Novel mesothelin specific chimeric antigen receptors (CAR) for solid tumors cancer immunotherapy

Abstract

To provide engineered immune cells expressing novel mesothelin (MSLN) specific chimeric antigen receptors (anti-mesothelin CAR), and to provide uses thereof in the treatment of solid tumors.SOLUTION: Provided is a mesothelin specific chimeric antigen receptor (CAR), comprising at least: an extracellular ligand binding-domain comprising VH and VL from a monoclonal anti-mesothelin antibody; a transmembrane domain; and a cytoplasmic domain comprising a CD3 zeta signalling domain and a co-stimulatory domain, the extra cellular ligand binding-domain being directed against the MSLN antigen polypeptide region comprising a specific sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to the field of cellular immunotherapy, and more specifically to engineered immune cells expressing a novel mesothelin (MLSN)-specific chimeric antigen receptor (anti-mesothelin CAR), useful in the treatment of solid tumors. [Background technology]

[0002] Background of the Invention Chimeric antigen receptors (CARs) are synthetic receptors that target T cells to cell surface antigens and enhance T cell function and persistence. Mesothelin is a cell surface antigen involved in tumor invasion and is highly expressed in mesothelioma, as well as lung, pancreatic, breast, ovarian, and other cancers. Encouragingly, recent clinical trials evaluating active immunization or immunoconjugates in patients with pancreatic adenocarcinoma or mesothelioma have demonstrated responses without toxicity. Overall, these findings and preclinical CAR therapy models using systemic or local T cell delivery indicate that mesothelin CAR therapy is advantageous in multiple solid tumors.

[0003] Given the potentially high efficiency of CAR therapy, it is important to identify appropriate antigens to target solid tumors in order to achieve tumor eradication with minimal or acceptable on-target / off-tumor toxicity to healthy tissues.

[0004] Solid tumor CAR targets currently under investigation are genetically engineered products, with the majority arising from gene mutations or splicing variants (EGFRvIII), glycosylation pattern variants (MUC1), cancer-testis antigen-derived peptides (MAGE), overexpressed differentiation antigens (CEA, PSMA, GD2, MUC16, HER2 / ERBB2, and mesothelin (MSLN)), or tumor-associated stroma (FAP and VEGFR).

[0005] Although overexpressed antigens are numerous and relatively frequent, concerns about "on-target / off-tumor" side effects arise because T cells are highly sensitive to even low-level antigen expression, which can exceed that of monoclonal antibodies. For example, the use of ERBB2 CAR T cells administered at high cell doses has resulted in fatal adverse events, which are thought to be due in part to low levels of ERBB2 expression in healthy lung epithelial cells and cardiovascular cells [Morgan, RA et al. (2010) Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther. 18:843-51 (Non-Patent Document 1)]. Therefore, optimal solid tumor antigen targets are those whose expression is restricted to tumor cells or occurs at extremely low levels in normal tissues that can be sacrificed.

[0006] MSLN has emerged as an attractive target for cancer immunotherapy due to its low expression in normal mesothelial cells and high expression in all solid tumors. Previously reported immunotherapies targeting MSLN have maintained favorable safety profiles. MSLN is a potential CAR target in several common solid tumors, including at least esophageal cancer, breast cancer, gastric cancer, bile duct adenocarcinoma, pancreatic cancer, colon cancer, lung cancer, thymic cancer, mesothelioma, ovarian cancer, and endometrial cancer [Morello, A. et al. (2016) Mesothelin-Targeted CARs: Driving T Cells to Solid Tumors. Cancer Discov. 6(2); 133-46 (Non-Patent Document 2)].

[0007] MSLN is a glycoprotein anchored to the cell membrane by a glycophosphatidylinositol (GPI) domain. It is initially synthesized as a 69-kDa cell surface protein. After amino-terminal cleavage by furin protease, a 40-kDa C-terminal fragment remains attached to the membrane, while a soluble 32-kDa N-terminal fragment called megakaryocyte-potentiating factor (MPF) is released [Pastan, I., Hassan, R. (2014) Discovery of mesothelin and exploiting it as a target for immunotherapy. Cancer. Res. 74:2907-12 (Non-Patent Document 3)]. A soluble form of MSLN has also been detected in the serum of patients with solid tumors and is called soluble MSLN-related protein (SMRP). SMRP is generated by alternative splicing or by proteolytic cleavage of the mature MSLN form induced by the TNFα-converting enzyme ADAM17.

[0008] The biological function of MSLN appears not to be essential in normal tissues, as MSLN knockout mice exhibit normal development, reproduction, and blood counts. In contrast, preclinical and clinical studies increasingly demonstrate that aberrant MSLN expression plays an active role in both tumor progression and aggressiveness by promoting cancer cell proliferation, contributing to local invasion and metastasis, and conferring resistance to cytotoxin-induced apoptosis. MSLN can act bidirectionally by directly activating intracellular pathways via its GPI domain or by interacting with its receptor, CA125 / MUC16. Overexpression of MSLN is sufficient to constitutively activate NFκB, MAPK, and PI3K intracellular pathways, promoting cell proliferation and resistance to apoptosis.

[0009] Physiologically, MSLN is expressed in mesothelial cells of the peritoneal and thoracic cavities and pericardium, and to a lesser extent on the surface of epithelial cells in the trachea, ovaries, rete testis, tonsils, and fallopian tubes. Overexpression of MSLN was first observed in mesothelioma and ovarian cancer, and subsequently in cancers of the lung, esophagus, pancreas, stomach, bile duct, endometrium, thymus, colon, and breast. Thus, MSLN overexpression accounts for an estimated 340,000 patient cases and 2 million affected individuals annually in the United States alone.

[0010] CARs generally consist of an ectodomain, hinge, transmembrane domain, and endodomain (typically including a CD3ζ-derived signaling domain and a costimulatory receptor) derived from a single-chain variable fragment (scFv). Second-generation CARs further enhance T cell function and persistence by incorporating a signaling domain that rescues and amplifies the activation signal provided by the CD3ζ cytoplasmic domain. Dual signaling prevents T cell anergy and increases persistence and function by enhancing T cell proliferation and cytokine (IFNγ and IL2) production and attenuating activation-induced cell death by recruiting PI3K, TRAF, and / or other pathways. Third-generation CARs typically contain three signaling domains, including the CD3ζ signaling domain and two costimulatory domains, such as CD28 and 4-1BB or CD28 and OX40. Compared to second-generation CARs, third-generation CARs have shown variable in vivo antitumor activity. Choosing an appropriate costimulatory domain is essential for maintaining CAR T cell activity and quantitating T cell persistence, but the ideal costimulatory domain may be context-dependent, as CAR function depends on multiple external factors, such as antigen density, CAR stoichiometry, CAR affinity, and the immune characteristics of the tumor microenvironment.

[0011] The spatial distance between CARs and their target antigens may be equally important for effective initiation of T cell signaling, but it depends on a completely different set of structural elements, including the location of the epitope on the target molecule and the spacer domain between the scFv and the T cell membrane. Several studies have demonstrated that CAR T cells can be activated more efficiently when the same epitope is expressed in a more membrane-proximal position than in a more membrane-distal position. For example, Hombach et al. demonstrated that CAR T cells recognizing the membrane-distal "N" epitope of carcinoembryonic antigen (CEA) were only moderately activated, whereas the same CAR T cells were activated more efficiently when recombinant CEA protein was engineered to express the N epitope in a membrane-proximal position [Hombach AA, et al. (2007) T cell activation by antibody-like immunoreceptors: the position of the binding epitope within the target molecule determines the efficiency of activation of redirected T cells. J Immunol. 178:4650-4657 (Non-Patent Document 4)]. This suggests that targeting some membrane-distal epitope on tumor cells may allow large phosphatases, such as CD45 and CD148, to enter the synapse and inhibit the phosphorylation events initiated by CAR binding. Engineered extracellular spacer sequences between the T cell membrane and the ligand-binding scFv to promote synapse formation could help overcome the spatial constraints imposed by the location of the target epitope.

[0012] A particular concern with MSLN CARs is interference from soluble MSLN, which could in principle occupy and block the scFv portion. However, activation of MSLN CAR T cells (cytokine secretion and cytotoxic activity) still appears to depend on cell surface MSLN expression (Carpenito C., et al. (2009) Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. PNAS. 106:3360-5).

[0013] Although the principle that T cells engineered to express novel synthetic CARs can effectively treat advanced, resistant cancers has been established, many questions remain before the full potential of this new therapeutic modality can be realized. Creating safer and more effective CARs for solid tumor cancer treatment will require moving beyond traditional empirical approaches to receptor design and cell engineering, ideally guided by our knowledge of TCR signaling, T cell biology, and manipulation of the tumor microenvironment. It is now clear that the binding affinity of CARs to target cells, the K on / K off ratio, and spatial constraints can affect the ability of CARs to optimally activate T cells to recognize tumors, particularly solid tumors [D'Aloia, MM, Zizzari, IG, Sacchetti, B. et al. (2018) CAR-T cells: the long and winding road to solid tumors. Cell Death Dis 9:282 (Non-Patent Document 6)].

[0014] In light of the above, selecting scFvs solely for their affinity to the MSLN antigen or their ability to induce T cell activation and proliferation in vitro does not appear to be sufficient to generate the most appropriate CAR T cells. Current data suggest that the optimal CAR affinity for individual target molecules cannot be determined a priori because there is currently no unbiased approach to identify the ideal affinity range that will yield the best in vivo results [Srivastava, S. and Ridell, RS (2015) Engineering CAR-T Cells: Design Concepts. Trends Immunol. 36(8): 494-502].

[0015] Additionally, the solid tumor microenvironment poses several obstacles for MSLN CAR-T cells, limiting their antitumor efficacy. To optimize CAR T cell efficiency, numerous approaches are being evaluated to tame the host tumor microenvironment or to create “armored” CAR T cells capable of overcoming immune barriers. Such strategies include (i) promoting CAR T-cell infiltration, (ii) enhancing CAR T-cell functional persistence, (iii) enhancing CAR T cells to overcome inhibitory signals encountered in the tumor microenvironment, and (iv) improving safety by preventing on-target / off-tumor toxicity. Among these approaches, combining specific CAR constructs with gene-edited cells appears to be the most promising. Riese et al. [Riese MJ, et al. Enhanced effector responses in activated CD8+ T cells deficient in diacylglycerol kinases. Cancer Res. 73:3566-77 (Non-Patent Document 8)], for example, demonstrated that genetic deletion of DGKζ greatly increased the antitumor activity of MSLN CAR T cells, as shown by enhanced in vitro secretion of effector cytokines, FASL, and TRAIL expression, and cytotoxic function.

[0016] Meanwhile, various strategies have been developed to address the risk of on-target / off-tumor toxicity and improve the safety of CAR T cells.

[0017] One such approach involves gene transfer of mRNA encoding the MSLN CAR, resulting in transient CAR expression for only a few days. Preclinical models have shown promise for this approach, with multiple infusions of mRNA CAR T cells producing robust antitumor effects in vivo [Zhao Y, et al. (2010) Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediated regression of human disseminated tumor. Cancer Res. 2070:9053-61 (Non-Patent Document 9)]. However, transient expression of the CAR may limit the long-term efficacy of the treatment. A clinical trial conducted at the University of Pennsylvania using autologous T cells electroporated with mRNA encoding a second-generation MSLN CAR (SS1-4-1BB CAR) resulted in modest clinical responses and transient increases in serum inflammatory cytokines such as IL12, IL6, G-CSF, MIP1β, MCP1, IL1RA, and RANTES.

[0018] Another approach to improving T cell safety is to utilize suicide genes to eliminate T cells in the event of an adverse event. In such situations, CAR T cells can be eliminated by drug-induced activation of suicide genes, such as herpes simplex thymidine kinase (HSV-TK), gene-inducible caspase-9, or the EGFR delta gene.

[0019] In a previous patent application, WO2016120216 (Patent Document 1), the applicant developed a suicide gene replacement system that involves inserting a foreign epitope into the CAR structure and allowing it to be recognized by a clinically approved antibody, such as rituximab, thereby enabling partial or complete elimination of CAR-positive immune cells infused into patients as needed. One advantage of this approach is that it does not require the co-expression of a suicide gene in addition to the CAR. However, inserting such an epitope may affect the overall structure of the CAR and may alter how the scFv interacts with its cognate antigen.

[0020] The objective of the present invention is to address some or all of the above limitations by providing safer, engineered immune CAR-positive cells that target MSLN-expressing cells, such as solid tumors, in vivo, with the intention of using them in allogeneic therapeutic strategies. [Prior art documents] [Patent documents]

[0021] [Patent Document 1] WO2016120216 [Non-patent literature]

[0022] [Non-Patent Document 1] Morgan, RA et al. (2010) Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther.18:843-51 [Non-patent document 2] Morello, A. et al. (2016) Mesothelin-Targeted CARs: Driving T Cells to Solid Tumors. Cancer Discov. 6(2); 133-46

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

[0023] The present invention relates primarily to mesothelin-specific chimeric antigen receptors (CARs) and their expression in immune cells, preferably T cells, for therapeutic use against malignant mesothelin-expressing cells or tissues.

[0024] Such CARs typically include: ·An extracellular ligand-binding domain containing VH and VL derived from a monoclonal anti-mesothelin antibody; Transmembrane domains; and The cytoplasmic domain containing the CD3 zeta signaling domain and the costimulatory domain The structure includes:

[0025] The extracellular ligand-binding domain of the CAR of the present invention preferably comprises one or more scFv segments derived from the antibody designated mesol, more particularly CDRs comprising SEQ ID NOs: 3, 4, 5, 6, 7 and / or 8 derived therefrom.

[0026] In a preferred aspect, the extracellular ligand binding domain of the CAR comprises: a variable heavy VH chain comprising CDRs from antibody meso1 having at least 90% identity to SEQ ID NO:3 (CDRH1-Meso1), SEQ ID NO:4 (CDRH2-meso1), and / or SEQ ID NO:5 (CDRH3-meso1), respectively; and a variable heavy VL chain comprising CDRs from antibody Meso1, each having at least 90% identity to SEQ ID NO:6 (CDRL1-meso1), SEQ ID NO:7 (CDRL2-meso1), and / or SEQ ID NO:8 (CDRL3-meso1), Includes.

[0027] The anti-mesothelin CARs of the present invention form exogenous polypeptide sequences that are expressed by immune cells and exposed on the cell surface, are encoded by polynucleotide sequences that are exogenous (relative to the native genome of the immune cell) and are preferably inserted into specific genomic loci, such as the TCR, B2m, and PD1 gene loci, using rare-cutting endonucleases.

[0028] In some embodiments, the CAR further comprises an additional exogenous polypeptide sequence comprising an epitope that can be targeted by a clinically approved ligand for in vivo clearance or targeted by other ligands for in vivo or in vitro detection or purification. Such additional exogenous polypeptide segments can be specifically recognized by rituximab, such as that referred to herein as "R2."

[0029] The present invention more specifically relates to immune cells or immune cell populations transformed with an anti-mesothelin CAR polynucleotide sequence, which contain the polynucleotide sequence and / or express the polypeptide anti-mesothelin CAR sequence.

[0030] Such modified immune cells or cell populations of the present invention can be further genetically modified, mutated, or gene-edited to improve therapeutic suitability or efficacy, for example, to improve persistence or longevity. In a preferred aspect, the modified immune cells of the present invention combine expression of an anti-mesothelin CAR sequence with other genetic modifications that reduce expression of its endogenous genes, such as TCR, HLA, and / or B2m genes. TGF-beta receptor

[0031] In a further preferred aspect, the engineered immune cells may be mutated to improve CAR-dependent immune activation, specifically by reducing or suppressing the expression of immune checkpoint proteins and / or their receptors, such as PD1 / PDL1.

[0032] In a further preferred aspect, the engineered immune cells can be mutated to improve CAR-dependent immune activation, specifically by reducing or suppressing the TGF-beta signaling pathway.

[0033] In other preferred aspects, additional exogenous genes, particularly inhibitors or decoys of TGF beta receptors, such as the sequence of a dominant-negative TGF beta receptor (dnTGFβRII), can also be inserted, co-transfected, or co-expressed with the anti-mesothelin CAR of the present invention.

[0034] Further examples of exogenous gene sequences are provided, the expression of which can be combined with the expression of an anti-mesothelin CAR to improve the therapeutic efficacy of immune cells, specifically: NK cell inhibitors, such as HLAG, HLAE, or ULBP1; CRS inhibitors, such as mutant IL6Ra, sGP130, or IL18-BP; or Cytochrome P450, CYP2D6-1, CYP2D6-2, CYP2C9, CYP3A4, CYP2C19, or CYP1A2, which sensitize said immune cells to drugs such as cyclophosphamide and / or isophosphamide; dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin, or methylguanine transferase (MGMT), mTORmut, or Lckmut, which confer drug resistance; Chemokines or cytokines, such as IL-2, IL-12, and IL-15; Tumor-associated macrophage (TAM) secretion inhibitors, such as CCR2 / CCL2 neutralizers, which enhance the therapeutic activity of immune cells is.

[0035] The engineered immune cells of the present invention are particularly suitable for treating conditions characterized by mesothelin-expressing cells, in particular solid tumors, such as typically esophageal cancer, breast cancer, gastric cancer, biliary adenocarcinoma, pancreatic cancer, colon cancer, lung cancer, thymic cancer, mesothelioma, ovarian cancer, and / or endometrial cancer.

[0036] The present invention therefore encompasses methods for producing modified cells, therapeutic cells obtained thereby, cell populations comprising such cells, and therapeutic compositions comprising same, as well as treatments that allow for addressing pathologies induced by mesothelin-expressing cells. [The present invention 1001] an extracellular ligand-binding domain comprising a VH and a VL derived from a monoclonal anti-mesothelin antibody; Transmembrane domains, and The cytoplasmic domain containing the CD3 zeta signaling domain and the costimulatory domain A mesothelin-specific chimeric antigen receptor (CAR) comprising at least The mesothelin-specific chimeric antigen receptor (CAR), wherein the extracellular ligand-binding domain is directed against the MSLN antigen polypeptide region SEQ ID NO:25. [The present invention 1002] the extracellular ligand-binding domain a variable heavy VH chain comprising CDRs from antibody Meso1, which have at least 90% identity to SEQ ID NO:3 (CDRH1-Meso1), SEQ ID NO:4 (CDRH2-Meso1), and SEQ ID NO:5 (CDRH3-Meso1), respectively; and a variable heavy VL chain comprising CDRs from antibody Meso1, which have at least 90% identity to SEQ ID NO:6 (CDRL1-Meso1), SEQ ID NO:7 (CDRL2-Meso1), and SEQ ID NO:8 (CDRL3-Meso1), respectively; 1001. A mesothelin-specific chimeric antigen receptor (CAR) of the present invention, comprising: [The present invention 1003] A mesothelin-specific chimeric antigen receptor of the present invention, wherein the extracellular ligand-binding domain comprises a VH chain and a VL chain having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with SEQ ID NO:9 (Meso1-VH) and SEQ ID NO:10 (Meso1-VL), respectively. [The present invention 1004] 1004. The mesothelin-specific chimeric antigen receptor (CAR) of any of claims 1001 to 1003, wherein the transmembrane domain is derived from the transmembrane region of the alpha chain, beta chain, or zeta chain of a T cell receptor, PD-1, 4-1BB, OX40, ICOS, CTLA-4, LAG3, 2B4, BTLA4, TIM-3, TIGIT, SIRPA, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. [The present invention 1005] 1004. A mesothelin-specific chimeric antigen receptor (CAR) of the present invention, wherein the transmembrane domain has at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with SEQ ID NO:6 derived from CD8α. [The present invention 1006] The mesothelin-specific chimeric antigen receptor (CAR) of any of claims 1001 to 1005, further comprising a hinge between the extracellular ligand-binding domain and the transmembrane domain. [The present invention 1007] 1006. The mesothelin-specific chimeric antigen receptor (CAR) of the present invention, wherein the hinge is selected from a CD8α hinge, an IgG1 hinge, and an FcγRIIIα hinge. [The present invention 1008] 1007. A mesothelin-specific chimeric antigen receptor (CAR) of the present invention, wherein the hinge has at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with SEQ ID NO: 16 (CD8α). [The present invention 1009] The CAR is A polypeptide structure comprising a CD8α hinge having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:16, and a CD8α transmembrane domain having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:17. A mesothelin-specific CAR according to any one of claims 1001 to 1008, comprising: [The present invention 1010] A mesothelin-specific CAR of any of 1001 to 1009, further comprising a safety switch comprising an epitope selected from Table 5. [The present invention 1011] A mesothelin-specific CAR of the present invention, wherein the safety switch comprises the epitope CPYSNPSLC (SEQ ID NO: 26) to which rituximab specifically binds. [The present invention 1012] A mesothelin-specific CAR of the present invention 1010 or 1011, comprising a safety switch R2 having at least 90% identity to SEQ ID NO:15. [The present invention 1013] A mesothelin-specific chimeric antigen receptor according to any one of claims 1001 to 1012, which comprises a costimulatory domain derived from 4-1BB or CD28. [The present invention 1014] 1013. A mesothelin-specific CAR of the present invention, wherein the costimulatory domain is derived from 4-1BB and / or has at least 80% identity with SEQ ID NO:18. [The present invention 1015] A mesothelin-specific CAR of any of claims 1001 to 1014, wherein the CD3 zeta signaling domain has at least 80% identity with SEQ ID NO:19. [The present invention 1016] A mesothelin-specific CAR of any of 1001 to 1015 of the present invention, further comprising a signal peptide. [The present invention 1017] A mesothelin-specific chimeric antigen receptor (CAR) according to any one of claims 1001 to 1016, which is a single-chain polypeptide. [The present invention 1018] A mesothelin-specific chimeric antigen receptor (CAR) of the present invention having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% overall amino acid sequence identity with SEQ ID NO:21 (Mesol CAR) or SEQ ID NO:22 (Mesol-R2 CAR). [The present invention 1019] A polynucleotide encoding any one of the chimeric antigen receptors of the present invention 1001 to 1018. [The present invention 1020] An expression vector comprising the polynucleotide of the present invention. [The present invention 1021] A modified immune cell comprising a polynucleotide of the present invention 1019 or an expression vector of the present invention 1020. [The present invention 1022] A modified immune cell that expresses any one of mesothelin-specific chimeric antigen receptors according to any one of claims 1001 to 1018 on its cell surface membrane. [The present invention 1023] The modified immune cell of the present invention 1021 or 1022, which is a T lymphocyte. [The present invention 1024] The modified immune cells of the present invention 1023, derived from primary cells or differentiated from stem cells such as iPS cells. [The present invention 1025] The modified immune cell of the present invention 1023 or 1024, which is derived from an inflammatory T lymphocyte, a cytotoxic T lymphocyte, or a helper T lymphocyte. [The present invention 1026] The modified immune cell of any of claims 1021 to 1025, wherein the expression of TCR is reduced or suppressed in said immune cell. [The present invention 1027] 1026. The modified immune cell of claim 1026, wherein at least one gene encoding TCR alpha or TCR beta in said cell is inactivated. [The present invention 1028] 1027. The modified immune cell of the present invention, wherein at least one gene encoding said TCR alpha or TCR beta is cleaved by a rare-cutting endonuclease. [The present invention 1029] The modified immune cell of claim 1027 or 1028, wherein a polynucleotide encoding the mesothelin-specific CAR is integrated into an endogenous locus, preferably the TCR alpha or TCR beta locus, under the transcriptional control of an endogenous promoter. [The present invention 1030] The modified immune cells of the present invention 1029 originating from a donor for allogeneic transplantation. [The present invention 1031] 1030. The modified immune cell of any of claims 1021 to 1030, which has been mutated to confer resistance to at least one immunosuppressant, such as an anti-CD52 antibody. [The present invention 1032] 1032. The modified immune cell of any of claims 1021 to 1031, which is further mutated to confer resistance to at least one chemotherapeutic agent, particularly a purine analogue agent. [The present invention 1033] 10. The modified immune cell of any of claims 1021 to 1032, which has been mutated to improve persistence or longevity in a patient, particularly in a gene encoding an MHCI component such as HLA or B2m. [The present invention 1034] The modified immune cell of any of claims 1021 to 1033, which has been mutated to improve CAR-dependent immune activation, specifically to reduce or suppress expression of immune checkpoint proteins and / or their receptors. [This invention 1035] The modified immune cell of any of claims 1021 to 1034, wherein the mesothelin-specific chimeric antigen receptor (CAR) is co-expressed in the cell with another exogenous gene sequence encoding an inhibitor or decoy of a TGF-beta receptor. [The present invention 1036] 1035. The modified immune cell of the present invention, wherein said TGF beta receptor decoy is a dominant negative TGF beta receptor, such as a TGF beta receptor having at least 80% polypeptide sequence identity with SEQ ID NO:24. [This invention 1037] an exogenous polynucleotide comprising a first polynucleotide sequence encoding the mesothelin-specific CAR, a second polynucleotide encoding a 2A self-cleaving peptide, and a third polynucleotide encoding the dominant-negative TGF beta receptor. 1036. The modified immune cell of the present invention, comprising: [The present invention 1038] The modified immune cell of any of claims 1021 to 1037, wherein the expression of at least one TGF beta receptor gene is reduced or inactivated. [This invention 1039] The modified immune cell of the present invention 1038, wherein the TGF beta receptor gene is TGFβRII. [The present invention 1040] the mesothelin-specific chimeric antigen receptor (CAR) NK cell inhibitors, such as HLAG, HLAE, or ULBP1; CRS inhibitors, such as mutant IL6Ra, sGP130, or IL18-BP; or Cytochrome P450, CYP2D6-1, CYP2D6-2, CYP2C9, CYP3A4, CYP2C19, or CYP1A2, which sensitize said immune cells to drugs such as cyclophosphamide and / or isophosphamide; dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin, or methylguanine transferase (MGMT), mTORmut, or Lckmut, which confer drug resistance; Chemokines or cytokines, such as IL-2, IL-12, and IL-15; Chemokine receptors, such as CCR2, CXCR2, or CXCR4; Secretion inhibitors of tumor-associated macrophages (TAMs), such as CCR2 / CCL2 neutralizing agents, that enhance the therapeutic activity of these immune cells 1039. The modified immune cell of any of claims 1021 to 1039, wherein said cell is co-expressed with another exogenous gene sequence selected from those encoding: [The present invention 1041] The modified immune cell of any of claims 1021 to 1040 for use in therapy. [The present invention 1042] The modified immune cell of any of claims 1021 to 1041 for use as a medicament for treating cancer. [This invention 1043] 10. The modified immune cell of any of claims 1021 to 1042 for use in therapy of a pre-malignant or malignant cancerous condition characterized by mesothelin-expressing cells. [This invention 1044] 10. The modified immune cell of any of claims 1021 to 1043 for use in therapy of a cancer condition selected from esophageal cancer, breast cancer, gastric cancer, bile duct adenocarcinoma, pancreatic cancer, colon cancer, lung cancer, thymic cancer, mesothelioma, ovarian cancer, and endometrial cancer. [This invention 1045] 1. A method for treating a patient having a condition characterized by mesothelin-expressing cells, comprising: modifying donor-derived immune cells to express a functional mesothelin-specific chimeric antigen receptor (CAR) according to any one of claims 1001 to 1020 of the present invention; administering the CAR-positive modified immune cells to a patient to eliminate mesothelin-expressing cells The method comprising: [The present invention 1046] 1045. A method of treating a patient according to claim 1045, comprising the additional step of treatment wherein said patient is lymphocyte depleted. [This invention 1047] 1046. The method for treating a patient of claim 1046, wherein said CAR-positive modified immune cells that deplete mesothelin-expressing cells are mutated to confer resistance to lymphocyte-depleting therapy. [This invention 1048] 1047. The method for treating a patient of claim 1047, wherein said CAR-positive modified immune cells that eliminate mesothelin-expressing cells are mutated in the CD52 gene. [Brief explanation of the drawings]

