Novel Mesothelin-Specific Chimeric Antigen Receptor (CAR) for Cancer Immunotherapy of Solid Tumors

Abstract

The present invention relates to engineered immune cells expressing novel mesothelin (MLSN)-specific chimeric antigen receptors (anti-mesothelin CARs) and their use in the treatment of solid tumors, particularly for allogeneic cellular immunotherapy.

Description

[Technical Field]

[0001] Field of Invention This invention relates to the field of cell immunotherapy, and more specifically to modified immune cells expressing a novel mesothelin (MLSN)-specific chimeric antigen receptor (anti-mesothelin CAR) that is useful for the treatment of solid tumors. [Background technology]

[0002] Background of the Invention Chimeric antigen receptors (CARs) are synthetic receptors that direct 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 in lung, pancreatic, breast, ovarian, and other cancers. Encouragingly, recent clinical trials evaluating active immunization or immunoconjugates in patients with splenic adenocarcinoma or mesothelioma have shown a non-toxic response. Overall, these findings, and preclinical CAR therapy models using systemic or local T cell delivery, suggest that mesothelin CAR therapy is advantageous in multiple solid tumors.

[0003] If CAR therapy is potentially highly efficient, then identifying appropriate antigens to capture solid tumors is crucial for achieving tumor eradication with minimal or acceptable on-target / off-tumor toxicity to healthy tissue.

[0004] The solid tumor CAR targets currently under investigation are genetically modified products, most of which arise from gene mutations or splicing variants (EGFRvIII), glycosylation pattern variants (MUC1), oncotesticular antigen-derived peptides (MAGE), overexpressed differentiation antigens (CEA, PSMA, GD2, MUC16, HER2 / ERBB2, and mesothelin (MSLN)), or tumor-associated stromas (FAP and VEGFR).

[0005] Overexpressed antigens are numerous and relatively frequent, but T cells are highly sensitive to even low levels of antigen expression, and the expression levels of such antigens can exceed those of monoclonal antibodies, raising concerns about "on-target / off-tumor" side effects. For example, a fatal adverse event occurred after administering ERBB2 CAR T cells at high cell doses, and part of the cause is thought to be the low level 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 Literature 1)]. Therefore, the optimal solid tumor antigen target is one whose expression is limited to tumor cells or occurs only at extremely low levels in normal tissues that can be sacrificed.

[0006] MSLNs have emerged as an attractive target for cancer immunotherapy due to their low expression in normal mesothelial cells and high expression in all solid tumors. Immunotherapy targeting MSLNs reported to date has maintained a favorable safety profile. MSLNs are a potential CAR target in several common solid tumors, including at least esophageal cancer, breast cancer, gastric cancer, cholangiocarcinoma, 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 Literature 2)].

[0007] MSLN is a glycoprotein anchored to the cell membrane by a glycophosphatidylinositol (GPI) domain. MSLN is initially synthesized as a 69 kDa cell surface protein. After the amino terminus is cleaved by frin proteases, the 40 kDa C-terminal fragment remains attached to the membrane, and a soluble 32 kDa N-terminal fragment called megakaryocyte-enhancing 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 Literature 3)]. Soluble MSLN has also been detected in the serum of solid tumor patients and is called soluble MSLN-associated protein (SMRP). SMRP is produced by alternative splicing or by protein cleavage of mature MSLN induced by TNFα-converting enzyme ADAM17.

[0008] The biological function of MSLNs appears to be insignificant in normal tissues, as MSLN knockout mice exhibit normal development, reproduction, and blood cell counts. However, preclinical and clinical studies have increasingly shown that abnormal MSLN expression plays an active role in both tumor malignancy and high tumor grade by promoting cancer cell proliferation, involvement in local invasion and metastasis, and conferring resistance to cytotoxic-induced apoptosis. MSLNs can act bidirectionally, either by directly activating intracellular pathways via their GPI domain or by interacting with their receptor, CA125 / MUC16. Overexpression of MSLNs alone is sufficient to essentially activate the NFκB, MAPK, and PI3K intracellular pathways, promoting cell proliferation and resistance to apoptosis.

[0009] Physiologically, MSLN is expressed in mesothelial cells of the abdominal and thoracic cavities and the pericardium, and in very small amounts on the surface of epithelial cells of the trachea, ovaries, reticular retina, tonsils, and fallopian tubes. MSLN overexpression 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 is estimated to affect 340,000 patients annually and 2 million symptomatic individuals in the United States alone.

[0010] CARs generally consist of an external domain, hinge, transmembrane domain, and internal domain (typically including a CD3ζ-derived signaling domain and a costimulatory receptor) derived from a single-stranded 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, as well as reducing activation-induced cell death through the recruitment of PI3K, TRAF, and / or other pathways. Third-generation CARs typically contain three signaling domains, including a CD3ζ signaling domain and two costimulatory domains, e.g., CD28 and 4-1BB or CD28 and OX40. Compared to second-generation CARs, third-generation CARs exhibit inconsistent in vivo antitumor activity. Selecting the appropriate co-stimulatory domain is essential for maintaining CAR T cell activity and calibrating T cell persistence. However, the ideal co-stimulatory domain can be context-dependent, as CAR function depends on multiple external factors, such as antigen density, CAR stoichiometry, CAR affinity, and the immunological properties of the tumor microenvironment.

