNA is capped by a cap-synthesizing complex associated with RNA polymerase. This enzyme complex catalyzes the chemical reactions required for mRNA cap formation. Synthesis proceeds as a multi-step biochemical reaction. The cap-forming moiety can be modified to modulate mRNA functionality, such as stability or translation efficiency.

[0166] As used herein, "in vitro transcribed RNA" refers to RNA, preferably mRNA, that has been synthesized in vitro. Generally, in vitro transcribed RNA is produced from an in vitro transcription vector. The in vitro transcription vector contains a template that is used to produce the in vitro transcribed RNA.

[0167] As used herein, "poly(A)" refers to a series of adenosines attached to mRNA by polyadenylation. In preferred embodiments of constructs for transient expression, the poly(A) is 50-5000 (SEQ ID NO: 30), preferably greater than 64, more preferably greater than 100, and most preferably greater than 300 or 400. The poly(A) sequence can be chemically or enzymatically modified to modulate mRNA functionality such as localization, stability, or translation efficiency.

[0168] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety or modified variants thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are adenylated at the 3' end. The 3' poly(A) tail is a long sequence (often several hundred) of adenine nucleotides added to pre-mRNA by the action of the enzyme polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the proteins that bind to it help protect the mRNA from exonucleolytic degradation. Polyadenylation is also important for transcription termination, mRNA export from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but can also occur later in the cytoplasm. After transcription is completed, the mRNA strand is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. Once the mRNA is cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

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

[0170] As used herein, the terms "treatment" and "treating" refer to a reduction or amelioration of the progression, severity, and / or duration of a proliferative disorder or an amelioration of one or more symptoms of a proliferative disorder (preferably, one or more discernible symptoms) resulting from the administration of one or more therapeutic agents (e.g., one or more therapeutic agents such as a CAR of the invention). In certain embodiments, the terms "treatment" and "treating" refer to an amelioration of at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which is not necessarily discernible by the patient. In other embodiments, the terms "treatment" and "treating" refer to arresting the progression of a proliferative disorder physically, e.g., by stabilization of a discernible symptom, physiologically, e.g., by stabilization of a physical parameter, or both. In other embodiments, the terms "treatment" and "treating" refer to a reduction or stabilization of tumor size or cancerous cell number.

[0171] The term "signal transduction pathway" refers to the biochemical associations between various signaling molecules that are responsible for transmitting a signal from one part of a cell to another part of the cell. The term "cell surface receptor" refers to molecules and complexes of molecules that can receive and transmit signals across the membrane of a cell.

[0172] The term "subject" is intended to include living organisms in which an immune response can be elicited (eg, mammals, humans).

[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 have been separated from other cell types with which they are normally associated in nature. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, the term simply refers to cells that have been separated from cells with which they are naturally associated. 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" refers to treatment. A therapeutic effect is achieved by reducing, suppressing, ameliorating, or eradicating a disease state.

[0175] As used herein, the term "prophylaxis" refers to the prevention or protective treatment of a disease or disease state.

[0176] The terms "cancer-associated antigen" or "tumor antigen" refer interchangeably to a molecule (generally a protein, carbohydrate, or lipid) that is expressed on the surface of cancer cells, either in whole or as fragments (e.g., MHC / peptide), and that is useful for preferential targeting of drugs to cancer cells. In some embodiments, a tumor antigen is a marker expressed by both normal and cancer cells, e.g., a cell lineage marker, e.g., CD19 on B cells. In some embodiments, a tumor antigen is a cell surface molecule that is overexpressed on cancer cells compared to normal cells, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold or more overexpression compared to normal cells. In some embodiments, a 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 compared to molecules expressed on normal cells. In some embodiments, a tumor antigen is synthesized exclusively on the cell surface of cancer cells, either in whole or as fragments (e.g., MHC / peptide), and is not synthesized or expressed on the surface of normal cells. In some embodiments, a CAR of the present invention comprises a CAR comprising an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presented peptide. Normally, peptides derived from endogenous proteins fill the pockets of major histocompatibility complex (MHC) type I molecules and bind to CD8 +They are recognized by the T cell receptor (TCR) on T lymphocytes. MHC-I type 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 viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified by screening libraries such as human scFv phage display libraries.

[0177] The terms "transfection" or "transformation" or "transduction" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transgenic" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

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

[0179] "Regulatable chimeric antigen receptor (RCAR)," as the term is used herein, refers to a set of polypeptides, generally two in the simplest embodiment, that, when present in an RCARX cell, has specificity for a target cell, generally a cancer cell, and provides the RCARX cell with controllable intracellular signal production or proliferation, which can optimize the immune effector properties of the RCARX cell. RCARX cells rely, at least in part, on an antigen-binding domain to provide specificity for target cells containing an antigen bound by the antigen-binding domain. In some embodiments, the RCAR contains a dimerization switch, which, upon the presence of a dimerization molecule, can link the intracellular signaling domain to the antigen-binding domain.

[0180] A "membrane anchor" or "membrane tethering domain," as that term is used herein, refers to a polypeptide or moiety, e.g., a myristoyl group, sufficient to anchor an extracellular or intracellular domain to the plasma membrane.

[0181] A "switch domain," as the term is used herein, e.g., when referring to an RCAR, refers to one that binds to another switch domain, generally a polypeptide-based one, in the presence of a dimerization molecule. The binding results in functional coupling between a first switch domain bound, e.g., fused, to a first switch domain and a second switch domain bound, e.g., fused, to a second switch domain. The first and second switch domains are collectively referred to as a dimerization switch. In some embodiments, the first and second switch domains are identical to each other, e.g., polypeptides with the same primary amino acid sequence, and are collectively referred to as a homodimerization switch. In some embodiments, the first and second switch domains are different from each other, e.g., polypeptides with different primary amino acid sequences, and are collectively referred to as a heterodimerization 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 dimerization molecule is a small molecule, e.g., a rapalog. In some embodiments, the switch domain is polypeptide-based, e.g., an scFv that binds a myc peptide, and the dimerization molecule is a polypeptide, fragment thereof, or multimer of a polypeptide, e.g., a myc ligand or multimer of a myc ligand that binds to one or more myc scFvs. In some embodiments, the switch domain is polypeptide-based, e.g., a myc receptor, and the dimerization molecule is an antibody or fragment thereof, e.g., a myc antibody.

[0182] "Dimerization molecule," as the term is used herein, e.g., when referring to RCAR, refers to a molecule that promotes association of a first switch domain and a second switch domain. In some embodiments, the dimerization molecule is not naturally present in a subject or is not present at concentrations that result in significant dimerization. In some embodiments, the dimerization molecule is a small molecule, e.g., rapamycin or a rapalog, e.g., RAD001.

[0183] The term "bioequivalent" refers to the amount of an agent other than a reference compound (e.g., RAD001) required to produce an effect equivalent to that produced by a reference dose or amount of the reference compound (e.g., RAD001). In some embodiments, the effect is a measure of the level of mTOR inhibition, e.g., by P70 S6 kinase inhibition, or phosphorylated S6 levels by Western blot, as assessed in an in vivo or in vitro assay, e.g., as measured by an assay described herein, e.g., the Boulay assay. In some embodiments, the effect is a change in the ratio of PD-1-positive / PD-1-negative T cells, as measured by cell sorting. In some embodiments, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of P70 S6 kinase inhibition as the reference dose or amount of the reference compound. In some embodiments, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of P70 S6 kinase inhibition as the reference dose or amount of the reference compound.

[0184] The term "low, immune-enhancing dose," when used in combination with an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 or rapamycin, or a catalytic mTOR inhibitor, is a dose of the mTOR inhibitor that partially, but not completely, inhibits mTOR activity, e.g., as measured by inhibition of P70 S6 kinase activity. For example, methods for assessing mTOR activity by inhibition of P70 S6 kinase are described herein. This dose is insufficient to produce complete immune suppression, but is sufficient to enhance the immune response. In some embodiments, the low, immune-enhancing dose of the mTOR inhibitor results in a decrease in the number of PD-1-positive T cells and / or an increase in the number of PD-1-negative T cells or an increase in the ratio of PD-1-negative T cells to PD-1-positive T cells. In some embodiments, the low, immune-enhancing dose of the mTOR inhibitor results in an increase in the number of naive T cells. In some embodiments, the low, immune-enhancing dose of the mTOR inhibitor results in one or more of the following: For example, the following markers on memory T cells, e.g., memory T cell precursors: CD62L 高 , CD127 高 , CD27+ and increased expression of one or more of BCL2; Decreased KLRG1 expression, e.g., in memory T cells, e.g., memory T cell precursors; and An increase in the number of memory T cell precursors, e.g., cells with one or a combination of the following characteristics: CD62L 高 Increased CD127 高 Increased CD27 + increased, KLRG1 decreased and BCL2 increased; wherein any of the above changes occur, for example, at least transiently, for example, as compared to an untreated subject.

[0185] Ranges: Throughout this disclosure, various aspects of the invention may be expressed 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. Accordingly, the description of a range should be construed as specifically disclosing all the possible subranges as well as individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be construed as specifically disclosing subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This 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 anti-mesothelin chimeric antigen receptors (CARs), e.g., human mesothelin CARs.

[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 protein. In one aspect, the present invention provides cells (e.g., T cells or NK cells) engineered to express a CAR, e.g., wherein the CAR T cells ("CART") exhibit anti-cancer properties. In one aspect, the cells are transformed with a CAR and express the CAR on the cell surface. In some embodiments, the cells (e.g., T cells or NK cells) are transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cells can stably express the CAR. In other embodiments, the cells (e.g., T cells or NK cells) are transduced with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR. In some such embodiments, the cells can transiently express a CAR.

[0188] In one aspect, the mesothelin protein-binding portion of the CAR is an scFv antibody fragment. In one aspect, such an antibody fragment is functional in that it retains equivalent binding affinity as the IgG antibody from which it is derived, i.e., it binds to the same antigen with comparable affinity. In one aspect, such an antibody fragment is functional in that it provides a biological response, including, but not limited to, activation of an immune response, inhibition of signal transduction from its target antigen, inhibition of kinase activity, etc., as understood by those of skill in the art. In one aspect, the mesothelin antigen-binding domain of the CAR is an scFv antibody fragment that is human or humanized relative to the mouse sequence of the scFv from which it is derived. In one embodiment, the human anti-mesothelin scFv antibody fragment comprises the light chain variable region and / or heavy chain variable region provided in Table 2, or a sequence having substantial identity thereto, e.g., 95-99% identity thereto.

[0189] In some aspects, the antibodies of the invention are incorporated into a chimeric antigen receptor (CAR). In one aspect, the CAR comprises a polypeptide sequence provided herein as 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 a sequence with 95-99% identity thereto.

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

[0191] In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 39. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 40. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 41. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 42. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 43. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 44. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 45. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 46. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 47. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 48. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 49. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 50. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 51. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 52. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 53. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 54. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 55. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 56. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 57. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 58. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 59. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 60.In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 61. In one aspect, the human mesothelin CAR molecule comprises an scFv portion provided in SEQ ID NO: 62.

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

[0193] In one aspect, the mesothelin CAR is selected from the sequence provided in one or more of SEQ ID NOs: 63-86. 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] Additionally, the present invention provides mesothelin CAR compositions and their use in medicaments or methods for the treatment of, among other diseases, cancer or any malignancy or autoimmune disease involving cells or tissues that express 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), wherein the CAR T cell ("CART") exhibits anti-tumor properties. A preferred antigen is mesothelin. In one aspect, the antigen binding domain of the CAR comprises a human anti-mesothelin antibody fragment. In one aspect, the antigen binding domain of the CAR comprises a human anti-mesothelin antibody fragment comprising an scFv. Thus, the present invention provides mesothelin CARs engineered into T cells or NK cells that comprise a human anti-mesothelin binding domain and methods of using them for adoptive therapy.

[0196] In one aspect, the mesothelin CAR comprises 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 signal domain, and any combination thereof. In one aspect, the mesothelin CAR comprises at least one intracellular signaling domain of one or more costimulatory molecules other than CD137 (4-1BB) or CD28, a CD3 zeta signal domain, and any combination thereof.

[0197] Additionally, the present invention provides mesothelin CAR compositions and their use in medicaments or methods for treating, among other diseases, cancer or any malignancy or autoimmune disease involving cells or tissues that express mesothelin.

[0198] Chimeric antigen receptor (CAR) The present invention encompasses recombinant nucleic acid constructs 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 aspect, the mesothelin is human mesothelin, and the sequence of the antibody fragment is adjacent to and in the same reading frame as the nucleic acid sequence encoding the intracellular signaling domain. The intracellular signaling domain can include a costimulatory signaling domain and / or a primary signaling domain, e.g., a zeta chain. The costimulatory signaling domain refers to a portion of the CAR that includes at least a portion of the intracellular domain of a costimulatory molecule.