[0037] [Figure 1]

[0039] Figure 1 shows the structure of a preferred version of an anti-mesothelin CAR of the present invention. A: A CAR comprising: V1 and V2, representing sequences comprising an ScFv that specifically binds to mesothelin, such as VH and VL or VL and VH derived from the mesol antibody; L: a linker; R1 and R2, representing a foreign epitope, such as the CD20 epitope recognized by a human approved monoclonal anti-CD20 antibody (e.g., rituximab); TM: a transmembrane domain; CO-STIM: a costimulatory domain; ITAM: a stimulatory domain containing an ITAM (immunoreceptor tyrosine-based activation motif (ITAM)). Such a CAR typically has at least 80% polypeptide sequence identity with SEQ ID NO: 21. B: A CAR without a foreign epitope, comprising: VH and VL or VL derived from the mesol antibody and; a (G4S)3 linker; a CD8α hinge domain; a CD8α transmembrane domain; a 4-1BB costimulatory domain; and a CD3z signaling domain. [Figure 2] 1 is a schematic diagram showing an anti-MSLN CAR-expressing immune cell of the present invention, with optional genetic features further introduced. A. Co-expression of an inactive variant of TGFβ receptor (e.g., dnTGFβRII) and / or genetic reduction or inactivation of TGFβ receptor expression to counteract tumor-induced immunosuppression. Reduction or inactivation of TCR (e.g., TCR alpha) expression to reduce immune T cell alloreactivity that contributes to GvHD. B. Genetic reduction or inactivation of TGFβ receptor, TCR, and / or CD52 expression using gene editing tools (such as TALEN). [Figure 3] Mesothelin protein expression on the surface of 293H, A2058, HeLa, and HPAC cells. Analysis of MSLN expression was performed by flow cytometry using a mouse monoclonal anti-human MSLN antibody as the primary antibody and a goat anti-mouse polyclonal antibody conjugated to APC as the secondary antibody. [Figure 4] Quantitative mesothelin protein expression on the surface of HeLa and HPAC cells. Analysis of MSLN expression levels was performed by flow cytometry using the fluorescence-based QIFIKIT. [Figure 5]Diagram depicting a serial killing assay performed to assess in vitro activation of anti-mesothelin CAR-positive cells. [Figure 6] CAR expression on the surface of primary [TCRalpha]neg T cells (UCART cells). Cryopreserved UCART cells generated from a single donor were stained with histidine-tagged recombinant human mesothelin protein and anti-histidine antibody conjugated to PE, or with biotinylated protein L and streptavidin conjugated to Vioblue, and analyzed by flow cytometry. [Figure 7] CD4 and CD8 expression by the CAR+ fraction of UCART cells. Cryopreserved UCART cells generated from a single donor were stained with anti-CD4 antibody conjugated to FITC and anti-CD8 antibody conjugated to BV510 and analyzed by flow cytometry. [Figure 8] IFNg production by UCART cells. Fresh UCART cells prepared from a single donor were cocultured with (A) HPAC (MSLN+) cells, (B) A2058 (MSLN-) cells, and (C) 293H (MSLN-) cells for 24 hours. IFNg produced in the culture supernatant was quantified by ELISA. [Figure 9] Figure 1 shows the percentage of cell lysis resulting from a serial killing assay of HPAC cells by primary [TCRalpha]neg T cells (UCART cells), the assay protocol of which is illustrated in Figure 5. Cryopreserved UCART cells generated from a single donor were co-cultured with HPAC cells at an E:T ratio of (A) 1:2 or (B) 1:8 for 15 days. [Figure 10] TCRαβ expression on the surface of UCART cells. Cryopreserved UCART cells generated from a single donor were stained with anti-TCRαβ antibody conjugated to PEVio770 and analyzed by flow cytometry. [Figure 11] Flow cytometry analysis of TCRαβ receptor expression on the surface of unmodified T cells, TRAC gene knockout T cells, and TRAC gene knockout and TCRαβ+ cell-depleted T cells. [Figure 12]Flow cytometry analysis of CD25 expression on the surface of unmodified T cells, TRAC gene knockout T cells, and TRAC gene knockout and TCRαβ+ cell-depleted T cells after exposure to medium (red line), + 0.1 μg / ml PHA-L (orange line), + 0.25 μg / ml PHA-L (green line), and + 2.5 μg / ml PHA-E and -L (blue line), respectively. [Figure 13] Measurement of UCART cell elimination by rituximab-mediated CDC with the foreign epitope polypeptide R2 contained in the P4-R2 CAR, Meso1-R2 CAR, and MESO2-R2 CAR. [Figure 14-1] Graph showing measurement of SMAD2-3 phosphorylation after TGFβ exposure of CART cells generated from two different donors. [Figure 14-2] Continuation of Figure 14-1. [Figure 15] Mean tumor volume (HPAC MSLN+ cells) in mice injected with three doses of UCARTmeso (TCR-negative anti-mesothelin P4-R2 CAR-positive cells) (1x106, 3x106, and 10x106 CAR+ cells / mouse). [Figure 16] Mean tumor volume in mice injected with three doses of UCARTmeso (TCR-negative anti-mesothelin Meso2-R2 CAR-positive cells) (1x106, 3x106, and 10x106 CAR+ cells / mouse). [Figure 17] Mean tumor volume in mice injected with three doses of UCARTmeso (TCR-negative anti-mesothelin Meso1-R2 CAR-positive cells) (1x106, 3x106, and 10x106 CAR+ cells / mouse). [Figure 18] Mean tumor volume ± standard deviation for mice injected with three doses of UCARTmeso cells (A: 1x106, B: 3x106, and C: 10x106 CAR+ cells / mouse). Comparison of different doses of Meso1-R2, P4-R2, and MESO2-R2, respectively. [Figure 19]Mean tumor volume in mice injected with two doses (3x106 and 10x106 CAR+ cells / mouse) of UCARTmeso cells that also express dnTGFBRII. [Figure 20] A. Comparison of CAR and dnTGFBRII detection in UCARTMeso cells expressing P4 or MESO1 constructs with dnTGFBRII. B. Percentages of CD4+ and CD8+ in the CAR-positive fraction of UCARTMeso cells produced in Example 5. [Figure 21] Percentage of Temra, Tem, Tcm; Tn / scm cells observed in the (A.) CAR+ CD4+ fraction or (B.) CAR+ CD8+ fraction of UCARTMeso cells produced in Example 5. [Figure 22] Percentage of H226 cell killing by different UCARTmeso cells in which the TGFBRII pathway was or was not inactivated by knockout (KO) or by expression of dominant-negative TGFBRII (dnTGFBRII). [Figure 23] IFNg production by UCARTMeso cells produced in Example 5 exposed (A.) or not (B.) to recombinant mesothelin protein. [Figure 24] Assessment of the sensitivity of UCARTmeso cells to TGFb. A. Percentage of pSMAD2 / 3 positive (gray) or negative (black) cells in the CAR-positive fraction of UCARTmeso produced in Example 5 after TGFb treatment. B. Percentage of growth inhibition of different UCARTmeso in the presence of TGFb and recombinant mesothelin protein. DETAILED DESCRIPTION OF THE INVENTION

[0038] Table 1: Amino acid sequences of the different domains that make up the P4, Meso1, and MESO2 scFvs of the CARs described in the Examples. Table 2: Amino acid sequences of different domains other than the scFv that make up the MSLN CAR of the present invention. Table 3: Examples of mAb-specific epitopes (and their corresponding mAbs) that can be used in the extracellular binding domain of the CAR of the invention for sorting and removal of modified cells. Table 4: Amino acid sequences of P4-R2, Meso1-R2, Meso1, and MESO2-R2 CARs. Table 5: Examples of mAb-specific epitopes (and their corresponding mAbs) that can be inserted into the extracellular binding domain of a CAR of the invention. Table 6: TALE nuclease target sequences for the TGFβRII gene. Table 7: CRISPR target sequence for TGFβRII gene. Table 8: Genomic sequences targeted by TALE nucleases (TALENs) to inactivate TCR and CD52. Table 9: Characteristics of modified T cell populations used in the examples. Table 10: Description of the six genetically modified T cells generated for the study provided in Example 5.

[0039] Detailed Description of the Invention Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of gene therapy, biochemistry, genetics, and molecular biology.

[0040] Although any methods and materials similar or equivalent to those described herein can be used in practicing or testing the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and, unless otherwise specified, are not intended to be limiting.

[0041] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art and are fully explained in the literature. For example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (MJ Gait ed., 1984); Mullis et al. U.S. Patent No. 4,683,195; Nucleic Acid Hybridization (BD Harries & SJ Higgins eds. 1984); Transcription And Translation (BD Hames & SJ Higgins eds. 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);Methods In ENZYMOLOGY series (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.Gene Transfer Vectors For Mammalian Cells (JH Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and CC Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

[0042] The present invention relates to a general method of treating solid tumors with adoptive immune cells directed against a specific epitope region of the transmembrane protein MSLN, specifically mesothelin spanning the polypeptide sequence SEQ ID NO:25 of this protein, and more specifically by using allogeneic CAR-T cells directed against this epitope, which have proven particularly effective.

[0043] Described herein are methods for producing engineered immune cells directed against human mesothelin (MSLN_human, referred to as Q13421 in the Uniprot database) displayed on the surface of malignant cells, more specifically, against a specific polypeptide region of mesothelin represented by SEQ ID NO: 25. As shown in the experimental section herein, highly efficient CAR T cells were produced by directing a CAR against this antigenic region comprising or consisting of SEQ ID NO: 25, specifically by using an scFv of the antibody Mesol, comprising SEQ ID NO: 9 and SEQ ID NO: 10.

[0044] The resulting modified immune cells, generally NK cells or T cells equipped with a CAR comprising SEQ ID NO:9 and / or SEQ ID NO:10, exhibit superior activation, potency, killing activity, cytokine release, and in vivo persistence compared to their counterparts with other conventional anti-mesothelin CARs.

[0045] Thus, the present invention relates to CAR immune cells that are specifically modified for the treatment of solid tumors and target specific epitopes contained in the sequence SEQ ID NO:25 of the MSLN protein present on the surface of malignant cells.

[0046] Design of MSLN-CAR expressed in immune cells : " Chimeric Antigen Receptor "(CAR)" refers to a recombinant receptor comprising a targeting moiety linked to one or more signaling domains in a single fusion molecule. Generally, the binding portion of a CAR consists of the antigen-binding domain of a single-chain antibody (scFv), which includes the light and heavy variable fragments of a monoclonal antibody connected by a flexible linker. Receptor-based binding moieties or ligand domains have also been successfully used. The signaling domains of CARs are typically derived from the cytoplasmic region of CD3 zeta or the gamma chain of an Fc receptor, and they are typically combined with signaling domains from costimulatory molecules such as CD28, OX-40 (CD134), ICOS, and 4-1BB (CD137) to enhance cell survival and proliferation. CARs are typically expressed in effector immune cells to redirect their immune activity to antigens expressed on the surface of tumor cells from various malignancies, such as lymphomas and solid tumors. One component of a CAR is any functional subunit of a CAR encoded by an exogenous polynucleotide sequence introduced into a cell. For example, this component may aid in the interaction with the target antigen, the intracellular stability or localization of the CAR.

[0047] In general, such a CAR: ·An extracellular ligand-binding domain containing VH and VL derived from a monoclonal anti-mesothelin antibody; Transmembrane domains; and a cytoplasmic domain containing a signaling domain, preferably a CD3 zeta signaling domain, and a costimulatory domain Includes.

[0048] The present invention more specifically relates to a CAR expressed in an immune cell, such as an NK cell or a T cell, wherein the CAR comprises an antigen-binding domain that specifically binds to SEQ ID NO:25.

[0049] In a preferred aspect, the mesothelin-specific chimeric antigen receptor (CAR) of the present invention has an extracellular ligand-binding domain comprising at least one CDR region from the variable heavy VH chain of antibody Mesol selected from CDRH1-Mesol (having identity to SEQ ID NO:3), CDRH2-Mesol (having identity to SEQ ID NO:4), and CDRH3-Mesol (having identity to SEQ ID NO:5), and / or a CDR region from the variable heavy VL chain of said antibody selected from CDRL1-Mesol (having identity to SEQ ID NO:6), CDRL2-Mesol (having identity to SEQ ID NO:7), and CDRL3-Mesol (having identity to SEQ ID NO:8).

[0050] Generally, the extracellular ligand-binding domain comprises: a variable heavy VH chain comprising CDRs from antibody Meso1, which have at least 90% identity to SEQ ID NO:3 (CDRH1-Meso1), SEQ ID NO:4 (CDRH2-Meso1), and SEQ ID NO:5 (CDRH3-Meso1), respectively; and / or a variable heavy VL chain comprising CDRs from antibody Meso1, which have at least 90% identity to SEQ ID NO:6 (CDRL1-Meso1), SEQ ID NO:7 (CDRL2-Meso1), and SEQ ID NO:8 (CDRL3-Meso1), respectively; Includes.

[0051] In a preferred embodiment of the invention, a mesothelin-specific chimeric antigen receptor has an extracellular ligand-binding domain comprising a VH chain having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with SEQ ID NO:9 (Mesol-VH), and a VL chain having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with SEQ ID NO:10 (Mesol-VL). Generally, framework residues within the framework regions can be substituted with the corresponding residue from the CDR donor antibody to alter, e.g., improve, antigen binding. These framework substitutions can be identified by methods well known in the art, such as modeling of CDR and framework residue interactions, thereby identifying framework residues important for antigen binding, and sequence comparison to identify exceptional framework residues at particular positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., (1988) Nature, 332:323, which are incorporated herein by reference in their entireties).

[0052] Various aspects of the present invention are provided through various features provided in the claims, in view of the common practice and knowledge of those skilled in the art. The details of the sequences contained in the CAR of the present invention are detailed in Tables 1, 2, 3, and 4, where each row or column should be considered an independent aspect of the present invention.

[0053] Table 1. Amino acid sequences of the different domains that make up the scFvs of P4, Meso1, and MESO2 TIFF2025186404000001.tif224163

[0054] Table 2: Amino acid sequences of domains other than the scFv that constitute the MSLN CAR of the present invention TIFF2025186404000002.tif89162

[0055] Table 3. Amino acid sequences of P4-R2, Meso1-R2, Meso1, and MESO2-R2 CARs TIFF2025186404000003.tif81161

[0056] Table 4. Full polypeptide sequences of MSLN CAR, dnTGFβRII, and MSLN epitope regions TIFF2025186404000004.tif227158TIFF2025186404000005.tif111158

[0057] The signal transduction domain or intracellular signal transduction domain of the CAR of the present invention is responsible for intracellular signal transduction after the extracellular ligand binding domain binds to the target, resulting in the activation of immune cells and immune responses.In other words, the signal transduction domain is responsible for activating at least one of the normal effector functions of the immune cells that CAR expresses.For example, the effector function of T cells can be cytolytic activity or helper activity, including cytokine secretion.Therefore, the term "signal transduction domain" refers to the part of a protein that transmits effector signal function signals to allow cells to perform specific functions.

[0058] Preferred examples of signaling domains used in CARs include cytoplasmic sequences of T cell receptors and co-receptors that act in concert to initiate signal transduction after binding to antigen receptors, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same functional capabilities. Signaling domains include two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and those that act antigen-independently to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that serve as binding sites for tyrosine kinases of the syk / zap70 class. Non-existent examples of ITAMs used in the present invention include those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In preferred embodiments, the signaling domain of the CAR may comprise a CD3 zeta signaling domain, which has an amino acid sequence having at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97%, or 99% sequence identity to an amino acid sequence selected from the group consisting of (SEQ ID NO:9).

[0059] In certain embodiments, the signaling domain of the CAR of the present invention comprises a costimulatory signal molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for a highly efficient immune response. A "costimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates T cell responses, such as, but not limited to, proliferation, activation, and differentiation, in addition to the primary signal provided by, for example, the binding of the TCR / CD3 complex to a peptide-loaded MHC molecule. Costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, and agonists that bind to Toll ligand receptors. Examples of costimulatory ligands include antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0060] In a preferred embodiment, the signaling domain of a CAR of the invention comprises a portion of a costimulatory signal molecule selected from the group consisting of fragments of 4-1BB (GenBank: AAA53133.) and CD28 (NP_006130.1). Specifically, the signaling domain of a CAR of the invention comprises an amino acid sequence that comprises at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97%, or 99% sequence identity to 4-1BB or CD28. Thus, a mesothelin-specific chimeric antigen receptor of the invention preferably comprises a CD3 zeta signaling domain that has at least 80% identity to SEQ ID NO:19, and generally comprises a costimulatory domain that has at least 80% identity to SEQ ID NO:18 (4-1BB).

[0061] The CAR of the present invention is generally expressed on the membrane surface of a cell. Therefore, such a CAR further comprises a transmembrane domain. A distinguishing feature of a suitable transmembrane domain is its ability to be expressed on the surface of a cell, preferably an immune cell in the present invention, specifically a lymphocyte or natural killer (NK) cell, and to interact with each other to direct the cellular response of the immune cell to a predetermined target cell. The transmembrane domain can be derived from a natural or synthetic source. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. As a non-existent example, the transmembrane polypeptide can be a subunit of a T cell receptor, such as α, β, γ, or ζ; a polypeptide constituting the CD3 complex; IL2 receptor p55 (α chain), p75 (β chain), or γ chain; a subunit chain of an Fc receptor, specifically Fcγ receptor III; or a CD protein. Alternatively, the transmembrane domain can be synthetic and mainly comprise hydrophobic residues such as leucine and valine. In a preferred embodiment, the transmembrane domain is derived from the human CD8 alpha chain (e.g., NP_001139345.1). The transmembrane domain may further comprise a hinge region between the extracellular ligand-binding domain and the transmembrane domain. As used herein, the term "hinge region" broadly refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. Specifically, the hinge region is used to provide additional flexibility and accessibility to the extracellular ligand-binding domain. The hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The hinge region may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or from all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be an entirely synthetic hinge sequence.In a preferred embodiment, the hinge domain comprises a portion of the human CD8 alpha chain, the FcγRIIIα receptor, or IgG1, respectively, or a hinge polypeptide exhibiting preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity to these polypeptides.

[0062] Thus, the mesothelin-specific chimeric antigen receptor (CAR) of the present invention comprises a hinge between the extracellular ligand-binding domain and the transmembrane domain, said hinge generally being selected from a CD8α hinge, an IgG1 hinge, and an FcγRIIIα hinge, or a polypeptide having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to these polypeptides, in particular to SEQ ID NO:16 (CD8α).

[0063] The CARs of the invention generally further comprise a transmembrane domain (TM), preferably selected from CD8α and 4-1BB, more preferably from CD8α-TM or a polypeptide exhibiting at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity to SEQ ID NO:17 (CD8α TM).

[0064] In a further embodiment, the mesothelin-specific CARs of the invention contain a safety switch that allows for convenient sorting, purification, and / or removal of the modified immune cells. While the methods of the invention are designed to be performed ex vivo, removal can be performed in vivo to control immune cell proliferation in the patient and potentially halt the therapeutic effect by using antibodies approved by regulatory agencies for human therapeutic use. Examples of mAb-specific epitopes (and their corresponding mAbs) that can be incorporated into the extracellular binding domain of the CARs of the invention are listed in Table 5.

[0065] Table 5. Examples of mAb-specific epitopes (and their corresponding mAbs) that can be inserted into the extracellular binding domain of the CAR of the invention TIFF2025186404000006.tif111139

[0066] Thus, mesothelin-specific CARs of the invention preferably contain a safety switch that includes at least one exogenous mAb epitope listed in Table 5. Preferably, mesothelin-specific CARs of the invention contain a safety switch that includes the epitope CPYSNPSLC (SEQ ID NO:26), to which rituximab specifically binds. More preferably, mesothelin-specific CARs contain a safety switch designated "R2," which has at least 90% identity to SEQ ID NO:15.

[0067] The mesothelin-specific CAR of the present invention also typically includes a signal peptide that aids in its expression on the surface of the engineered cell. Chimeric antigen receptors (CARs) typically form single polypeptide chains, although they may also be produced in multi-chain formats, e.g., WO2014039523.

[0068] As illustrated in the Examples, preferred CARs of the invention are MSLN-CAR-Mesol-R2 or MSLN-CAR-Mesol, which have at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% overall amino acid sequence identity with SEQ ID NO:21 (Mesol-R2) or SEQ ID NO:22 (Mesol), respectively.

[0069] The preferred polypeptide structure of the MSLN-CAR of the present invention is illustrated in FIG.

[0070] More generally, the CARs of the present invention are produced by assembling various polynucleotide sequences encoding consecutive fragments of the CAR polypeptide in a vector and transfecting them into immune cells for expression, as described in the art and as outlined, for example, in [Boyiadzis, MM, et al. (2018) Chimeric antigen receptor (CAR) T therapies for the treatment of hematologic malignancies: clinical perspective and significance. J. Immunotherapy Cancer 6, 137].

[0071] The present invention relates to the polynucleotides and vectors, as well as any intermediate steps intervening in the process of producing the immune cells referred to herein.

[0072] "Vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. "Vector" in the present invention includes, but is not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules that may consist of chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acids. Preferred vectors are those capable of autonomous replication (episomal vectors) and / or vectors capable of expressing nucleic acids to which they are linked (expression vectors). Many suitable vectors are known to those of skill in the art and are commercially available. Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses (AAV)), coronaviruses, negative-strand RNA viruses, such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses, such as picornaviruses and alphaviruses, and double-stranded DNA viruses, such as adenoviruses, herpesviruses (e.g., herpes simplex viruses 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox viruses). Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include avian leukosis and sarcoma viruses, mammalian types C, B, and D viruses, the HTLV-BLV complex, lentiviruses, and spumaviruses (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0073] In particular, the present invention provides, inter alia, expression vectors in the form of lentiviral or AAV vectors that comprise a polynucleotide sequence encoding a CAR described herein.

[0074] Such lentiviral vectors can comprise a polynucleotide sequence encoding a CAR of the invention operably linked to a promoter (such as a spleen focus-forming virus promoter (SFFV)). "Operably linked" means that the described components are juxtaposed in a relationship permitting their intended function. When a gene (such as a polynucleotide sequence encoding a CAR) is "operably linked" to a promoter and its transcription is under the control of the promoter, transcription results in the production of a product encoded by the gene.

[0075] Lentiviral vectors of the present invention typically contain regulatory elements such as 5' and 3' long terminal repeats (LTRs), but may also contain other structural and functional genetic elements originally derived from lentiviruses. Such structural and functional genetic elements are well known in the art. Lentiviral vectors may contain, for example, the gag, pol, and env genes. Preferably, however, lentiviral vectors of the present invention do not contain the gag, pol, and env genes. As additional regulatory elements, lentiviral vectors may contain one or more (e.g., two or more) packaging signals (e.g., packaging signal ψ), a primer binding site, a transactivation response region (TAR), and a rev response region (RRE).

[0076] The 5'-terminal repeat (LTR) and 3'-terminal repeat (LTR), typically located on either side of the lentiviral genome, possess promoter / enhancer activity and are essential for the correct expression of full-length lentiviral vector transcripts. LTRs usually contain the repeat sequence U3RU5, present at both the 5' and 3' ends of the double-stranded DNA molecule. This sequence is a combination of a 5'R-U5 segment and a 3'U3-R segment of single-stranded RNA, where the R repeat occurs at both ends of the RNA, but U5 (unique sequence 5) occurs only at the 5' end of the RNA, and U3 (unique sequence 3) occurs only at the 3' end of the RNA. The safety of lentiviral vectors can be improved by removing the U3 sequence, resulting in "self-activating" vectors that are completely devoid of the viral promoter and enhancer sequences originally present in the LTR. Therefore, the vector can infect and integrate into the host genome once but is not further inherited, thereby increasing the safety of the vector for use as a gene delivery vector.