[0011] While the spatial distance between CARs and their target antigens may be equally important for the effective initiation of T cell signaling, 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 more proximal to the membrane than distally. For example, Hombach et al. demonstrated that CAR T cells recognizing the distal "N" epitope of carcinoembryonic antigen (CEA) were only moderately activated, but when recombinant CEA protein was produced and the N epitope was expressed proximal to the membrane, the same CAR T cells were activated more efficiently [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 Literature 4)]. This suggests that targeting a distal membrane epitope on tumor cells may allow large phosphatases such as CD45 and CD148 to enter the synapse, enabling inhibition of phosphorylation events initiated by CAR binding. Modifying the extracellular spacer sequence between the T cell membrane and 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 respect to MSLN CARs is interference from soluble MSLN, which in principle can occupy and block the scFv region. However, the activation of MSLN CAR T cells (cytokine secretion and cytotoxic activity) still appears to depend on MSLN expression on the cell surface [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 (Non-patent Literature 5)].

[0013] While the principle that genetically modified T cells expressing novel synthetic CARs can effectively treat advanced, resistant cancers has been established, many questions remain about realizing the full potential of this new therapeutic approach. To create safer and more effective CARs for the treatment of solid tumors, it is necessary to break away from conventional 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 CAR-target cell binding affinity, Kon / Koff ratio, and spatial constraints can influence the ability of CARs to optimally activate T cells to recognize tumors, especially 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 Literature 6)].

[0014] In light of the above, selecting scFvs solely based on their affinity for the MSLN antigen or their ability to induce T cell activation and proliferation in vitro does not appear sufficient to generate the most appropriate CAR T cells. Current data suggest that the optimal CAR affinity for individual target molecules cannot be deductively determined because there is currently no unbiased approach to identify the ideal affinity range that yields the best results in vivo [Srivastava, S. and Ridell, RS (2015) Engineering CAR-T Cells: Design Concepts. Trends Immunol. 36(8): 494-502 (Non-Patent Literature 7)].

[0015] In addition, the solid tumor microenvironment can impair MSLN CAR-T cells in several ways, limiting their antitumor efficacy. To optimize the efficiency of CAR T cells, several approaches are being evaluated to tame the host tumor microenvironment or to create "armed" CAR T cells that can overcome immune barriers. Such strategies include (i) promoting CAR T cell infiltration, (ii) enhancing the persistence of CAR T cell function, (iii) strengthening CAR T cells to overcome inhibitory signals encountered in the tumor microenvironment, and (iv) improving safety by preventing on-target / off-tumor toxicity. Of these approaches, combining specific CAR structures 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 Literature 8)] demonstrated, for example, that gene deletion of DGKζ significantly increases the antitumor activity of MSLN CAR T cells, as shown by in vitro secretion of effector cytokines, expression of FASL and TRAIL, and enhancement of cytotoxic function.

[0016] On the other hand, various strategies are being developed to improve the safety of CAR T cells by addressing the risks of on-target / off-tumor toxicity.

[0017] One such approach involves gene transfer of mRNA encoding an MSLN CAR, resulting in temporary expression of the CAR for a few days. Preclinical models have shown the potential of this approach, with multiple infusions of mRNA CAR T cells producing robust in vivo antitumor effects [Zhao Y, et al. (2010) Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. Cancer Res. 2070:9053-61 (Non-patent Literature 9)]. However, the temporary expression of the CAR may limit the long-term efficacy of the treatment. In a clinical trial conducted at the University of Pennsylvania, autologous T cells into which mRNA encoding a second-generation MSLN CAR (SS1-4-1BB CAR) was introduced via electroporation resulted in a moderate clinical response and a transient increase in serum inflammatory cytokines, such as IL12, IL6, G-CSF, MIP1β, MCP1, IL1RA, and RANTES.

[0018] Another approach to enhance T cell safety involves using suicide genes to eliminate T cells in the event of adverse events. In such situations, CAR T cells can be eliminated by drug-induced activation of suicide genes, such as herpesthymidine kinase (HSV-TK), gene-induced caspase-9, or the EGFRΔ gene.

[0019] In a previous patent application, WO2016120216 (Patent Document 1), the applicant developed a suicide gene alternative system that involves inserting an exogenous epitope into the CAR structure and making it recognizable by clinically approved antibodies such as rituximab, thereby allowing for the removal of some or all of the CAR-positive immune cells injected into the patient 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 within the cell. However, inserting such an epitope may affect the overall structure of the CAR, potentially altering how scFv interacts with its homologous antigens.