[0199] In a particular aspect, a CAR construct of the invention comprises an scFv domain selected from the group consisting of SEQ ID NOs: 39-62, wherein the scFv can be preceded by an optional leader sequence such as provided in SEQ ID NO: 1, and can be followed by an optional hinge sequence such as provided in SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5, a transmembrane region such as provided in SEQ ID NO: 6, an intracellular signaling domain comprising SEQ ID NO: 7 or SEQ ID NO: 8, and a CD3 zeta sequence comprising SEQ ID NO: 9 or SEQ ID NO: 10, wherein these domains are contiguous and in the same reading frame to form a single fusion protein. Also encompassed by the present invention is a nucleotide sequence encoding a polypeptide 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 a sequence having 95 to 99% identity thereto. Also encompassed by the present invention are nucleotide sequences encoding each of the scFv fragments 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, 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 thereto, and each of the polypeptides of the domains of SEQ ID NOs:1, 2, and 6-9, as well as the encoded mesothelin CAR fusion proteins of the invention. In one aspect, exemplary mesothelin CAR constructs include an optional leader sequence, an extracellular mesothelin-binding domain, a hinge, a transmembrane domain, and an intracellular stimulatory domain.In one aspect, the mesothelin CAR construct comprises an optional leader sequence, a mesothelin binding domain, a hinge, a transmembrane domain, an intracellular costimulatory domain, and an intracellular stimulatory domain. Exemplary mesothelin CAR constructs comprising a human scFv domain are provided as SEQ ID NOs: 87-110.

[0200] Full-length CAR sequences are also provided herein 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. An exemplary leader sequence is provided as SEQ ID NO:1. An exemplary hinge / spacer sequence is provided as SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5. An exemplary transmembrane domain sequence is provided as SEQ ID NO:6. An exemplary sequence of the intracellular signaling domain of 4-1BB protein is provided as SEQ ID NO:7. An exemplary sequence of the intracellular signaling domain of CD27 is provided as SEQ ID NO:8. An exemplary 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 antimesothelin binding domain, e.g., as described herein, contiguous with and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. In one aspect, the antimesothelin binding domain is selected from one or more of SEQ ID NOs: 87-110. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 87. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 88. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 89. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 90. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 91. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 92. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 93. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 94. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 95. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 96. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 97. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 98. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 99. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 100. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 101. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 102. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 103. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 104. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 105. In one aspect, the antimesothelin binding domain comprises SEQ ID NO: 106. In one aspect, the anti-mesothelin binding domain comprises SEQ ID NO: 107. In one aspect, the anti-mesothelin binding domain comprises SEQ ID NO: 108. In one aspect, the anti-mesothelin binding domain comprises SEQ ID NO: 109. In one aspect, the anti-mesothelin binding domain comprises SEQ ID NO: 110.In one aspect, the present invention provides a recombinant DNA construct comprising a transgene encoding a CAR, wherein the transgene comprises a nucleic acid sequence encoding an anti-mesothelin binding domain described herein, e.g., a human anti-mesothelin binding domain selected from one or more of SEQ ID NOs: 87-110, wherein the sequence is contiguous with and in the same reading frame as the nucleic acid sequence encoding the intracellular signaling domain. Exemplary intracellular signaling domains that can be used in a CAR include, but are not limited to, one or more intracellular signaling domains, such as, for example, CD3 zeta, CD28, 4-1BB, etc. In certain examples, a CAR can comprise any combination of CD3 zeta, CD28, 4-1BB, etc. In one aspect, the nucleic acid sequence of a CAR construct of the invention is selected from one or more of SEQ ID NOs: 111-134. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 111. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 112. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 113. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 114. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 115. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 116. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 117. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 118. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 119. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 120. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 121. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 122. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 123. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 124. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 125. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 126. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 127. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 128. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 129.In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 130. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 131. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 132. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 133. In one aspect, the nucleic acid sequence of the CAR construct is SEQ ID NO: 134.

[0202] Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as screening libraries from cells which express the gene, extracting the gene from a vector known to contain it, or isolating it directly from cells and tissues which contain it, using standard techniques. Alternatively, the desired nucleic acid can be produced synthetically rather than cloned.

[0203] The present invention includes retroviral and lentiviral vector constructs expressing 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 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 bases in length that contains 3' and 5' untranslated sequences ("UTR"), a 5' cap and / or internal ribosome entry site (IRES), the nucleic acid to be expressed, and a poly(A) tail (SEQ ID NO: 35). The RNA produced in this manner efficiently transduces different types of cells. In one embodiment, the template contains the sequence for the CAR. In some embodiments, the RNA CAR vector is transduced into T cells by electroporation.

[0204] antigen-binding domain In one aspect, the CAR of the present invention comprises 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 can be selected to recognize an antigen that acts as a cell surface marker on the target cell associated with a particular disease state.

[0205] In one aspect, a CAR-mediated immune effector cell response can be directed to a cell expressing a desired antigen, wherein the CAR comprises an antigen binding domain that specifically binds to the desired antigen. In one aspect, the portion of the CAR that comprises the antigen binding domain comprises an antigen binding domain that targets mesothelin. In one aspect, the antigen binding domain targets human mesothelin.

[0206] The antigen-binding domain can be any domain that binds to an antigen, including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies and functional fragments thereof, including single-domain antibodies such as the heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VHH) of camelid-derived nanobodies, and alternative scaffolds known in the art to function as antigen-binding domains, such as recombinant fibronectin domains. In certain instances, it is beneficial for the antigen-binding domain to be derived from the same species as the CAR will ultimately be used in. For example, for use in humans, it may be beneficial for the antigen-binding domain of the CAR to comprise human or humanized residues for the antigen-binding domain of an antibody or antibody fragment. Thus, in one aspect, the antigen-binding domain comprises a human antibody or antibody fragment.

[0207] In one embodiment, the anti-mesothelin binding domain does not compete, or competes poorly, for binding to human mesothelin with an antigen binding domain comprising an amino acid sequence comprising SEQ ID NO: 279, e.g., murine SS1 scFv, e.g., in a competition 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 an LC CDR1, LC CDR2, and LC CDR3 of an anti-mesothelin light chain amino acid sequence selected from SEQ ID NO: 43 or SEQ ID NO: 49 and an HC CDR1, HC CDR2, and HC CDR3 of an anti-mesothelin heavy chain amino acid sequence selected from SEQ ID NO: 43 or SEQ ID NO: 49, e.g., in a competition assay described herein. In one embodiment, the anti-mesothelin binding domain competes for binding to human mesothelin with an antigen-binding domain comprising an LC CDR1 selected from SEQ ID NO: 203 or SEQ ID NO: 209, an LC CDR2 selected from SEQ ID NO: 227 or SEQ ID NO: 233, and an LC CDR3 selected from SEQ ID NO: 251 or SEQ ID NO: 257; and an HC CDR1 selected from SEQ ID NO: 138 or SEQ ID NO: 144, an HC CDR2 selected from SEQ ID NO: 156 or SEQ ID NO: 162, and an HC CDR3 selected from SEQ ID NO: 179 or SEQ ID NO: 185, e.g., in a competition assay described herein.

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

[0211] In some embodiments, the competitive assay is an SPR-based assay. Briefly, an antigen, for example, human mesothelin, is immobilized on a surface. A control antibody is injected over the antigen layer through a microfluidic system. Binding of the control antibody to the antigen results in a signal increase, e.g., a control signal, generally expressed in response units (RU). After a desired time, a test antibody is injected over the antigen layer. If the test antibody binds to a different region or epitope on the antigen, a further signal increase, e.g., an increase in signal (e.g., RU) 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 compared to the highest signal, e.g., the control signal, detected by binding of the control antibody. If the test antibody binds to the same region or epitope of the antigen, there will be little or no increase in signal, e.g., an increase in signal, e.g., less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% RU, compared to the highest signal, e.g., control signal, detected upon binding of the control antibody. Using this SPR-based competition assay, an antibody is said to compete with the control antibody when an increase in signal, e.g., less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% RU, is detected compared to the control signal detected upon binding of the control antibody to the antigen. An antibody is said to not compete or to compete poorly with a control antibody when an increase in signal, e.g., RU, 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 compared to a control signal detected by binding of a control antibody to the antigen.

[0212] Identification of the epitope bound by the antigen-binding domain described herein can be accomplished by various methods known in the art. For example, a crystal structure containing the antigen-binding domain bound or complexed with an antigen can be solved. In another example, an assay, such as a protection assay, can be performed to identify the region of the antigen that contributes to the epitope or to identify the epitope. A representative protection assay, a hydrogen / deuterium exchange (HDX) mass spectrometry assay, is further described in Example 18. HDX mass spectrometry can be performed to identify the putative epitope on human MSLN, e.g., hMSLN, for mouse SS1, e.g., SEQ ID NO: 279, and the M5 scFv described herein, e.g., SEQ ID NO: 43. 296~588 , for example, SEQ ID NO: 278. hMSLN 296~588 For example, SEQ ID NO:278 represents amino acids 296-588 of human mesothelin, e.g., 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. The amino acid sequence of human mesothelin, amino acids 296-588, is provided below (SEQ ID NO:278): [ka]

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

[0214] In one embodiment, the anti-mesothelin binding domain described herein is an antigen binding domain comprising a sequence including SEQ ID NO: 279, e.g., binds to an epitope of human mesothelin that is different from the epitope of human mesothelin targeted by mouse SS1, e.g., SEQ ID NO: 278.

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

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

[0217] In one embodiment, the anti-mesothelin binding domain comprises one or more (e.g., all three) of light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of a human anti-mesothelin binding domain selected from SEQ ID NOs: 39 to 62, and one or more (e.g., all three) of heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of a human anti-mesothelin binding domain selected from SEQ ID NOs: 39 to 62. In one embodiment, the human anti-mesothelin binding domain comprises a light chain variable region described herein (e.g., in Table 2) and / or a heavy chain variable region described herein (e.g., in Table 2). In one embodiment, the anti-mesothelin binding domain comprises an scFv comprising the light chain variable region and heavy chain variable region of the amino acid sequences in Table 2. In certain embodiments, the anti-mesothelin binding domain (e.g., scFV) comprises a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) in the amino acid sequence of a light chain variable region shown in Table 2, or a sequence having 95 to 99% identity to an amino acid sequence of Table 2; and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) in the amino acid sequence of a heavy chain variable region shown in Table 2, but not more than 30, 20, or 10 modifications (e.g., substitutions) in the amino acid sequence of a heavy chain variable region shown in Table 2, or a sequence having 95 to 99% identity to an 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, and a light chain variable region comprising an amino acid sequence described herein, e.g., in Table 2 or 3, is linked to a heavy chain variable region comprising an amino acid sequence described herein, e.g., in Table 2 or 3, via a linker, e.g., a linker described herein. In one embodiment, the humanized anti-mesothelin binding domain comprises a (Gly4-Ser)n linker (SEQ ID NO: 26) (n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4). The light and heavy chain variable regions of an ScFv can be, for example, in either of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0219] In one aspect, the antigen-binding domain portion comprises one or more sequences selected from SEQ ID NOs: 39 to 62. In one aspect, the CAR is one or more sequences selected from SEQ ID NOs: 63 to 86.

[0220] In one aspect, antibodies of the present invention may exist in a variety of other forms, including, for example, Fab, Fab', F(ab'), Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFvs), Fd fragments consisting of a VH domain and a CH1 domain, linear antibodies, single domain antibodies such as sdAbs (either VL or VH), camelid VHH domains, multispecific antibodies formed from antibody fragments such as bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope-binding fragments of antibodies. In one aspect, the antibody fragments provided herein are scFvs. In one example, human scFvs 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 the polypeptide components. The polypeptide components vary so that different amino acid sequences are represented. The polypeptide components can be of any length, for example, from 3 amino acids to over 300 amino acids. A single display library can contain more than one polypeptide component, for example, two polypeptide chains of a Fab. In one exemplary embodiment, a display library can be used to identify anti-mesothelin binding domains. During selection, the polypeptide component of each library member is probed with mesothelin or a fragment thereof, and if the polypeptide component binds to mesothelin, the display library member is identified, typically by being retained on a support.

[0222] The retained display library members are removed from the support and analyzed. Analysis can involve amplification followed by selection under similar or different conditions. For example, positive and negative selection can be alternating. Analysis can also include 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 formats can be used for display libraries. An example is phage display. In phage display, protein components are generally covalently linked to a bacteriophage coat protein. The linkage results from translation of a nucleic acid encoding the protein component fused to the coat protein. Linkages include flexible peptide linkers, protease sites, or amino acids incorporated as a result of suppression of a stop codon. Phage display is described, for example, in U.S. Pat. No. 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. 23(3)344-8. Bacteriophage displaying the protein component can be amplified and obtained using standard phage preparation methods, such as PEG precipitation from the growth medium. After selection of individual display phages, nucleic acids encoding the selected protein component can be isolated after amplification from cells infected with the selected phage or from the phage itself. Individual colonies or plaques can be picked, and the nucleic acid isolated and sequenced.

[0224] Other display formats include cell-based display (see, e.g., WO03 / 029456), protein-nucleic acid fusions (see, e.g., U.S. Pat. No. 6,207,446), ribosome display (see, 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 E. coli periplasmic display (J Immunol Methods. 2005 Nov 22; PMID: 16337958).

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

[0226] In one embodiment, the non-human animal contains at least a portion of a human immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production that contain large fragments of the human Ig locus. Hybridoma technology can be used to produce and select antigen-specific monoclonal antibodies (Mabs) derived from genes with the desired specificity. For example, the XENOMOUSE TM See, Green et al., 1994, Nat. Gen. 7:13-21; US ​​2003-0070185, WO 96 / 34096 (published October 31, 1996) and PCT application PCT / US96 / 05928 (filed April 29, 1996).