[0077] In some embodiments, the lentiviral vector is a self-inactivating (SIN) lentiviral vector. In certain embodiments, the lentiviral vector comprises a 3'LTR, wherein the 3'LTR enhancer-promoter sequence (i.e., the U3 sequence) is modified (e.g., deleted).

[0078] In some embodiments, the lentiviral vector comprises the following elements in 5' to 3' order: · 5' long terminal repeat (5'LTR); · promoters (e.g. EF1-alpha promoter); a polynucleotide sequence encoding a chimeric antigen receptor of the invention; and / or 3' long terminal repeat (3' LTR), preferably a 3' self-inactivating LTR It includes a polynucleotide sequence comprising one or more of:

[0079] In certain embodiments, the lentiviral vector comprises, in 5' to 3' order, the following elements: · 5' long terminal repeat (5'LTR); · promoters (e.g. EF1-alpha promoter); The car of the present invention may include a safety switch such as R2. a polynucleotide sequence encoding the 2A peptide; a polynucleotide sequence encoding one or any additional polypeptides, such as dnTGFβR, to be co-expressed with the CAR; and / or 3' long terminal repeat (3' LTR), preferably a 3' self-inactivating LTR The polynucleotide sequence may further comprise at least one of:

[0080] Alternatively, the lentiviral vector may comprise, in 5' to 3' order, the following elements: · 5' long terminal repeat (5'LTR); · promoters (e.g. EF1-alpha promoter); a polynucleotide sequence encoding one or any additional polypeptides, such as dnTGFβR, to be co-expressed with the CAR; a polynucleotide sequence encoding the 2A peptide; The car of the present invention may include a safety switch such as R2. and / or 3' long terminal repeat (3' LTR), preferably a 3' self-inactivating LTR It may include at least one of:

[0081] Generally, the resulting vector forms a single transcription unit operably linked to the promoter of item (b), and all are transcribed under the control of said promoter.

[0082] AAV vectors, particularly vectors from the AAV6 family [Wang, J., et al. (2015) Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat Biotechnol 33, 1256-1263], are particularly useful for introducing the MSLN-CAR of the present invention into the genome via site-specific homologous recombination. Site-specific homologous recombination is typically induced in immune cells by expression of a rare-cutting endonuclease, such as a TALEN, as previously taught for other CARs treating hematologic cancers in EP3276000 and WO2018073391. Site-specific integration of CARs may offer several advantages, including more stable integration, integration that places the transgene under the transcriptional control of the endogenous promoter at the selected locus, and integration that can inactivate the endogenous locus. These latter aspects are discussed in more detail in the following section on genome engineering of therapeutic immune cells.

[0083] As one object of the present invention, an AAV vector is provided that includes a polynucleotide sequence encoding an MSLN-CAR, as described above, and, optionally, another sequence encoding a cis-regulatory element (e.g., a 2A peptide cleavage site) or an internal ribosome entry site (IRES), allowing for the simultaneous expression of a third sequence encoding a product that improves the therapeutic efficacy of the engineered immune cells. Provided as an example herein is overexpression of dnTGFβRII, which has been shown to reduce SMAD2-3 phosphorylation, resulting in reduced TGFβ-induced cell elimination in the tumor environment.

[0084] " therapeutic propertiesThe term "therapeutic properties" encompasses various ways in which such cells can be improved with respect to their use in therapeutic treatment. This means that genetic engineering can confer a therapeutic benefit on the cells (i.e., therapeutic efficacy) or facilitate their use or production. For example, genetic engineering can result in effector cells that have better survival rates, faster growth, a shorter cell cycle, improved immune activity, are more functional, are more differentiated, are more specific for target cells, are more sensitive or resistant to drugs, or are more sensitive to glucose deprivation, oxygen, or amino acid deprivation (i.e., are better able to adapt to the tumor microenvironment). Progenitor cells may be more productive, may be tolerated by the recipient patient, and may be more likely to produce cells that differentiate into the desired effector cells. These examples of "therapeutic properties" are provided by way of non-limiting example.

[0085] Genome engineering of MSLN CAR immune cells for cell therapy More specifically, the present invention includes cells and reagents having the following characteristics:

[0086] Effector cells: Effector cells are relatively short-lived, activated cells that defend the body during immune responses. Activated T cells include cytotoxic T cells and helper T cells, which are the preferred effector cells that carry out cellular responses. The category of effector T cells is broad, encompassing various T cell types that actively respond to stimuli, including costimulation. This includes helper, killer, regulatory, and other potential T cell types.

[0087] "Immune cells" refer to cells of hematopoietic origin that are functionally involved in the initiation of innate and / or adaptive immune responses, typically CD3- or CD4-positive cells. The immune cells of the present invention may be dendritic cells, killer dendritic cells, mast cells, NK cells, B cells, or T cells selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes.

[0088] "Primary cells" refer to cells obtained directly from biological tissue (e.g., biopsy material) and grown in vitro for a limited time, i.e., allowing a limited number of population doublings, to become established as a cell line. Primary cells are the opposite of persistently tumorigenic or artificially immortalized cell lines. Non-existent examples of such cell lines include CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; and Molt4 cells.

[0089] Primary immune cells can be obtained from several sources, including, but not limited to, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of inflammation, ascites, pleural effusion, spleen tissue, and tumor-infiltrating lymphocytes from tumors. In some embodiments, the immune cells can be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with an infectious disease. In other embodiments, the cells are part of a mixed population of immune cells exhibiting different phenotypic characteristics, including CD4, CD8, and CD56 positive cells. Primary immune cells can be provided from a donor or patient by various methods known in the art, such as leukapheresis, as outlined by Schwartz J. et al. (Guidelines on the use of therapeutic apheresis in clinical practice—evidence-based approach from the Writing Committee of the American Society for Apheresis: the sixth special issue (2013) J Clin Apher. 28(3):145-284).

[0090] Stem cell-derived immune cells, specifically immune cells derived from induced pluripotent stem cells (iPS) [Yamanaka, K. et al. (2008). "Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors". Science. 322 (5903): 949-53], are also considered primary immune cells of the present invention. Lentiviral expression of reprogramming factors has been used to induce pluripotent cells from human peripheral blood cells [Staerk, J. et al. (2010). "Reprogramming of human peripheral blood cells to induced pluripotent stem cells". Cell Stem Cell. 7 (1): 20-4] [Loh, YH. et al. (2010). "Reprogramming of T cells from human peripheral blood". Cell Stem Cell. 7 (1): 15-9].

[0091] In a preferred embodiment of the present invention, immune cells are derived from human embryonic stem cells by techniques known in the art that do not destroy the human fetus [Chung et al. (2008) Human Embryonic Stem Cell lines generated without embryo destruction, Cell Stem Cell 2(2):113-117].

[0092] "Genetic engineering" means any method aimed at introducing, modifying, and / or removing genetic material from cells. Gene editing " refers to genetic engineering that allows for the addition, removal, or modification of genetic material at specific locations (locuses) within the genome, including punctual mutations. Gene editing generally involves sequence-specific reagents.

[0093] By "sequence-specific reagent" is meant any active molecule capable of specifically recognizing a selected polynucleotide sequence, termed a "target sequence," generally at least 9 bp, more preferably at least 10 bp, and even more preferably at least 12 bp in length at a genomic locus, with a view to altering expression of said locus. The expression can be altered by mutations, deletions, or insertions in coding or regulatory polynucleotide sequences, by epigenetic changes such as by methylation or histone modifications, or by interfering with the level of transcription by interaction with transcription factors or polymerases.

[0094] Examples of sequence-specific reagents are endonucleases, RNA guides, RNAi, methylases, exonucleases, histone deacetylases, endonucleases, end-processing enzymes, such as exonucleases, more particularly cytidine deaminases, such as those that combine with the CRISPR / cas9 system to perform base editing (i.e., nucleotide substitution), but without necessarily relying on nuclease cleavage, as described, for example, in Hess, GT et al. [Methods and applications of CRISPR-mediated base editing in eukaryotic genomes (2017) Mol Cell. 68(1): 26-43].

[0095] In a preferred aspect of the invention, the sequence-specific reagent is preferably a sequence-specific nuclease reagent, such as an RNA guide coupled to a guided endonuclease.

[0096] The present invention aims to improve the therapeutic potential of immune cells by gene editing methods, in particular by targeted gene integration.

[0097] "Targeted gene integration" means any known site-specific method that allows for the insertion of, replacing, or modifying a genomic coding sequence into a living cell.

[0098] In a preferred aspect of the invention, the targeted gene integration involves the insertion or replacement of at least one exogenous nucleotide, preferably a sequence of several nucleotides (i.e., a polynucleotide), more preferably a coding sequence, by homologous recombination at the locus of the targeted gene.

[0099] By "DNA target," "DNA target sequence," "target DNA sequence," "nucleic acid target sequence," "target sequence," or "processing site" is intended a polynucleotide sequence that can be targeted and processed by the sequence-specific nuclease reagents of the invention. These terms refer to a specific DNA location within a cell, preferably a genomic location, but also to a portion of genetic material that can exist independently of the body of genetic material, such as, for example, a plasmid, episome, virus, transposon, or part of an organelle such as a mitochondrion, as an exemplary example. An exemplary RNA-guided target sequence is a genomic sequence that can hybridize with a guide RNA that directs an RNA-guided endonuclease to a desired locus.

[0100] "Slow-cutting endonucleases" are any sequence-specific endonuclease reagent so long as their recognition sequence is in the range of approximately 10-50 contiguous base pairs, preferably 12-30 bp, more preferably 14-20 bp.

[0101] In a preferred aspect of the present invention, the endonuclease reagent is a homing endonuclease, e.g., as described in Arnould S., et al. [WO2004067736], a zinc finger nuclease (ZFN), e.g., as described in Urnov F., et al. [Highly efficient endogenous human gene correction using designed zinc-finger nucleases (2005) Nature 435:646-651], a TALE nuclease, e.g., as described in Mussolino et al. [A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity (2011) Nucl. Acids Res. 39(21):9283-9293], or a TALE nuclease, e.g., as described in Boissel et al. [MegaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering (2013) Nucleic Acids Research 42(4):2591-2601].

[0102] In another embodiment, the endonuclease reagent is RNA-guided, used in conjunction with an RNA-guided endonuclease such as Cas9 or Cpf1, among others, according to the teachings of Doudna, J., and Chapentier, E., [The new frontier of genome engineering with CRISPR-Cas9 (2014) Science 346 (6213):1077] (incorporated herein by reference).

[0103] In a preferred aspect of the invention, the endonuclease reagent is expressed transiently in the cell, i.e., the reagent is not integrated into the genome or persists long term, as is the case with RNA, more particularly mRNA, proteins, or mixed complexes of proteins and nucleic acids (e.g., ribonucleoproteins).

[0104] To enhance stability, the mRNA form of the endonuclease is preferably synthesized with a cap by techniques known in the art, such as those described in Kore AL, et al. [Locked nucleic acid (LNA)-modified dinucleotide mRNA cap analogue: synthesis, enzymatic incorporation, and utilization (2009) J Am Chem Soc. 131(18):6364-5].

[0105] In general, the electroporation process used to transduce primary immune cells such as PBMCs is typically carried out in a closed chamber containing parallel plate electrodes, as described in WO2004083379 (particularly page 23, line 25 to page 29, line 11), which is incorporated herein by reference, and a substantially uniform pulsed electric field greater than 100 volts / cm and less than 5000 volts / cm is generated between the parallel plate electrodes throughout the treatment volume. One such electroporation chamber preferably has a volume equal to the quotient of the electrode gap squared (cm2) relative to the chamber volume (cm2). 3 ) and divide by the shape factor (cm -1 ), which has a view factor of 0.1 cm -1 wherein the suspension of cells and sequence-specific reagents is in a medium adjusted to have a conductivity in the range of 0.01 to 1.0 millisiemens. Generally, the cell suspension is subjected to one or more pulsed electric fields. In this method, the processing volume of the suspension is scalable, and the cell processing time within the chamber is substantially uniform.

[0106] For example, Mussolino et al. As reported in [facilitate targeted genome editing in human cells with high specificity and low cytotoxicity (2014) Nucl. Acids Res. 42(10): 6762-6773], TALE nucleases have proven to be particularly suitable sequence-specific nuclease reagents for therapeutic applications due to their relatively high specificity, especially when acting in heterodimeric form, i.e., as a pair of a "right" (also called "5'" or "forward") and a "left" (also called "3'" or "reverse") monomer.

[0107] As mentioned above, the sequence-specific reagent is preferably in the form of a nucleic acid, e.g., DNA or RNA encoding a slow-cutting endonuclease, its subunits, but may also be part of a conjugate comprising polynucleotides and polypeptides, such as so-called "ribonucleoproteins." Such conjugates can be formed with reagents such as Cas9 or Cpf1 (RNA-guided endonucleases) as described in Zetsche, B. et al. [Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System (2015) Cell 163(3): 759-771] and Gao F. et al. [DNA-guided genome editing using the Natronobacterium gregoryi Argonaute (2016) Nature Biotech], which contain an RNA or DNA guide that can be complexed with the respective nuclease.

[0108] "Exogenous sequence" refers to any nucleotide or nucleic acid sequence not originally present at a selected locus. This sequence may be homologous to or a copy of a genomic sequence, or may be a foreign sequence introduced into a cell. Conversely, "endogenous sequence" refers to a genomic sequence of a cell originally present at a locus. The exogenous sequence preferably encodes a polypeptide whose expression confers a therapeutic advantage over sister cells that do not have the exogenous sequence integrated into the locus. An endogenous sequence that has been gene-edited by inserting a nucleotide or polynucleotide according to the methods of the present invention to express a different polypeptide is broadly referred to as an exogenous coding sequence.

[0109] By using the above reagents and techniques, the present invention provides: providing immune cells, preferably primary cells, from a donor or a patient; expressing the MSLN-CAR in such cells as described above, generally by introducing the MSLN-CAR coding sequence into the genome of the cells via a viral vector; introducing a sequence-specific reagent, such as a rare-cutting endonuclease, into such immune cells to induce an alteration (mutation or coding sequence insertion) in the endogenous gene locus; and / or introducing an exogenous coding sequence into said cells in order to improve the therapeutic efficacy of said cells, in particular their immunological properties; By implementing one or more of these, a method for producing therapeutic cells will be developed.

[0110] In some aspects of the invention, immune cells are derived from the patient or a matched donor, and the MSLN CAR is expressed in the cells, allowing for the implementation of so-called "autologous" infusion of the engineered immune cells. The immune cells may also be derived from stem cells, such as iPS cells, derived from such a patient or matched donor, or from tumor-infiltrating lymphocytes (TILL).

[0111] In some aspects of the invention, methods are directed to providing "off-the-shelf" compositions of immune cells, said immune cells being modified for allogeneic therapeutic treatment.

[0112] "Allogeneic" means that the cells are derived from a donor, produced or differentiated from stem cells with a view to being infused into a patient with a different haplotype.

[0113] Such immune cells are generally modified to be less alloreactive and / or more durable in relation to the patient host. More specifically, the method involves reducing or inactivating the expression of TCR in T cells or stem cells derived from T cells. This can be achieved by various sequence-specific reagents, such as gene silencing or gene editing methods (nucleases, base editing, RNAi, etc.).

[0114] Applicant has previously made available robust protocols and gene editing strategies for producing allogeneic therapeutic-grade T cells from PBMCs, particularly by providing highly safe and specific endonuclease reagents in the form of TALE nucleases (TALEN®), which encode donor-derived [TCR]. negThe production of so-called "universal T cells" has been achieved and successfully infused into patients with mild graft-versus-host disease (GVhD) [Poirot et al. (2015) Multiplex Genome-Edited T-cell Manufacturing Platform for "Off-the-Shelf" Adoptive T-cell Immunotherapies. Cancer. Res. 75 (18): 3853-3864] [Qasim, W. et al. (2017) Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Science Translational 9(374)]. Additionally, inactivation of TCR or β2m components in primary T cells can be combined with inactivation of additional genes encoding checkpoint inhibitor proteins, as described, for example, in WO2014184744.

[0115] In a preferred embodiment, the present invention provides a method of modifying an immune cell, in which at least one gene encoding a TCR alpha or TCR beta is inactivated, preferably by expression of a rare-cutting endonuclease, while an exogenous polynucleotide encoding an MSLN-CAR is introduced into the genome of the cell for stable expression. Preferably, the exogenous sequence is integrated into the locus encoding the TCR alpha or TCR beta, more preferably under the transcriptional control of the endogenous promoter of the TCR alpha or TCR beta.

[0116] In a further embodiment, the modified immune cells can be further modified to confer resistance to at least one immunosuppressant, for example, by inactivating CD52, the target of an anti-CD52 antibody (e.g., alemtuzumab), which is described, for example, in WO2013176915 for the treatment of hematological cancers.

[0117] While the use of anti-CD52 lymphocyte depleting agents has so far been limited to liquid tumor cancers [Quasim W. et al. (2019) Allogeneic CAR T cell therapies for leukemia Am J Hematol. 94:S50-S54.], one key aspect of the present invention is the use of genetically modified lymphocytes that have been rendered resistant to lymphocyte depletion regimens for the treatment of solid tumors.

[0118] The present invention also provides modified lymphocytes comprising chimeric antigen receptors directed against solid tumors, particularly against mesothelin-positive cells, which are used in the cancer treatment of solid tumors in conjunction with or following lymphocyte-depleting treatment steps.

[0119] Such lymphocyte depleting regimens may include anti-CD52 reagents, such as alemtuzumab or purine analogs used in the treatment of hematological cancers.

[0120] In preferred embodiments, modified lymphocytes comprising the MSLN-CAR described herein are made resistant to such lymphodepletion regimens by inhibiting or interfering with the expression of the molecule targeted by the lymphodepletion reagent, such as, for example, the antigen CD52 in the case of alemtuzumab.

[0121] In a further embodiment, the modified immune cells can be further modified to confer resistance to chemotherapeutic agents, particularly purine analogue agents, and / or by inactivating DCK, for example as described in WO201575195.

[0122] As mentioned above, treating solid tumor cancers with genetically modified lymphocytes equipped with chimeric antigen receptors that are resistant to chemotherapy or lymphocyte-depleting regimens is an important aspect of the present invention. Such regimens may include antibodies targeting antigens present on the surface of immune cells, such as CD52, CD3, CD4, CD8, CD45, or other specific markers, as well as less specific drugs such as purine analogs (e.g., fludarabine and / or chlorofarabine) and glucocorticoids. One aspect of the present invention is to render modified lymphocytes resistant to such regimens by inactivating or reducing the expression of a gene encoding at least one molecular target of these lymphocyte-depleting agents, such as the purine analog-metabolizing gene DCK or the gene encoding the glucocorticoid receptor (GR).

[0123] Thus, the present invention more specifically focuses on CAR-positive cells and renders them less alloreactive and resistant to lymphodepletion regimens by reducing, inactivating, or impairing the expression of TCR, CD52, and / or DCK and / or GR for their allogeneic use in the treatment of solid tumors.

[0124] In further embodiments, the modified immune cells can be further modified to improve persistence or longevity in the patient, specifically by inactivating genes encoding MHC-I components such as HLA or β2m, as described in WO2015136001 or Liu, X. et al. [CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells (2017) Cell Res 27:154-157].

[0125] In a preferred aspect of the present invention, the engineered immune cells are mutated to improve their CAR-dependent immune activation, specifically to reduce or suppress the expression of immune checkpoint proteins and / or their receptors, e.g., PD1 or CTLA4 as described in WO2014184744.

[0126] In a further aspect, the modified immune cell can be further modified to obtain the co-expression of another exogenous gene sequence within said cell, said exogenous gene sequence being: NK cell inhibitors, such as HLAG, HLAE, or ULBP1; CRS inhibitors, such as mutant IL6Ra, sGP130, or IL18-BP; Cytochrome P450, CYP2D6-1, CYP2D6-2, CYP2C9, CYP3A4, CYP2C19, or CYP1A2, which sensitize said immune cells to drugs such as cyclophosphamide and / or isophosphamide; dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin, or methylguanine transferase (MGMT), mTORmut, or Lckmut, which confer drug resistance; Chemokines or cytokines, such as IL-2, IL-12, and IL-15; a chemokine receptor, such as CCR2, CXCR2, or CXCR4; and / or Tumor-associated macrophage (TAM) secretion inhibitors, such as CCR2 / CCL2 neutralizers, which enhance the therapeutic activity of immune cells The nucleotide sequence is selected from those encoding:

[0127] The present application claims an immune cell in which at least one exogenous sequence encoding an MSLN-CAR described herein and another exogenous sequence encoding a human polypeptide selected from the list above are co-expressed in the modified immune cell to produce a therapeutic composition for solid tumors.

[0128] Combining MSLN-CAR expression with TGFbRII signaling pathway disruption in therapeutically engineered immune cells The present invention more specifically combines the expression of an exogenous sequence encoding the aforementioned MSLN-CAR with another exogenous sequence encoding an inhibitor of the TGF beta receptor, in particular an inhibitor of TGFβRII (Uniprot - P37173).

[0129] TGF-beta receptors have been described as having a major role in the tumor microenvironment [Papageorgis, P. et al. (2015). Role of TGF-beta in regulation of the tumor microenvironment and drug delivery (Review). International Journal of Oncology, 46, 933-943].

[0130] Although the exact role of TGF beta receptors in tumorigenesis remains controversial, the present inventors have found that co-expressing a mesothelin-specific chimeric antigen receptor (CAR) with another exogenous gene sequence encoding an inhibitor of TGFBRII signaling, and / or inactivating or reducing TGF beta receptor signaling using sequence-specific reagents, results in improved therapeutic efficacy of engineered immune cells. Specifically, the present inventors have used two different approaches to inhibit the TGF beta RII signaling pathway, which may be used in combination: Expression of an inactive ligand of TGFβRII, such as a dominant-negative TGFβRII (SEQ ID NO:26), as described by Hiramatsu, K., et al. [Expression of dominant-negative TGF-β receptors inhibits cartilage formation in conditional transgenic mice (2011) J. Bone. Miner. Metab. 29: 493], or a similar inactive form of TGFβRII having at least 80%, preferably at least 90%, more preferably at least 95% identity to the polypeptide sequence SEQ ID NO:26. and / or Inactivation of the endogenous gene sequence of TGFβRII, specifically by using slow-cutting endonucleases such as TALE nucleases or RNA-guided endonucleases (e.g., Cas9 or Cpf1).

[0131] Anti-TGFβRII IgG1 monoclonal antibodies such as LY3022859, which inhibit receptor-mediated signaling activation [Tolcher, AW et al. (2017) A phase 1 study of anti-TGFβ receptor type-II monoclonal antibody LY3022859 in patients with advanced solid tumors Cancer Chemother Pharmacol.79(4):673-680] can also be used in combination with the CARs of the invention to inhibit TGFbeta receptor signaling.

[0132] The present application discloses herein a set of TALE nucleases that are specifically specific for a set of target sequences within the TGFβRII gene. These TALE nucleases exhibited the highest TGFβRII knockout efficiency while exhibiting minimal off-target cleavage, resulting in a large population of modified viable cells sufficient for administration to several patients. These preferred TALE nucleases and their corresponding target sequences are listed in Table 6.

[0133] Table 6: TALE nuclease target sequences for the TGFβRII gene TIFF2025186404000007.tif72158

[0134] RNA guides were also designed for TGFβRII gene inactivation using Cas9 nuclease reagents, and the corresponding target sequences are disclosed in Table 7.

[0135] Table 7. CRISPR target sequence for TGFβRII gene TIFF2025186404000008.tif106161TIFF2025186404000009.tif243161TIFF2025186404000010.tif243161TIFF2025186404000011.tif22161

[0136] The present invention therefore encompasses the use of TALE nucleases or RNA-guided endonucleases designed to bind to any of the target sequences SEQ ID NO:X-Y set forth in Tables 5 or 6 to inactivate or reduce expression of TGFβRII for the production of therapeutic immune cells within the teachings of the present specification.

[0137] The present invention also relates to modified immune cells that contain an exogenous polynucleotide encoding a nuclease as described above to inactivate or reduce expression of the endogenous TGFβRII gene in the modified immune cells.

[0138] The present application therefore reports modified immune cells, in particular CAR immune cells, into which an exogenous sequence encoding an inhibitor of the TGF-beta receptor, more particularly a sequence encoding a dominant-negative TGF-beta receptor, has been introduced, and such cells are more particularly dedicated to the treatment of solid tumors, in particular MSLN-positive tumors.

[0139] Therefore, the present application also claims a vector, particularly a viral vector, such as a lentiviral or AAV vector as described in the art, comprising at least a polynucleotide sequence encoding a dominant-negative TGFβRII and, optionally, a mesothelin-specific chimeric antigen receptor. In a preferred embodiment, the vector comprises a first polynucleotide sequence encoding the dominant-negative TGFβRII, a second polynucleotide sequence encoding a 2A self-cleaving peptide, and a third polynucleotide sequence encoding the mesothelin-specific chimeric antigen receptor.

[0140] Targeted insertion into immune cells can be greatly improved by using AAV vectors, in particular vectors of the AAV6 family, or the chimeric vector AAV2 / 6 previously described by Sharma A., et al. [Transduction efficiency of AAV 2 / 6, 2 / 8 and 2 / 9 vectors for delivering genes in human corneal fibroblasts. (2010) Brain Research Bulletin. 81 (2-3): 273-278].

[0141] One aspect of the present invention is therefore the transduction of an AAV vector comprising an MSLN-CAR coding sequence in human primary immune cells in conjunction with the expression of a sequence-specific endonuclease reagent, such as a TALE endonuclease, to increase gene integration at the aforementioned locus.