[0020] The objective of the present invention is to address some or all of the above limitations by providing safer, modified immuneCAR-positive cells that target MSLN-expressing cells, such as solid tumors, in vivo, with use in allogeneic therapeutic strategies in mind. [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] Pastan, I., Hassan, R. (2014) Discovery of mesothelin and exploiting it as a target for immunotherapy. Cancer. Res. 74:2907-12 [Non-Patent Document 4] 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 5] 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 [Non-Patent Document 6] D’Aloia, M.M., Zizzari, I.G., Sacchetti, B. et al. (2018) CAR-T cells: the long and winding road to solid tumors. Cell Death Dis 9:282 [Non-Patent Document 7] Srivastava, S. and Ridell, RS (2015) Engineering CAR-T Cells: Design Concepts. Trends Immunol. 36(8): 494-502 [Non-Patent Document 8] Riese MJ, et al. Enhanced effector responses in activated CD8+ T cells deficient in diacylglycerol kinases. Cancer Res. 73:3566-77 [Non-Patent Document 9] Zhao Y, et al. (2010) Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. Cancer Res. 2070:9053-61 [Overview of the Initiative]

[0023] This invention primarily relates to mesothelin-specific chimeric antigen receptors (CARs), which are expressed in immune cells, preferably T cells, for therapeutic purposes against cells or tissues expressing malignant mesothelin.

[0024] Such CARs are typically, • Extracellular ligand-binding domains containing VH and VL derived from monoclonal anti-mesothelin antibodies; • Transmembrane domain; and • Cytoplasmic domain containing the CD3 zeta signaling domain, and costimulatory domain This shows a structure that includes this.

[0025] The extracellular ligand-binding domain of the CAR of the present invention preferably comprises one or more CDRs containing an antibody-derived scFv segment called meso1, more specifically SEQ ID NO:3,4,5,6,7 and / or8 derived therefrom.

[0026] In a favorable scenario, the extracellular ligand-binding domain of the CAR is • Variable weight VH chains containing antibody meso1-derived CDRs having at least 90% identity with SEQ ID NO:3 (CDRH1-Meso1), SEQ ID NO:4 (CDRH2-meso1), and / or SEQ ID NO:5 (CDRH3-meso1), respectively, and Variable weight VL chains containing antibody Meso1-derived CDRs having at least 90% identity with SEQ ID NO:6 (CDRL1-meso1), SEQ ID NO:7 (CDRL2-meso1), and / or SEQ ID NO:8 (CDRL3-meso1), respectively. Includes.

[0027] The anti-mesothelin CARs of the present invention form exogenous polypeptide sequences, which are expressed by immune cells and exposed to the cell surface. These are encoded by exogenous polynucleotide sequences (relative to the immune cell's original genome) and are preferably inserted into specific genomic loci, such as the TCR, B2m, and PD1 gene loci, using low-frequency cleavage endonucleases.

[0028] In some embodiments, the CAR further comprises an additional exogenous polypeptide sequence containing an epitope, which can be targeted to a clinically approved ligand and removed in vivo, or targeted to another ligand and detected or purified in vivo or in vitro. Such additional exogenous polypeptide segments can be specifically recognized by a rituximab, such as one referred to herein as "R2".

[0029] More specifically, the present invention relates to immune cells or a population of immune cells transformed with an anti-mesothelin CAR polynucleotide sequence, wherein the immune cells or population of immune cells 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 may be further genetically modified, mutated, or gene-edited to improve therapeutic suitability or efficacy, for example, to improve persistence or lifespan. In a preferred aspect, the modified immune cells of the present invention combine the expression of an anti-mesothelin CAR sequence with other genetic modifications that reduce the expression of its endogenous genes, such as the TCR, HLA, and / or B2m gene.

[0031] In a further favorable scenario, modified 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 favorable scenario, modified immune cells may be mutated to improve CAR-dependent immune activation, specifically by reducing or suppressing the TGF-beta signaling pathway.

[0033] In other favorable scenarios, further exogenous genes, specifically TGF beta receptor inhibitors or decoys, such as the sequence of a dominant-negative TGF beta receptor (dnTGFβRII), may be inserted, co-generated, or co-expressed together with the anti-mesothelin CAR of the present invention.

[0034] Further examples of exogenous gene sequences are provided, and their expression can be combined with the expression of anti-mesothelin CARs 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 cause hypersensitivity of the immune cells to drugs such as cyclophosphamide and / or isophosphamide; • Drug-inducing agents that induce drug resistance: dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin, or methylguanine transferase (MGMT), mTORmut, or Lckmut; Chemokines or cytokines such as IL-2, IL-12, and IL-15; • Inhibitors of tumor-associated macrophage (TAM) secretion, such as CCR2 / CCL2 antagonists, that enhance the therapeutic activity of immune cells. That is the case.