[0227] In some instances, scFvs can be produced by methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced, for example, by linking the VH and VL domains using a flexible polypeptide linker. The scFv molecule can include a linker of optimized length and / or amino acid composition (e.g., a Ser-Gly linker). Linker length can significantly affect how the variable regions of an scFv fold and interact. In fact, when short polypeptide linkers (e.g., 5-10 amino acids) are used, intrachain folding is prevented. Interchain folding is also required for two variable regions to join together to form a functional epitope-binding site. For examples of linker orientations and sizes, see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT Publication No. WO2006 / 020258, WO2007 / 024715, which is incorporated herein by reference.

[0228] The scFv may comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues between the VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises the amino acids glycine and serine. In other embodiments, the linker sequence is (Gly4Ser) n(where n is a positive integer greater than or equal to 1) (SEQ ID NO: 135). In one embodiment, the linker can be (Gly4Ser)4 (SEQ ID NO: 27) or (Gly4Ser)3 (SEQ ID NO: 28). Variation in linker length can retain or enhance activity, resulting in superior efficacy in activity tests.

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

[0230] The improved thermal stability of the anti-mesothelin binding domain, e.g., scFv, contributes to the overall subsequent 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 a conventional antibody. In one embodiment, the anti-mesothelin binding domain, e.g., scFv, has a thermal stability that is improved by 1°C compared to a conventional antibody. In another embodiment, the anti-mesothelin binding domain, e.g., scFv, has a thermal stability that is improved by 2°C compared to a conventional antibody. In other embodiments, the anti-mesothelin binding domain, e.g., scFv, has a thermal stability that is improved by 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 compared to a conventional antibody. For example, a comparison can be made between the scFv molecules disclosed herein and the scFv molecules or Fab fragments of the antibodies from which the scFv VH and VL are derived. Thermal stability can be measured using methods known in the art. For example, in one embodiment, Tm can be measured. Methods for measuring Tm and other methods for determining protein stability are described in further detail below.

[0231] Mutations in the scFv (generated by direct mutagenesis of soluble scFv) alter the stability of the scFv and improve the overall stability of the scFv and CART constructs. The stability of the humanized scFv is compared to the murine 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 other embodiments, the anti-mesothelin binding domain, e.g., scFv, has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 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 mutant scFvs can be determined using the assays described in the Examples.

[0233] binding affinity A wide variety of methods for determining binding affinity are known in the art. A typical method for determining binding affinity is to use surface plasmon resonance, which allows for the analysis of real-time biomolecular specific interactions by detecting changes in protein concentration in a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). Surface plasmon resonance is an optical phenomenon. For further description, 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 Johnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0234] In one aspect, the portion of the CAR composition of the invention comprising an antibody or fragment thereof comprises an amino acid sequence homologous to an amino acid sequence described herein, wherein the antibody or fragment thereof retains the desired functional properties of the anti-mesothelin antibody fragment of the invention. In one specific aspect, the CAR composition of the 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 invention comprising an antibody or antibody fragment is engineered by modifying one or more amino acids within one or both of the variable regions (i.e., VH and / or VL), e.g., within one or more CDR regions and / or within one or more framework regions. In one specific aspect, the CAR composition of the invention comprises an antibody fragment. In a further aspect, the antibody fragment comprises an scFv.

[0236] It will be understood by those skilled in the art that antibodies or antibody fragments of the present invention can be further modified so that their amino acid sequence differs (e.g., from wild-type) but the desired activity does not. For example, additional nucleotide substitutions can be made in the protein, leading to amino acid substitutions at "non-essential" amino acid residues. For example, non-essential amino acid residues in the molecule can be substituted with other amino acid residues from the same side chain family. In other embodiments, a string of amino acids can be replaced with a structurally similar string that differs in the order and / or composition of side chain family members, i.e., conservative substitutions can be made in which amino acid residues are replaced with amino acid residues having similar side chains.

[0237] Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), 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] Percent identity, in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences that are the same. Two sequences are "substantially identical" if they have a specified percentage of amino acid residues or nucleotides that are the same (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 over a specified region, or, when not specified, over the entire sequence, when compared and aligned for maximum correspondence over a comparison window, or designated region, using one of the following sequence comparison algorithms, or by manual alignment and visual inspection. Optionally, identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0239] For sequence comparison, generally one sequence acts as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. Methods for aligning sequences for comparison are well known in the art. 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, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computer-controlled implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology).

[0240] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0241] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4:11-17, as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can also be determined using the Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453 algorithm, as incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossom 62 matrix or a 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 aspect, the present invention contemplates modifications of the amino acid sequence of a starting antibody or fragment (e.g., scFv) to produce a functionally equivalent molecule. For example, the VH or VL of an anti-mesothelin binding domain, e.g., an scFv, contained in a CAR can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the starting VH or VL framework region of the anti-mesothelin binding domain, e.g., scFv. The present invention contemplates modifications of the entire CAR construct, for example, modifications in one or more of the amino acid sequences of various domains of the CAR construct, to produce functionally equivalent molecules. 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%, or 99% identity with the starting CAR construct.

[0243] Transmembrane domain With regard to the transmembrane domain, in various embodiments, a CAR can be designed to include a transmembrane domain that is associated with the extracellular domain of the CAR. The transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids associated with the extracellular region of the protein from which the transmembrane domain was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is used in association with one of the other domains of the CAR; for example, in one embodiment, the transmembrane domain can be derived from the same protein from which the signaling domain, costimulatory domain, or hinge domain is derived. In another aspect, the transmembrane domain is not derived from the same protein as any of the other domains of the CAR. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domain of the same or a different surface membrane protein, for example, to minimize interaction with other members of the receptor complex. In one aspect, the transmembrane domain can homodimerize with other CARs on the cell surface of a CAR-expressing cell. In a different aspect, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interaction with the binding domain of a natural binding partner present in the same CAR-expressing cell.

[0244] The transmembrane domain can be derived from natural or recombinant sources. When the origin is natural, the domain can be derived from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain can transmit signals to the intracellular domain whenever the CAR is bound to the target. Transmembrane domains of particular use in the present invention can include at least the transmembrane domains of, for example, the alpha, beta, or zeta chains of the 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 some embodiments, the transmembrane domain is selected from the group consisting of, for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITG It may include at least the transmembrane domains 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 certain examples, the transmembrane domain can be linked to the extracellular region of the CAR, e.g., the antigen-binding domain of the CAR, via a hinge, e.g., a hinge from a human protein. For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge, or a CD8a hinge. In one embodiment, the hinge or spacer comprises (e.g., consists of) the amino acid sequence of SEQ ID NO:2. In one aspect, the transmembrane domain comprises (e.g., consists of) the transmembrane domain of SEQ ID NO:6.

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

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

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

[0249] In some embodiments, the hinge or spacer is [ka] and a hinge encoded by the nucleotide sequence of

[0250] In one aspect, the transmembrane domain may be engineered to contain predominantly hydrophobic residues such as leucine and valine, hi one aspect, triplets of phenylalanine, tryptophan and valine can be found at each end of the engineered transmembrane domain.

[0251] Optionally, a short oligo- or polypeptide linker, 2-10 amino acids in length, can form the linkage between the transmembrane domain and the cytoplasmic signaling region of the CAR. A glycine-serine doublet provides a particularly suitable linker. For example, in one aspect, the linker is [ka] In some embodiments, the linker comprises the amino acid sequence [ka] It is encoded by the nucleotide sequence

[0252] In one aspect, the hinge or spacer comprises a KIR2DS2 hinge and portions thereof.

[0253] Cytoplasmic domain The cytoplasmic domain or region of a CAR comprises an intracellular signaling domain. The intracellular signaling domain is generally responsible for activating at least one of the normal effector functions of the immune cell into which the CAR is introduced. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the entire chain, so long as it transmits the effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0254] Examples of intracellular signaling domains for use in the CARs of the present invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any recombinant sequences that have the same functional capabilities.

[0255] It is known that signals generated by the TCR alone are insufficient for full activation of T cells; secondary and / or costimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains).

[0256] The primary cytoplasmic signaling domain controls the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0257] Examples of ITAM-containing primary intracellular signaling domains that are 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, a CAR of the present invention comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3 zeta.

[0258] In one embodiment, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain, that has altered (e.g., increased or decreased) activity compared to the native ITAM domain. In one embodiment, the primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and / or truncated ITAM-containing primary intracellular signaling domain. In some embodiments, the primary signaling domain comprises one, two, three, four, or more ITAM motifs.

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

[0260] The intracellular domain of the CAR may comprise a CD3 zeta signaling domain alone, or 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 comprise a CD3 zeta chain portion and a costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the CAR that comprises 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 necessary for the efficient response of lymphocytes to antigens. Examples of such molecules include 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 a ligand that specifically binds to 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 costimulatory 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, a short oligo- or polypeptide linker, e.g., 2 to 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length, can be formed between the intracellular signaling domains. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, e.g., alanine, glycine, can be used as a suitable linker.

[0262] In one aspect, the intracellular signaling domain is designed to comprise two or more, e.g., two, three, four, five, or more, costimulatory signaling domains. In some embodiments, the two or more, e.g., two, three, four, five, or more, costimulatory signaling domains are separated by a linker molecule, e.g., a linker molecule described herein. In one embodiment, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

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

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

[0265] In one aspect, a CAR-expressing cell described herein can further comprise a second CAR, e.g., a second CAR to the same target (mesothelin) or a different target, e.g., comprising a different antigen binding domain (e.g., a target other than mesothelin on a stromal cell, e.g., FAP; a target other than mesothelin on a prostate cancer cell, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1, or MAD-CT-2; a target other than mesothelin on an ovarian cancer cell, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor alpha, claudin 6, GloboH, or sperm protein 17; e.g., a target other than mesothelin on a lung cancer cell, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4, or Trop-2). In one embodiment, the CAR-expressing cells comprise a first CAR that targets a first antigen and comprises an intracellular signaling domain with a costimulatory signaling domain but without a primary signaling domain, and a second CAR that targets a second, different antigen and comprises an intracellular signaling domain with a primary signaling domain but without a costimulatory signaling domain. Arrangement of a costimulatory signaling domain, e.g., 4-1BB, CD28, CD27, or OX-40, on the first CAR and a primary signaling domain, e.g., CD3 zeta, on the second CAR can restrict CAR activity to cells where both targets are expressed.In one embodiment, the CAR-expressing cells comprise a first mesothelin CAR that comprises a mesothelin-binding domain, a transmembrane domain, and a costimulatory domain, and a second CAR that targets an antigen other than mesothelin (e.g., a target other than mesothelin on a stromal cell, e.g., FAP; a target other than mesothelin on a prostate cancer cell, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1, or MAD-CT-2; a target other than mesothelin on an ovarian cancer cell, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor alpha, claudin 6, GloboH, or sperm protein 17; e.g., a target other than mesothelin on a lung cancer cell, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4, or Trop-2) and comprises an antigen-binding domain, a transmembrane domain, and a primary signaling domain. In other embodiments, the CAR-expressing cells comprise a first mesothelin CAR comprising a mesothelin-binding domain, a transmembrane domain, and a primary signaling domain, and a second CAR that targets an antigen other than mesothelin (e.g., a target other than mesothelin on a stromal cell, e.g., FAP; a target other than mesothelin on a prostate cancer cell, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1, or MAD-CT-2; a target other than mesothelin on an ovarian cancer cell, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor alpha, claudin 6, GloboH, or sperm protein 17; e.g., a target other than mesothelin on a lung cancer cell, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4, or Trop-2) and comprises an antigen-binding domain for the antigen, a transmembrane domain, and a costimulatory signaling domain.

[0266] In one embodiment, the CAR-expressing cells comprise a mesothelin CAR and an inhibitory CAR described herein. In one embodiment, the inhibitory CAR comprises an antigen-binding domain that binds to an antigen found on normal cells, e.g., normal cells that also express mesothelin, but not on cancer cells. In one embodiment, the inhibitory CAR comprises the antigen-binding domain, transmembrane domain, and intracellular domain of an inhibitory molecule. For example, the intracellular domain of the inhibitory CAR can 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 comprises two or more different CARs, the antigen-binding domains of the different CARs can be such that the antigen-binding domains do not interact with each other. For example, a cell expressing a first CAR and a second CAR can have the antigen-binding domain of the first CAR, e.g., as a fragment, e.g., scFv, that does not associate with the antigen-binding domain of the second CAR, e.g., the antigen-binding domain of the second CAR is a VHH.

[0268] In some embodiments, the antigen-binding domain comprises a single-domain antigen-binding (SDAB) molecule, including molecules whose complementarity-determining regions are part of a single-domain polypeptide. Examples include, but are not limited to, heavy chain variable domains, binding molecules that naturally lack light chains, single domains derived from traditional four-chain antibodies, engineered domains, and single-domain scaffolds other than those derived from antibodies. SDAB molecules can be any of the art or future single-domain molecules. SDAB molecules can be derived from any species, including, but not limited to, mouse, human, camel, llama, lamprey, fish, shark, goat, rabbit, and cow. The term also includes naturally occurring single-domain antibody molecules from species other than camelids and sharks.