[0142] In a preferred aspect of the present invention, the sequence-specific endonuclease reagent may be introduced into the cell by gene transfer, more preferably by electroporation of mRNA encoding said sequence-specific endonuclease reagent.

[0143] The insertion of an exogenous nucleic acid sequence can result in the introduction of genetic material, the modification or replacement of an endogenous sequence, more preferably "in frame" with the endogenous gene sequence at that locus.

[0144] In another aspect of the invention, 10 per cell 5 ~10 7 , preferably 10 6 ~10 7 , more preferably about 5.10 6 The viral genome is transduced.

[0145] In another aspect of the invention, cells can be treated with a proteasome inhibitor, such as bortezomib, or an HDAC inhibitor to further favor homologous recombination.

[0146] For purposes of the present invention, the AAV vector used in the method may comprise a promoterless exogenous coding sequence, said coding sequence being any of the sequences mentioned herein.

[0147] The present invention also provides an efficient method for obtaining primary immune cells that can be edited at various gene loci, more specifically those involved in host-graft interaction and recognition. Other loci can also be edited with a view to improving the activity, survival, or longevity of the modified primary cells, particularly primary T cells.

[0148] Figure 2 maps the major cellular functions that can be modified by gene editing of the present invention to improve the efficacy of modified immune cells. Any of the gene inactivations listed under each function can be combined with each other to achieve a synergistic effect on the overall therapeutic efficacy of the immune cells.

[0149] The present invention more particularly provides combinations of genetic modifications (genotypes) in immune cells that are useful in the treatment of solid tumors, in particular: [MSLN-CAR] + , [MSLN-CAR] + [dnTGFβRII] + [MSLN-CAR] + [dnTGFβRII] + [TCR] - , [MSLN-CAR] + [dnTGFβRII] + [TGFβRII] - [TCR] - , [MSLN-CAR] + [TGFβRII] - , [MSLN-CAR] + [TGFβRII] - [TCR] - , [MSLN-CAR] + [β2m] - , [MSLN-CAR]+ [dnTGFβRII] + [β2m] - 、 ·[MSLN-CAR] + [TGFβRII] - [β2m] - 、 ·[MSLN-CAR] + [dnTGFβRII] + [β2m] - [TCR] - 、 ·[MSLN-CAR] + [TGFβRII] - [β2m] - [TCR] - 、 ·[MSLN-CAR] + [dnTGFβRII] + [TGFβRII] - [β2m] - [TCR] - 、 ·[MSLN-CAR] + [PD1] - 、 ·[MSLN-CAR] + [TGFβRII] - [PD1] - 、 ·[MSLN-CAR] + [dnTGFβRII] + [PD1] - 、 ·[MSLN-CAR] + [dnTGFβRII] + [TGFβRII] - [PD1] - 、 ·[MSLN-CAR] + [dnTGFβRII] + [β2m] - [PD1] - 、 ·[MSLN-CAR] + [TGFβRII] - [PD1] - [TCR] - 、 ·[MSLN-CAR] +[dnTGFβRII] + [TGFβRII] - [PD1] - [TCR] - , [MSLN-CAR] + [PD1] - [β2m] - , [MSLN-CAR] + [TGFβRII] - [PD1] - [β2m] - , [MSLN-CAR] + [dnTGFβRII] + [PD1] - [β2m] - , [MSLN-CAR] + [dnTGFβRII] + [TGFβRII] - [PD1] - [β2m] - , [MSLN-CAR] + [dnTGFβRII] + [β2m] - [PD1] - [TCR] - , [MSLN-CAR] + [TGFβRII] - [PD1] - [TCR] - [β2m] - , [MSLN-CAR] + [dnTGFβRII] + [TGFβRII] - [PD1] - [TCR] - [β2m] - This will rapidly improve the efficacy of immune cells against MSLN-positive malignant cells such as

[0150] As mentioned above, the present invention is also particularly focused on the use of CAR-positive cells in the treatment of solid tumors, which may be used in conjunction with or following lymphocyte-depleting regimens in the allogeneic setting, as the cells are rendered resistant to lymphocyte-depleting agents. Such cells preferably exhibit the following genotype: Partial or complete resistance to anti-CD52 antibodies: [MSLN-CAR] + [CD52] - [TCR] - , [MSLN-CAR] + [CD52] - [TCR] - [β2m] - ,, [MSLN-CAR] + [TGFβRII] - [CD52] - [TCR] - , [MSLN-CAR] + [TGFβRII] - [CD52] - [TCR] - [β2m] - , [MSLN-CAR] + [dnTGFβRII] + [CD52] - [TCR] - , [MSLN-CAR] + [dnTGFβRII] + [CD52] - [TCR] - [β2m] - , Partial or complete resistance to purine analogues: [MSLN-CAR] + [DCK] - [TCR] - , [MSLN-CAR] + [DCK] - [TCR] - [β2m] - ,, [MSLN-CAR] +[TGFβRII] - [DCK] - [TCR] - , [MSLN-CAR] + [TGFβRII] - [DCK] - [TCR] - [β2m] - , [MSLN-CAR] + [dnTGFβRII] + [DCK] - [TCR] - , [MSLN-CAR] + [dnTGFβRII] + [DCK] - [TCR] - [β2m] - , Partial or complete resistance to glucocorticoids: [MSLN-CAR] + [GR] - [TCR] - , [MSLN-CAR] + [GR] - [TCR] - [β2m] - ,, [MSLN-CAR] + [TGFβRII] - [GR] - [TCR] - , [MSLN-CAR] + [TGFβRII] - [GR] - [TCR] - [β2m] - , [MSLN-CAR] + [dnTGFβRII] + [GR] - [TCR] - , [MSLN-CAR] + [dnTGFβRII] + [GR] - [TCR] - [β2m]- ,

[0151] Further improving therapeutic immune cells by expressing transgenes at inactivated loci The above preferred genotypes may be obtained by targeted gene integration, preferably at the PD1, TCR (TCR alpha and / or TCR beta), or TGFβRII locus, but also at further selected loci as described below.

[0152] "Targeted gene integration" refers to any known site-specific method that allows a living cell to be inserted, replaced, or modified with a genomic sequence. Targeted gene integration usually involves the mechanism of homologous recombination or NHEJ (non-homologous end joining), enhanced by endonuclease sequence-specific reagents, resulting in the insertion or replacement of at least one exogenous nucleotide, preferably a sequence of several nucleotides (i.e., a polynucleotide), more preferably a coding sequence, at a predetermined locus.

[0153] The method of the present invention may be combined with other methods involving gene conversion, such as viral transduction, and may also be combined with other transgene expression methods that do not necessarily involve integration.

[0154] In one aspect, the method of the invention comprises: a) polynucleotide sequences whose expression is involved in the decrease in glycolysis and calcium signaling in response to low glucose conditions, such as SERCA3, which increases calcium signaling, miR101 and mir26A, which increase glycolysis, and BCAT, which mobilizes glycolytic stores; and / or b) a polynucleotide sequence whose expression upregulates immune checkpoint proteins (e.g., TIM3, CEACAM, LAG3, TIGIT), such as IL27RA, STAT1, STAT3; and / or c) a polynucleotide sequence, the expression of which mediates interaction with HLA-G, such as ILT2 or ILT4; and / or d) polynucleotide sequences whose expression is involved in downregulating T cell proliferation, such as SEMA7A, SHARPIN, which attenuate Treg proliferation, STAT1, which reduces apoptosis, PEA15, which increases IL-2 secretion, and RICTOR, which supports CD8 memory differentiation; and / or e) a polynucleotide sequence, the expression of which is involved in the downregulation of T cell activation, such as mir21; and / or f) polynucleotide sequences whose expression is involved in signal transduction pathways in response to cytokines, such as JAK2 and AURKA; and / or g) Polynucleotide sequences whose expression is involved in T cell elimination, such as DNMT3, miRNA31, MT1A, MT2A, and PTGER2. into an endogenous locus of an immune cell, preferably by expressing a sequence-specific reagent within said cell that specifically targets said selected endogenous locus.

[0155] The transgene or exogenous polynucleotide sequence is preferably inserted such that its expression is under the transcriptional control of at least one endogenous promoter present in one of the loci.

[0156] Targeting one such locus by performing gene integration is beneficial to further improve the efficacy of the therapeutic immune cells of the present invention.

[0157] Examples of such exogenous sequences or transgenes that can be expressed or overexpressed at a selected locus are given below.

[0158] Expression of a transgene that confers resistance to drugs or immunoablative agents In one aspect of this method, the exogenous sequence integrated into the genomic locus of the immune cell encodes a molecule that confers resistance to a drug on said immune cell.

[0159] Examples of preferred exogenous sequences are variants of dihydrofolate reductase (DHFR) that confer resistance to folate analogs such as methotrexate, variants of inosine monophosphate dehydrogenase 2 (IMPDH2) that confer resistance to IMPDH inhibitors such as mycophenolic acid (MPA) or its prodrug mycophenolate mofetil (MMF), variants of calcineurin or methylguanine transferase (MGMT) that confer resistance to calcineurin inhibitors such as FK506 and / or CsA, variants of mTOR such as mTORmut that confer resistance to rapamycin, and variants of Lck such as Lckmut that confer resistance to imatinib and Gleevec.

[0160] The term "drug" is used herein to refer to a compound or its derivatives, preferably a standard chemotherapeutic agent, which is generally used to interact with cancer cells and thereby reduce the proliferation or survival state of the cells. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, ifosamide), antimetabolites (e.g., purine nucleoside antimetabolites, such as clofarabine, fludarabine, or 2'-deoxyadenosine, methotrexate (MTX), 5-fluorouracil, or derivatives thereof), antitumor antibiotics (e.g., mitomycin, adriamycin), plant-derived antitumor agents (e.g., vincristine, vindesine, taxol), cisplatin, carboplatin, etoposide, etc. Such agents may further include, but are not limited to, the anti-cancer agents TRIMETHOTRIXATE™ (TMTX), TEMOZOLOMIDE™, RALTRITREXED™, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzyguanidine (6-BG), bis-chloronitrosourea (BCNU), and CAMPTOTHECIN™, or a therapeutic derivative of any of these.

[0161] As used herein, an immune cell is rendered "resistant or tolerant" to a drug when the cell or cell population is modified so that it is able to proliferate, at least in vitro, in culture medium containing a 50% maximal inhibitory concentration (IC50) of the drug (the IC50 being determined for an unmodified cell or cell population).

[0162] In a particular embodiment, said drug resistance can be conferred to an immune cell by expression of at least one "drug resistance coding sequence," which refers to a nucleic acid sequence that confers "resistance" to an agent, such as one of the chemotherapeutic agents mentioned above. The drug resistance coding sequence of the present invention can encode resistance to antimetabolites, methotrexate, vinblastine, cisplatin, alkylating agents, anthracyclines, cytotoxic antibiotics, antiimmunophilins, analogs or derivatives thereof, etc. (Takebe, N., SC Zhao, et al. (2001) "Generation of dual resistance to 4-hydroperoxycyclophosphamide and methotrexate by retroviral transfer of the human aldehyde dehydrogenase class 1 gene and a mutated dihydrofolate reductase gene", Mol. Ther. 3(1): 88-96) (Zielske, SP, JS Reese, et al. (2003) "In vivo selection of MGMT(P140K) lentivirus-transduced human NOD / SCID repopulating cells without pretransplant irradiation conditioning", J. Clin. Invest. 112(10): 1561-70) (Nivens, MC, T. Felder, et al. (2004) "Engineered resistance to camptothecin and antifolates by retroviral coexpression of tyrosyl DNA phosphodiesterase-I and thymidylate synthase" Cancer Chemother Pharmacol 53(2): 107-15), (Bardenheuer, W., K. Lehmberg, et al. (2005)."Resistance to cytarabine and gemcitabine and in vitro selection of transduced cells after retroviral expression of cytidine deaminase in human hematopoietic progenitor cells". Leukemia 19(12): 2281-8), (Kushman, ME, SL Kabler, et al. (2007) "Expression of human glutathione S-transferase P1 confers resistance to benzo[a]pyrene or benzo[a]pyrene-7,8-dihydrodiol mutagenesis, macromolecular alkylation and formation of stable N2-Gua-BPDE adducts in stably transfected V79MZ cells co-expressing hCYP1A1" Carcinogenesis 28(1): 207-14). .

[0163] The expression of such drug resistance exogenous sequences in immune cells according to the invention more particularly allows the use of said immune cells in treatment schemes of cell therapy in which cell therapy is combined with chemotherapy or in patients who have received these drug treatments.

[0164] Several drug resistance coding sequences have been identified that can potentially be used to confer drug resistance in the present invention. One example of a drug resistance coding sequence can be, for example, a mutant or variant of dihydrofolate reductase (DHFR). DHFR is an enzyme involved in regulating the amount of tetrahydrofolate in cells and is essential for DNA synthesis. Folic acid analogs, such as methotrexate (MTX), inhibit DHFR and are therefore used clinically as anti-cancer drugs. Various mutant forms of DHFR that have increased resistance to inhibition by therapeutic antifolates have been described. In certain embodiments, the drug resistance coding sequence of the present invention can be a nucleic acid sequence encoding a mutant form of human wild-type DHFR (GenBank: AAH71996.1), which contains at least one mutation that confers resistance to antifolate therapy, such as methotrexate. In certain embodiments, mutant forms of DHFR contain at least one mutated amino acid at position G15, L22, F31, or F34, preferably at position L22 or F31 (Schweitzer et al. (1990) "Dihydrofolate reductase as a therapeutic target" Faseb J 4(8): 2441-52; International Patent Application WO94 / 24277; and U.S. Patent No. 6,642,043). In certain embodiments, the DHFR mutant form contains two mutated amino acids at positions L22 and F31. Correspondences of amino acid positions described herein are often expressed as the amino acid position in the wild-type DHFR polypeptide form. In certain embodiments, the serine residue at position 15 is preferably replaced with a tryptophan residue. In another specific embodiment, the leucine residue at position 22 is preferably replaced with an amino acid that prevents the mutant DHFR from binding to antifolates, preferably with an uncharged amino acid residue such as phenylalanine or tyrosine. In another specific embodiment, the phenylalanine residue at position 31 or 34 is preferably replaced with a small hydrophilic amino acid such as alanine, serine, or glycine.

[0165] Another example of a drug-resistant coding sequence may be a mutant or variant of ionisine-5'-monophosphate dihydrogenase II (IMPDH2), the rate-limiting enzyme in the de novo synthesis of guanosine nucleotides. The mutant or variant of IMPDH2 is an IMPDH inhibitor-resistant gene. The IMPDH inhibitor may be mycophenolic acid (MPA) or its prodrug, mycophenolate mofetil (MMF). The mutant IMPDH2 may contain at least one, preferably two, mutations in the MAP binding site of wild-type human IMPDH2 (Genebank: NP_000875.2), thereby significantly increasing resistance to IMPDH inhibitors. The mutations in these variants are preferably at positions T333 and / or S351 (Yam, P., M. Jensen, et al. (2006) "Ex vivo selection and expansion of cells based on expression of a mutated inosine monophosphate dehydrogenase 2 after HIV vector transduction: effects on lymphocytes, monocytes, and CD34+ stem cells" Mol. Ther. 14(2): 236-44) (Jonnalagadda, M., et al. (2013) "Engineering human T cells for resistance to methotrexate and mycophenolate mofetil as an in vivo cell selection strategy." PLoS One 8(6): e65519).

[0166] Another drug-resistant coding sequence is a mutant form of calcineurin. Calcineurin (PP2B - NCBI: ACX34092.1) is a ubiquitously expressed serine / threonine protein phosphatase that is involved in many biological processes and is important for T cell activation. Calcineurin is a heterodimer composed of a catalytic subunit (CnA; three isoforms) and a regulatory subunit (CnB; two isoforms). After binding to the T cell receptor, calcineurin dephosphorylates the transcription factor NFAT, allowing it to translocate to the nucleus and activate key target genes, such as IL-2. FK506 complexed with FKBP12 or cyclosporin A (CsA) complexed with CyPA prevents NFAT from accessing the active site of calcineurin, preventing its dephosphorylation and thereby inhibiting T cell activation (Brewin et al. (2009) "Generation of EBV-specific cytotoxic T cells that are resistant to calcineurin inhibitors for the treatment of posttransplantation lymphoproliferative disease" Blood 114(23): 4792-803). In certain embodiments, the mutant forms may contain at least one mutated amino acid of the wild-type calcineurin heterodimer at positions V314, Y341, M347, T351, W352, L354, or K360, preferably a double mutation at positions T351 and L354, or V314 and Y341.In particular embodiments, the valine residue at position 341 may be replaced with a lysine or arginine residue, the tyrosine residue at position 341 may be replaced with a phenylalanine residue; the methionine at position 347 may be replaced with a glutamic acid, arginine, or tryptophan residue; the threonine at position 351 may be replaced with a glutamic acid residue; the tryptophan residue at position 352 may be replaced with a cysteine, glutamic acid, or alanine residue, the serine at position 353 may be replaced with a histidine or asparagine residue, the leucine at position 354 may be replaced with an alanine residue; and the lysine at position 360 may be replaced with an alanine or phenylalanine residue. In another specific embodiment, the mutant form may contain at least one mutated amino acid of wild-type calcineurin heterodimer b at position V120, N123, L124, or K125, preferably a double mutation at positions L124 and K125. In specific embodiments, the valine at position 120 may be replaced with a serine, aspartic acid, phenylalanine, or leucine residue; the asparagine at position 123 may be replaced with tryptophan, lysine, phenylalanine, arginine, histidine, or serine; the leucine at position 124 may be replaced with a threonine residue; the lysine at position 125 may be replaced with an alanine, glutamic acid, tryptophan, or two residues, such as leucine-arginine or isoleucine-glutamic acid, may be added after the lysine at position 125 of the amino acid sequence. The amino acid position correspondences described herein are often expressed as the amino acid position in the form of the wild-type human calcineurin heterodimer b polypeptide (NCBI: ACX34095.1).

[0167] Another drug resistance coding sequence is O(6)-methylguanine methyltransferase (MGMT - UniProtKB: P16455), which encodes human alkylguanine transferase (hAGT). AGT is a DNA repair protein that confers resistance to the cytotoxic effects of alkylating agents such as nitrosoureas and temozolomide (TMZ). 6-benzylguanine (6-BG) is an AGT inhibitor that enhances the toxicity of nitrosoureas and is co-administered with TMZ to enhance the cytotoxic effects of the same agent. Some MGMT mutant forms encoding variants of AGT are highly resistant to inactivation by 6-BG but retain the ability to repair DNA damage (Maze, R. et al. (1999) "Retroviral-mediated expression of the P140A, but not P140A / G156A, mutant form of O6-methylguanine DNA methyltransferase protects hematopoietic cells against O6-benzylguanine sensitization to chloroethylnitrosourea treatment" J. Pharmacol. Exp. Ther. 290(3): 1467-74). In certain embodiments, AGT mutant forms can contain a mutant amino acid at position P140 of wild-type AGT. In a preferred embodiment, the proline at position 140 is replaced with a lysine residue.

[0168] Another drug resistance coding sequence can be the multidrug resistance protein (MDR1) gene. This gene encodes a membrane glycoprotein known as P-glycoprotein (P-GP), which is involved in the transport of metabolic by-products across the cell membrane. The P-GP protein exhibits broad specificity for several structurally unrelated chemotherapeutic agents. Thus, expression of a nucleic acid sequence encoding MDR-1 (Genebank NP_000918) can confer drug resistance to cells.

[0169] Other drug resistance coding sequences may contribute to the production of cytotoxic antibiotics, such as those derived from the ble or mcrA genes. Ectopic expression of the ble gene or mcrA in immune cells confers a selective advantage upon exposure to the chemotherapeutic agents bleomycin and mitomycin C, respectively (Belcourt, MF (1999) "Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA". PNAS. 96(18):10489-94).

[0170] Another drug resistance coding sequence may be derived from a gene encoding a mutated version of a drug target, such as a mutated variant of mTOR (mTOR mut) that confers resistance to rapamycin, as described by Lorenz MC et al. (1995) "TOR Mutations Confer Rapamycin Resistance by Preventing Interaction with FKBP12-Rapamycin" The Journal of Biological Chemistry 270, 27531-27537, or a specific mutated variant of Lck (Lckmut) that confers resistance to Gleevec, as described by Lee KC et al. (2010) "Lck is a key target of imatinib and dasatinib in T-cell activation", Leukemia, 24: 896-900.

[0171] As described above, the genetic modification step of the method can include introducing into a cell an exogenous nucleic acid comprising at least one sequence encoding a drug resistance coding sequence and a portion of an endogenous gene, thereby allowing homologous recombination to occur between the endogenous gene and the exogenous nucleic acid. In certain embodiments, the endogenous gene can be a wild-type "drug resistance" gene, and after homologous recombination, the wild-type gene is replaced with a mutant form of the gene that confers resistance to the drug.

[0172] Transgene expression enhances immune cell persistence in vivo In one aspect of this method, the exogenous sequence integrated into the immune cell genomic locus encodes a molecule that enhances immune cell persistence, particularly in vivo persistence in a tumor environment.

[0173] By "enhanced persistence" is meant increasing the survival time of the immune cells, particularly in terms of lifespan, after the modified immune cells have been injected into a patient. For example, enhanced persistence occurs when the average survival time of the modified cells is significantly longer than that of unmodified cells, at least 10%, preferably 20%, more preferably 30%, and even more preferably 50% longer.

[0174] This is particularly relevant when the immune cells are allogeneic. This can be achieved by creating local immune protection by introducing coding sequences that ectopically express and / or secrete immunosuppressive polypeptides at or across the cell membrane. Such polypeptides, specifically a diverse panel of immunosuppressive peptides derived from immune checkpoint antagonists, viral envelopes, or NKG2D ligands, can enhance the persistence and / or engraftment of allogeneic immune cells in the patient's body.

[0175] In one embodiment, the immunosuppressive polypeptide encoded by the exogenous coding sequence is a ligand for cytotoxic T-lymphocyte antigen 4 (CTLA-4, also known as CD152, GenBank Accession No. AF414120.1). The ligand polypeptide is preferably an anti-CTLA-4 immunoglobulin, such as CTLA-4a Ig and CTLA-4b Ig, or a functional variant thereof.

[0176] In one embodiment, the immunosuppressive polypeptide encoded by the exogenous coding sequence is a PD1 antagonist, e.g., PD-L1 (also known as CD274, programmed cell death 1 ligand; see UniProt for human polypeptide sequence Q9NZQ7), which encodes a 290-amino acid type I transmembrane protein consisting of a 30-amino acid Ig V-like domain, an Ig C-like domain, a hydrophobic transmembrane domain, and a cytoplasmic tail. Such membrane-bound forms of PD-L1 ligands are considered, for purposes of the present invention, to be either the native (wild-type) form or truncated forms, e.g., by removal of the intracellular domain or one or more mutations (Wang S et al., 2003, J Exp Med. 2003;197(9):1083-1091). It should be noted that PD1 is not considered, for purposes of the present invention, to be a membrane-bound form of a PD-L1 ligand. In another embodiment, the immunosuppressive polypeptide is a secreted form. Such recombinant secreted PD-L1 (or soluble PD-L1) can be produced by fusing the extracellular domain of PD-L1 to the Fc portion of immunoglobulin (Haile ST et al., 2014, Cancer Immunol. Res. 2(7): 610-615; Song MY et al., 2015, Gut. 64(2):260-71). This recombinant PD-L1 can neutralize PD-1 and abrogate PD-1-mediated T cell inhibition. PD-L1 ligands can be coexpressed with CTLA4 Ig to further enhance the persistence of both.

[0177] In another embodiment, the exogenous sequence encodes a non-human MHC homolog, particularly a viral MHC homolog, or a chimeric β2m polypeptide, as described in Margalit A. et al. (2003) "Chimeric β2 microglobulin / CD3ζ polypeptides expressed in T cells convert MHC class I peptide ligands into T cell activation receptors: a potential tool for specific targeting of pathogenic CD8+ T cells" Int. Immunol. 15 (11): 1379-1387.

[0178] In one embodiment, the foreign sequence encodes an NKG2D ligand. Some viruses, such as cytomegalovirus, have acquired mechanisms to evade NK cell-mediated immune surveillance and interfere with the NKG2D pathway by secreting proteins capable of binding to NKG2D ligands and preventing their surface expression (Welte, SA et al. (2003) "Selective intracellular retention of virally induced NKG2D ligands by the human cytomegalovirus UL16 glycoprotein", Eur. J. Immunol., 33, 194-203). Tumor cells have also evolved mechanisms to evade NKG2D responses by secreting NKG2D ligands such as ULBP2, MICB, or MICA (Salih HR, Antropius H, Gieseke F, Lutz SZ, Kanz L, et al. (2003) Functional expression and release of ligands for the activating immunoreceptor NKG2D in leukemia. Blood 102: 1389-1396).

[0179] In one embodiment, the exogenous sequence encodes a cytokine receptor, such as an IL-12 receptor. IL-12 is a well-known activator of immune cell activation (Curtis JH (2008) "IL-12 Produced by Dendritic Cells Augments CD8+ T Cell Activation through the Production of the Chemokines CCL1 and CCL171". The Journal of Immunology. 181 (12): 8576-8584).

[0180] In one embodiment, the foreign sequence encodes an antibody directed against the inhibitory peptide or protein. The antibody is preferably secreted in soluble form by immune cells. In this regard, nanobodies derived from sharks and camels are advantageous because they are structured as single-chain antibodies (Muyldermans S. (2013) "Nanobodies: Natural Single-Domain Antibodies" Annual Review of Biochemistry 82: 775-797). They are also thought to be more easily fused to secretory signal polypeptides and soluble hydrophilic domains.

[0181] Various aspects of enhancing the persistence of the expanded cells described above, as further detailed below, are particularly preferred when the exogenous coding sequence is introduced by disrupting the endogenous gene encoding β2m or another MHC component.