[0035] The modified immune cells of the present invention are particularly suitable for treating mesothelin-expressing cells, specifically solid tumors, such as those typically characterized by esophageal cancer, breast cancer, gastric cancer, cholangiocarcinoma, 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 containing such cells, therapeutic compositions containing the same, and therapeutic methods that enable addressing pathologies induced in mesothelin-expressing cells. [Invention 1001] • Extracellular ligand-binding domains including VH and VL derived from monoclonal anti-mesothelin antibodies, • Transmembrane domain, and • Cytoplasmic domain containing the CD3 zeta signaling domain, and costimulatory domain A mesothelin-specific chimeric antigen receptor (CAR) comprising at least the following: The mesothelin-specific chimeric antigen receptor (CAR) wherein the extracellular ligand-binding domain is directed toward the MSLN antigen polypeptide region SEQ ID NO:25. [Invention 1002] The extracellular ligand-binding domain, • Variable weight VH chains containing antibody Meso1-derived CDRs having at least 90% identity with SEQ ID NO:3 (CDRH1-Meso1), SEQ ID NO:4 (CDRH2-Meso1), and SEQ ID NO:5 (CDRH3-Meso1), respectively, and Variable weight VL chains containing antibody Meso1-derived CDRs, each having at least 90% identity with SEQ ID NO:6 (CDRL1-Meso1), SEQ ID NO:7 (CDRL2-Meso1), and SEQ ID NO:8 (CDRL3-Meso1). The present invention 1001 includes a mesothelin-specific chimeric antigen receptor (CAR). [Invention 1003] The mesothelin-specific chimeric antigen receptor of the present invention 1001, 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. [Invention 1004] A mesothelin-specific chimeric antigen receptor (CAR) according to any of the present invention 1001 to 1003, wherein the transmembrane domain is derived from the transmembrane region of the alpha, beta, or zeta chain of the 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. [Invention 1005] The mesothelin-specific chimeric antigen receptor (CAR) of the present invention 1004, 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. [Invention 1006] A mesothelin-specific chimeric antigen receptor (CAR) according to any one of the present invention 1001 to 1005, further comprising a hinge between the extracellular ligand-binding domain and the transmembrane domain. [Invention 1007] The mesothelin-specific chimeric antigen receptor (CAR) of the present invention 1006, wherein the hinge is selected from the CD8α hinge, the IgG1 hinge, and the FcγRIIIα hinge. [Invention 1008] The mesothelin-specific chimeric antigen receptor (CAR) of the present invention 1007, 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). [Invention 1009] The aforementioned CAR A polypeptide structure comprising a CD8α hinge having at least 80% identity with the amino acid sequence shown in SEQ ID NO:16, and a CD8α transmembrane domain having at least 80% identity with the amino acid sequence shown in SEQ ID NO:17. A mesothelin-specific CAR having any of the present invention 1001 to 1008. [Invention 1010] A mesothelin-specific CAR according to any one of the invention 1001 to 1009 further comprises a safety switch containing an epitope selected from Table 5. [Invention 1011] The safety switch is a mesothelin-specific CAR of the present invention 1010, comprising the epitope CPYSNPSLC (SEQ ID NO:26) to which rituximab specifically binds. [Invention 1012] A mesothelin-specific CAR of the present invention 1010 or 1011, comprising a safety switch R2 having at least 90% identity with SEQ ID NO:15. [Invention 1013] A mesothelin-specific chimeric antigen receptor according to any of the present invention 1001 to 1012, comprising a costimulatory domain derived from 4-1BB or CD28. [Invention 1014] The aforementioned co-stimulatory domain is derived from 4-1BB and / or has at least 80% identity with SEQ ID NO:18, the mesothelin-specific CAR of the present invention 1013. [Invention 1015] The CD3 zeta signaling domain has at least 80% identity with SEQ ID NO:19, and is a mesothelin-specific CAR according to any of the present invention 1001 to 1014. [Invention 1016] A mesothelin-specific CAR according to any of invention 1001 to 1015, further comprising a signal peptide. [Invention 1017] A single-chain polypeptide, a mesothelin-specific chimeric antigen receptor (CAR) according to any of the invention's 1001-1016. [Invention 1018] A mesothelin-specific chimeric antigen receptor (CAR) of the present invention 1017, 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 (Meso1 CAR) or SEQ ID NO:22 (Meso1-R2 CAR). [Invention 1019] A polynucleotide encoding any of the chimeric antigen receptors described in invention 1001 to 1018. [Invention 1020] An expression vector containing the polynucleotide of the present invention 1019. [Invention 1021] Modified immune cells comprising the polynucleotide of Invention 1019 or the expression vector of Invention 1020. [Invention 1022] Modified immune cells expressing any of the mesothelin-specific chimeric antigen receptors described in invention 1001 to 1018 on their cell surface membrane. [Invention 1023] Modified immune cells according to invention 1021 or 1022, which are T lymphocytes. [Invention 1024] Modified immune cells according to Invention 1023, derived from primary cells or differentiated from stem cells such as iPS cells. [Invention 1025] Modified immune cells according to invention 1023 or 1024, derived from inflammatory T lymphocytes, cytotoxic T lymphocytes, or helper T lymphocytes. [Invention 1026] Modified immune cells according to any of items 1021 to 1025 of the present invention, wherein the expression of TCRs in the immune cells is reduced or suppressed. [Invention 1027] Modified immune cells according to the present invention 1026, wherein at least one gene encoding TCR alpha or TCR beta is inactivated in the aforementioned cells. [Invention 1028] Modified immune cells according to the present invention 1027, wherein at least one gene encoding TCR alpha or TCR beta is cleaved by a low-frequency cleavage endonuclease. [Invention 1029] Modified immune cells according to invention 1027 or 1028, wherein the polynucleotide encoding the mesothelin-specific CAR is incorporated into an endogenous locus, preferably a TCR alpha or TCR beta locus, under transcriptional control of an endogenous promoter. [Invention 1030] Modified immune cells according to Invention 1029, originating from a donor for allogeneic transplantation. [Invention 1031] Modified immune cells according to any of the inventions 1021-1030, which have been mutated to confer resistance to at least one immunosuppressant, such as an anti-CD52 antibody. [Invention 1032] Modified immune cells of any of the invention 1021-1031, further mutated to confer resistance to at least one chemotherapy drug, specifically a purine analog drug. [Invention 1033] Modified immune cells according to any of the invention 1021-1032, which are mutated in the patient's body, specifically in genes encoding MHCI components such as HLA or B2m, to improve their persistence or lifespan. [Invention 1034] Modified immune cells according to any of the inventions 1021-1033, which are mutated to improve CAR-dependent immune activation, specifically to reduce or suppress the expression of immune checkpoint proteins and / or their receptors. [Invention 1035] A modified immune cell according to any of items 1021 to 1034 of the present invention, wherein the mesothelin-specific chimeric antigen receptor (CAR) is co-expressed in the cell with another exogenous gene sequence encoding a TGF beta receptor inhibitor or decoy. [Invention 1036] Modified immune cells of the present invention 1035, wherein the decoy of the TGF beta receptor 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. [Invention 1037] An exogenous polynucleotide comprising a first polynucleotide sequence encoding the mesothelin-specific CAR, a second polynucleotide encoding the 2A self-cleaving peptide, and a third polynucleotide encoding the dominant-negative TGF beta receptor. Modified immune cells according to Invention 1036, including the modified immune cells of Invention 1036. [Invention 1038] Modified immune cells according to any of items 1021 to 1037 of the present invention, wherein the expression of at least one TGF beta receptor gene is reduced or inactivated. [Invention 1039] A modified immune cell according to the present invention 1038, wherein the TGF beta receptor gene is TGFβRII. [Invention 1040] The aforementioned 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 cause hypersensitivity of the immune cells to drugs such as cyclophosphamide and / or isophosphamide; • Drug-inducing agents that induce drug resistance: dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin, or methylguanine transferase (MGMT), mTORmut, or Lckmut; Chemokines or cytokines such as IL-2, IL-12, and IL-15; Chemokine receptors such as CCR2, CXCR2, or CXCR4; • Inhibitors of tumor-associated macrophages (TAMs), such as CCR2 / CCL2 antagonists, that enhance the therapeutic activity of the aforementioned immune cells. A modified immune cell according to any of items 1021 to 1039 of the present invention, which is co-expressed in the cell with another exogenous gene sequence selected from those encoding the above. [Invention 1041] Modified immune cells according to any of Invention 1021-1040 for use in therapy. [Invention 1042] Modified immune cells according to any of invention 1021 to 1041 for use as a drug for cancer treatment. [Invention 1043] Modified immune cells according to any of invention 1021-1042 for use in the therapy of pre-malignant or malignant cancer conditions characterized by mesothelin-expressing cells. [Invention 1044] Modified immune cells of any of invention 1021-1043 for use in the therapy of cancer conditions 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. [Invention 1045] A method for treating a patient having a condition characterized by mesothelin-expressing cells, - A step of modifying donor-derived immune cells to express any of the functional mesothelin-specific chimeric antigen receptors (CARs) described in items 1001 to 1020 of the present invention; • The process of administering the CAR-positive modified immune cells to the patient to remove mesothelin-expressing cells. The method, including the method described above. [Invention 1046] A method for treating a patient according to the present invention 1045, comprising an additional treatment step in which the patient undergoes lymphocyte removal. [Invention 1047] A method for treating a patient of the present invention 1046, wherein the CAR-positive modified immune cells that remove mesothelin-expressing cells are mutated to give resistance to lymphocyte depletion therapy. [Invention 1048] A method for treating a patient according to the present invention 1047, comprising mutating the CD52 gene in the CAR-positive modified immune cells that remove mesothelin-expressing cells. [Brief explanation of the drawing]