[0269] In one aspect, SDAB molecules can be derived from the variable regions of immunoglobulins found in fish, such as those derived from the immunoglobulin isotype known as novel antigen receptors (NARs) found in shark serum. Methods for producing single domain molecules derived from the variable regions of NARs ("IgNARs") are described in WO 03 / 014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

[0270] In another aspect, SDAB molecules are naturally occurring single-domain antigen-binding molecules known as heavy chains lacking light chains.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 a heavy chain molecule naturally lacking light chains is known herein as a VHH or nanobody to distinguish it from the conventional VH of four-chain immunoglobulins.Such VHH molecules can be derived from Camelidae species, such as camel, llama, dromedary, alpaca and guanaco.Species other than Camelidae may naturally produce heavy chain molecules lacking light chains, and such VHHs are within the scope of the present invention.

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

[0272] It has also been discovered that in cells having multiple chimeric membrane-embedded receptors containing antigen-binding domains, interactions between the receptor's multiple antigen-binding domains can be undesirable, for example, because they inhibit the ability of one or more antigen-binding domains to bind to their cognate antigen. Accordingly, disclosed herein are cells having first and second non-naturally occurring chimeric membrane-embedded receptors containing antigen-binding domains in which such interactions have been minimized. Also disclosed herein are nucleic acids encoding first and second non-naturally occurring chimeric membrane-embedded receptors containing antigen-binding domains in which such interactions have been minimized, as well as methods for producing such cells and nucleic acids. In some embodiments, one of the antigen-binding domains of the first and second non-naturally occurring chimeric membrane-embedded receptors comprises an scFv, and the other comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence.

[0273] In some embodiments, the present invention includes a first CAR and a second CAR, wherein the antigen-binding domain of one of the first CAR and the second CAR does not comprise a variable light chain domain and a variable heavy chain domain. In some embodiments, the antigen-binding domain of one of the first CAR and the second CAR is an scFv, and the other is not an scFv. In some embodiments, the antigen-binding domain of one of the first CAR and the second CAR comprises a single VH domain, such as a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen-binding domain of one of the first CAR and the second CAR comprises a nanobody. In some embodiments, the antigen-binding domain of one of the first CAR and the second CAR comprises a camelid VHH domain.

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

[0275] In some embodiments, when displayed on the cell surface, binding of the antigen binding domain of the first CAR to its cognate antigen is not substantially diminished by the presence of the second CAR, ie, binding of the antigen binding domain of the first CAR to its cognate 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 cognate antigen in the absence of the second CAR.

[0276] In some embodiments, when displayed on the cell surface, the antigen binding domains of the first CAR and the second CAR bind to each other less than when both are scFv antigen binding domains, hi some embodiments, the antigen binding domains of the first CAR and the second CAR bind to each other 85%, 90%, 95%, 96%, 97%, 98% or 99% less than when both are scFv antigen binding domains.

[0277] In other aspects, the CAR-expressing cells described herein can further express other agents, e.g., agents that enhance the activity or fitness of the CAR-expressing cells. For example, in one embodiment, the agent can be an agent that inhibits a molecule that regulates or controls, e.g., inhibits, T cell function. In some embodiments, the molecule that regulates or controls T cell function is an inhibitory molecule. Inhibitory molecules, e.g., PD1, in some embodiments, reduce the ability of CAR-expressing cells to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In some embodiments, an agent, such as an inhibitory nucleic acid, for example, a dsRNA, for example, an siRNA or shRNA, as described herein; or an inhibitory protein or inhibitory system, for example, a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription activator-like effector nuclease (TALEN) or a zinc finger endonuclease (ZFN), can be used to inhibit the expression of a molecule that regulates or controls, for example, inhibits, T cell function in CAR-expressing cells. In some embodiments, the agent is an shRNA, for example, an shRNA described herein. In some embodiments, an agent that regulates or controls, for example, inhibits, T cell function is inhibited in CAR-expressing cells. For example, a dsRNA molecule that inhibits the expression of a molecule that regulates or controls, for example, inhibits, T cell function is bound to a nucleic acid encoding a component, for example, all components, of a CAR.

[0278] In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, linked to a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide 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 TGFRbetaT, 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 described herein (e.g., a costimulatory domain (e.g., as described herein, e.g., 41BB, CD27, or CD28) and / or a primary signaling domain (e.g., a co-stim ... In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and / or a 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 al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43).PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immune suppression can be reversed by inhibiting the local interaction of PD1 and PD-L1.

[0279] In one embodiment, the agent comprises the extracellular domain (ECD) of an inhibitory molecule, for example, programmed death 1 (PD1), which can be fused to a transmembrane domain and an intracellular signaling domain such as 41BB and CD3 zeta (also referred to herein as a PD1 CAR). In one embodiment, the PD1 CAR improves T cell persistence when used in combination with a mesothelin CAR described herein. In one embodiment, the CAR is a PD1 CAR comprising the extracellular domain of PD1 shown underlined in SEQ ID NO:24 and a signal sequence that is 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, the PD1 CAR without the N-terminal signal sequence comprises the amino acid sequence provided below (SEQ ID NO: 22). [ka]

[0281] In one embodiment, the agent comprises a nucleic acid sequence encoding a PD1 CAR, e.g., a PD1 CAR described herein, linked to an N-terminal signal sequence. In one embodiment, the nucleic acid sequence of a PD1 CAR is shown below, with the PD1 ECD underlined in SEQ ID NO: 23 below. [ka]

[0282] In another aspect, the present invention provides a population of CAR-expressing cells, e.g., CAR T cells. In some embodiments, 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 can include a first cell expressing a CAR with an anti-CD19 binding domain described herein and a second cell expressing a CAR with a different anti-CD19 binding domain, e.g., an 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 can include, e.g., a first cell expressing a CAR comprising an anti-mesothelin binding domain, as described herein, and a second cell expressing a CAR comprising an antigen binding domain for a target other than mesothelin (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 alpha, claudin 6, GloboH, or sperm protein 17; e.g., a target other than mesothelin 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, a first cell expressing a CAR comprising a primary intracellular signaling domain and a second cell expressing a CAR comprising a secondary signaling domain.

[0283] In another aspect, the invention provides a population comprising at least one cell expressing a CAR having an anti-mesothelin binding domain described herein, and a second cell population expressing another agent, e.g., an agent that enhances the activity or function of the CAR-expressing cell. For example, in one embodiment, the agent can be an agent that modulates or regulates, e.g., inhibits, T cell function. In some embodiments, the molecule that modulates or regulates T cell function is an inhibitory molecule, e.g., an agent described herein. An inhibitory molecule, for example, in some embodiments, can reduce the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide of an inhibitory molecule such as, e.g., 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 a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., a costimulatory domain (e.g., 41BB, CD27, or CD28, e.g., as described herein) and / or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of the extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and / or a CD3 zeta signaling domain described herein).

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

[0285] Regulatable Chimeric Antigen Receptors In some embodiments, a controllable CAR (RCAR), in which CAR activity can be controlled, is desired to optimize the safety and efficacy of CAR therapy. There are many ways in which CAR activity can be controlled. For example, using a caspase fused to a dimerization domain, e.g., 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 the simplest embodiment, in which the components of a standard CAR described herein, e.g., an antigen-binding domain and an intracellular signaling domain, are separated on separate polypeptides or members. In some embodiments, the set of polypeptides comprises a dimerization switch that can link the polypeptides to each other due to the presence of a dimerization molecule, e.g., linking the antigen-binding domain to the intracellular signaling domain.

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

[0287] In some embodiments, the first and second switch domains can form an intracellular or extracellular dimerization switch. In some embodiments, the dimerization switch can be a homodimerization switch, e.g., when the first and second switch domains are the same, or a heterodimerization switch, e.g., when the first and second switch domains are different from one another.

[0288] In some embodiments, an RCAR can comprise a "multi-switch." The multi-switch comprises a heterodimerization switch domain or a homodimerization switch domain. The multi-switch comprises multiple switch domains, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, regardless of the first member, e.g., an antigen-binding member, and the second member, e.g., an intracellular signaling member. In an embodiment, the first member can comprise multiple first switch domains, e.g., FKBP-based switch domains, and the second member can comprise multiple second switch domains, e.g., FRB-based switch domains. In an embodiment, the first member can comprise a first and a second switch domain, e.g., an FKBP-based switch domain and an FRB-based switch domain, and the second member can comprise a first and a second switch domain, e.g., an FKBP-based switch domain and an FRB-based switch domain.

[0289] In some embodiments, the intracellular signaling member comprises one or more intracellular signaling domains, e.g., a primary intracellular signaling domain and one or more costimulatory signaling domains.

[0290] In some embodiments, the antigen binding member can comprise one or more intracellular signaling domains, e.g., one or more costimulatory signaling domains. In some embodiments, the antigen binding member comprises multiple, e.g., two or three, costimulatory signaling domains described herein, e.g., selected from 41BB, CD28, CD27, ICOS, and OX40, and in some embodiments, does not comprise a primary intracellular signaling domain. In some embodiments, the antigen binding member comprises the following costimulatory signaling domains, from extracellular to intracellular: 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 an antigen binding domain, a transmembrane domain, and two costimulatory domains and a first switch domain, e.g., as described herein; and (2) an intracellular signaling domain comprising a transmembrane domain or membrane tethering domain and at least one primary intracellular signaling domain and a second switch domain.

[0291] Some embodiments provide RCARs in which the antigen binding member is not tethered to the surface of the CAR cell. This allows cells having an intracellular signaling member to be conveniently paired with one or more antigen binding domains without transforming the cell with a sequence encoding the antigen binding member. In such embodiments, the RCAR comprises: 1) an intracellular signaling member comprising a first switch domain, a transmembrane domain, and 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 comprise a transmembrane domain or a membrane tethering domain, and optionally does not comprise an intracellular signaling domain, comprising an antigen binding domain and a second switch domain, e.g., as described herein. In some embodiments, the RCAR can further comprise: 3) 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 dual specificity activation and targeting capabilities. In this embodiment, the antigen-binding member can comprise multiple, for example, two, three, four, or five, antigen-binding domains, such as scFvs, where each antigen-binding domain binds to a target antigen, for example, a different antigen, or the same antigen, for example, the same or different epitopes on the same antigen. In some embodiments, the multiple antigen-binding domains are in tandem, and optionally, a linker or hinge region is disposed between each of the antigen-binding domains. Suitable linkers and hinge regions are described herein.

[0293] One embodiment provides an RCAR with a configuration that allows proliferation switching. In this embodiment, the RCAR comprises: 1) an intracellular signaling member that optionally comprises a transmembrane domain or membrane tethering 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 that does not comprise a switch domain or does not comprise a switch domain that dimerizes with a switch domain on the intracellular signaling member, e.g., an antigen binding member that comprises an antigen binding domain, a transmembrane domain, and a primary intracellular signaling domain, e.g., a CD3 zeta domain, as described herein. In some embodiments, the antigen binding member does not comprise a costimulatory signaling domain. In some embodiments, the intracellular signaling member comprises a switch domain from a homodimerization switch. In some embodiments, the intracellular signaling member comprises a first switch domain of a heterodimerization switch, and the RCAR comprises a second intracellular signaling member that comprises a second switch domain of the heterodimerization switch. In such embodiments, the second intracellular signaling member comprises the same intracellular signaling domain as the intracellular signaling member. In some embodiments, the dimerization switch is intracellular. In some embodiments, the dimerization switch is extracellular.

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

[0295] Also provided herein are cells comprising the RCARs described herein. Any cells engineered to express RCAR can be used as RCARX cells. In some embodiments, the RCARX cells are T cells and are referred to as RCAR T cells. In some embodiments, the RCARX cells are NK cells and are referred to as RCARN cells.

[0296] Also provided herein are nucleic acids and vectors comprising RCAR-encoding sequences. Sequences encoding various components of an RCAR can be located on the same nucleic acid molecule, e.g., the same plasmid or vector, e.g., a viral vector, e.g., a lentiviral vector. In some embodiments, (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., vector. Production of the corresponding proteins can be achieved, for example, by using separate promoters or by using a bicistronic transcription product (which can produce two proteins by cleavage of a single translation product or by translation of two separate protein products). In some embodiments, a sequence encoding a cleavable peptide, e.g., a P2A or F2A sequence, is located between (i) and (ii). In some embodiments, a sequence encoding an IRES, e.g., an EMCV or EV71 IRES, is located between (i) and (ii). In these embodiments, (i) and (ii) are transcribed as a single RNA. In some embodiments, a first promoter is operably linked to (i) and a second promoter is operably linked to (ii) such that (i) and (ii) are transcribed as separate mRNAs.

[0297] Alternatively, the sequences encoding the various components of the RCAR can be located on different nucleic acid molecules, e.g., different plasmids or vectors, e.g., viral vectors, e.g., lentiviral vectors. For example, (i) a sequence encoding an antigen-binding member can be located on a first nucleic acid, e.g., a first vector, and (ii) a sequence encoding an intracellular signaling member can be present on a second nucleic acid, e.g., a second vector.