[0182] Transgene expression to enhance the therapeutic activity of immune cells In one aspect of this method, the exogenous sequence integrated into the immune cell genomic locus encodes a molecule that enhances the therapeutic activity of the immune cell.

[0183] "Enhanced therapeutic activity" means that immune cells or cell populations modified according to the present invention are more aggressive against a selected type of target cell than unmodified cells or cell populations. The target cells preferably consist of a predetermined type of cell or cell population characterized by a common surface marker. As used herein, "therapeutic potential" reflects therapeutic activity measured by in vitro experiments. Generally, sensitive cancer cell lines, such as Daudi cells, are used to assess whether immune cells are more or less active against the cells by measuring cell lysis or reduced proliferation. This can also be assessed by measuring the levels of immune cell degranulation or chemokine and cytokine production. Experiments can also be performed in mice by injecting tumor cells and observing the resulting tumor growth. Enhanced activity is considered significant if the number of cells generated by immune cells in these experiments is reduced by more than 10%, preferably more than 20%, more preferably more than 30%, and even more preferably more than 50%.

[0184] In one aspect of the invention, the exogenous sequence encodes a chemokine or cytokine, such as IL-12. Expression of IL-12 is particularly advantageous, as this cytokine has been widely documented to promote immune cell activation (Colombo MP et al. (2002) "Interleukin-12 in anti-tumor immunity and immunotherapy" Cytokine Growth Factor Rev. 13(2):155-68).

[0185] In a preferred aspect of the invention, the exogenous coding sequence encodes or promotes secreted factors that act on other immune cell populations, such as regulatory T cells, to counteract their inhibitory effects on said immune cells.

[0186] In one aspect of the present invention, the exogenous sequence encoding an inhibitor of regulatory T cell activity is a polypeptide inhibitor of forkhead / winged helix transcription factor 3 (FoxP3), more preferably a cell-penetrating peptide inhibitor of FoxP3, such as that designated P60 (Casares N. et al. (2010) "A peptide inhibitor of FoxP3 impairs regulatory T cell activity and improves vaccine efficacy in mice." J Immunol 185(9):5150-9).

[0187] "Inhibitor of regulatory T cell activity" refers to a molecule secreted by a T cell or a precursor of said molecule, which allows the T cell to escape the down-regulatory activity of regulatory T cells on the T cell. Generally, such inhibitors of regulatory T cell activity have the effect of reducing FoxP3 transcriptional activity in said cells.

[0188] In one aspect of the present invention, the exogenous sequence encodes a tumor-associated macrophage (TAM) secretion inhibitor, such as a CCR2 / CCL2 neutralizing agent. Tumor-associated macrophages (TAM) are important modifiers of the tumor microenvironment. Clinical pathology studies have shown that the accumulation of TAM in tumors correlates with poor clinical outcomes. Consistent with this evidence, experimental and animal studies support the view that TAM may provide a favorable microenvironment for promoting tumor development and progression (Theerawut C. et al. (2014) "Tumor-Associated Macrophages as Major Players in the Tumor Microenvironment" Cancers (Basel) 6(3): 1670-1690). Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP1 - NCBI NP_002973.1), is a small cytokine belonging to the CC chemokine family, secreted by macrophages and exerting chemoattractant effects on monocytes, lymphocytes, and basophils. CCR2 (CC chemokine receptor type 2 - NCBI NP_001116513.2) is the receptor for CCL2.

[0189] The coding sequence inserted into the locus generally encodes a polypeptide that improves the therapeutic potential of the modified immune cell, but the inserted sequence may also be a nucleic acid that can direct or block the expression of other genes, such as an interfering RNA or guide RNA. The polypeptide encoded by the inserted sequence may act directly or indirectly, such as a signal transduction factor or transcription factor.

[0190] Modified immune cells and immune cell populations The present invention also relates to various modified immune cells that can be obtained in isolated form or as part of a cell population by one of the methods described herein.

[0191] In preferred aspects of the invention, the modified cells are primary immune cells, such as NK cells or T cells, which are generally part of a cell population that may include various types of cells, generally a patient- or donor-derived population isolated by leukapheresis from PBMCs (peripheral blood mononuclear cells).

[0192] The present invention encompasses immune cells comprising any combination of various exogenous coding sequences and gene inactivations, each of which is independently described above. Particularly preferred among these combinations are those that combine expression of the CAR under the transcriptional control of an endogenous promoter that is active upon immune cell activation, specifically one promoter present in one TCR locus, specifically the TCR alpha promoter.

[0193] Another preferred combination is the insertion of an exogenous sequence encoding a CAR, or one of its components, under the transcriptional control of the hypoxia inducible factor 1 gene promoter (Uniprot: Q16665).

[0194] The present invention also relates to pharmaceutical compositions comprising the modified primary immune cells or immune cell populations described above for the treatment of infectious diseases or cancer, and to methods of treating a patient in need of treatment, said methods comprising: providing a population of primary immune cells modified by the method of the invention as described above; optionally, purifying or sorting the modified primary immune cells; activating the modified primary immune cell population when or after the cells are infused into the patient. Includes.

[0195] T cell activation and proliferation Whether before or after genetic modification, the immune cells of the present invention can be activated or expanded, even if they can be activated or expanded independently of an antigen binding mechanism. In particular, T cells can be activated and expanded using methods such as those described in, for example, 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. T cells can be proliferated in vitro or in vivo. T cells generally proliferate by contact with agents that stimulate the CD3 TCR complex and costimulatory molecules on the T cell surface, generating a T cell activation signal. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to generate a T cell activation signal.

[0196] As a non-existent example, a T cell population can be stimulated in vitro, for example, by contact with a surface-immobilized anti-CD3 antibody or its antigen-binding fragment, or anti-CD2 antibody, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. Co-stimulation of accessory molecules on the T cell surface is achieved using a ligand that binds to the accessory molecule. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under appropriate conditions to stimulate T cell proliferation. Suitable conditions for T cell culture include an appropriate medium (e.g., minimal essential medium, or RPMI Media 1640 or X-vivo 5 (Lonza)), which may contain factors necessary for proliferation and viability, such as serum (e.g., fetal bovine or human fetal serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFp, and TNF, or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Media may include RPMI 1640, A1M-V, DMEM, MEM, α-MEM, F-12, X-Vivo 1, and X-Vivo 20, or Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and may be serum-free or supplemented with an appropriate amount of serum (or plasma), a set of specific hormones, and / or cytokines in amounts sufficient for T cell growth and proliferation. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and are not included in cultures of cells to be infused into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2). T cells exposed to various stimulation times may exhibit different characteristics.

[0197] In another particular aspect, the cells can be expanded in co-culture with tissue or cells. The cells can also be expanded in vivo, for example, in the blood of a subject after administration of the cells to the subject.

[0198] Therapeutic Compositions and Uses The methods of the present invention described above allow for the production of modified primary immune cells within a limited time frame of about 15-30 days, preferably 15-20 days, and most preferably 18-20 days, which therefore retain their maximum immunotherapeutic potential, particularly with regard to cytotoxic activity.

[0199] These cells form cell populations, preferably derived from a single donor or patient, which can be expanded in closed culture recipients to comply with the most stringent requirements of manufacturing practices and can be frozen prior to infusion into patients, providing an "off-the-shelf" or "ready-to-use" therapeutic composition.

[0200] In the present invention, a large number of cells from the same leukapheresis source can be obtained, which is important for obtaining a sufficient dose to treat patients. Although differences between cell populations from various donors can be observed, the number of immune cells obtained by leukapheresis is generally about 10 8 ~10 10 PBMCs contain several types of cells, including granulocytes, monocytes, and lymphocytes, of which 30–60% are T cells. Primary T cells from a single donor are generally 10 8 ~10 9 The method of the present invention generally involves about 10 8 T cells, more commonly about 10 9 T cells, more commonly about 10 10 More than 10 T cells, usually 11 Finally, a population of modified cells is obtained, reaching a super-population of T cells.

[0201] The present invention therefore more particularly relates to therapeutically effective primary immune cell populations, wherein at least 30%, preferably 50%, more preferably 80% of the cells of said population have been modified according to any one of the methods described herein.

[0202] In a preferred aspect of the invention, more than 50% of the immune cells in the population are TCR-negative T cells. In a more preferred aspect of the invention, more than 50% of the immune cells in the population are CAR-positive T cells. Modified immune cells, cell populations, therapeutic compositions, and uses.

[0203] Such a composition or cell population can therefore be used as a medicament in patients in need thereof, in particular for cancer treatment, in particular for lymphoma treatment, but also for solid tumors, such as melanoma, neuroblastoma, glioma, or carcinoma, such as lung, breast, rectal, prostate, or ovarian tumors.

[0204] The present invention more particularly relates to primary TCR-negative T cell populations of single donor origin, wherein at least 20%, preferably 30%, more preferably 50% of the cells in said population have been modified with sequence-specific reagents at at least two, preferably three different loci.

[0205] In another aspect, the invention relates to a method of treating a patient in need thereof, said method comprising: (a) determining the specific antigen markers present on the surface of a patient's tumor biopsy; (b) providing a population of modified primary immune cells modified by one of the methods of the invention described above, preferably expressing a recombinant receptor directed against said specific antigen marker; (c) administering the modified primary immune cell population to the patient. Contains at least one of the following:

[0206] Generally, the cell population comprises primarily CD4 and CD8 positive immune cells, such as T cells, which can undergo robust in vivo T cell proliferation and survive for extended periods in vitro and in vivo.

[0207] Treatments involving the modified primary immune cells of the present invention can be palliative, curative, or prophylactic. They can be part of an autologous or allogeneic immunotherapeutic treatment.

[0208] In another aspect, the isolated cells of the present invention or cell lines derived from the isolated cells can be used to treat solid tumors, particularly solid tumors such as, typically, esophageal, breast, gastric, biliary, pancreatic, colon, lung, thymic, mesothelioma, ovarian, and / or endometrial cancers.

[0209] Adult tumors / cancers and pediatric tumors / cancers are also included.

[0210] Treatment with the modified immune cells of the present invention may be combined with one or more cancer therapies selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiation therapy.

[0211] In a preferred embodiment of the invention, the treatment may be administered to a patient undergoing immunosuppressive therapy. Indeed, the invention preferably relies on cells or cell populations that have been made resistant to such immunosuppressive agents by virtue of the inactivation of a gene encoding a receptor for at least one immunosuppressive agent. In this respect, the immunosuppressive therapy should aid in the selection and expansion of the T cells of the invention in the patient.

[0212] Administration of the cells or cell populations of the present invention can be carried out in any convenient manner, including aerosol inhalation, injection, oral ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one aspect, the cell compositions of the present invention are preferably administered by intravenous injection.

[0213] The administration of cells or cell populations was 10 per kg of body weight. 4 ~10 9 cells, preferably 10 per kg of body weight 5 ~10 6 The present invention therefore provides for the administration of 10 cells originating from a single donor or patient sampling. 6 ~10 8 In some embodiments, more than 10, typically more than 50, more typically more than 100, and usually more than 1000 doses containing gene-edited cells may be provided.

[0214] The cells or cell population may be administered in one or more doses. In another embodiment, the effective amount of cells is administered as a single dose. In another embodiment, the effective amount of cells is administered as two or more doses over a period of time. The timing of administration is within the discretion of the attending physician and depends on the patient's clinical condition. The cells or cell population may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determining the optimal range of effective amounts of a given cell type for a particular disease or condition is within the skill of the art. An effective amount refers to an amount that provides therapeutic or prophylactic benefit. The dosage depends on the recipient's age, health, and weight, the type of concurrent treatment, if any, the frequency of treatment, and the nature of the desired effect.

[0215] In another embodiment, the effective amount of cells or a composition comprising the cells is administered parenterally. The administration can be intravenous. The administration can be by direct tumor injection.

[0216] In certain embodiments of the invention, the cells are administered to the patient in conjunction with (e.g., before, simultaneously with, or after) any number of relevant treatment modalities, including, but not limited to, antiviral therapy, cidofovir, and treatment with agents such as interleukin-2, cytarabine (also known as ARA-C), or nataliziimab treatment for MS patients, or efaliztimab treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, or other antibody therapies, cytoxin, fludaribine, cyclosporine, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or p70S6 kinase (rapamycin), which is important for growth factor-induced signal transduction (Henderson, Naya et al. 1991; Liu, Albers et al. 1992; Bierer, Hollander et al. 1993). In a further embodiment, the cell compositions of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) bone marrow transplantation, chemotherapy such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or T cell depletion therapy using antibodies such as OKT3 or CAMPATH. In another embodiment, the cell compositions of the present invention are administered following B cell depletion therapy, such as an agent reactive with CD20, e.g., Rituxan. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In certain embodiments, following transplantation, the subject receives an infusion of the expanded immune cells of the present invention. In additional embodiments, the expanded cells are administered before or after surgery.

[0217] The present invention also specifically relates to a general method of treating a solid tumor in a patient, said method comprising immunodepleting said patient with a lymphocyte-depleting regimen and injecting genetically modified lymphocytes that have been rendered resistant to the lymphocyte-depleting agent used in the lymphocyte-depleting regimen and that specifically target said solid tumor, preferably CAR-positive T cells, more preferably comprising an MSLN-CAR as described herein.

[0218] Lymphocyte depletion regimens preferably include antibodies directed against antigens such as CD52, CD3, CD4, CD8, CD45, or other specific markers present on the surface of immune cells, or drugs such as purine analogs (e.g., fludarabine and / or chlorofarabine) and glucocorticoids.

[0219] In a preferred embodiment of the invention, the method comprises subjecting the patient to a lymphocyte depletion regimen comprising an antibody directed against CD52 and administering modified CAR T cells comprising an MSLN-CAR with reduced, deficient or inactivated expression of CD52.

[0220] In a preferred embodiment of the present invention, lymphocyte-depleting therapy may include an anti-CD52 antibody, such as alemtuzumab, either alone or in combination. For example, lymphocyte-depleting regimens may include cyclophosphamide, typically for 1-3 days, fludarabine for 1-5 days, and alemtuzumab for 1-5 days. Generally, lymphocyte-depleting regimens may include cyclophosphamide at 50-70 mg / kg / day, fludarabine at 20-40 mg / m2 / day, and alemtuzumab at 0.1-0.5 mg / kg / day, either alone or in combination.

[0221] To this end, the present invention provides a composition for lymphocyte depletion in patients afflicted with solid tumors, comprising a composition comprising an anti-CD52 antibody in combination with an engineered lymphocyte population that is insensitive to the antibody and targets MSLN, preferably comprising cells that express an MSLN-CAR and have impaired CD52 expression, wherein an allele of the CD52 gene has been inactivated, preferably by a rare-cutting endonuclease, such as a TALE nuclease or an RNA-guided endonuclease, as described above.

[0222] The present invention also provides a medical kit for use in the treatment of solid tumor cancer, comprising the lymphocyte depleting composition and the modified cell population resistant thereto.

[0223] By "cytolytic activity" or "cytotoxic activity" or "cytotoxicity" is meant the percentage of cytolysis of target cells conferred by immune cells.

[0224] The method for determining cytotoxicity is described below.

[0225] For adherent target cells: 2.10 4 Specific target antigen (STA)-positive or STA-negative cells were plated in a 96-well plate at 0.1 ml per well. The day after plating, STA-positive and STA-negative cells were labeled with CellTrace CFSE and incubated at 4 x 10 for 4 hours. 5 T cells were co-cultured, then harvested, stained with a fixable viability dye (eBioscience), and analyzed using a MACSQuant flow cytometer (Miltenyi).

[0226] For suspension target cells: Label STA-positive and STA-negative cells with CellTrace CFSE and CellTrace Violet, respectively. Approximately 2 x 10 4 2 x 10 ROR1-positive cells 4 STA-negative cells and 4 x 10 cells in 0.1 ml per well in a 96-well plate 5After 4 hours of incubation, cells were harvested, stained with a fixable viability dye (eBioscience), and analyzed using a MACSQuant flow cytometer (Miltenyi).

[0227] The percentage of specific lysis can be calculated using the following formula: TIFF2025186404000012.tif25128

[0228] By "increased cytotoxicity" is meant that the % cytolysis of target cells conferred by the modified immune cells is increased by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% or more compared to the % cytolysis of target cells conferred by unmodified immune cells.

[0229] "Identity" refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing positions within each sequence, which may be aligned for comparison. If a position in the compared sequences is occupied by the same base, the molecules are identical at that position. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at the same position shared by the nucleic acid sequences. Various alignment algorithms and / or programs can be used to calculate the identity between two sequences, including FASTA or BLAST, which are available as part of the GCG Sequence Analysis Package (University of Wisconsin, Madison, Wisconsin), and can be used, for example, with default settings. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98%, or 99% identity to the specific polypeptides described herein and preferably exhibiting substantially the same function, as well as polynucleotides encoding such polypeptides, are contemplated.

[0230] The term "subject" or "patient," as used herein, generally refers to a mammal, preferably a primate, and more preferably a human.

[0231] The foregoing specification of the invention provides manners and methods of making and using the invention, and will enable any person skilled in the art to make and use the invention, and this enabling information is provided with particularity in relation to the subject matter of the appended claims, which subject matter forms a part of this specification.

[0232] Where numerical limits or ranges are recited herein, the endpoints are included. Also, every value and subrange within the numerical limit or range is specifically included, as if such values ​​were expressly written.

[0233] Having broadly described the invention, a further understanding can be obtained by reference to specific embodiments, which are provided herein for illustrative purposes only and are not intended to limit the scope of the invention as claimed. [Example]

[0234] Mesothelin (MSLN) is a glycophosphatidylinositol (GPI)-linked cell surface protein that is normally expressed in mesothelioma cells lining the pleura, peritoneum, and pericardium. The MSLN gene encodes a 71-kDa precursor protein, which is processed into a 31-kDa cleaved protein called MPF (megakaryocyte-potentiating factor) and a 40-kDa membrane-bound protein, mesothelin.

[0235] Mesothelin has been reported to be highly expressed in several types of malignant tumors, including malignant mesothelioma, ovarian cancer, pancreatic adenocarcinoma, and lung adenocarcinoma (Morello et al., 2016; O'Hara et al., 2016). In some cases, mesothelin expression has been associated with increased tumor grade and poor clinical outcomes.

[0236] Description of the MSLN-specific CAR used in this study We generated three second-generation CARs, each composed of scFv P4, meso1, and MESO2, containing the CD8α hinge / transmembrane domain, and the 4-1BB and CD3ζ activation domains, and tested the first mesoCARs against target cell lines expressing different levels of mesothelin (MSLN). + T cells were screened for in vitro chimeric antigen receptor (CAR) expression and antitumor activity. In a specific version, a suicide switch, "R2," was introduced into the CAR construct. As previously described in WO2016120216, the "R2" polypeptide, containing two CD20 mimotopes, was placed between the scFv and hinge to confer sensitivity to anti-CD20 therapeutic antibodies, such as rituximab.

[0237] A schematic diagram of the CAR structure is shown in Figure 1.

[0238] The different sequences contained in each CAR are detailed in Tables 1, 2, and 3, and their complete amino acid sequences are shown in Table 4.

[0239] 1 - In vitro assay CARs were screened for expression in primary T cells derived from PBMCs and against three target cell lines expressing different levels of MSLN: ·Epithelial cervical adenocarcinoma HeLa (ATCC (registered trademark) CCL-2), epithelial splenic adenocarcinoma HPAC (ATCC® CRL-2119), and 293H or A2058 cells, mesothelin-negative cells were assayed for their antitumor activity against each of the following:

[0240] The expression of mesothelin on the surface of these cells was assessed by flow cytometry as shown in Figures 3 and 4 .

[0241] 2 - Production of Gene-Edited MSLN-UCART Cells On day 0, frozen human peripheral blood mononuclear cells (PBMCs) from Hemacare (Northridge, CA 91325, USA) were thawed, washed, counted, and resuspended in X-vivo 15 medium supplemented with 5% AB serum. The cells were then transferred to an incubator set at 37°C and 5% CO2. On day 1, PBMCs were counted, analyzed by flow cytometry to assess the % CD3+ cells, centrifuged, and resuspended in X-vivo 15 medium supplemented with 5% AB serum, 350 UI / ml IL2, and MACS GMP T Cell TransAct (60 μl / million CD3+ cells). Cells were then transferred to an incubator set at 37°C and 5% CO2. On day 4, T cells and rLV vectors carrying polynucleotide sequences encoding anti-MSLN CARs were resuspended in X-vivo 15 medium supplemented with 5% AB serum and 350 UI / ml IL2 and seeded onto retronectin-coated plates. The plates were then transferred to an incubator set at 37°C and 5% CO2. On day 5, T cells were washed and resuspended in X-vivo 15 medium supplemented with 5% AB serum and 350 UI / ml IL-2, and then transferred to an incubator set at 37°C and 5% CO2. On day 6, T cells were co-electroporated with mRNA encoding the right and left arms of the TRAC and CD52 TALENs, respectively, as previously described [Poirot et al. (2013) Blood. 122 (21): 1661] to efficiently inactivate the TCRα and CD52 genes and prevent TCRαβ expression on the surface of primary T cells. TALEN is the registered name for TALE nucleases designed by Cellectis (rue de la Croix Jarry 8, 75013 Paris, France). The genomic target sequences of these TALE nucleases are listed in Table 8 below. Gene transfection was performed using the AgilePulse method. Cells were then transferred to an incubator set at 37°C and 5% CO2. On day 7, T cells were washed and resuspended in X-vivo 15 medium supplemented with 5% AB serum and 350 UI / ml IL-2, and then transferred to an incubator set at 37°C and 5% CO2. T cells were expanded in the GRex device between days 7 / 8 and 18. When using the GRex 6 multiwell cell culture plate, half of the culture medium was removed on days 11 and 15 and replaced with fresh medium containing IL2, and fresh IL2 was added on day 13. When using the GRex 100M, fresh IL2 was added on days 11, 13, and 15 without a medium change. During the expansion period, cell cultures were incubated at 37°C in 5% CO2. On day 18, all UCART cells were cryopreserved for later use in in vitro and in vivo assays.

[0242] Table 8. Genomic sequences targeted by TALE nucleases (TALENs) TIFF2025186404000013.tif58156

[0243] 2.1 Analysis of MSLN-CAR expression Three MSLN-specific UCART cell products were generated using the P4-R2, Meso1-R2, and MESO2-R2 CARs, and these different UCART cell products were then evaluated in vitro.

[0244] The first in vitro study performed on the four UCART cell products aimed to determine the phenotype of UCART cells at day 18. To this end, the expression of CAR and TCRαβ on the surface of UCART cells, as well as the expression of CD4 and CD8 on the surface of the CAR+ fraction of UCART cells, were analyzed by flow cytometry. CAR surface expression was assessed using either His-tagged recombinant human mesothelin protein or biotinylated protein L, both of which recognize the scFv portion of CAR or biotinylated rituximab, which recognizes the R2 suicide switch portion of CAR. Surface expression of the TCRαβ receptor was assessed using an anti-TCRαβ antibody conjugated with PE-vio770. Surface expression of CD4 and CD8 was assessed using a CD4 antibody conjugated with FITC and a CD8 antibody conjugated with BV510.

[0245] Surface expression of CAR was assessed by flow cytometry using either His-tagged recombinant human mesothelin protein, which recognizes the scFv portion of CAR, or biotinylated rituximab, which recognizes the R2 suicide switch portion of CAR.

[0246] As shown in Figure 6, at least 50% of UCART cells modified with either P4-R2, Meso1-R2, or MESO2-R2 were CAR-positive. However, the P4-R2 CAR showed higher expression levels than the Meso1-R2 and MESO2-R2 CARs.

[0247] As shown in Figure 7, at least 51% of the CAR+ fraction of UCART cells modified with P4-R2, Meso1-R2, and MESO2-R2 CARs was CD4+.

[0248] 2.2 IFNg production and killing activity The second in vitro study performed on the three UCART cell products was aimed at analyzing UCART cell function. The ability of UCART cells to produce cytokines after 24 hours of coculture with HPAC (MSLN+) or 293H (MSLN-) cells was assessed by quantifying IFNg in cell culture supernatants using standard ELISA procedures.

[0249] As shown in Figure 8, UCART cells modified with the P4-R2 CAR and Meso1-R2 CAR produced more than 40,000 pg / ml and 50,000 pg / ml of IFNg, respectively, after coculture with the MSLN+ cell line, and less than 1,500 pg / ml and 200 pg / ml of IFNg, respectively, after coculture with the MSLN- cell line. Although UCART cells modified with the Meso1-R2 CAR produced similar levels of IFNg compared to UCART cells modified with the P4-R2 CAR after coculture with the MSLN+ cell line, cells equipped with the Meso1-R2 CAR produced extremely low levels of IFNg after coculture with the MSLN- cell line. UCART cells modified with the MESO2-R2 CAR produced only 15,000 pg / ml or less of IFNg after coculture with the MSLN+ cell line.

[0250] Next, the ability of UCART cells to carry out sequential killing of HPAC cells was assessed over a 15-day period, including six rounds of exposure to MSLN+ (HPAC) cells at ratios of 1:2 and 1:8.

[0251] As shown in Figure 9, CART cells modified with P4-R2, Meso1-R2, and MESO2-R2 CARs exhibited similar levels of killing activity against HPAC cells after one round of exposure. However, after several rounds of exposure, T cells equipped with Meso1-R2 and P4-R2 exhibited much higher continuous killing activity than CART cells equipped with MESO2-R2 CAR, while CART cells modified with Meso1-R2 exhibited more sustained activity than CART cells modified with P4-R2.

[0252] Importantly, none of these CART cell products exhibited significant killing activity against mesothelin-negative A2058 and 293H cells.