[0037] [Figure 1]Structural diagram of a preferred version of the anti-mesothelin CAR of the present invention. A: CAR comprising: V1 and V2 representing sequences containing ScFv that specifically bind to mesothelin, e.g., VH and VL or VL and VH derived from a meso1 antibody; L: linker; R1 and R2 representing exogenous epitopes such as CD20 epitope recognition recognized by a human approved monoclonal anti-CD20 antibody (e.g., rituximab); TM: transmembrane domain; CO-STIM: costimulatory domain; ITAM: stimulatory domain containing ITAM (immunosensory activating tyrosine motif (ITAM)). Such CARs typically have at least 80% polypeptide sequence identity with SEQ ID NO: 21. B: CAR without exogenous epitopes comprising: VH and VL or VL and; (G4S)3 linker; CD8α hinge domain; CD8α transmembrane domain; 4-1BB costimulatory domain; and CD3z signaling domain. [Figure 2] This is a schematic diagram showing anti-MSLN CAR-expressing immune cells of the present invention, with further introduction of optionally selected genetic characteristics. A. Co-expression of an inactive variant of the TGFβ receptor (e.g., dnTGFβRII) and / or reduction or inactivation of TGFβ receptor expression to counteract tumor immunosuppression. Reduction or inactivation of TCR (e.g., TCR alpha) expression to reduce immune T cell alloreactivity that causes GvHD. B. Reduction or inactivation of TGFβ receptor, TCR and / or CD52 expression using gene editing tools (e.g., TALEN). [Figure 3] Mesothelin protein expression on the surface of 293H, A2058, HeLa, and HPAC cells. MSLN expression was analyzed by flow cytometry using a mouse monoclonal anti-human MSLN antibody as the primary antibody and a goat anti-mouse polyclonal antibody conjugated with APC as the secondary antibody. [Figure 4] Quantitative mesothelin protein expression on the surface of HeLa and HPAC cells. MSLN expression levels were analyzed by flow cytometry using fluorescence-based QIFIKIT. [Figure 5]This figure illustrates a serial killing assay performed to evaluate in vitro activation of anti-mesothelin CAR-positive cells. [Figure 6] Surface CAR expression of primary [TCRalpha]neg T cells (UCART cells). Cryopreserved UCART cells generated from a single donor were stained with an anti-histidine antibody conjugated with histidine-tagged recombinant human mesothelin protein and PE, or with streptavidin conjugated with biotinylated protein L and Vioblue, and analyzed by flow cytometry. [Figure 7] CD4 and CD8 expression in CAR+ fractions of UCART cells. Cryopreserved UCART cells from a single donor were stained with anti-CD4 antibody conjugated with FITC and anti-CD8 antibody conjugated with BV510, and analyzed by flow cytometry. [Figure 8] IFNg production by UCART cells. Fresh UCART cells, generated from a single donor, were co-cultured for 24 hours with (A) HPAC (MSLN+) cells, (B) A2058 (MSLN-) cells, and (C) 293H (MSLN-) cells. IFNg produced in the culture supernatant was quantified by ELISA. [Figure 9] This graph shows the cell lysis percentage results of a serial HPAC cell killing assay using primary [TCRalpha]neg T cells (UCART cells), and the assay protocol is illustrated in Figure 5. Cryopreserved UCART cells generated from a single donor were co-cultured with HPAC cells for 15 days at an E:T ratio of (A) 1:2 or (B) 1:8. [Figure 10] TCRαβ expression on the surface of UCART cells. Cryopreserved UCART cells prepared from a single donor were stained with an 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 culture 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). [Figure 13] Measurement of UCART cell removal by rituximab-mediated CDC using exogenous epitope polypeptide R2 contained in P4-R2 CAR, Meso1-R2 CAR, and MESO2-R2 CAR. [Figure 14-1] A graph showing the measurement of SMAD2-3 phosphorylation after TGFβ exposure in CART cells generated from two different donors. [Figure 14-2] A continuation of Figure 14-1. [Figure 15] Mean tumor volume (HPAC MSLN+ cells) of 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 of 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 of 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 in mice injected with three doses of UCARTmeso cells (A: 1x10⁶, B: 3x10⁶, and C: 10x10⁶ CAR+ cells / mouse). Comparison of different doses for Meso1-R2, P4-R2, and MESO2-R2. [Figure 19]Mean tumor volume of mice injected with two doses of UCARTmeso cells (3x10⁶ and 10x10⁶ CAR+ cells / mouse) that also express dnTGFBRII. [Figure 20] A. Comparison of CAR and dnTGFBRII detection in UCARTMeso cells expressing P4 or MESO1 constructs containing dnTGFBRII. B. Percentages of CD4+ and CD8+ in the CAR-positive fraction of UCARTMeso cells produced in Example 5. [Figure 21] Percentages of Temra, Tem, and 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 inactivated or not inactivated by knockout (KO) or dominant-negative TGFBRII (dnTGFBRII) expression. [Figure 23] IFNg production by UCARTMeso cells produced in Example 5, either (A) exposed to recombinant mesothelin protein or (B) not exposed. [Figure 24] Evaluation of UCARTmeso cell sensitivity 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 proliferation inhibition of different UCARTmeso in the presence of TGFb and recombinant mesothelin protein. [Modes for carrying out the invention]