[0298] Dimerization switch Dimerization switches can be non-covalent or covalent. In non-covalent dimerization switches, the dimerization molecule promotes non-covalent interactions between the switch domains. In covalent dimerization switches, the dimerization molecule promotes covalent interactions between the switch domains.

[0299] In some embodiments, the RCAR comprises an FKBP / FRAP or FKBP / FRB-based dimerization switch. FKBP12 (FKBP or FK506-binding protein) is an abundant cytoplasmic protein that serves as the initial intracellular target of the natural product immunosuppressant rapamycin. Rapamycin binds to FKBP and the large PI3K homolog FRAP (RAFT, mTOR). FRB is a 93-amino acid portion of FRAP that is sufficient for binding of 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, an FKBP / FRAP, e.g., an FKBP / FRB-based switch can use a dimerization molecule, e.g., rapamycin or a rapamycin analog.

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

[0302] In some embodiments, the FKBP switch domain can comprise an FRB-binding fragment of FKBP, for example, the underlined portion of SEQ ID NO: 382, ​​which is as follows: [ka]

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

[0304] "FKBP / FRAP, e.g., FKPP / FRB-based switch," as that term is used herein, includes an FKBP-binding fragment or FKBP analog, e.g., RAD001, having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the FKBP sequence of SEQ ID NO: 382 or 383, or 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid sequence. and a second switch domain comprising an FKBP-binding fragment or FRB analog and having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the FRB sequence of SEQ ID NO: 384, or differing by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue. In one embodiment, an RCAR described herein comprises one switch domain comprising the amino acid residues disclosed in SEQ ID NO: 382 (or SEQ ID NO: 383) and one switch domain comprising 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, e.g., increased affinity, for a dimerization molecule, e.g., rapamycin or a rapalog, e.g., RAD001. In certain embodiments, the modified FRB switch domain comprises one or more, e.g., two, three, four, five, six, seven, eight, nine, ten, or more mutations selected from mutations at amino acid positions L2031, E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105, and F2108, where the wild-type amino acid is mutated to any other naturally occurring amino acid. In some embodiments, the mutant FRB comprises a mutation at 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 some embodiments, the mutant FRB comprises a mutation at T2098, where T2098 is mutated to phenylalanine (T2098F) or leucine (T2098L) (e.g., SEQ ID NO: 387). In some embodiments, the mutant FRB comprises mutations at 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 some embodiments, the mutant FRB comprises an E2032I and a T2098L mutation (e.g., SEQ ID NO: 389). In some embodiments, the mutant FRB comprises an E2032L and a T2098L mutation (e.g., SEQ ID NO: 340).

[0306] [Table 1]

[0307] Other suitable dimerization switches include a GyrB-GyrB-based dimerization switch, a gibberellin-based dimerization switch, a tag / binder dimerization switch, and a halotag / snaptag dimerization switch. Following the guidance provided herein, such switches and suitable dimerization molecules will be apparent to those of skill in the art.

[0308] dimerization molecule Association between the switch domains is promoted by a dimerization molecule. In the presence of a dimerization molecule, interaction or association between the switch domains allows signaling between a polypeptide associated with, e.g., fused to, a first switch domain and a polypeptide associated with, e.g., fused to, a second switch domain. In the presence of a non-limiting level of a dimerization molecule, signaling increases by 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 5-fold, 10-fold, 50-fold, or 100-fold, e.g., as measured in a system described herein.

[0309] Rapamycin and rapamycin analogs (sometimes referred to as rapalogs), e.g., RAD001, can be used as dimerization molecules in the FKBP / FRB-based dimerization switches described herein. In some embodiments, the dimerization molecule is selected from rapamycin (sirolimus), RAD001 (everolimus), zotarolimus, temsirolimus, AP-23573 (ridaforolimus), biolimus, and AP21967. Additional rapamycin analogs suitable for use with FKBP / FRB-based dimerization switches are further described in the chapter entitled "Combination Therapies" or the subsection entitled "Representative mTOR Inhibitors."

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

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

[0312] In one embodiment, in vitro transcribed RNA CAR 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 source can be directly converted into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase by PCR. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable source of DNA. The desired template for in vitro transcription is the CAR of the present invention. For example, the template for RNA CAR comprises an extracellular region comprising the single-chain variable domain of an anti-tumor antibody; a hinge region, a transmembrane domain (for example, the transmembrane domain of CD8a); and a cytoplasmic region comprising 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 contains an open reading frame. The DNA can be derived from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the nucleic acid can include some or all of the 5' and / or 3' untranslated regions (UTRs). The nucleic acid can 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 can alternatively be an artificial DNA sequence that is not normally expressed in naturally occurring organisms. An exemplary artificial DNA sequence is one that includes portions of genes ligated together to form an open reading frame encoding a fusion protein. The portions of DNA ligated together can be from a single organism or from more than one organism.

[0314] PCR is used to generate templates for in vitro transcription of mRNA for use in gene transfer. Methods for performing PCR are well known in the art. Primers used in PCR contain regions that are substantially complementary to regions of DNA used as templates for PCR. The term "substantially complementary" refers to a sequence of nucleotides in which most or all of the bases in the primer sequence are complementary, or in which one or more bases are non-complementary or mismatched. A substantially complementary sequence 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 portion of the DNA template. For example, primers can be designed to amplify a portion of a nucleic acid that is normally transcribed in cells (open reading frame), including the 5' UTR and 3' UTR. Primers can also be designed to amplify a portion of a nucleic acid encoding a specific desired domain. In one embodiment, primers are designed to amplify the coding region of a 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 the art. A "forward primer" is a primer that contains a region of nucleotides that are substantially complementary to nucleotides on a DNA template that are upstream of the DNA sequence to be amplified. The term "upstream" refers to a position 5' to the DNA sequence to be amplified relative to the coding strand. A "reverse primer" refers to a primer that contains a region of nucleotides that are substantially complementary to a double-stranded DNA template that is downstream of the DNA sequence to be amplified. The term "downstream" refers to a position 3' to the DNA sequence to be amplified relative to the coding strand.

[0315] Any DNA polymerase useful in PCR can be used in the methods disclosed herein. Reagents and polymerases are commercially available from a number of sources.

[0316] Chemical structures capable of promoting stability and / or translation efficiency may also be used. The RNA preferably comprises a 5'UTR and a 3'UTR. In one embodiment, the 5'UTR is 1 to 3,000 nucleotides long. The lengths of the 5'UTR and 3'UTR sequences added to the coding region can be varied by various methods, including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this method, those skilled in the art can modify the 5'UTR and 3'UTR lengths required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0317] The 5'UTR and 3'UTR can be the naturally occurring endogenous 5'UTR and 3'UTR of the desired nucleic acid. Alternatively, a UTR sequence that is not endogenous to the desired nucleic acid can be added by incorporating the UTR sequence into the forward and reverse primers or by some other modification of the template. The use of a UTR sequence that is not endogenous to the desired nucleic acid can 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 characteristics of UTRs that are well known in the art, 3'UTRs can be selected or designed to increase the stability of transcribed RNA.

[0318] In one embodiment, the 5'UTR can contain the Kozak sequence of the endogenous nucleic acid. Alternatively, when adding a non-endogenous 5'UTR to the desired nucleic acid by PCR as described above, a consensus Kozak sequence can be redesigned by adding a 5'UTR sequence. While the Kozak sequence can increase the translation efficiency of some RNA transcripts, it is not believed to be necessary for all RNAs to enable efficient translation. The necessity of the Kozak sequence for many mRNAs is known in the art. In another embodiment, the 5'UTR is the 5'UTR of an RNA virus whose RNA genome is stable in cells. In another embodiment, various nucleotide analogs can be used in the 3' or 5'UTR to prevent exonuclease degradation of the mRNA.

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

[0320] In a preferred embodiment, the mRNA has a cap on both the 5' end and a 3' poly(A) tail, which determines ribosome binding, translation initiation, and stability in cells. In a circular DNA template, such as plasmid DNA, RNA polymerase produces a long concatemeric product that is not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the end of the 3' UTR, even if polyadenylated after transcription, results in a normal-sized mRNA that is not effective for eukaryotic 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 traditional method for incorporating a polyA / T stretch into a DNA template is molecular cloning. However, integration of a polyA / T sequence into plasmid DNA can cause the plasmid to become unstable, 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 the cloning-free construction of DNA templates with a polyA / T 3' stretch is highly desirable.

[0323] The poly(A) / T segment of the transcription DNA template can be generated by any other method, including, but not limited to, DNA ligation or in vitro recombination, during or after PCR using a reverse primer containing a poly(T) tail, such as a 100T tail (SEQ ID NO: 31) (the size can range from 50 to 5000T (SEQ ID NO: 32)). The poly(A) tail also stabilizes the RNA and reduces 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 poly(A) tail of RNA can be further extended after in vitro transcription using a poly(A) polymerase, such as E. coli poly(A) polymerase (E-PAP). In one embodiment, extending the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides (SEQ ID NO: 34) results in an approximately two-fold increase in RNA translation efficiency. Furthermore, linking different chemical groups to the 3' end can increase mRNA stability. Such linkages can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further enhance RNA stability.

[0325] A 5' cap also provides stability to an RNA molecule. In a preferred embodiment, the RNA produced by the methods 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] The RNA produced by the methods disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and promotes translation initiation. Any solute suitable for cell electroporation may be included, including factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and detergents.

[0327] RNA can be introduced into target cells using any of a number of different methods, including, but not limited to, commercially available methods, including 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 biolistic particle delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

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

[0329] Accordingly, 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 stimulatory domain. In one embodiment, the anti-mesothelin binding domain is an anti-mesothelin binding domain described herein, e.g., an anti-mesothelin binding domain comprising 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 costimulatory 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 the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain comprises 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, SEQ ID NO:3, 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 costimulatory 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, 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. In one embodiment, the costimulatory domain comprises the sequence of SEQ ID NO: 7 or a sequence having 95-99% identity thereto. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of 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 thereto, and the sequence of SEQ ID NO:9 or SEQ ID NO:10, or a sequence having 95-99% identity thereto, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.In another aspect, the invention provides a leader sequence of SEQ ID NO:1, an scFv domain having 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, 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 a sequence with 95-99% identity thereto), an scFv domain having a sequence selected from the group consisting of SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 (or a sequence having 95 to 99% identity thereto), a transmembrane domain having the sequence of SEQ ID NO:6 (or a sequence having 95 to 99% identity thereto), a 4-1BB costimulatory domain having the sequence of SEQ ID NO:7 (or a sequence having 95 to 99% identity thereto) or a CD27 costimulatory domain having the sequence of SEQ ID NO:8 (or a sequence having 95 to 99% identity thereto), and a CD3 zeta stimulatory domain having the sequence of SEQ ID NO:9 or SEQ ID NO:10 (or a sequence having 95 to 99% identity thereto).

[0330] In another aspect, the invention relates to an isolated polypeptide molecule encoded by the nucleic acid molecule. In one embodiment, the isolated polypeptide molecule comprises a sequence selected from the group consisting of SEQ ID NO: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, and SEQ ID NO:86, or a sequence having 95-99% identity thereto.

[0331] In another aspect, the invention pertains 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 comprising a stimulatory domain, wherein the nucleic acid encoding the anti-mesothelin-binding domain comprises a sequence selected from the group consisting of SEQ ID NO:111; SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, or a sequence with 95-99% identity thereto.

[0332] In one embodiment, the encoded CAR 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), and 4-1BB (CD137). In one embodiment, the costimulatory domain comprises 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 the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment, the transmembrane domain comprises the sequence of SEQ ID NO: 6. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and a 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, wherein the sequences comprising the intracellular signaling domain are 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 another aspect, the invention pertains 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 with 95-99% identity thereto, a hinge region of SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5, a transmembrane domain having a sequence of SEQ ID NO: 6, a 4-1BB costimulatory domain having a sequence of SEQ ID NO: 7 or a CD27 costimulatory domain having a sequence of SEQ ID NO: 8, and a CD3 zeta stimulatory domain having a sequence of SEQ ID NO: 9 or SEQ ID NO: 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 with 95-99% identity thereto.

[0334] The present invention further provides a vector comprising a CAR transgene. In one aspect, the CAR vector can be directly transduced into cells, such as T cells or NK cells. In one aspect, the vector is a cloning or expression vector, such as, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral constructs, and lentiviral vector constructs. In one aspect, the vector can express the CAR construct in mammalian T cells or NK cells. In one aspect, the mammalian T cells are human T cells or human NK cells.

[0335] The present invention also includes CAR-encoding RNA constructs that can be directly transfected into cells, e.g., T cells or NK cells. Methods for producing mRNA for use in transfection include in vitro transcription (IVT) of a template using specially designed primers, followed by poly(A) addition, to produce a construct generally 50-2000 bases in length, containing 3' and 5' untranslated sequences ("UTR"), a 5' cap and / or internal ribosome entry site (IRES), the gene to be expressed, and a poly(A) tail. The RNA thus produced can be efficiently transfected into different cell types. In one aspect, the template contains the sequence for the CAR.

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

[0337] vector The present invention also provides a vector into which the DNA of the present invention is inserted. Retrovirus-derived vectors, such as lentivirus, are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentivirus vectors have the added advantage over oncoretrovirus-derived vectors, such as murine leukemia viruses, of being able to transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0338] In one embodiment, the vector comprising the nucleic acid encoding the desired CAR of the present invention is a DNA, RNA, plasmid, adenoviral vector, lentiviral vector, or retroviral vector.