[0253] 3. Functional validation of gene characteristics of UCART MSLN cells 3.1 Knockout of TRAC and / or CD52 genes As shown in Figure 10, less than 20% of UCART cells modified with P4-R2, Meso1-R2, and MESO2-R2 CARs remained TCRαβ+, suggesting that the level of TALEN-mediated inactivation of the TCRα gene was highly efficient in the cell population.

[0254] Furthermore, flow cytometry data shown in Figure 11 demonstrate that depletion of TCRαβ+ cells eliminates unmodified cells and promotes [CAR] + [TCR] - This was an efficient process for selecting UCART cells, with 84% of unmodified T cells being TCRαβ+, compared with 8% for TRAC gene knockout T cells and 0.2% for TRAC gene knockout and TCRαβ+ cell-depleted T cells.

[0255] To fully demonstrate that the absence of detection of TCRαβ receptors on the surface of TRAC gene knockout and TCRαβ+ cell-depleted cells is associated with the absence of expression of functional TCRαβ receptors, unmodified T cells, TRAC gene knockout T cells, and TRAC gene knockout and TCRαβ+ cell-depleted T cells were exposed to phytohemagglutinin (PHA) for 24 hours and analyzed by flow cytometry to observe the expression of the activation marker CD25.

[0256] As shown in Figure 12, only unmodified T cells expressed significant levels of CD25 on their surface after exposure to PHA. These data clearly demonstrate that knockout of the TRAC gene in T cells prevents the expression of functional TCRαβ receptors on the cell surface.

[0257] CD52 gene inactivation was assessed by incubating engineered cells in 50 μg / ml of anti-CD52 monoclonal antibody (or rat IgG as a control) with or without 30% rabbit complement (Cedarlane) for 7 days. After 2 hours of incubation at 37°C, cells were labeled with fluorescent dye-conjugated anti-CD52 antibody along with a fluorescent viability dye (eBioscience) and analyzed by flow cytometry to measure the frequency of CD52-positive and CD52-negative cells among live cells. Alternatively, cells were cultured with the antibody to select for resistance.

[0258] 3.2 UCART cell ablation using rituximab against the R2 polypeptide Another in vitro study performed on the UCART cell product aimed to evaluate the ability of the CAR+ fraction of UCART cells to be eliminated following treatment with rituximab.

[0259] UCART cells modified with P4-R2, Meso1-R2, and MESO2-R2 CARs were cocultured with HPAC cells for 2 days and exposed to medium, rituximab (RTX), baby rabbit complement (BRC), or a mixture of rituximab and baby rabbit complement for 2 hours. The percentage of CAR+ cells was then analyzed by flow cytometry. As shown in Figure 13, UCART cells modified with P4-R2, Meso1-R2, and MESO2-R2 were efficiently eliminated after treatment with rituximab and baby rabbit complement. In contrast, UCART cells modified with P4 naked (MSLN CAR with the P4 structure but without the R2 sequence) were not eliminated.

[0260] 3.3 Inactivation of the TGFβ signaling pathway in CAR T cells Another property conferred to MesoCAR T cells was to render them resistant to the tumor microenvironment by inactivating the TGFb signaling pathway. Two different strategies were investigated to inactivate TGFbRII gene expression: by knockout or by overexpressing a dominant-negative form of the TGFbRII gene (dnTGFbRII).

[0261] 3.3.1. Inactivation of TGFbRII by KO Activated T cells were electroporated with 10 μg of mRNA encoding the right and left arms of two TALENs targeting the TGFbRII gene (SEQ ID NO: 155 (pCLS32939) and SEQ ID NO: 156 (pCLS32940), or SEQ ID NO: 157 (pCLS32967) and SEQ ID NO: 158 (pCLS32968)). Three days after gene transfer, T cells were harvested, gDNA was extracted, and PCR was performed to amplify the amplicon. Analysis of PCR products by deep sequencing showed that transfection with TALENs encoded by pCLS32939 and pCLS32940, or TALENs encoded by pCLS32967 and pCLS32968, yielded 96.62% and 97.28% gene editing (i.e., insertion and / or deletion), respectively, demonstrating high efficiency of TGFbRII knockout.

[0262] 3.3.2. TGFbRII inactivation by overexpression of dnTGFbRII As described in Example 2, MSLN-CAR T cells were produced using two different donors and rLV vectors encoding different MSLN CARs, with or without dnTGFbRII (SEQ ID NO:24) separated by a 2A cleavage peptide. After the production process, these different MSLN-CAR T cells were thawed and seeded at 3 million cells / ml with 70 UI / ml of IL-2. One day after thawing, the cells were exposed to 5 ng / ml of TGFb (R&D systems). After one hour, the cells were stained for CAR expression by cell surface staining using biotinylated recombinant mesothelin protein (LakePharma) and Brilliant Violet 421 streptavidin (BD). In addition, intracellular staining of MSLN-CAR T cells was performed using anti-phospho-SMAD2 / 3 (BD) conjugated with PE according to the manufacturer's instructions. Cells were analyzed by flow cytometry, and the status of phosphorylated SMAD2 / 3 was determined in CAR-positive and CAR-negative subpopulations (Figure 14). The results demonstrate that in the absence of dnTGFbRII, cells were SMAD2 / 3 phosphorylation-positive (Figure 14, left panel), but in the presence of dnTGFbRII, only CAR-positive cells could show decreased SMAD2 / 3 phosphorylation (Figure 14, right panel). These results indicated that TGFb signaling may be impaired in MSLN-CART cells expressing dnTGFbRII.

[0263] 4 - In vivo experiments Preliminary in vivo studies were performed to define and validate the animal / tumor model and administration route of CAR T cells. To evaluate the in vivo antitumor activity of T cells expressing the three selected mesoCAR constructs, NSG mice injected subcutaneously with HPAC tumor cells were selected as the animal / tumor model. Although meso1-R2 had lower levels of cell surface expression, cytotoxicity, and IFNγ secretion, it was included in the study and compared with the P4-R2 and MESO2-R2 CARs.

[0264] We then evaluated the in vivo antitumor activity of human T cells expressing mesoCAR candidates and P4-CAR. Briefly, NSG mice were transfected with MSLN + The cell line was transplanted (SC injection of HPAC cells) followed by mesoCAR + Treated with T cells (IV injection, 3 doses). mesoCAR + T cell activity was assessed by monitoring tumor growth. + T cells demonstrated different levels of in vivo antitumor activity against HPAC tumor cells. T cells expressing the MESO2-R2 CAR were significantly more potent than the other mesoCARs evaluated. + Compared to T cells, it showed lower activity.

[0265] 4.1 Rationale for choosing an animal model mesoCAR + Because T cells are human-specific, standard immunocompetent animal models are not available, as human T cells are rapidly targeted and eliminated by the xenogeneic immune response. The animal model of choice is the highly immunodeficient NSG mouse strain (NOD.Cg-Prkdc from the Jackson laboratory). scid Il2rg tm1Wjl / SzJ strain), which is a human MSLN + This is to allow the transplantation of both tumor cells and human CAR T cells.

[0266] 4.2 Establishment of animal / tumor models 4.2.1 Transplantation of Hela and HPAC In this study, we investigated two mesothelin-expressing tumor cell lines, epithelial cervical adenocarcinoma HeLa (ATCC (登録商標) HeLa cells (CCL-2), and epithelial splenic adenocarcinoma HPAC (ATCC® CRL-2119) were used. The objective of this first study was to evaluate parameters of tumor engraftment and tumor growth after subcutaneous (SC) injection of HeLa and HPAC cells into NSG mice (6-8 weeks old).

[0267] Briefly, on day 0, mice were randomly divided into four groups of six mice each based on their individual body weight and injected with HeLa cells (1 × 10 6 or 10x10 6 cells / mouse) or HPAC cells (2 x 10 6 or 10x10 6 The tumor cells were injected SC (1000 x 1000 cells / mouse). The amount of tumor cells was selected according to the literature [Abate-Daga, D., et al. (2014). A Novel Chimeric Antigen Receptor Against Prostate Stem Cell Antigen Mediates Tumor Destruction in a Humanized Mouse Model of Pancreatic. Cancer. Hum. Gene Ther; Arjomandnejad et al. (2014). HeLa cell line xenograft tumor as a suitable cervical cancer model: Growth kinetic characterization and immunohistochemistry array. Arch. Iran. Med.; Kusakawa et al. (2015). Characterization of in vivo tumorigenicity tests using severe immunodeficient NOD / Shi-scid IL2Rγ null mice for detection of tumorigenic cellular impurities in human cell-processed therapeutic products. Regen. Ther.].

[0268] Body weight, survival, and behavior were monitored daily. Tumor volume was measured three times a week. Surviving mice were sacrificed on day 61 (study termination). Necropsies (microscopic examination) were performed on all sacrificed test animals and, when possible, on all euthanized moribund or dead animals found.

[0269] Both HPAC and Hela tumors grew in NSG mice. HPAC tumors grew faster than Hela tumors. 1x10 6 and 10x10 6 The mean tumor volume V (500 mm) of mice injected with HeLa cells 3 ) are 519 mm 3 and 498 mm 3 and the average time to reach it was 55 days and 49 days. 6 and 10x10 6 The mean tumor volume V (500 mm) of mice injected with HPAC cells was 3 ) are 557 mm 3 and 500 mm 3 and the mean time to reach this was 24 and 23 days.

[0270] 4.2.2 Mesothelin expression in HeLa and HPAC tumors in NSG mice The second study objective was to assess mesothelin expression levels in both HPAC and HeLa tumors after subcutaneous injection and growth of tumor cells in NSG mice.

[0271] Briefly, on day 0, mice were randomly divided into two groups of three mice each based on their individual body weight and injected with HeLa cells (10 × 10 6 cells / mouse) or HPAC cells (2 x 10 6 The mice were injected SC with 1000 x 1000 cells / mouse. Body weight, survival, and behavior were monitored daily. Tumor volume was measured three times a week. When tumor volume reached 300–500 mm, 3 Tumors were harvested when the tumor size reached 100 mg / kg. Tumor samples were analyzed for mesothelin expression by immunohistochemistry (IHC). The last mouse was sacrificed on day 63 (end of study).

[0272] Both HPAC and HeLa tumors grew in NSG mice. As observed in previous studies, HPAC tumors grew more rapidly than HeLa tumors. Mice injected with HeLa or HPAC cells had an average tumor volume of 300 mm. 3 were reached in 40 and 28 days, respectively.

[0273] In addition, mesothelin expression in tumor cells was checked by IHC on tumors recovered from the mice. Both tumors expressed mesothelin.

[0274] Based on these confirmed data, mesoCAR + To evaluate the antitumor activity of T cells, HPAC cells (2 x 10 6 We decided to use rhesus monkey cells (SC injection).

[0275] 4.2.3 Implantation of HPAC-luc-GFP tumor cells HPAC cells expressing firefly luciferase and GFP (HPAC-luc-GFP) were generated at Cellectis, and tumor engraftment and tumor growth of HPAC-luc-GFP cells were evaluated in NSG mice as previously described.

[0276] Briefly, on day 0, three mice received a SC injection of HPAC-luc-GFP cells (2 x 10 6 Cells / mouse). Body weight, survival, and behavior were monitored daily. Tumor volume was measured three times a week, and bioluminescence imaging was performed on days 7, 14, and 24. Survival and behavior were monitored daily.

[0277] These conditions provided sensitivity for this assay, so wild-type HPAC cells (SC injection, 2 x 10 6 mesoCAR (cells / mouse) + We decided to assess T cell activity in vivo.

[0278] 4.3 Evaluation of antitumor activity of UCARTmeso candidates against HPAC tumors in NSG mice The objective of this study was to compare the antitumor activity of three UCART meso candidates (P4-R2, MESO2-R2, and meso1-R2) in NSG mice bearing subcutaneous HPAC tumors, using treatment conditions defined in a pilot study. + T cells were assessed (1, 3, and 10x10 6 CAR-positive cells / mouse).

[0279] UCARTmeso and control T cells were produced using PBMCs from the same donor. UCARTmeso and control T cells were not purified for TCRαβ-negative cells. The characteristics of the T cells used are described in Table 9.

[0280] Table 9. Characteristics of T cells used in this study TIFF2025186404000014.tif49153Day 18: End of production process (before freezing); % CAR + : CD45 on day 18 + CD45 against + / CAR + Percentage of CARs (measured by flow cytometry using recombinant MSLN protein for P4-R2 CARs and L-protein for Meso1-R2 CARs and MESO2-R2 CARs) % CD4 + : CD45 on day 18 + CD45 against + / CAR + / CD4 + Percentage (%) % CD8 + : CD45 on day 18 + CD45 against + / CAR + / CD8 + Percentage (%) % TCRαβ - : CD45 on day 18 + CD45 against + / TCRαβ - Percentage (%)

[0281] Briefly, 85 NSG mice received a subcutaneous injection of HPAC tumor cells (2X10 6 On day 0, 80 tumor-bearing mice were randomly divided into 16 groups of 5 mice each based on tumor volume.

[0282] Human T cells (UCARTmeso cells and KO TRAC / NT cell controls) were injected on day 0 (groups 1-15) or day 12 (group 16). For UCARTmeso cells, the total number of cells injected was determined according to the percentage of CAR-positive cells within the batch, with the indicated number of CAR-positive cells (1, 3, or 10x10) injected per mouse. 6 CAR-positive cells) were injected.

[0283] The antitumor activity of UCARTmeso candidate CARs (P4-R2, Meso1-R2, and MESO2-R2) was assessed by measuring tumor volume (Figures 15, 16, and 17). All UCARTmeso cells exhibited antitumor activity, although the level of activity varied among different CAR T cells.

[0284] As shown in Figure 18, the higher the dose (10x10 6 ), antitumor activity was observed for all CAR candidates. 6 MESO2-R2 CAR + The cells were unable to control HPAC tumor growth.

[0285] conclusion We developed an animal / tumor model and treatment conditions that allowed us to evaluate the antitumor activity of mesothelin-targeting CAR T cells. Using this animal model, we evaluated the in vivo activity of three CAR candidates, MESO2-R2, Meso1-R2, and P4-R2, and found that all CAR T cells demonstrated antitumor activity against HPAC cells.

[0286] However, the activity of MESO2-R2 CAR+ T cells was lower than that of Meso1-R2-expressing CAR+ T cells.

[0287] Among CAR T cells with selected properties (R2 suicide switch and TRAC KO), the Meso1-R2 CAR T cell candidate shows the highest in vivo activity at the three doses evaluated.

[0288] 4.4 - Evaluation of the antitumor activity of dnTGFBRII-expressing UCARTmeso candidates The purpose of this study was to compare the antitumor activity of two UCART meso candidates (P4-R2, MESO1-R2) that also express dnTGFBRII in NSG mice. Briefly, on day 0, 4–6 mice / group were inoculated with HPAC cells (2 x 10 6 The mice were injected SC with 1000 x 1000 cells / mouse. Body weight, survival, and behavior were monitored daily. Tumor volume was measured three times a week. Survival and behavior were monitored daily.

[0289] All dnTGFBRII-expressing UCARTmeso were produced using PBMCs derived from the same donor and administered at two doses of CAR + T cells were assessed (3 and 10x10 6 CAR-positive cells / mouse).

[0290] The antitumor activity of dnTGFBRII-expressing UCARTmeso candidates (P4-R2, MESO1-R2) was evaluated by measuring tumor volume (Figure 19). Both UCARTmeso cells exhibited antitumor activity, although the level of activity varied, with the MESO1 construct exhibiting greater antitumor activity.

[0291] 5 - Comparison of TGFBRII gene inactivation (KO TGFBRII) approach and dnTGFBRII gene overexpression approach for mesothelin-targeted UCART (UCART MESO) The investigation presented in this study aimed to select the best approach for UCART MESO by comparing the TGFBRII KO approach with the dnTGFBRII gene overexpression approach.

[0292] To conduct this study, six types of genetically modified T cells (defined in Table 10) were generated and tested.

[0293] Table 10. Description of the six genetically modified T cells generated for this study. TIFF2025186404000015.tif75158

[0294] 5.1 Different UCART MESO production On day 0, frozen human peripheral blood mononuclear cells (PBMCs) from Hemacare (Northridge, CA 91325, USA) were thawed, washed, counted, and resuspended in X-vivo 15 medium supplemented with 5% AB serum. The cells were then transferred to an incubator set at 37°C and 5% CO2. On day 1, PBMCs were counted, analyzed by flow cytometry to assess the % CD3+ cells, centrifuged, and resuspended in X-vivo 15 medium supplemented with 5% AB serum, 350 UI / ml IL2, and MACS GMP T Cell TransAct (60 μl / million CD3+ cells). Cells were then transferred to an incubator set at 37°C and 5% CO2. On day 4, T cells and rLV vectors carrying polynucleotide sequences encoding different anti-mesothelin CARs, P4-CAR (SEQ ID NO:161) and MESO1 CAR (SEQ ID NO:22), with or without the dnTGFBRII gene (SEQ ID NO:24), were resuspended in X-vivo 15 medium supplemented with 5% AB serum and 350 UI / ml IL2 and plated onto retronectin-coated plates. The plates were then transferred to an incubator set at 37°C and 5% CO2. On day 5, T cells were washed and resuspended in X-vivo 15 medium supplemented with 5% AB serum and 350 UI / ml IL-2, and then transferred to an incubator set at 37°C and 5% CO2. On day 6, T cells were co-electroporated with mRNA encoding the right and left arms of the TRAC TALEN, with or without mRNA encoding the right and left arms of the TGFBRII TALEN (SEQ ID NO:157 and SEQ ID NO:158). Gene transfer was performed using the AgilePulse method. Cells were then transferred to an incubator set at 37°C and 5% CO2. On day 7, T cells were washed and resuspended in X-vivo 15 medium supplemented with 5% AB serum and 350 UI / ml IL-2, and then transferred to an incubator set at 37°C and 5% CO2. T cells were expanded in the GRex device between days 7 / 8 and 18. During the expansion period, cell cultures were incubated at 37°C under 5% CO2, with occasional changes of culture medium. On day 18, all UCART cells were cryopreserved for later use in in vitro and in vivo assays.

[0295] 5.2 Evaluation of UCART cell populations On day 18, UCART cells were analyzed by flow cytometry. CAR surface expression was assessed using biotinylated recombinant mesothelin protein, which recognizes the scFv portion of CAR and streptavidin conjugated with PE. dnTGFBRII expression was assessed using anti-TGFBRII (Abcam) and an anti-mouse IgG antibody conjugated with APC. CD4 and CD8 surface expression was assessed using a CD4 antibody conjugated with FITC and a CD8 antibody conjugated with BV510. In addition, the stemness of UCART cells in CAR+ CD4+ or CAR+ CD8+ positive cells was analyzed using anti-CD62L conjugated with PECy7 and anti-CD45RA conjugated with APC.

[0296] As shown in Figure 20A, P4 expression was detected in 58% of UCART cells, and MESO1 expression was detected in 37% of UCART cells. In addition, when transduced with the P4-dnTGFBRII and MESO1-dnTGFBRII constructs, dnTGFBR2 was detected in 32% and 17% of UCART cells. These results may reflect the lower expression of dnTGFBRII placed downstream of the 2A peptide and CAR.

[0297] Interestingly, the percentage of CD8+ cells among CAR-positive cells ranged from 27% to 35% when UCART cells expressed the P4 CAR construct, whereas the percentage of CD8+ cells (among CAR-positive cells) ranged from 36% to 51% when expressing the MESO1 construct (Figure 20B).

[0298] Additionally, stemness analysis revealed that in CAR+CD4+ cells (Figure 21A), naive (Tn) and memory stem (Tscm) T cells for P4CAR ranged from 1% to 3%, while this subset was higher and varied by as much as 6% for T cells expressing the MESO1 CAR. This effect was also observed, albeit to a lesser extent, in CAR+CD8+ cells, where this T cell subset varied from 15% to 19% or 19% to 22% for P4 or MESO1 constructs, respectively (Figure 21B).

[0299] These results demonstrate that the MESO1 CAR, even though expressed or detected less, results in a higher proportion of CD8+ (i.e., cytotoxic) and higher proportions of Tn and Tscm subsets compared to the P4CAR. Importantly, inactivation of TGFBRII, whether by overexpression or KO of the dnTGFBRII construct, had no effect on any of the phenotypes analyzed.

[0300] 5.3 Evaluation of UCART cytotoxicity and IFNγ production After 16 hours of post-thaw recovery, the genetically modified T cells shown in Table 9 were mixed with H226-Luc / GFP cells at effector-to-target (E:T) ratios of 1:3, 1:1, 3:1, or 10:1. The co-cultures were then incubated overnight at 37°C, and bioluminescence was measured to quantify H226 cell lysis. Alternatively, after the post-thaw recovery period, these genetically modified T cells were resuspended in culture medium and plated at a density of 200,000 cells / well into 96-well plates either untreated or pre-coated with 75 ng / well of His-tagged recombinant mesothelin protein. After a 24-hour incubation period, cell supernatants were collected and analyzed by ELISA to quantify IFNg production.

[0301] As shown in Figure 22, MESO1-expressing UCART was able to induce higher cytotoxicity than P4-expressing UCART at the lowest doses (1:3 and 1:1 ratios).

[0302] Figure 23A demonstrates that recombinant mesothelin protein was able to induce IFNg secretion in all UCAR T cells produced. This production ranged from 40,000 pg / ml to a maximum of 90,000 pg / ml, and no obvious effect of CAR construct or TGFB pathway inhibition was observed. However, when IFNg secretion was analyzed in the absence of recombinant mesothelin (Figure 23B), it was surprising to find that the MESO1 CAR construct produced less IFNg than the P4 construct. This result suggests that UCAR T cells expressing the MESO1 CAR construct are less stimulated in the absence of antigen, implying that the MESO1 CAR has reduced "autoactivation." This is an important characteristic in therapeutic settings, as "autoactivation" tends to eliminate CAR T cells.

[0303] 5.4 Assessment of TGFb sensitivity To determine the impact of TGFBRII pathway inactivation, either by KO or dnTGFBRII overexpression, the genetically modified T cells listed in Table 9 were exposed to TGFb for 1 hour and analyzed by flow cytometry to assess the pSMAD2 / 3-positive versus pSMAD2 / 3-negative fraction of CAR-positive cells. In another set of experiments, produced UCART cells were exposed to recombinant mesothelin protein in the presence or absence of TGFb and counted after 7 days to assess proliferation.

[0304] Figure 24A shows that without TGFβ pathway inhibition, over 95% of the CAR-positive fraction of UCART cells was pSMAD2 / 3 positive. When inhibited by dnTGFBRII overexpression, 67% and 60% of the CAR-positive fraction of UCART cells expressing the P4 or MESO1 constructs were pSMAD2 / 3 negative. Interestingly, when expressing the P4 or MESO1 constructs, inactivation by KO resulted in 85% and 83% pSMAD2 / 3 negative cells, respectively. This result demonstrates that TGFBRII KO has a stronger ability to reduce SMAD2 / 3 phosphorylation.

[0305] Figure 24B shows that TGFb can inhibit antigen-mediated proliferation of UCART expressing either the P4 or MESO1 construct to the same extent. Interestingly, inhibition of the TGFb pathway, either by KO or by dnTGFBRII overexpression, can reduce or even eliminate such inhibition.