[0038] Table 1: Amino acid sequences of the different domains constituting P4, Meso1, and MESO2 scFv of the CAR described in the examples. Table 2: Amino acid sequences of different domains other than scFv that constitute the MSLN CAR of the present invention. Table 3: Examples of mAb-specific epitopes (and their corresponding mAbs) that may be used in the extracellular binding domain of the CAR of the present invention for the segregation 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 the CAR of the present invention. Table 6: TALE nuclease target sequences related to the TGFβRII gene. Table 7: CRISPR target sequences related to the TGFβRII gene. Table 8: Genomic sequences targeted by TALE nucleases (TALENs) to inactivate TCR and CD52. Table 9: Characteristics of the modified T cell population used in the examples. Table 10: Description of the six types of genetically modified T cells prepared for the study provided in Example 5.

[0039] Detailed description of the invention Unless otherwise defined herein, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the fields of gene therapy, biochemistry, genetics, and molecular biology.

[0040] Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described herein. All publications, patent applications, patents, and other references referred herein are incorporated herein by reference in their entirety. In case of any conflict, including definitions, this specification shall prevail. Furthermore, the materials, methods, and examples are illustrative and not intended to limit unless otherwise noted.

[0041] In carrying out this invention, unless otherwise specified, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology will be used, which are within the scope of the skills of the art. Such techniques are well described 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 The 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.See also: 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 (DM 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 for treating solid tumors using adoptive immune cells directed to a specific epitope region of mesothelin across the polypeptide sequence SEQ ID NO:25 of the transmembrane protein MSLN, and more specifically, to allogeneic CAR-T cells that have been shown to be particularly efficient and directed to this epitope.