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

[0340] In summary, the expression of natural or synthetic nucleic acid encoding CAR is generally achieved by operably linking the nucleic acid encoding CAR polypeptide or a part thereof to a promoter, and incorporating the construct into an expression vector.The vector can be suitable for replication and integration in eukaryotes.Typical cloning vectors include transcription and translation terminators, initiation sequences and promoters that are useful for controlling the expression of desired nucleic acid sequences.

[0341] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, the entire contents of which are incorporated herein by reference. In another embodiment, the present invention provides a gene therapy vector.

[0342] Nucleic acids can be cloned into a wide variety of vectors, including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particularly desirable vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0343] Furthermore, expression vectors can be provided to cells in the form of viral vectors.Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY, and 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, suitable vectors include a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and US Patent No. 6,326,193).

[0344] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Using methods known in the art, a selected gene can be introduced into a vector and packaged into retroviral particles. The recombinant virus is then isolated and delivered to target cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0345] Additional promoter elements, such as enhancers, control the frequency of transcription initiation. Typically, these are located in the region 30–110 bp upstream of the start site, although many promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, allowing promoter function to be preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements may function cooperatively or independently to activate 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 a CAR transgene 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, e.g., Milone et al., Mol. Ther. 17(8):1453-1464 (2009). In one aspect, the EF1 promoter comprises the sequence provided in SEQ ID NO: 11.

[0347] Another example of a promoter is the immediate-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 linked to it. However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters, including, but not limited to, 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 contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can activate expression of an operably linked polynucleotide sequence when expression is desired and shut off such expression when expression is not desired. Examples of inducible promoters include, but are not limited to, a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

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

[0349] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that are not present in or expressed by recipient organisms or tissues and encode polypeptides whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at an appropriate time after DNA is introduced into recipient cells. Suitable reporter genes include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be produced using known techniques or obtained commercially. Generally, the construct with the minimal 5' flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate the ability of agents to modulate promoter-driven transcription.

[0350] In one embodiment, the vector can further comprise a nucleic acid encoding a second CAR. In one embodiment, the second CAR comprises an antigen-binding domain for, for example, a target other than mesothelin on stromal cells, such as FAP; a target other than mesothelin on prostate cancer cells, such as 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, such as Tn, PRSS21, CD171, Lewis Y, folate receptor alpha, claudin 6, GloboH, or sperm protein 17; or a target other than mesothelin on lung cancer cells, such as 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 comprises an intracellular signaling domain with a costimulatory signaling domain but without a primary signaling domain, and a nucleic acid sequence encoding a second CAR that targets a second, different antigen and comprises an intracellular signaling domain with a primary signaling domain but without a costimulatory signaling domain. In one embodiment, the vector comprises a nucleic acid encoding a first mesothelin CAR comprising a mesothelin-binding domain, a transmembrane domain, and a costimulatory domain, and a nucleic acid encoding a second CAR that targets an antigen other than mesothelin (e.g., a target other than mesothelin on a stromal cell, e.g., FAP; a target other than mesothelin on a prostate cancer cell, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1, or MAD-CT-2; a target other than mesothelin on an ovarian cancer cell, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor alpha, claudin 6, GloboH, or sperm protein 17; e.g., a target other than mesothelin on a lung cancer cell, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4, or Trop-2) and comprises an antigen-binding domain, a transmembrane domain, and a primary signaling domain.In other embodiments, the vector comprises a nucleic acid encoding a first mesothelin CAR comprising a mesothelin-binding domain, a transmembrane domain, and a primary signaling domain, and a nucleic acid encoding a second CAR that targets an antigen other than mesothelin (e.g., a target other than mesothelin on a stromal cell, e.g., FAP; a target other than mesothelin on a prostate cancer cell, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1, or MAD-CT-2; a target other than mesothelin on an ovarian cancer cell, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor alpha, claudin 6, GloboH, or sperm protein 17; e.g., a target other than mesothelin on a lung cancer cell, e.g., VEGF, HER3, IGF-1R, EGFR, DLL4, or Trop-2) and comprises an antigen-binding domain for the antigen, a transmembrane domain, and a costimulatory signaling domain.

[0351] In one embodiment, the vector comprises a nucleic acid encoding a mesothelin CAR described herein and a nucleic acid encoding an inhibitory CAR. In one embodiment, the inhibitory CAR comprises an antigen-binding domain that binds to an antigen found on normal cells, such as normal cells that also express CLL, but not on cancer cells. In one embodiment, the inhibitory CAR comprises the antigen-binding domain, transmembrane domain, and intracellular domain of an inhibitory molecule. For example, the intracellular domain of the inhibitory CAR can 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 a mesothelin CAR described herein and an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, e.g., as described herein.

[0353] Methods for introducing and expressing genes into cells are known in the art. In relation to expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, the expression vector 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, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or foreign nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY. A preferred method for introducing polynucleotides into host cells is 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, especially retroviral vectors, have become the most widely used method for gene insertion into mammalian, e.g., human cells.Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, and adeno-associated virus, etc.See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0356] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A typical colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle). Other methods for advanced targeted delivery of nucleic acids are available, such as delivery of polynucleotides in targeted nanoparticles or other suitable submicron-sized delivery systems.

[0357] When a non-viral delivery system is utilized, a representative delivery vehicle is a liposome. The use of lipid formulations for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo) is contemplated. In other aspects, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated within the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, bound to the liposome by a linking molecule that binds both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained as a suspension in lipid, contained in or complexed with a micelle, or otherwise associated with lipid. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure such as a micelle, or have a "collapsed" structure. Alternatively, they may simply be dispersed in solution, forming aggregates that may not be uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include the 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] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids 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. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the production of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. Phospholipids form spontaneously when suspended in an excess of aqueous solution. The lipid components undergo self-reorganization, subsequently forming closed structures that trap water between the lipid bilayers and dissolve solutes (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions that assume structures other than the usual vesicular structure in solution are also encompassed. For example, lipids may exist as micellar structures or simply heterogeneous aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0359] Regardless of the method used to introduce foreign nucleic acid into host cells or otherwise expose the cells to an inhibitor of the invention, a variety of assays can be performed to confirm the presence of the recombinant DNA sequence within the host cells. Such assays include "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR, and PCR; "biochemical" assays that detect the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot), or by the assays described herein to identify agents within the scope of the invention.

[0360] The present invention further provides a vector comprising a CAR-encoding nucleic acid molecule. In one aspect, the CAR vector can be directly transduced into a cell, e.g., a T cell or an NK cell. In one aspect, the vector is a cloning vector or an expression vector, such as, but not limited to, one or more of a plasmid (e.g., an expression plasmid, a cloning vector, a minicircle, a minivector, a double minute chromosome), a retroviral construct, and a lentiviral vector construct. In one aspect, the vector is capable of expressing the CAR construct in a mammalian T cell. In one aspect, the mammalian T cell is a human T cell. In one aspect, the mammalian cell is a human NK cell.

[0361] Origin of cells Prior to expansion and genetic modification, the source of the cells (e.g., T cells or NK cells) is obtained from a subject. The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some aspects of the invention, any number of T cell lines available in the art can be used. In some aspects of the invention, T cells are isolated using Ficoll immunoglobulin (FICULT) immunoglobulin (IGI) or immunoglobulin (IGI) ... TMCells can be obtained from a unit of blood collected from a subject using any number of methods known to those of skill in the art, such as cell separation. In one preferred aspect, cells from an individual's circulating blood are obtained by apheresis. The apheresis product generally contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In one aspect of the invention, cells are washed with phosphate-buffered saline (PBS). In another aspect, the wash solution lacks calcium, may lack magnesium, or may lack many, if not all, divalent cations. An initial activation step in the absence of calcium can extend activation. As one of skill in the art will readily recognize, washing steps can be accomplished by methods known to those of skill in the art, such as using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate, or a Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, saline solution with or without PlasmaLyte A or other buffering agents. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0362] In one aspect, T cells are lysed by lysing red blood cells and monocytes, e.g., by PERCOLL TM CD3 is isolated from peripheral blood lymphocytes by depletion through gradient centrifugation or counterflow centrifugal 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., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a period of time sufficient for positive selection of the desired T cells. In one aspect, the period is about 30 minutes. In a further aspect, the period ranges from 30 minutes to 36 hours or longer, and all integer values ​​therebetween. In a further aspect, the period is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. In yet another preferred aspect, the 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 times, 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 from immunocompromised individuals. Additionally, the use of long incubation times can improve the isolation of CD8 + The efficiency of T cell capture may be increased. Therefore, by simply shortening or lengthening the time that T cells are allowed to bind to the CD3 / CD28 beads and / or by increasing or decreasing the bead-to-T cell ratio (described further herein), subpopulations of T cells can be preferentially selected for or eliminated at the initiation of culture or at other time points during treatment. Furthermore, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, subpopulations of T cells can be preferentially selected for or eliminated at the initiation of culture or at other desired time points. Those skilled in the art will recognize that multiple rounds of selection can also be used in the context of the present invention. In some aspects, it may be desirable to use "unselected" cells during the activation and expansion process using a selection procedure. "Unselected" cells can also be subjected to further selection.

[0363] Enrichment of a T cell population by negative selection can be achieved by combining antibodies directed against surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, negative selection can enrich for CD4 + To enrich for cells, the monoclonal antibody cocktail generally includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. + , CD25 + , CD62Lhi, GITR + and FoxP3 + Alternatively, in one aspect, T regulatory cells are depleted using anti-C25 conjugated beads or other similar selection methods.

[0364] In one embodiment, a T cell population can be selected that expresses one or more of 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, e.g., other cytokines. Methods for screening cell expression can be determined, for example, by the methods described in PCT Publication WO2013 / 126712.

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

[0366] In one embodiment, T cell population is Ikaros-deficient.Ikaros-deficient cell refers to the cell that does not express Ikaros RNA or protein or has reduced or inhibited Ikaros activity, and Ikaros-deficient cell can be produced by genetic methods, for example, by administering RNA interference agent, such as siRNA, shRNA, miRNA, to reduce or block Ikaros expression.Alternatively, Ikaros-deficient cell can be produced by treating with Ikaros inhibitor, for example, lenalidomide.

[0367] In some embodiments, the T cell population is DGK- and Ikaros-deficient, e.g., does not express DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity. Such DGK- and Ikaros-deficient cells can be produced by any of the methods described herein.

[0368] To isolate a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain aspects, it may be desirable to significantly reduce the volume in which the beads and cells are combined (e.g., increase the concentration of cells) to ensure maximum contact between the cells and beads. For example, in one aspect, a concentration of 2 billion cells / ml is used. In one aspect, 1 billion cells / ml is used. In a further aspect, greater than 100 million cells / ml is used. In a further aspect, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In a further aspect, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In a further aspect, a concentration of 125 or 150 million cells / ml can be used. The use of high concentrations can result in 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 a desired target antigen, such as CD28-negative T cells, or cells from samples containing many tumor cells (e.g., leukemia blood, tumor tissue, etc.). Such a population of cells may have therapeutic value and is desirable to obtain. For example, the use of high cell concentrations can be used to capture CD8 T cells that normally have weak CD28 expression. + Allows for more efficient selection of T cells.

[0369] In a related aspect, it may be desirable to use a low concentration of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), particle-cell interactions are minimized. Cells that express high amounts of the desired antigen that binds to the particles are selected for. For example, CD4 + T cells express high levels of CD28 and, at dilute concentrations, CD8 + In one aspect, the concentration of cells used is 5×10e 6 In another aspect, the concentration used is about 1 x 10 5 / ml ~ 1 × 10 6 / ml and any integer value therebetween.

[0370] In other aspects, the cells may be incubated on a rotator for various lengths of time at various speeds at 2-10° C. or at room temperature.

[0371] Stimulatory T cells may also be frozen after a washing step. Without wishing to be bound by theory, the freezing and subsequent thawing step provides a more uniform product by removing granulocytes and, to some extent, monocytes in the cell population. After a washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and are useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, or 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing media, including, for example, Hespan and PlasmaLyte A. The cells are then frozen at -80°C at a rate of 1°C / min and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as immediate uncontrolled freezing at -20°C or in liquid nitrogen, can be used.

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

[0373] Also contemplated in the context of the present invention is the collection of a blood sample or apheresis product from a subject at a time before the expanded cells described herein are needed. That is, the source of the cells to be expanded can be collected at any time necessary, 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, a blood sample or apheresis is taken from a generally healthy subject. In some aspects, a blood sample or apheresis is taken from a generally healthy subject who is at risk for developing a disease but has not yet developed the disease, and desired cells are isolated and frozen for subsequent use. In some aspects, T cells can be expanded, frozen, and used at a later time. In some aspects, a sample is isolated from a patient shortly after diagnosis of a particular disease described herein, but prior to treatment. In a further aspect, the cells are isolated from a blood sample or apheresis from the subject prior to any number of appropriate treatment modalities, including, but not limited to, treatment with such means as natalizumab, efalizumab, antivirals, chemotherapeutics, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228 and irradiation. These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase, which is important in 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 aspect, cells are isolated from a patient and frozen for subsequent use in conjunction with (e.g., before, concurrently with, or after) bone marrow or stem cell transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), T cell ablative therapy using cyclophosphamide or antibodies such as OKT3 or CAMPATH. In one aspect, cells can be pre-isolated and frozen for subsequent treatment after B cell ablative therapy, such as an agent reactive with CD20, e.g., Rituxan.