[0306] Sequence information SEQUENCE LISTING <110> Cellectis <120> NEW MESOTHELIN SPECIFIC CHIMERIC ANTIGEN RECEPTORS (CAR) FOR SOLID TUMORS CANCER IMMUNOTHERAPY <150> DK PA 2019 70835 <151> 2019-12-23 <160> 161 <170> PatentIn version 3.5 <210> 1 <211> 127 <212> PRT <213> artificial <220> <223> P4 heavy chain variable region <400> 1 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Val Ser Val Lys Ser Arg Met Ser Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Gly Met Met Thr Tyr Tyr Tyr Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ile Leu Gly 115 120 125 <210> 2 <211> 116 <212> PRT <213> artificial <220> <223> P4 light chain variable region <400> 2 Gln Pro Val Leu Thr Gln Ser Ser Ser Leu Ser Ala Ser Pro Gly Ala 1 5 10 15 Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn Val Gly Pro 20 25 30 Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr 35 40 45 Leu Leu Asn Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Val 65 70 75 80 Leu Leu Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 85 90 95 Met Ile Trp His Ser Ser Ala Ala Val Phe Gly Gly Gly Thr Gln Leu 100 105 110 Thr Val Leu Ser 115 <210> 3 <211> 5 <212> PRT <213> artificial <220> <223> CDRH1-meso1 <400> 3 Watch Tyr Tyr Trp Watch 1 5 <210> 4 <211> 16 <212> PRT <213> artificial <220> <223> CDRH2-meso1 <400> 4 Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 5 <211> 8 <212> PRT <213> artificial <220> <223> CDRH3- meso1 <400> 5 Val Asp Tyr Lys Ala Phe Asp Ile 1 5 <210> 6 <211> 11 <212> PRT <213> artificial <220> <223> CDRL1- meso1 <400> 6 Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu His 1 5 10 <210> 7 <211> 7 <212> PRT <213> artificial <220> <223> CDRL2- meso1 <400> 7 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 8 <211> 9 <212> PRT <213> artificial <220> <223> CDRL3- meso1 <400> 8 Leu Gln His Tyr Ser Tyr Pro Trp Thr 1 5 <210> 9 <211> 116 <212> PRT <213> artificial <220> <223> meso1 heavy chain variable region <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Val Asp Tyr Lys Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser 115 <210> 10 <211> 107 <212> PRT <213> artificial <220> <223> meso1 light chain variable region <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Tyr Ser Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 11 <211> 121 <212> PRT <213> artificial <220> <223> meso2 heavy chain variable region <400> 11 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Met Pro Ser Asp Ser Tyr Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly His Gly Met Tyr Gly Gly Ala Leu Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 12 <211> 111 <212> PRT <213> artificial <220> <223> meso2 light chain variable region <400> 12 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Ser 20 25 30 Arg Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Lys Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser His Asp Pro 85 90 95 Ser Gly Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 <210> 13 <211> 15 <212> PRT <213> artificial <220> <223> Linker <400> 13 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 14 <211> 21 <212> PRT <213> artificial <220> <223> CD8α signal peptide <400> 14 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 15 <211> 40 <212> PRT <213> artificial <220> <223> R2 suicide switch <400> 15 Ser Asp Pro Gly Ser Gly Gly Gly Gly Ser Cys Pro Tyr Ser Asn Pro 1 5 10 15 Ser Leu Cys Ser Gly Gly Gly Gly Ser Cys Pro Tyr Ser Asn Pro Ser 20 25 30 Leu Cys Ser Gly Gly Gly Gly Ser 35 40 <210> 16 <211> 45 <212> PRT <213> artificial <220> <223> CD8α hinge <400> 16 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 17 <211> 24 <212> PRT <213> artificial <220> <223> CD8α transmembrane domain <400> 17 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 18 <211> 42 <212> PRT <213> artificial <220> <223> 4-1BB co-stimulatory domain <400> 18 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 19 <211> 112 <212> PRT <213> artificial <220> <223> CD3ζ signalling domain <400> 19 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 20 <211> 542 <212> PRT <213> artificial <220> <223> P4-R2 CAR full sequence <400> 20 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu 20 25 30 Val Thr Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp 35 40 45 Ser Val Ser Ser Asn Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro 50 55 60 Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp 65 70 75 80 Tyr Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Met Ser Ile Asn Pro 85 90 95 Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro 100 105 110 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Met Met Thr Tyr Tyr 115 120 125 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Gly Ile Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gln Pro Val Leu Thr Gln Ser Ser Ser Leu Ser Ala Ser 165 170 175 Pro Gly Ala Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn 180 185 190 Val Gly Pro Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro 195 200 205 Pro Gln Tyr Leu Leu Asn Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly 210 215 220 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn 225 230 235 240 Ala Gly Val Leu Leu Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp 245 250 255 Tyr Tyr Cys Met Ile Trp His Ser Ser Ala Ala Val Phe Gly Gly Gly 260 265 270 Thr Gln Leu Thr Val Leu Ser Ser Asp Pro Gly Ser Gly Gly Gly Gly 275 280 285 Ser Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser Gly Gly Gly Gly Ser 290 295 300 Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser Gly Gly Gly Gly Ser Thr 305 310 315 320 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 325 330 335 Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 340 345 350 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 355 360 365 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 370 375 380 Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 385 390 395 400 Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 405 410 415 Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 420 425 430 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 435 440 445 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 450 455 460 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 465 470 475 480 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 485 490 495 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 500 505 510 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 515 520 525 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 530 535 540 <210> 21 <211> 522 <212> PRT <213> artificial <220> <223> meso1-R2 CAR full sequence <400> 21 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu 20 25 30 Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly 35 40 45 Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys 50 55 60 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr 65 70 75 80 Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys 85 90 95 Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Val Asp Tyr Lys Ala Phe Asp Ile Trp Gly 115 120 125 Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 145 150 155 160 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 165 170 175 Ala Ser Gln Gly Ile Arg Asn Asp Leu His Trp Tyr Gln Gln Lys Pro 180 185 190 Gly Lys Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser 195 200 205 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 210 215 220 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 225 230 235 240 Leu Gln His Tyr Ser Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val 245 250 255 Glu Ile Lys Ser Asp Pro Gly Ser Gly Gly Gly Gly Ser Cys Pro Tyr 260 265 270 Ser Asn Pro Ser Leu Cys Ser Gly Gly Gly Gly Ser Cys Pro Tyr Ser 275 280 285 Asn Pro Ser Leu Cys Ser Gly Gly Gly Gly Ser Thr Thr Thr Pro Ala 290 295 300 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 305 310 315 320 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 325 330 335 Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 340 345 350 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys 355 360 365 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 370 375 380 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 385 390 395 400 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 405 410 415 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 420 425 430 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 435 440 445 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 450 455 460 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 465 470 475 480 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 485 490 495 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 500 505 510 Ala Leu His Met Gln Ala Leu Pro Pro Arg 515 520 <210> 22 <211> 482 <212> PRT <213> artificial <220> <223> meso1 CAR full sequence <400> 22 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu 20 25 30 Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly 35 40 45 Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys 50 55 60 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr 65 70 75 80 Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys 85 90 95 Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Val Asp Tyr Lys Ala Phe Asp Ile Trp Gly 115 120 125 Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 145 150 155 160 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 165 170 175 Ala Ser Gln Gly Ile Arg Asn Asp Leu His Trp Tyr Gln Gln Lys Pro 180 185 190 Gly Lys Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser 195 200 205 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 210 215 220 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 225 230 235 240 Leu Gln His Tyr Ser Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val 245 250 255 Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 260 265 270 Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 275 280 285 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 290 295 300 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 305 310 315 320 Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu 325 330 335 Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 340 345 350 Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 355 360 365 Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 370 375 380 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 385 390 395 400 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 405 410 415 Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 420 425 430 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 435 440 445 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 450 455 460 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 465 470 475 480 Pro Arg <210> 23 <211> 531 <212> PRT <213> artificial <220> <223> meso2-R2 full sequence <400> 23 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30 Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr 35 40 45 Ser Phe Thr Asn Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys 50 55 60 Gly Leu Glu Trp Met Gly Val Ile Met Pro Ser Asp Ser Tyr Thr Arg 65 70 75 80 Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser 85 90 95 Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr 100 105 110 Ala Met Tyr Tyr Cys Ala Arg Tyr Gly His Gly Met Tyr Gly Gly Ala 115 120 125 Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val 145 150 155 160 Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala 165 170 175 Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Ser Arg Leu Ala 180 185 190 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly 195 200 205 Ala Ser Lys Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly Ser Gly 210 215 220 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp 225 230 235 240 Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser His Asp Pro Ser Gly Thr 245 250 255 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Ser Asp Pro Gly 260 265 270 Ser Gly Gly Gly Gly Ser Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser 275 280 285 Gly Gly Gly Gly Ser Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser Gly 290 295 300 Gly Gly Gly Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 305 310 315 320 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 325 330 335 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 340 345 350 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 355 360 365 Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu 370 375 380 Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 385 390 395 400 Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 405 410 415 Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 420 425 430 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 435 440 445 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 450 455 460 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 465 470 475 480 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 485 490 495 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 500 505 510 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 515 520 525 Pro Pro Arg 530 <210> 24 <211> 199 <212> PRT <213> artificial <220> <223> dominant negative dnTGFβRII <400> 24 Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val 20 25 30 Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 35 40 45 Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 50 55 60 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 65 70 75 80 Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 85 90 95 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 100 105 110 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 115 120 125 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 130 135 140 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu 145 150 155 160 Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu 165 170 175 Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn 180 185 190 Arg Gln Gln Lys Leu Ser Ser 195 <210> 25 <211> 291 <212> PRT <213> homo sapiens <220> <223> MSLN target antigen region <400> 25 Glu Val Glu Lys Thr Ala Cys Pro Ser Gly Lys Lys Ala Arg Glu Ile 1 5 10 15 Asp Glu Ser Leu Ile Phe Tyr Lys Lys Trp Glu Leu Glu Ala Cys Val 20 25 30 Asp Ala Ala Leu Leu Ala Thr Gln Met Asp Arg Val Asn Ala Ile Pro 35 40 45 Phe Thr Tyr Glu Gln Leu Asp Val Leu Lys His Lys Leu Asp Glu Leu 50 55 60 Tyr Pro Gln Gly Tyr Pro Glu Ser Val Ile Gln His Leu Gly Tyr Leu 65 70 75 80 Phe Leu Lys Met Ser Pro Glu Asp Ile Arg Lys Trp Asn Val Thr Ser 85 90 95 Leu Glu Thr Leu Lys Ala Leu Leu Glu Val Asn Lys Gly His Glu Met 100 105 110 Ser Pro Gln Ala Pro Arg Arg Pro Leu Pro Gln Val Ala Thr Leu Ile 115 120 125 Asp Arg Phe Val Lys Gly Arg Gly Gln Leu Asp Lys Asp Thr Leu Asp 130 135 140 Thr Leu Thr Ala Phe Tyr Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu 145 150 155 160 Glu Leu Ser Ser Val Pro Pro Ser Ser Ile Trp Ala Val Arg Pro Gln 165 170 175 Asp Leu Asp Thr Cys Asp Pro Arg Gln Leu Asp Val Leu Tyr Pro Lys 180 185 190 Ala Arg Leu Ala Phe Gln Asn Met Asn Gly Ser Glu Tyr Phe Val Lys 195 200 205 Ile Gln Ser Phe Leu Gly Gly Ala Pro Thr Glu Asp Leu Lys Ala Leu 210 215 220 Ser Gln Gln Asn Val Ser Met Asp Leu Ala Thr Phe Met Lys Leu Arg 225 230 235 240 Thr Asp Ala Val Leu Pro Leu Thr Val Ala Glu Val Gln Lys Leu Leu 245 250 255 Gly Pro His Val Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val 260 265 270 Arg Asp Trp Ile Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu Gly 275 280 285 Leu Gly Leu 290 <210> 26 <211> 9 <212> PRT <213> artificial <220> <223> Mimotope R2 (Rituximab) <400> 26 Cys Pro Tyr Ser Asn Pro Ser Leu Cys 1 5 <210> 27 <211> 24 <212> PRT <213> artificial <220> <223> Palivizumab epitope <400> 27 Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp 1 5 10 15 Gln Lys Lys Leu Met Ser Asn Asn 20 <210> 28 <211> 12 <212> PRT <213> artificial <220> <223> Cetuximab epitope 1 <400> 28 Cys Gln Phe Asp Leu Ser Thr Arg Arg Leu Lys Cys 1 5 10 <210> 29 <211> 12 <212> PRT <213> artificial <220> <223> Cetuximab epitope 2 <400> 29 Cys Gln Tyr Asn Leu Ser Ser Arg Ala Leu Lys Cys 1 5 10 <210> 30 <211> 12 <212> PRT <213> artificial <220> <223> Cetuximab epitope 3 <400> 30 Cys Val Trp Gln Arg Trp Gln Lys Ser Tyr Val Cys 1 5 10 <210> 31 <211> 12 <212> PRT <213> artificial <220> <223> Cetuximab epitope 4 <400> 31 Cys Met Trp Asp Arg Phe Ser Arg Trp Tyr Lys Cys 1 5 10 <210> 32 <211> 25 <212> PRT <213> artificial <220> <223> Nivolumab epitope 1 <400> 32 Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp 1 5 10 15 Lys Leu Ala Ala Phe Pro Glu Asp Arg 20 25 <210> 33 <211> 19 <212> PRT <213> artificial <220> <223> Nivolumab epitope 2 <400> 33 Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln 1 5 10 15 Ile Lys Glu <210> 34 <211> 24 <212> PRT <213> artificial <220> <223> QBEND-10 Epitope <400> 34 Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser Thr Asn Val Ser 1 5 10 15 Pro Ala Lys Pro Thr Thr Thr Ala 20 <210> 35 <211> 12 <212> PRT <213> artificial <220> <223> Alemtuzumab epitope <400> 35 Gly Gln Asn Asp Thr Ser Gln Thr Ser Ser Pro Ser 1 5 10 <210> 36 <211> 49 <212> DNA <213> homo sapiens <220> <223> T003387 target sequence TGFbetaRII <400> 36 ttttgtttcc ccatcagaat ataacaccag caatcctgac ttgttgcta 49 <210> 37 <211> 49 <212> DNA <213> homo sapiens <220> <223> T003401 target sequence TGFbetaRII <400> 37 tccctatgag gagtatgcct cttggaagac agagaaggac atcttctca 49 <210> 38 <211> 49 <212> DNA <213> homo sapiens <220> <223> T003400 target sequence TGFbetaRII <400> 38 tccctatgag gagtatgcct cttggaagac agagaaggac atcttctca 49 <210> 39 <211> 49 <212> DNA <213> homo sapiens <220> <223> T003405 target sequence TGFbetaRII <400> 39 tgtgggaggc ccaagatgcc catcgtgcac agggacctca agagctcca 49 <210> 40 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 40 acagtgatca cactccatgt ggg 23 <210> 41 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 41 gcagaagctg agttcaacct ggg 23 <210> 42 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 42 aggttaggtc gttcttcacg agg 23 <210> 43 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 43 aaagcgacct ttccccacca ggg <210> 44 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 44 tggatgacct ggctaacagt ggg <210> 45 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 45 cctgggaac cggcaagacg cgg <210> 46 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 46 acagatatgg caactcccag tgg <210> 47 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 47 gtggaggtga gcaatccccc ggg 23 <210> 48 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 48 acctacagga gtacctgacg cgg 23 <210> 49 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 49 gtgatcacac tccatgtggg agg 23 <210> 50 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 50 gctggtgtta tattctgatg ggg 23 <210> 51 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 51 cacagtgatc acactccatg tgg 23 <210> 52 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 52 cacatggagt gtgatcactg tgg 23 <210> 53 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 53 cagagtaggg tccagacgca ggg 23 <210> 54 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 54 gcttctgctg ccggttaacg cgg 23 <210> 55 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 55 gtggatgacc tggctaacag tgg <210> 56 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 56 gggaaagccc aaagtcacac agg <210> 57 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 57 aatatgacta gcaacaagtc agg <210> 58 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 58 atggagtgtg atcactgtgg agg <210> 59 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 59 atcaccgcct tccacgccaa ggg 23 <210> 60 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 60 aggagcggaa gacggagttg ggg 23 <210> 61 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 61 ccacgccaag ggcaacctac agg 23 <210> 62 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 62 caagatgccc atcgtgcaca ggg 23 <210> 63 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 63 throw ctcccagtgg tgg <210> 64 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 64 agcagaagct gagttcaacc tgg <210> 65 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 65 cctgtaggtt gcccttggcg tgg <210> 66 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 66 gtgagcaatc ccccggggcga ggg <210> 67 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 67 acagagtagg gtccagacgc agg 23 <210> 68 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 68 tagcaacaag tcaggattgc tgg 23 <210> 69 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 69 ccaagatgcc catcgtgcac agg 23 <210> 70 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 70 tgtggaggtg agcaatcccc cgg 23 <210> 71 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 71 tgcctcttgg aagacagaga agg <210> 72 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 72 gcccattgag ctggacaccc tgg <210> 73 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 73 ctgagttcaa cctgggaac cgg <210> 74 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 74 tgggaggacc tgcgcaagct ggg <210> 75 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 75 gtactcctgt aggttgccct tgg 23 <210> 76 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 76 tcttccgctc ctcagccgtc agg 23 <210> 77 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 77 ctgggcagct ccctcgcccg ggg 23 <210> 78 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 78 cattgagctg gacaccctgg tgg 23 <210> 79 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 79 tggcaactcc cagtggtggc agg 23 <210> 80 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 80 caactcccag tggtggcagg agg 23 <210> 81 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 81 cgagcactgt gccatcatcc tgg 23 <210> 82 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 82 gcggtcatct tccaggatga tgg 23 <210> 83 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 83 agagctgctg cccattgagc tgg 23 <210> 84 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 84 accagggtgt ccagctcaat ggg 23 <210> 85 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 85 ctggacaccc tggtggggaa agg 23 <210> 86 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 86 caaagcgacc tttccccacc agg 23 <210> 87 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 87 gacggctgag gagcggaaga cgg 23 <210> 88 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 88 tactctgtct gtggatgacc tgg 23 <210> 89 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 89 cggcaagacg cggaagctca tgg 23 <210> 90 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 90 cccaaagtca cacaggcagc agg 23 <210> 91 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 91 taggttgccc ttggcgtgga agg 23 <210> 92 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 92 tgaggagcgg aagacggagt tgg 23 <210> 93 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 93 cctgtgcacg atgggcatct tgg 23 <210> 94 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 94 gggagctgcc cagcttgcgc agg 23 <210> 95 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 95 ttgaactcag cttctgctgc cgg <210> 96 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 96 ccaagaggca tactcctcat agg <210> 97 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 97 23. catgagcttc cgcgtcttgc cgg <210> 98 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 98 cggagttggg gaaacaatac tgg <210> 99 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 99 gctcctcagc cgtcaggaac tgg 23 <210> 100 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 100 caccagggtg tccagctcaa tgg 23 <210> 101 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 101 ctagtcatat ttcaagtgac agg 23 <210> 102 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 102 gctgggcagc tccctcgccc ggg 23 <210> 103 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 103 tgtcagagcg gtcatcttcc agg 23 <210> 104 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 104 ctgggaggac ctgcgcaagc tgg 23 <210> 105 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 105 agctgggcag ctccctcgcc cgg 23 <210> 106 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 106 tgtgttgtgg ttgatgttgt tgg 23 <210> 107 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 107 cctgctgcct gtgtgacttt ggg 23 <210> 108 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 108 acctgctgcc tgtgtgactt tgg 23 <210> 109 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 109 gacgcggcat gtcatcagct ggg 23 <210> 110 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 110 ggtcatccac agacagagta ggg 23 <210> 111 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 111 agtcaagatc tttccctatg agg 23 <210> 112 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 112 gaacatactc cagttcctga cgg 23 <210> 113 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 113 acgtggagct gatgtcagag cgg 23 <210> 114 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 114 ggggaaaggt cgctttgctg agg 23 <210> 115 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 115 tctggaccct actctgtctg tgg 23 <210> 116 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 116 tgggcagctc cctcgcccgg ggg 23 <210> 117 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 117 agctgatgac atgccgcgtc agg 23 <210> 118 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 118 gccgcgtcag gtactcctgt agg 23 <210> 119 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 119 caagaggcat actcctcata ggg <210> 120 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 120 ggtgagcaat cccccggggcg agg <210> 121 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 121 ttgctggtgt father tgg <210> 122 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 122 cctatgagga gtatgcctct tgg 23 <210> 123 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 123 tgctggtgtt atattctgat ggg 23 <210> 124 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 124 cgaggatatt ggagctcttg agg 23 <210> 125 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 125 gttgatgttg ttggcacacg tgg 23 <210> 126 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 126 tgacgcggca tgtcatcagc tgg 23 <210> 127 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 127 ttgagctgga caccctggtg ggg 23 <210> 128 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 128 ttcagagcag tttgagacag tgg 23 <210> 129 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 129 gcggcatgtc atcagctggg agg 23 <210> 130 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 130 aggtcatcca cagacagagt agg 23 <210> 131 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 131 gaagatgatg atgacagata tgg 23 <210> 132 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 132 tgacctggct aacagtgggc agg <210> 133 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 133 tctactgcta cggcgttaac cgg <210> 134 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 134 gtccttctct gtcttccaag agg <210> 135 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 135 attgagctgg acaccctggt ggg 23 <210> 136 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 136 gtcgttcttc acgaggatat tgg 23 <210> 137 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 137 catcagcctc ctgccaccac tgg 23 <210> 138 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 138 agttcctgac ggctgaggag cgg 23 <210> 139 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 139 actccagttc ctgacggctg agg 23 <210> 140 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 140 cttgaggtcc ctgtgcacga tgg 23 <210> 141 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 141 ttgaggtccc tgtgcacgat ggg 23 <210> 142 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 142 ccgcgtcttg ccggtttccc agg 23 <210> 143 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 143 gttgcccttg gcgtggaagg cgg 23 <210> 144 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 144 ctttgggctt tccctgcgtc tgg 23 <210> 145 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 145 gatcaccgcc ttccacgcca agg 23 <210> 146 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 146 tgaagtgttc tgcttcagct tgg 23 <210> 147 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 147 ctgtgcacga tgggcatctt ggg 23 <210> 148 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 148 ggcatcttgg gcctcccaca tgg 23 <210> 149 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 149 ttacctgccc actgttagcc agg 23 <210> 150 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 150 atcagcctcc tgccaccact ggg 23 <210> 151 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 151 tcgctttgct gaggtctata agg 23 <210> 152 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 152 agtcacacag gcagcaggtt agg 23 <210> 153 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 153 gaggagcgga agacggagtt ggg 23 <210> 154 <211> 23 <212> DNA <213> homo sapiens <220> <223> CRISPR target sequences for TGFβRII gene <400> 154 tgggcagcag ctctgtgttg tgg 23 <210> 155 <211> 2781 <212> DNA <213> artificial <220> <223> R-TALEN TGFbRII pCLS32939 <400> 155 atgggcgatc ctaaaaagaa acgtaaggtc atcgatatcg ccgatctacg cacgctcggc 60 tacagccagc agcaacagga gaagatcaaa ccgaaggttc gttcgacagt ggcgcagcac 120 cacgaggcac tggtcggcca cgggtttaca cacgcgcaca tcgttgcgtt aagccaacac 180 ccggcagcgt tagggaccgt cgctgtcaag tatcaggaca tgatcgcagc gttgccagag 240 gcgacacacg aagcgatcgt tggcgtcggc aaacagtggt ccggcgcacg cgctctggag 300 gccttgctca cggtggcggg agagttgaga ggtccaccgt tacagttgga cacaggccaa 360 cttctcaaga ttgcaaaacg tggcggcgtg accgcagtgg aggcagtgca tgcatggcgc 420 aatgcactga cgggtgcccc gctcaacttg accccccagc aggtggtggc catcgccagc 480 aatggcggtg gcaagcaggc gctggagacg gtccagcggc tgttgccggt gctgtgccag 540 gcccacggct tgacccccca gcaggtggtg gccatcgcca gcaatggcgg tggcaagcag 600 gcgctggaga cggtccagcg gctgttgccg gtgctgtgcc aggcccacgg cttgaccccc 660 cagcaggtgg tggccatcgc cagcaatggc ggtggcaagc aggcgctgga gacggtccag 720 cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cccagcaggt ggtggccatc 780 gccagcaata atggtggcaa gcaggcgctg gagacggtcc agcggctgtt gccggtgctg 840 tgccaggccc acggcttgac cccccagcag gtggtggcca tcgccagcaa tggcggtggc 900 aagcaggcgc tggagacggt ccagcggctg ttgccggtgc tgtgccaggc ccacggcttg 960 accccccagc aggtggtggc catcgccagc aatggcggtg gcaagcaggc gctggagacg 1020 gtccagcggc tgttgccggt gctgtgccag gcccacggct tgacccccca gcaggtggtg 1080 gccatcgcca gcaatggcgg tggcaagcag gcgctggaga cggtccagcg gctgttgccg 1140 gtgctgtgcc aggcccacgg cttgaccccg gagcaggtgg tggccatcgc cagccacgat 1200 ggcggcaagc aggcgctgga gacggtccag cggctgttgc cggtgctgtg ccaggcccac 1260 ggcttgaccc cggagcaggt ggtggccatc gccagccacg atggcggcaa gcaggcgctg 1320 gagacggtcc agcggctgtt gccggtgctg tgccaggccc acggcttgac cccggagcag 1380 gtggtggcca tcgccagcca cgatggcggc aagcaggcgc tggagacggt ccagcggctg 1440 ttgccggtgc tgtgccaggc ccacggcttg accccggagc aggtggtggc catcgccagc 1500 cacgatggcg gcaagcaggc gctggagacg gtccagcggc tgttgccggt gctgtgccag 1560 gcccacggct tgaccccgga gcaggtggtg gccatcgcca gcaatattgg tggcaagcag 1620 gcgctggaga cggtgcaggc gctgttgccg gtgctgtgcc aggcccacgg cttgaccccc 1680 cagcaggtgg tggccatcgc cagcaatggc ggtggcaagc aggcgctgga gacggtccag 1740 cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cggagcaggt ggtggccatc 1800 gccagccacg atggcggcaa gcaggcgctg gagacggtcc agcggctgtt gccggtgctg 1860 tgccaggccc acggcttgac cccggagcag gtggtggcca tcgccagcaa tattggtggc 1920 aagcaggcgc tggagacggt gcaggcgctg ttgccggtgc tgtgccaggc ccacggcttg 1980 acccctcagc aggtggtggc catcgccagc aatggcggcg gcaggccggc gctggagagc 2040 attgttgccc agttatctcg ccctgatccg gcgttggccg cgttgaccaa cgaccacctc 2100 gtcgccttgg cctgcctcgg cggggcgtcct gcgctggatg cagtgaaaaa gggattgggg 2160 gatcctatca gccgttccca gctggtgaag tccgagctgg aggagaaga atccgagttg aggcacaagc tgaagtacgt gccccacgag tacatcgagc tgatcgagat cgcccggaac 2280. agcacccagg accgtatcct ggagatgaag gtgatggagt tcttcatgaa ggtgtacggc tacaggggca agcacctggg cggctccagg aagcccgacg gcgccatcta caccgtgggc tcccccatcg actacggcgt gatcgtggac accaaggcct actccggcgg ctacaacctg 2460 cccatcggcc aggccgacga aatgcagagg tacgtggagg agaaccagac caggaacaag cacatcaacc ccaacgagtg gtggaaggtg tacccctcca gcgtgaccga gttcaagttc ctgttcgtgt ccggccactt caagggcaac tacaaggccc agctgaccag gctgaaccac atcaccaact gcaacggcgc cgtgctgtcc gtggaggagc tcctgatcgg cggcgagatg 2700 atcaaggccg gcaccctgac cctggaggag gtgaggagg agttcaacaa cggcgagatc aacttcgcgg ccgactgata a <210> 156 <211> 2781 <212> DNA <213> artificial <220> <223> L-TALEN TGFbRII pCLS32940 <400> 156 atgggcgatc ctaaaaagaa acgtaaggtc atcgatatcg ccgatctacg cacgctcggc 60 tacagccagc agcaacagga gaagatcaaa ccgaaggttc gttcgacagt ggcgcagcac 120 cacgaggcac tggtcggcca cgggtttaca cacgcgcaca tcgttgcgtt aagccaacac 180 ccggcagcgt tagggaccgt cgctgtcaag tatcaggaca tgatcgcagc gttgccagag 240 gcgacacacg aagcgatcgt tggcgtcggc aaacagtggt ccggcgcacg cgctctggag 300 gccttgctca cggtggcggg agagttgaga ggtccaccgt tacagttgga cacaggccaa 360 cttctcaaga ttgcaaaacg tggcggcgtg accgcagtgg aggcagtgca tgcatggcgc 420 aatgcactga cgggtgcccc gctcaacttg accccggagc aggtggtggc catcgccagc 480 aatattggtg gcaagcaggc gctggagacg gtgcaggcgc tgttgccggt gctgtgccag 540 gcccacggct tgacccccca gcaggtggtg gccatcgcca gcaataatgg tggcaagcag 600 gcgctggaga cggtccagcg gctgttgccg gtgctgtgcc aggcccacgg cttgaccccg 660 gagcaggtgg tggccatcgc cagccacgat ggcggcaagc aggcgctgga gacggtccag 720 cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cggagcaggt ggtggccatc 780 gccagcaata ttggtggcaa gcaggcgctg gagacggtgc aggcgctgtt gccggtgctg 840 tgccaggccc acggcttgac cccggagcag gtggtggcca tcgccagcaa tattggtggc 900 aagcaggcgc tggagacggt gcaggcgctg ttgccggtgc tgtgccaggc ccacggcttg 960 accccggagc aggtggtggc catcgccagc cacgatggcg gcaagcaggc gctggagacg 1020 gtccagcggc tgttgccggt gctgtgccag gcccacggct tgaccccgga gcaggtggtg 1080 gccatcgcca gcaatattgg tggcaagcag gcgctggaga cggtgcaggc gctgttgccg 1140 gtgctgtgcc aggcccacgg cttgaccccg gagcaggtgg tggccatcgc cagcaatatt 1200 ggtggcaagc aggcgctgga gacggtgcag gcgctgttgc cggtgctgtg ccaggcccac 1260 ggcttgaccc cccagcaggt ggtggccatc gccagcaata atggtggcaa gcaggcgctg 1320 gagacggtcc agcggctgtt gccggtgctg tgccaggccc acggcttgac cccccagcag 1380 gtggtggcca tcgccagcaa tggcggtggc aagcaggcgc tggagacggt ccagcggctg 1440 ttgccggtgc tgtgccaggc ccacggcttg accccggagc aggtggtggc catcgccagc 1500 cacgatggcg gcaagcaggc gctggagacg gtccagcggc tgttgccggt gctgtgccag 1560 gcccacggct tgaccccgga gcaggtggtg gccatcgcca gcaatattgg tggcaagcag 1620 gcgctggaga cggtgcaggc gctgttgccg gtgctgtgcc aggcccacgg cttgaccccc 1680 cagcaggtgg tggccatcgc cagcaataat ggtggcaagc aggcgctgga gacggtccag 1740 cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cccagcaggt ggtggccatc 1800 gccagcaata atggtggcaa gcaggcgctg gagacggtcc agcggctgtt gccggtgctg 1860 tgccaggccc acggcttgac cccggagcag gtggtggcca tcgccagcaa tattggtggc 1920 aagcaggcgc tggagacggt gcaggcgctg ttgccggtgc tgtgccaggc ccacggcttg 1980 acccctcagc aggtggtggc catcgccagc aatggcggcg gcaggccggc gctggagagc 2040 attgttgccc agttatctcg ccctgatccg gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggggcgtcct gcgctggatg cagtgaaaaa gggattgggg 2160 gatcctatca gccgttccca gctggtgaag tccgagctgg aggagaaga atccgagttg aggcacaagc tgaagtacgt gccccacgag tacatcgagc tgatcgagat cgcccggaac 2280. agcacccagg accgtatcct ggagatgaag gtgatggagt tcttcatgaa ggtgtacggc tacaggggca agcacctggg cggctccagg aagcccgacg gcgccatcta caccgtgggc tcccccatcg actacggcgt gatcgtggac accaaggcct actccggcgg ctacaacctg 2460 cccatcggcc aggccgacga aatgcagagg tacgtggagg agaaccagac caggaacaag cacatcaacc ccaacgagtg gtggaaggtg tacccctcca gcgtgaccga gttcaagttc ctgttcgtgt ccggccactt caagggcaac tacaaggccc agctgaccag gctgaaccac atcaccaact gcaacggcgc cgtgctgtcc gtggaggagc tcctgatcgg cggcgagatg 2700 atcaaggccg gcaccctgac cctggaggag gtgaggagg agttcaacaa cggcgagatc aacttcgcgg ccgactgata a 2781 <210> 157 <211> 2781 <212> DNA <213> artificial <220> <223> R-TALEN TGFbRII pCLS32967 <400> 157 atgggcgatc ctaaaaagaa acgtaaggtc atcgatatcg ccgatctacg cacgctcggc 60 tacagccagc agcaacagga gaagatcaaa ccgaaggttc gttcgacagt ggcgcagcac 120 cacgaggcac tggtcggcca cgggtttaca cacgcgcaca tcgttgcgtt aagccaacac 180 ccggcagcgt tagggaccgt cgctgtcaag tatcaggaca tgatcgcagc gttgccagag 240 gcgacacacg aagcgatcgt tggcgtcggc aaacagtggt ccggcgcacg cgctctggag 300 gccttgctca cggtggcggg agagttgaga ggtccaccgt tacagttgga cacaggccaa 360 cttctcaaga ttgcaaaacg tggcggcgtg accgcagtgg aggcagtgca tgcatggcgc 420 aatgcactga cgggtgcccc gctcaacttg accccggagc aggtggtggc catcgccagc 480 cacgatggcg gcaagcaggc gctggagacg gtccagcggc tgttgccggt gctgtgccag 540 gcccacggct tgaccccgga gcaggtggtg gccatcgcca gccacgatgg cggcaagcag 600 gcgctggaga cggtccagcg gctgttgccg gtgctgtgcc aggcccacgg cttgaccccg 660 gagcaggtgg tggccatcgc cagccacgat ggcggcaagc aggcgctgga gacggtccag 720 cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cccagcaggt ggtggccatc 780 gccagcaatg gcggtggcaa gcaggcgctg gagacggtcc agcggctgtt gccggtgctg 840 tgccaggccc acggcttgac cccggagcag gtggtggcca tcgccagcaa tattggtggc 900 aagcaggcgc tggagacggt gcaggcgctg ttgccggtgc tgtgccaggc ccacggcttg 960 accccccagc aggtggtggc catcgccagc aatggcggtg gcaagcaggc gctggagacg 1020 gtccagcggc tgttgccggt gctgtgccag gcccacggct tgacccccca gcaggtggtg 1080 gccatcgcca gcaataatgg tggcaagcag gcgctggaga cggtccagcg gctgttgccg 1140 gtgctgtgcc aggcccacgg cttgaccccg gagcaggtgg tggccatcgc cagcaatatt 1200 ggtggcaagc aggcgctgga gacggtgcag gcgctgttgc cggtgctgtg ccaggcccac 1260 ggcttgaccc cccagcaggt ggtggccatc gccagcaata atggtggcaa gcaggcgctg 1320 gagacggtcc agcggctgtt gccggtgctg tgccaggccc acggcttgac cccccagcag 1380 gtggtggcca tcgccagcaa taatggtggc aagcaggcgc tggagacggt ccagcggctg 1440 ttgccggtgc tgtgccaggc ccacggcttg accccggagc aggtggtggc catcgccagc 1500 aatattggtg gcaagcaggc gctggagacg gtgcaggcgc tgttgccggt gctgtgccag 1560 gcccacggct tgacccccca gcaggtggtg gccatcgcca gcaataatgg tggcaagcag 1620 gcgctggaga cggtccagcg gctgttgccg gtgctgtgcc aggcccacgg cttgaccccc 1680 cagcaggtgg tggccatcgc cagcaatggc ggtggcaagc aggcgctgga gacggtccag 1740 cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cggagcaggt ggtggccatc 1800 gccagcaata ttggtggcaa gcaggcgctg gagacggtgc aggcgctgtt gccggtgctg 1860 tgccaggccc acggcttgac cccccagcag gtggtggcca tcgccagcaa tggcggtggc 1920 aagcaggcgc tggagacggt ccagcggctg ttgccggtgc tgtgccaggc ccacggcttg 1980 acccctcagc aggtggtggc catcgccagc aatggcggcg gcaggccggc gctggagagc attgttgccc agttatctcg ccctgatccg gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggggcgtcct gcgctggatg cagtgaaaaa gggattgggg 2160 gatcctatca gccgttccca gctggtgaag tccgagctgg aggagaaga atccgagttg aggcacaagc tgaagtacgt gccccacgag tacatcgagc tgatcgagat cgcccggaac 2280. agcacccagg accgtatcct ggagatgaag gtgatggagt tcttcatgaa ggtgtacggc tacaggggca agcacctggg cggctccagg aagcccgacg gcgccatcta caccgtgggc tcccccatcg actacggcgt gatcgtggac accaaggcct actccggcgg ctacaacctg 2460 cccatcggcc aggccgacga aatgcagagg tacgtggagg agaaccagac caggaacaag cacatcaacc ccaacgagtg gtggaaggtg tacccctcca gcgtgaccga gttcaagttc ctgttcgtgt ccggccactt caagggcaac tacaaggccc agctgaccag gctgaaccac atcaccaact gcaacggcgc cgtgctgtcc gtggaggagc tcctgatcgg cggcgagatg 2700 atcaaggccg gcaccctgac cctggaggag gtgaggagga agttcaacaa cggcgagatc 2760 aacttcgcgg ccgactgata a 2781 <210> 158 <211> 2781 <212> DNA <213> artificial <220> <223> L-TALEN TGFbRII pCLS32968 <400> 158 atgggcgatc ctaaaaagaa acgtaaggtc atcgatatcg ccgatctacg cacgctcggc 60 tacagccagc agcaacagga gaagatcaaa ccgaaggttc gttcgacagt ggcgcagcac 120 cacgaggcac tggtcggcca cgggtttaca cacgcgcaca tcgttgcgtt aagccaacac 180 ccggcagcgt tagggaccgt cgctgtcaag tatcaggaca tgatcgcagc gttgccagag 240 gcgacacacg aagcgatcgt tggcgtcggc aaacagtggt ccggcgcacg cgctctggag 300 gccttgctca cggtggcggg agagttgaga ggtccaccgt tacagttgga cacaggccaa 360 cttctcaaga ttgcaaaacg tggcggcgtg accgcagtgg aggcagtgca tgcatggcgc 420 aatgcactga cgggtgcccc gctcaacttg accccccagc aggtggtggc catcgccagc 480 aataatggtg gcaagcaggc gctggagacg gtccagcggc tgttgccggt gctgtgccag 540 gcccacggct tgaccccgga gcaggtggtg gccatcgcca gcaatattgg tggcaagcag 600 gcgctggaga cggtgcaggc gctgttgccg gtgctgtgcc aggcccacgg cttgaccccc 660 cagcaggtgg tggccatcgc cagcaataat ggtggcaagc aggcgctgga gacggtccag 720 cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cggagcaggt ggtggccatc 780 gccagcaata ttggtggcaa gcaggcgctg gagacggtgc aggcgctgtt gccggtgctg 840 tgccaggccc acggcttgac cccggagcag gtggtggcca tcgccagcaa tattggtggc 900 aagcaggcgc tggagacggt gcaggcgctg ttgccggtgc tgtgccaggc ccacggcttg 960 accccccagc aggtggtggc catcgccagc aataatggtg gcaagcaggc gctggagacg 1020 gtccagcggc tgttgccggt gctgtgccag gcccacggct tgaccccgga gcaggtggtg 1080 gccatcgcca gcaatattgg tggcaagcag gcgctggaga cggtgcaggc gctgttgccg 1140 gtgctgtgcc aggcccacgg cttgaccccc cagcaggtgg tggccatcgc cagcaatggc 1200 ggtggcaagc aggcgctgga gacggtccag cggctgttgc cggtgctgtg ccaggcccac 1260 ggcttgaccc cccagcaggt ggtggccatc gccagcaata atggtggcaa gcaggcgctg 1320 gagacggtcc agcggctgtt gccggtgctg tgccaggccc acggcttgac cccccagcag 1380 gtggtggcca tcgccagcaa tggcggtggc aagcaggcgc tggagacggt ccagcggctg 1440 ttgccggtgc tgtgccaggc ccacggcttg accccggagc aggtggtggc catcgccagc 1500 cacgatggcg gcaagcaggc gctggagacg gtccagcggc tgttgccggt gctgtgccag 1560 gcccacggct tgaccccgga gcaggtggtg gccatcgcca gccacgatgg cggcaagcag 1620 gcgctggaga cggtccagcg gctgttgccg gtgctgtgcc aggcccacgg cttgaccccc 1680 cagcaggtgg tggccatcgc cagcaatggc ggtggcaagc aggcgctgga gacggtccag 1740 cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cccagcaggt ggtggccatc 1800 gccagcaatg gcggtggcaa gcaggcgctg gagacggtcc agcggctgtt gccggtgctg 1860 tgccaggccc acggcttgac cccggagcag gtggtggcca tcgccagcca cgatggcggc 1920 aagcaggcgc tggagacggt ccagcggctg ttgccggtgc tgtgccaggc ccacggcttg acccctcagc aggtggtggc catcgccagc aatggcggcg gcaggccggc gctggagagc attgttgccc agttatctcg ccctgatccg gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggggcgtcct gcgctggatg cagtgaaaaa gggattgggg 2160 gatcctatca gccgttccca gctggtgaag tccgagctgg aggagaaga atccgagttg aggcacaagc tgaagtacgt gccccacgag tacatcgagc tgatcgagat cgcccggaac 2280. agcacccagg accgtatcct ggagatgaag gtgatggagt tcttcatgaa ggtgtacggc tacaggggca agcacctggg cggctccagg aagcccgacg gcgccatcta caccgtgggc tcccccatcg actacggcgt gatcgtggac accaaggcct actccggcgg ctacaacctg 2460 cccatcggcc aggccgacga aatgcagagg tacgtggagg agaaccagac caggaacaag cacatcaacc ccaacgagtg gtggaaggtg tacccctcca gcgtgaccga gttcaagttc ctgttcgtgt ccggccactt caagggcaac tacaaggccc agctgaccag gctgaaccac atcaccaact gcaacggcgc cgtgctgtcc gtggaggagc tcctgatcgg cggcgagatg 2700 atcaaggccg gcaccctgac cctggaggag gtgaggagga agttcaacaa cggcgagatc 2760 aacttcgcgg ccgactgata a 2781 <210> 159 <211> 49 <212> DNA <213> artificial <220> <223> TRAC TALEN target sequence <400> 159 ttgtcccaca gatatccaga accctgaccc tgccgtgtac cagctgaga 49 <210> 160 <211> 49 <212> DNA <213> artificial <220> <223> CD52 TALEN target sequence <400> 160 ttcctcctac tcaccatcag cctcctggtt atggtacagg taagagcaa 49 <210> 161 <211> 502 <212> PRT <213> artificial <220> <223> P4 CAR full sequence <400> 161 Put Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Wing Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu 20 25 30 Val Thr Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp 35 40 45 Ser Val Ser Ser Asn Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro 50 55 60 Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp 65 70 75 80 Tyr Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Met Ser Ile Asn Pro 85 90 95 Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro 100 105 110 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Met Met Thr Tyr Tyr 115 120 125 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Gly Ile Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gln Pro Val Leu Thr Gln Ser Ser Ser Leu Ser Ala Ser 165 170 175 Pro Gly Ala Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn 180 185 190 Val Gly Pro Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro 195 200 205 Pro Gln Tyr Leu Leu Asn Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly 210 215 220 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn 225 230 235 240 Ala Gly Val Leu Leu Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp 245 250 255 Tyr Tyr Cys Met Ile Trp His Ser Ser Ala Ala Val Phe Gly Gly Gly 260 265 270 Thr Gln Leu Thr Val Leu Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 275 280 285 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 290 295 300 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 305 310 315 320 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 325 330 335 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 340 345 350 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 355 360 365 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 370 375 380 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 385 390 395 400 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 405 410 415 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 420 425 430 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 435 440 445 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 450 455 460 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 465 470 475 480 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 485 490 495 Gln Ala Leu Pro Pro Arg 500