[0043] This specification describes a method for producing modified immune cells directed to a specific polypeptide region of human mesothelin (MSLN_human, referred to as Q13421 in the Uniprot database), more specifically, to a mesothelin represented by SEQ ID NO:25, which is presented on the surface of malignant cells. As shown in the experimental section of this specification, highly efficient CAR T cells were produced by directing CARs to this antigen region, which includes or consists of SEQ ID NO:25, specifically by using scFv of antibody Meso1 containing SEQ ID NO:9 and SEQ ID NO:10.

[0044] The modified immune cells obtained in this invention are generally NK cells or T cells equipped with CARs containing SEQ ID NO:9 and / or SEQ ID NO:10, and exhibit superior activation, potency, killing activity, cytokine release, and in vivo persistence compared to their equivalents with other conventional anti-mesothelin CARs.

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

[0046] Design of MSLN-CAR expressed in immune cells : " Chimeric antigen receptor A recombinant receptor (CAR) is a single fusion molecule containing a target-directed moiety bound to one or more signaling domains. Generally, the binding moiety of a CAR consists of the antigen-binding domain of a single-chain antibody (scFv) and includes lightly variable and heavily variable fragments of a monoclonal antibody linked by a flexible linker. Receptor-based binding moieties or ligand domains have also been successfully utilized. The signaling domains of CARs are generally derived from the cytoplasmic region of CD3 zeta or the gamma chain of an Fc receptor, and are commonly combined with signaling domains derived from co-stimulatory molecules such as CD28, OX-40 (CD134), ICOS, and 4-1BB (CD137) to enhance cell survival and proliferation. CARs are generally expressed in effector immune cells, redirecting 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 the CAR, encoded by an exogenous polynucleotide sequence introduced into the cell. For example, this component may be useful for interaction with target antigens, intracellular stability, or localization of CARs.

[0047] Generally, such CARs are • Extracellular ligand-binding domains containing VH and VL derived from monoclonal anti-mesothelin antibodies; • Transmembrane domain; and • A signaling domain, preferably a cytoplasmic domain including a CD3 zeta signaling domain, and a costimulatory domain. Includes.

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

[0049] In a preferred scenario, the mesothelin-specific chimeric antigen receptor (CAR) of the present invention has an extracellular ligand-binding domain comprising at least one CDR region derived from the variable weight VH chain of an antibody Meso1 selected from CDRH1-Meso1 (identical to SEQ ID NO:3), CDRH2-Meso1 (identical to SEQ ID NO:4), and CDRH3-Meso1 (identical to SEQ ID NO:5), and / or a CDR region derived from the variable weight VL chain of the antibody selected from CDRL1-Meso1 (identical to SEQ ID NO:6), CDRL2-Meso1 (identical to SEQ ID NO:7), and CDRL3-Meso1 (identical to SEQ ID NO:8).

[0050] Generally, the extracellular ligand-binding domain is • Variable weight VH chains containing antibody Meso1-derived CDRs having at least 90% identity with SEQ ID NO:3 (CDRH1-Meso1), SEQ ID NO:4 (CDRH2-Meso1), and SEQ ID NO:5 (CDRH3-Meso1), and / or Variable weight VL chains containing antibody Meso1-derived CDRs, each having at least 90% identity with SEQ ID NO:6 (CDRL1-Meso1), SEQ ID NO:7 (CDRL2-Meso1), and SEQ ID NO:8 (CDRL3-Meso1). Includes.

[0051] In a preferred embodiment of the present invention, the mesothelin-specific chimeric antigen receptor has an extracellular ligand-binding domain, which includes 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 (Meso1-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 (Meso1-VL). Generally, antigen binding can be modified, for example, by substituting framework residues within the framework region with corresponding residues derived from a CDR donor antibody. These framework substitutions can be identified by methods well known in the art, such as modeling the interaction between CDRs and framework residues, thereby identifying framework residues that are important for antigen binding and for sequence comparison to identify exceptional framework residues at specific locations [see, for example, Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., (1988) Nature, 332:323. These are incorporated herein by reference].

[0052] Various aspects of the present invention are provided through the various features provided in the claims, in view of the ordinary practices and knowledge of those skilled in the art. Details of the sequences included 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 constituting the scFv of P4, Meso1, and MESO2 TIFF0007830327000001.tif224163

[0054] (Table 2) Amino acid sequences of different domains other than scFv that constitute the MSLN CAR of the present invention TIFF0007830327000002.tif89162

[0055] (Table 3) Amino acid sequences of P4-R2, Meso1-R2, Meso1, and MESO2-R2 CARs TIFF0007830327000003.tif81161

[0056] (Table 4) Complete polypeptide sequences of MSLN CAR, dnTGFβRII, and MSLN epitope region TIFF0007830327000004.tif227158TIFF0007830327000005.tif111158

[0057] The signaling domain or intracellular signaling domain of the CAR of the present invention is responsible for intracellular signaling after the extracellular ligand-binding domain has bound to a target, resulting in the activation of immune cells and immune responses. In other words, the signaling domain is responsible for the activation of at least one of the normal effector functions of the immune cells on which the CAR expresses. For example, the effector function of a T cell may include cytokine secretion, cytolytic activity, or helper activity. Therefore, the term "signaling domain" refers to a portion of a protein that transmits effector signaling function signals to cause cells to perform specific functions.