[0374] In a further aspect of the present invention, T cells are obtained directly from a patient after treatment that leaves the subject with functional T cells. In this regard, it has been observed that following certain cancer treatments, particularly treatment with drugs that damage the immune system, the quality of the T cells obtained is optimal or perhaps even improved in their ability to be expanded ex vivo immediately following treatment, during the period when the patient is typically in the recovery phase from treatment. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a state favorable for enhanced engraftment and in vivo expansion. Therefore, in the context of the present invention, it is contemplated that blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, may be harvested during this recovery period. Furthermore, in certain aspects, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create conditions favorable for the repopulation, recirculation, regeneration, and / or proliferation of specific cell types in the subject, particularly for a defined time frame following treatment. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0375] In some embodiments, the NK cells are obtained from a subject. In other embodiments, the NK cells are an NK cell line, e.g., the NK-92 cell line (Conkwest).

[0376] Similar Car In embodiments described herein, the immune effector cells can be allogeneic immune effector cells, e.g., T cells or NK cells. For example, the cells can be allogeneic T cells, e.g., allogeneic T cells that lack expression of a functional T cell receptor (TCR) and / or human leukocyte antigen (HLA), e.g., HLA class I and / or HLA class II.

[0377] T cells that lack a functional TCR can be, for example, engineered to not express any functional TCR on their surface, engineered to not express one or more subunits that comprise a functional TCR, or engineered to produce only a small amount of functional TCR on their surface. Alternatively, T cells can express a substantially impaired TCR, for example, by expression of mutated or truncated forms of one or more subunits of the TCR. The term "substantially impaired TCR" means that the TCR does not elicit a harmful immune response in the host.

[0378] The T cells described herein can be engineered, for example, to not express functional HLA on their surface. For example, the T cells described herein are engineered to downregulate cell surface expression of HLA, e.g., HLA class I and / or HLA class II.

[0379] In some embodiments, the T cells may lack a functional TCR and a functional HLA, e.g., HLA class I and / or HLA class II.

[0380] Modified T cells lacking expression of a functional TCR and / or HLA can be obtained by any suitable means, including knockout or knockdown of one or more subunits of the TCR or HLA. For example, T cells can include knockdown of the TCR and / or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR) transcription activator-like effector nuclease (TALEN), or zinc finger endonuclease (ZFN).

[0381] In some embodiments, the allogeneic cells are cells that do not express or express at low levels an inhibitory molecule, e.g., by any of the methods described herein. For example, the cells are cells that do not express or express at low levels an inhibitory molecule that can, for example, reduce the ability of a CAR-expressing cell 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. Inhibition of inhibitory molecules, e.g., by inhibition at the DNA, RNA, or protein level, can optimize CAR-expressing cell performance. In some embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription activator-like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), e.g., as described herein, can be used.

[0382] siRNA and shRNA for inhibiting TCR or HLA In certain embodiments, TCR expression and / or HLA expression can be inhibited using siRNA or shRNA targeting nucleic acids encoding TCR and / or HLA in T cells.

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

[0384] Representative shRNAs that downregulate the expression of components of the TCR 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" or "CRISPR for TCR and / or HLA" or "CRISPR for inhibiting TCR and / or HLA" refers to a system comprising a set of clustered regularly interspaced short palindromic repeats or repeats of such a set. As used herein, "Cas" refers to a CRISPR-associated protein. A "CRISPR / Cas" system is a CRISPR- and Cas-derived system that can be used to silence or mutate TCR and / or HLA genes.

[0386] Naturally occurring CRISPR / Cas systems are found in approximately 40% of sequenced eubacterial 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 foreign genetic elements 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, enhancing, or altering specific genes) in eukaryotic organisms such as mice or primates. Wiedenheft et al. (2012) Nature 482:331-8. This is achieved by introducing into eukaryotic cells a plasmid containing a specially designed CRISPR and one or more appropriate Cass.

[0388] CRISPR sequences, sometimes referred to as CRISPR loci, contain alternating repeats and spacers. In naturally occurring CRISPRs, the spacers usually contain sequences foreign to the bacterium, such as plasmid or phage sequences, while in TCR and / or HLA CRISPR / Cas systems, the spacers are derived from TCR or HLA gene sequences.

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

[0390] Because they occur naturally in many different types of bacteria, the exact location of CRISPRs and 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, Cse (Cas subtype, E. coli) proteins (e.g., CasA) form a functional complex, Cascade, which processes CRISPR RNA transcripts into spacer repeat units that retain 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. In Pyrococcus furiosus and other prokaryotes, Cmr (Cas RAMP module) proteins form a functional complex with small CRISPR RNAs, which recognize and cleave complementary target RNAs. A simpler CRISPR system relies on the protein Cas9, a nuclease with two active cleavage sites, one for each strand of the double helix. The combination of Cas9 and modified CRISPR locus RNA can be used in a system for gene editing. Pennisi (2013) Science 341:833-836.

[0391] Therefore, the CRISPR / Cas system can be used to edit TCR and / or HLA genes (add or remove base pairs) or introduce premature stops, thereby reducing TCR and / or HLA expression. Alternatively, the CRISPR / Cas system can be used like RNA interference, blocking TCR and / or HLA genes in a reversible manner. In mammalian cells, for example, RNA can guide Cas proteins to the TCR and / or HLA promoter, sterically blocking RNA polymerase.

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

[0393] TALENs to inhibit TCR and / or HLA "TALEN" or "TALEN against HLA and / or TCR" or "TALEN for inhibiting HLA and / or TCR" refers to a transcription activator-like effector nuclease, an artificial nuclease that can be used to edit HLA genes and / or TCR genes.

[0394] TALENs are artificially produced by fusing a TAL effector DNA binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) can be engineered to bind to any desired DNA sequence, including parts of HLA or TCR genes. By combining engineered TALEs with DNA cleavage domains, 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] TALEs are proteins secreted by Xanthomonas bacteria. The DNA-binding domain contains a highly conserved 33-34 amino acid sequence, with the exception of repeated amino acids 12 and 13. These two regions are highly variable and show a strong correlation with specific nucleotide recognition. Thus, they can be engineered to bind to desired DNA sequences.

[0396] To produce a TALEN, a TALE protein is fused to a nuclease (N), which is a wild-type or mutant FokI endonuclease. Several mutations of FokI have been made for its use in TALENs, which 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 unique DNA-binding domains for the target genome site with precise orientation and spacing. Both the number of amino acid residues between the TALE DNA-binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites are thought to be 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 in cells to generate double-strand breaks (DSBs). If the repair mechanism improperly repairs the break by non-homologous end joining, mutations can be introduced at the break site. For example, improper repair can introduce frameshift mutations. Alternatively, foreign DNA can be introduced into cells together with TALENs, and depending on the sequence of the foreign DNA and chromosomal sequence, this process can be used to correct defects in HLA or TCR genes or introduce such defects into wild-type HLA or TCR genes, thereby reducing the expression of HLA or TCR.

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

[0400] Zinc finger nucleases for inhibiting HLA and / or TCR "ZFN" or "zinc finger nuclease" or "ZFN against HLA and / or TCR" or "ZFN for inhibiting HLA and / or TCR" refers to a zinc finger nuclease, an artificial nuclease that can be used to edit HLA genes 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. Zinc fingers, for example, contain Cys2His2 and can recognize approximately 3-bp sequences. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides that recognize approximately 6-, 9-, 12-, 15-, or 18-bp sequences. A variety of selection and modular assembly techniques are available to generate zinc fingers (and combinations thereof) that recognize specific sequences, including phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells.

[0403] Like TALENs, ZFNs must dimerize to cleave DNA. Therefore, a pair of ZFNs is required to target non-palindromic DNA sites. Two individual ZFNs must bind to opposite strands of DNA with their nucleases appropriately spaced apart. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95:10570-5.

[0404] Also like TALENs, ZFNs can create double-strand breaks in DNA that, if improperly repaired, create frameshift mutations, reducing the expression and amount 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 HLA and / or TCR sequences can be constructed using any method known in the art. See, for example, Provasi (2011) Nature Med. 18: 807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al. (2008) Mol. Ther. 16: 1200-7; and Guo et al. (2010) J. Mol. Biol. 400: 96; U.S. Publication No. 2011 / 0158957; U.S. Publication No. 2012 / 0060230.

[0406] Cell activation and proliferation Cells can generally be activated and expanded using methods described, for example, 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 Application Publication No. 20060121005.

[0407] Generally, T cells of the present invention can be expanded by contacting them with a surface to which an agent that stimulates a CD3 / TCR complex-associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells is bound. In particular, T cell populations can be stimulated as described herein, such as by contacting them with an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody immobilized on a surface, or by contacting them with a protein kinase C activator (e.g., bryostatin) combined with a calcium ionophore. For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate T cell proliferation. CD4 + T cells or CD8 + To stimulate the proliferation of any of the T cells, anti-CD3 antibodies and anti-CD28 antibodies are used. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), and can be used as well as other methods generally known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0408] In certain aspects, the primary stimulatory signal and the costimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal are in solution or bound to a surface. When bound to a surface, the agents may be bound to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one agent may be bound to a surface and the other agent in solution. In one aspect, the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or bound to a surface. In one aspect, either agent may be in solution. In one aspect, the agents may be in soluble form and then crosslinked 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, e.g., U.S. Patent Application Publication Nos. 20040101519 and 20060034810, for artificial antigen presenting cells (aAPCs) contemplated for use in the activation and expansion of T cells in the present invention.

[0409] In one aspect, the two agents are immobilized on beads, either on the same bead, i.e., "cis," or on separate beads, i.e., "trans." For example, the agent providing the primary activation signal is an anti-CD3 antibody or antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof, and both agents are co-immobilized on the same bead with the same molecular weight. In one aspect, the CD4 +A 1:1 ratio of each antibody bound to beads for T cell proliferation and T cell growth is used. In certain aspects of the invention, a ratio of anti-CD3:CD28 antibodies bound to beads is used such that an increase in T cell proliferation is observed compared to the proliferation seen using a 1:1 ratio. In one specific aspect, an increase of about 1 to about 3 fold is observed compared to the proliferation seen using a 1:1 ratio. In one aspect, the ratio of CD3:CD28 antibodies bound to beads ranges from 100:1 to 1:100, and all integer values ​​therebetween. In one aspect of the invention, more anti-CD28 antibody than anti-CD3 antibody is bound to the particles, i.e., the CD3:CD28 ratio is less than 1. In some aspects 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, a 1:100 CD3:CD28 ratio of antibodies bound to beads is used. In one aspect, a 1:75 CD3:CD28 ratio of antibodies bound to beads is used. In a further aspect, a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1:30 CD3:CD28 ratio of antibody bound to beads is used. In one preferred aspect, a 1:10 CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1:3 CD3:CD28 ratio of antibody bound to beads is used. In a further aspect, a 3:1 CD3:CD28 ratio of antibody bound to beads is used.

[0410] Particle-to-cell ratios ranging from 1:500 to 500:1, and any integer value therebetween, can be used to stimulate T cells or other target cells. As one of skill in the art will readily appreciate, the particle-to-cell ratio can depend on the particle size relative to the target cells. For example, small beads can only bind a few cells, while larger beads can bind many more. In one aspect, the particle-to-cell ratio ranges from 1:100 to 100:1, and any integer value therebetween, and in a further aspect, the ratio includes 1:9 to 9:1, and any integer value therebetween can also be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 conjugated particles to T cells that results in T cell stimulation can vary as described above; however, certain 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. In a further aspect, the particle-to-cell ratio can vary depending on the day of stimulation. For example, in one aspect, the particle to cell ratio is 1:1 to 10:1 on the first day, and additional particles are added to the cells daily or every other day thereafter for up to 10 days, to a final ratio of 1:1 to 1:10 (based on the cell count on the day of addition). In one specific aspect, the particle to cell ratio is 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fourth days of stimulation. In one aspect, particles are added daily or every other day from 1:1 on the first day of stimulation to a final ratio of 1:5 on the third and fourth days of stimulation. In one aspect, the particle to cell ratio is 2:1 on the first day of stimulation and 1:10 on the third and fourth days of stimulation. In one aspect, particles are added daily or every other day from 1:1 on the first day to a final ratio of 1:10 on the third and fourth days of stimulation. Those of skill in the art will recognize that a variety of other ratios may be suitable for use in the present invention. In particular, the ratio will depend on particle size and cell size and type. In one aspect, the most typical ratios to use are around 1:1, 2:1 and 3:1 on the first day.

[0411] In a further aspect of the invention, cells, such as T cells, are combined with drug-coated beads, the beads and cells are subsequently separated, and the cells are then cultured. In another aspect, the drug-coated beads and cells are not separated but are cultured together prior to culturing. I...