Claims

1. an extracellular ligand-binding domain comprising VH and VL derived from a monoclonal anti-mesothelin antibody; - a transmembrane domain, and - A cytoplasmic domain containing the CD3 zeta signaling domain and a costimulatory domain A mesothelin-specific chimeric antigen receptor (CAR) comprising at least The mesothelin-specific chimeric antigen receptor (CAR), wherein the extracellular ligand-binding domain is directed against the MSLN antigen polypeptide region SEQ ID NO:

25.

2. the extracellular ligand-binding domain a variable heavy VH chain comprising CDRs from antibody Meso1, having at least 90% identity to SEQ ID NO:3 (CDRH1-Meso1), SEQ ID NO:4 (CDRH2-Meso1), and SEQ ID NO:5 (CDRH3-Meso1), respectively; and a variable heavy VL chain comprising CDRs from antibody Meso1, which have at least 90% identity to SEQ ID NO:6 (CDRL1-Meso1), SEQ ID NO:7 (CDRL2-Meso1), and SEQ ID NO:8 (CDRL3-Meso1), respectively; 2. The mesothelin-specific chimeric antigen receptor (CAR) of claim 1, comprising:

3. The mesothelin-specific chimeric antigen receptor of claim 1, wherein the extracellular ligand-binding domain comprises a VH chain and a VL chain having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with SEQ ID NO:9 (Meso1-VH) and SEQ ID NO:10 (Meso1-VL), respectively.

4. 4. The mesothelin-specific chimeric antigen receptor (CAR) of any one of claims 1 to 3, wherein the transmembrane domain is derived from the transmembrane region of the alpha, beta, or zeta chain of a T cell receptor, PD-1, 4-1BB, OX40, ICOS, CTLA-4, LAG3, 2B4, BTLA4, TIM-3, TIGIT, SIRPA, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.

5. 5. The mesothelin-specific chimeric antigen receptor (CAR) of claim 4, wherein the transmembrane domain has at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with SEQ ID NO:6 derived from CD8α.

6. The mesothelin-specific chimeric antigen receptor (CAR) of any one of claims 1 to 5, further comprising a hinge between the extracellular ligand-binding domain and the transmembrane domain.

7. 7. The mesothelin-specific chimeric antigen receptor (CAR) of claim 6, wherein the hinge is selected from a CD8α hinge, an IgG1 hinge, and an FcγRIIIα hinge.

8. The mesothelin-specific chimeric antigen receptor (CAR) of claim 7, wherein the hinge has at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with SEQ ID NO: 16 (CD8α).

9. The CAR is A polypeptide structure comprising a CD8α hinge having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:16, and a CD8α transmembrane domain having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:

17. The mesothelin-specific CAR of any one of claims 1 to 8, comprising:

10. The mesothelin-specific CAR of any one of claims 1 to 9, further comprising a safety switch comprising an epitope selected from Table 5.

11. The mesothelin-specific CAR of claim 10, wherein the safety switch comprises the epitope CPYSNPSLC (SEQ ID NO:26) to which rituximab specifically binds.

12. The mesothelin-specific CAR of claim 10 or 11, comprising a safety switch R2 having at least 90% identity to SEQ ID NO:

15.

13. 13. The mesothelin-specific chimeric antigen receptor of any one of claims 1 to 12, comprising a costimulatory domain derived from 4-1BB or CD28.

14. The mesothelin-specific CAR of claim 13, wherein the costimulatory domain is derived from 4-1BB and / or has at least 80% identity to SEQ ID NO:

18.

15. The mesothelin-specific CAR of any one of claims 1 to 14, wherein the CD3 zeta signaling domain has at least 80% identity to SEQ ID NO:

19.

16. The mesothelin-specific CAR of any one of claims 1 to 15, further comprising a signal peptide.

17. The mesothelin-specific chimeric antigen receptor (CAR) of any one of claims 1 to 16, which is a single-chain polypeptide.

18. The mesothelin-specific chimeric antigen receptor (CAR) of claim 17, having an overall amino acid sequence identity of at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% with SEQ ID NO:21 (Mesol CAR) or SEQ ID NO:22 (Mesol-R2 CAR).

19. A polynucleotide encoding the chimeric antigen receptor of any one of claims 1 to 18.

20. 20. An expression vector comprising the polynucleotide of claim 19.

21. 21. A modified immune cell comprising the polynucleotide of claim 19 or the expression vector of claim 20.

22. A modified immune cell that expresses the mesothelin-specific chimeric antigen receptor of any one of claims 1 to 18 on its cell surface membrane.

23. 23. The modified immune cell of claim 21 or 22, which is a T lymphocyte.

24. 24. The modified immune cell of claim 23, wherein the modified immune cell is derived from a primary cell or differentiated from a stem cell, such as an iPS cell.

25. 25. The modified immune cell of claim 23 or 24, wherein the modified immune cell is derived from an inflammatory T lymphocyte, a cytotoxic T lymphocyte, or a helper T lymphocyte.

26. 26. The modified immune cell of any one of claims 21 to 25, wherein expression of a TCR is reduced or suppressed in said immune cell.

27. 27. The modified immune cell of claim 26, wherein at least one gene encoding TCR alpha or TCR beta in said cell is inactivated.

28. 28. The modified immune cell of claim 27, wherein at least one gene encoding the TCR alpha or TCR beta is cleaved with a rare-cutting endonuclease.

29. The modified immune cell of claim 27 or 28, wherein a polynucleotide encoding the mesothelin-specific CAR is integrated into an endogenous locus, preferably the TCR alpha or TCR beta locus, under the transcriptional control of an endogenous promoter.

30. 30. The modified immune cells of claim 29, originating from a donor for allogeneic transplantation.

31. 31. The modified immune cell of any one of claims 21 to 30, which has been mutated to confer resistance to at least one immunosuppressant, such as an anti-CD52 antibody.

32. 32. The modified immune cell of any one of claims 21 to 31, further mutated to confer resistance to at least one chemotherapeutic agent, particularly a purine analogue agent.

33. 33. The modified immune cell of any one of claims 21-32, which has been mutated to improve persistence or longevity in a patient, particularly in genes encoding MHCI components such as HLA or B2m.

34. 34. The modified immune cell of any one of claims 21-33, wherein the modified immune cell has been mutated to improve CAR-dependent immune activation, specifically to reduce or suppress expression of immune checkpoint proteins and / or their receptors.

35. 35. The modified immune cell of any one of claims 21-34, wherein said mesothelin-specific chimeric antigen receptor (CAR) is co-expressed in said cell with another exogenous gene sequence encoding an inhibitor or decoy of a TGF beta receptor.

36. 36. The modified immune cell of claim 35, wherein the TGF beta receptor decoy is a dominant-negative TGF beta receptor, such as a TGF beta receptor having at least 80% polypeptide sequence identity with SEQ ID NO:

24.

37. an exogenous polynucleotide comprising a first polynucleotide sequence encoding the mesothelin-specific CAR, a second polynucleotide encoding a 2A self-cleaving peptide, and a third polynucleotide encoding the dominant-negative TGF beta receptor.

37. The modified immune cell of claim 36, comprising:

38. 38. The modified immune cell of any one of claims 21-37, wherein expression of at least one TGF beta receptor gene is reduced or inactivated.

39. 39. The modified immune cell of claim 38, wherein the TGF beta receptor gene is TGFβRII.

40. the mesothelin-specific chimeric antigen receptor (CAR) NK cell inhibitors, such as HLAG, HLAE, or ULBP1; - CRS inhibitors, such as mutant IL6Ra, sGP130, or IL18-BP; or - cytochrome P450, CYP2D6-1, CYP2D6-2, CYP2C9, CYP3A4, CYP2C19, or CYP1A2, which confers hypersensitivity of said immune cells to drugs such as cyclophosphamide and / or isophosphamide; dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin, or methylguanine transferase (MGMT), mTORmut, or Lckmut, which confer drug resistance; Chemokines or cytokines, such as IL-2, IL-12, and IL-15; Chemokine receptors, such as CCR2, CXCR2, or CXCR4; - secretion inhibitors of tumor-associated macrophages (TAMs), such as CCR2 / CCL2 neutralizing agents, which enhance the therapeutic activity of said immune cells 40. The modified immune cell of any one of claims 21-39, wherein said cell is co-expressed with another exogenous gene sequence selected from those encoding:

41. 41. The modified immune cell of any one of claims 21 to 40 for use in therapy.

42. 42. The modified immune cell of any one of claims 21 to 41 for use as a medicament for treating cancer.

43. 43. The modified immune cell of any one of claims 21-42 for use in therapy of a pre-malignant or malignant cancer condition characterized by mesothelin-expressing cells.

44. 44. The modified immune cell of any one of claims 21-43 for use in therapy of a cancer condition selected from esophageal cancer, breast cancer, gastric cancer, bile duct adenocarcinoma, pancreatic cancer, colon cancer, lung cancer, thymic cancer, mesothelioma, ovarian cancer, and endometrial cancer.

45. 1. A method for treating a patient having a condition characterized by mesothelin-expressing cells, comprising: - modifying immune cells from a donor to express a functional mesothelin-specific chimeric antigen receptor (CAR) according to any one of claims 1 to 20; administering the CAR-positive modified immune cells to a patient to eliminate mesothelin-expressing cells. The method comprising:

46. 46. ​​The method of treating a patient of claim 45, comprising an additional therapeutic step in which the patient is lymphodepleted.

47. 47. The method for treating a patient of claim 46, wherein the CAR-positive modified immune cells that deplete mesothelin-expressing cells are mutated to confer resistance to lymphocyte-depleting therapy.

48. 48. The method for treating a patient of claim 47, wherein the CAR-positive modified immune cells that eliminate mesothelin-expressing cells are mutated in the CD52 gene.

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