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR is (i) an antibody or antibody fragment comprising a human anti-mesothelin binding domain; (ii) a transmembrane domain; and (iii) an intracellular signaling domain comprising a stimulatory domain. and the anti-mesothelin binding domain comprises (a) a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 213, a light chain complementarity determining region 2 (LC CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 237, a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 261, a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 147, a heavy chain complementarity determining region 2 (HC CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 166, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 189; or (b) a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 215, a light chain complementarity determining region 2 (LC CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 239, a light chain complementarity determining region 3 (LC CDR3) comprising the amino acid sequence set forth in SEQ ID NO: 263, a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence set forth in SEQ ID NO: 147, a heavy chain complementarity determining region 2 (HC CDR2) comprising the amino acid sequence set forth in SEQ ID NO: 167, and a heavy chain complementarity determining region 3 (HC CDR3) comprising the amino acid sequence set forth in SEQ ID NO:

191. An isolated nucleic acid molecule comprising:

2. The isolated nucleic acid molecule of claim 1 , wherein the anti-mesothelin binding domain is an scFv.

3. The anti-mesothelin binding domain is (i) an amino acid sequence comprising the light chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (ii) an amino acid sequence having 95-99% identity to the light chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (iii) an amino acid sequence having at least 90% identity to the light chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (iv) an amino acid sequence comprising the heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (v) an amino acid sequence having 95-99% identity to the heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (vi) an amino acid sequence having at least 90% identity to the heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (vii) an amino acid sequence comprising the light chain variable region and the heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (viii) an amino acid sequence having 95-99% identity to the light chain variable region and the heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; or (ix) an amino acid sequence having at least 90% identity to the light chain variable region and heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55 3. The isolated nucleic acid molecule of claim 1 or 2, comprising:

4. (a) the anti-mesothelin binding domain comprises SEQ ID NO:53, SEQ ID NO:55, or a sequence having 95-99% identity to SEQ ID NO:53 or SEQ ID NO:55; or (b) the nucleic acid sequence encoding the anti-mesothelin binding domain comprises SEQ ID NO:101, SEQ ID NO:103, or a sequence having 95-99% identity to SEQ ID NO:101 or SEQ ID NO:

103. An isolated nucleic acid molecule according to any one of claims 1 to 3.

5. (a) the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154; (b) the transmembrane domain comprises SEQ ID NO:6; (c) the transmembrane domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 6; (d) the transmembrane domain comprises an amino acid sequence having 95-99% identity to the amino acid sequence of SEQ ID NO:6; or (e) the nucleic acid sequence encoding the transmembrane domain comprises the sequence of SEQ ID NO: 17 or a sequence having 95-99% identity to SEQ ID NO: 17; An isolated nucleic acid molecule according to any one of claims 1 to 4.

6. the anti-mesothelin binding domain is connected to the transmembrane domain by a hinge region; (a) the hinge region comprises SEQ ID NO:2 or a sequence having 95-99% identity to SEQ ID NO:2; or (b) the hinge region comprises an amino acid sequence encoded by SEQ ID NO: 13, or the hinge region comprises an amino acid sequence encoded by a sequence having 95-99% identity to the nucleic acid sequence of SEQ ID NO: 13; An isolated nucleic acid molecule according to any one of claims 1 to 5.

7. 7. The isolated nucleic acid molecule of any one of claims 1 to 6, further comprising a sequence encoding a costimulatory domain, (a) the costimulatory domain is a functional signaling domain derived 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), or the costimulatory domain comprises the sequence of SEQ ID NO: 7; (b) the costimulatory domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 7; (c) the costimulatory domain comprises a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 7; (d) the nucleic acid sequence encoding the costimulatory domain comprises the sequence of SEQ ID NO: 18, or the sequence encoding the costimulatory domain comprises a sequence having at least 90% identity to the nucleic acid sequence of SEQ ID NO: 18; or (e) the costimulatory domain comprises a sequence having 95-99% identity to the nucleic acid sequence of SEQ ID NO: 18; Isolated nucleic acid molecule.

8. The isolated nucleic acid molecule of any one of claims 1 to 7, wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and / or a functional signaling domain of CD3 zeta.

9. The intracellular signaling domain is (i) the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; (ii) an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; (iii) an amino acid sequence having 95 to 99% identity with the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; (iv) the amino acid 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; or (v) an amino acid sequence encoded by the sequence of SEQ ID NO: 18 or a sequence having 95 to 99% identity thereto, and an amino acid sequence encoded by the sequence of SEQ ID NO: 20 or SEQ ID NO: 21 or a sequence having 95 to 99% identity thereto 9. The isolated nucleic acid molecule of claim 8, comprising:

10. The isolated nucleic acid molecule of any one of claims 1 to 9, further comprising a leader sequence.

11. 11. The isolated nucleic acid molecule of claim 10, wherein the leader sequence comprises the amino acid sequence of SEQ ID NO:

1.

12. 12. An isolated nucleic acid molecule according to any one of claims 1 to 11, (a) the CAR comprises an amino acid sequence selected from SEQ ID NO: 77 or SEQ ID NO: 79, or a sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 79; or (b) the nucleic acid sequence encoding the CAR comprises SEQ ID NO: 125, SEQ ID NO: 127, or a sequence having 95-99% identity to SEQ ID NO: 125 or SEQ ID NO: 127; Isolated nucleic acid molecule.

13. An isolated chimeric antigen receptor (CAR) molecule comprising a human anti-mesothelin binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the human anti-mesothelin binding domain (i) a light chain complementarity determining region 1 (LC CDR1) of SEQ ID NO: 213, a light chain complementarity determining region 2 (LC CDR2) of SEQ ID NO: 237, and a light chain complementarity determining region 3 (LC CDR3) of SEQ ID NO: 261; and a heavy chain complementarity determining region 1 (HC CDR1) of SEQ ID NO: 147, a heavy chain complementarity determining region 2 (HC CDR2) of SEQ ID NO: 166, and a heavy chain complementarity determining region 3 (HC CDR3) of SEQ ID NO: 189; or (ii) an LC CDR1 of SEQ ID NO:215, an LC CDR2 of SEQ ID NO:239, an LC CDR3 of SEQ ID NO:263; and an HC CDR1 of SEQ ID NO:147, an HC CDR2 of SEQ ID NO:167, and an HC CDR3 of SEQ ID NO:

191.

1. An isolated CAR molecule comprising:

14. The isolated CAR molecule of claim 13, wherein the human anti-mesothelin binding domain is an scFv.

15. a human anti-mesothelin binding domain, (i) an amino acid sequence comprising the light chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (ii) an amino acid sequence having 95-99% identity to the light chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (iii) an amino acid sequence having at least 90% identity to the light chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (iv) an amino acid sequence comprising the heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (v) an amino acid sequence having 95-99% identity to the heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (vi) an amino acid sequence having at least 90% identity to the heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (vii) an amino acid sequence comprising the light chain variable region and the heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (viii) an amino acid sequence having 95-99% identity to the light chain variable region and the heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; or (ix) an amino acid sequence having at least 90% identity to the light chain variable region and heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55 15. The isolated CAR molecule of claim 13 or 14, comprising:

16. The transmembrane domain (i) a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154; (ii) the amino acid sequence of SEQ ID NO:6; (iii) an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 6; or (iv) an amino acid sequence having 95 to 99% identity with the amino acid sequence of SEQ ID NO: 6 The isolated CAR molecule of any one of claims 13 to 15, comprising:

17. The isolated CAR molecule of any of claims 13 to 16, wherein the human anti-mesothelin binding domain is connected to the transmembrane domain by a hinge region, and the hinge region comprises SEQ ID NO: 2 or a sequence having 95 to 99% identity thereto.

18. 18. The isolated CAR molecule of any one of claims 13 to 17, wherein the intracellular signaling domain comprises a costimulatory domain. (a) 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 (ICOS), and 4-1BB (CD137); (b) the amino acid sequence of SEQ ID NO: 7; (c) an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 7; or (d) an amino acid sequence having 95 to 99% identity with the amino acid sequence of SEQ ID NO: 7 1. An isolated CAR molecule comprising:

19. The isolated CAR molecule of any of claims 13 to 18, wherein the intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of CD3 zeta.

20. Intracellular signaling domains (i) the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9; (ii) the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 10; (iii) an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; or (iv) an amino acid sequence having 95 to 99% identity with the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10 20. The isolated CAR molecule of claim 19, comprising:

21. 21. The isolated CAR molecule of claim 20, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

22. The isolated CAR molecule of any one of claims 13 to 19, further comprising a leader sequence.

23. The isolated CAR molecule of claim 22, wherein the leader sequence comprises the amino acid sequence of SEQ ID NO: 1 or a sequence having 95 to 99% identity to the amino acid sequence of SEQ ID NO:

1.

24. The isolated CAR molecule of any of claims 13 to 23, comprising an amino acid sequence selected from SEQ ID NO: 77 or SEQ ID NO: 79, or a sequence having 95 to 99% identity to the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO:

79.

25. An antibody or antibody fragment comprising a human anti-mesothelin binding domain comprising light chain (LC) and heavy chain (HC) CDRs, wherein the human anti-mesothelin binding domain (a) an LC CDR1 of SEQ ID NO:213, an LC CDR2 of SEQ ID NO:237, and an LC CDR3 of SEQ ID NO:261; and an HC CDR1 of SEQ ID NO:147, an HC CDR2 of SEQ ID NO:166, and an HC CDR3 of SEQ ID NO:189; or (b) an LC CDR1 of SEQ ID NO:215, an LC CDR2 of SEQ ID NO:239, an LC CDR3 of SEQ ID NO:263; and an HC CDR1 of SEQ ID NO:147, an HC CDR2 of SEQ ID NO:167, and an HC CDR3 of SEQ ID NO:

191. An antibody or antibody fragment comprising:

26. (i) the light chain variable region and heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (ii) (a) an amino acid sequence comprising the light chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (b) an amino acid sequence having 95-99% identity to the light chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (c) an amino acid sequence having at least 90% identity to the light chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55 a light chain variable region comprising: (iii) (d) an amino acid sequence comprising the heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; (e) an amino acid sequence having 95-99% identity to the heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55; or (f) an amino acid sequence having at least 90% identity to the heavy chain variable region of SEQ ID NO: 53 or SEQ ID NO: 55 a heavy chain variable region comprising 26. The human anti-mesothelin binding domain of claim 25, comprising:

27. A vector comprising the nucleic acid molecule of any one of claims 1 to 12, which is selected from the group consisting of a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

28. 28. The vector of claim 27, wherein the vector comprises DNA or RNA.

29. 29. An immune effector cell comprising the vector of claim 27 or 28.

30. 29. A method for producing immune effector cells, comprising transducing immune effector cells in vitro with the vector of claim 27 or 28.

31. 13. A method for producing a population of RNA-engineered cells, comprising introducing in vitro transcribed or synthetic RNA into cells in vitro, wherein the RNA comprises a nucleic acid molecule according to any one of claims 1 to 12.

32. 13. A cell comprising the nucleic acid molecule of any one of claims 1 to 12 for use in a method for providing anti-cancer immunity in a mammal, said method comprising administering to the mammal an effective amount of the cell.

33. 13. A cell comprising a nucleic acid molecule according to any one of claims 1 to 12 for use in a method for treating a mammal having a disease associated with expression of mesothelin, the method comprising administering to the mammal an effective amount of the cell.

34. 34. The cell of claim 33, wherein the disease is a mesothelin-associated 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.

35. 30. The cell of claim 29 for use as a medicament or for use in treating a disease that expresses mesothelin.

36. 30. The cell of claim 29, further expressing a polypeptide comprising a first polypeptide comprising at least a portion of an inhibitory molecule bound to a second polypeptide comprising an intracellular signaling domain.

37. 35. The cell of claim 33 or 34, wherein the nucleic acid is introduced into a T cell or NK cell using in vitro transcription, and the subject receives two or more administrations of the cells containing the nucleic acid, wherein the administrations are spaced apart by no more than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days, after the previous administration.

38. Use of a cell comprising a nucleic acid molecule according to any one of claims 1 to 12 in the manufacture of a medicament for providing anti-cancer immunity in a mammal.

39. 13. Use of a cell containing a nucleic acid molecule according to any one of claims 1 to 12 in the manufacture of a medicament for treating a mammal having a disease associated with the expression of mesothelin.

40. 40. The use of claim 39, wherein the disease is a mesothelin-associated 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.

41. Use of the isolated nucleic acid molecule of any one of claims 1 to 12, the isolated CAR molecule of any one of claims 13 to 24, the anti-mesothelin binding domain of claim 25 or 26, the vector of claim 27 or 28, or the cell of claim 29 in the manufacture of a medicament for the treatment of a disease expressing mesothelin.

42. 30. Use of the cells described in claim 29 in the manufacture of a medicament, wherein the cells further express an inhibitory molecule comprising a first polypeptide comprising at least a portion of the inhibitory molecule bound to a second polypeptide comprising a positive signal from an intracellular signaling domain.

43. 41. The use according to claim 39 or 40, wherein the nucleic acid is introduced into T cells or NK cells using in vitro transcription.

44. 44. The use of claim 39, 40 or 43, wherein the cells are formulated for two or more administrations, wherein the interval between administrations is within 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days after the previous administration.

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