[0058] Preferred examples of signaling domains used in CARs may be cytoplasmic sequences of T cell receptors and co-receptors that, after binding to antigen receptors, act in coordination to initiate signaling, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capabilities. The signaling domain comprises two distinct classes of cytoplasmic signaling sequences, which are sequences that initiate antigen-dependent primary activation and sequences that act antigen-independently to provide secondary or co-stimulatory signals. The primary cytoplasmic signaling sequence may comprise a signaling motif known as an immunoreceptor-activated tyrosine motif or (of) ITAM. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that serve as binding sites for syk / zap70 class tyrosine kinases. Non-specific 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 a preferred embodiment, the signaling domain of CAR may include a CD3 zeta signaling domain which has an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity with 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 includes a costimulatory signaling molecule. The costimulatory molecule is a cell surface molecule that is not an antigen receptor, or a ligand thereof, which is necessary for a highly efficient immune response. A "costimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a related costimulatory molecule on a T cell, thereby providing a signal that mediates T cell responses, such as 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. Co-stimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intracellular adhesion molecules (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, agonists that bind to Toll ligand receptors or Examples include antibodies and ligands that specifically bind to B7-H3. Costimulatory ligands also include, among others, ligands that specifically bind to antibodies that specifically bind to costimulatory molecules present on T cells, such as, but are 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 CD83.

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

[0061] The CARs of the present invention are generally expressed on the membrane surface of cells. Therefore, such CARs further include a transmembrane domain. A distinguishing feature of a suitable transmembrane domain is its ability to be expressed on the surface of cells, preferably immune cells in this invention, specifically lymphocytes or natural killer (NK) cells, and to interact with them to direct the cellular response of the immune cells toward a predetermined target cell. The transmembrane domain may originate from either a natural or synthetic source. The transmembrane domain may originate from any membrane-bound or transmembrane protein. Non-existent examples include transmembrane polypeptides such as α, β, γ, or ζ subunits of T cell receptors, polypeptides constituting the CD3 complex, IL2 receptor p55 (α chain), p75 (β chain), or γ chain, Fc receptor subunit chains, specifically Fcγ receptor III, or CD proteins. Alternatively, the transmembrane domain may be synthetic and may primarily contain hydrophobic residues such as leucine and valine. In a preferred embodiment, the transmembrane domain is derived from a human CD8 alpha chain (e.g., NP_001139345.1). The transmembrane domain may further include 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 the extracellular ligand-binding domain with further flexibility and accessibility. The hinge region may contain 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 native molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or from all or part of the antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a native hinge sequence, or it may be a synthetic hinge sequence entirely.In a preferred embodiment, the hinge domain comprises a portion of a human CD8 alpha chain, an FcγRIIIα receptor, or IgG1, or a hinge polypeptide exhibiting at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity with these polypeptides.

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

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

[0064] In a further embodiment, the mesothelin-specific CAR of the present invention includes a safety switch convenient for fractionating, purifying, and / or removing modified immune cells. The method of the present invention is designed to be carried out ex vivo, but removal can be carried out in vivo to control the proliferation of immune cells in the patient's body and potentially halt the therapeutic effect by using antibodies approved by regulatory authorities for therapeutic use in humans. Examples of mAb-specific epitopes (and their corresponding mAbs) that can be incorporated into the extracellular binding domain of the CAR of the present 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 present invention TIFF0007830327000006.tif111139

[0066] Therefore, the mesotheline-specific CAR of the present invention preferably includes a safety switch containing at least one exogenous mAb epitope listed in Table 5. Preferably, the mesotheline-specific CAR of the present invention preferably includes a safety switch containing the epitope CPYSNPSLC (SEQ ID NO: 26) to which rituximab specifically binds. More preferably, the mesotheline-specific CAR includes a safety switch called "R2" having at least 90% identity with SEQ ID NO: 15.

[0067] The mesothelin-specific CARs of the present invention generally also include signal peptides that help them to be expressed on the surface of modified cells. Chimeric antigen receptors (CARs) generally form single-chain polypeptides, but they can also be produced in a multi-chain form, such as WO2014039523.

[0068] As illustrated in the examples, preferred CARs of the present invention are MSLN-CAR-Meso1-R2 or MSLN-CAR-Meso1, 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(Meso1-R2) or SEQ ID NO:22(Meso1), respectively.

[0069] A preferred polypeptide structure of the MSLN-CAR of the present invention is illustrated in Figure 1.

[0070] More generally, the CARs of the present invention are produced by assembling various polynucleotide sequences encoding sequential fragments of CAR polypeptides within a vector, and then introducing and expressing them in immune cells, as described in the art or 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 polynucleotides and vectors, as well as any intermediate steps involved in the process of producing immune cells as referred to herein.

[0072] A "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. In this invention, "vectors" include, but are not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules that may consist of chromosomes, non-chromosomes, semi-synthetic, or synthetic nucleic acids. Preferred vectors are self-replicating vectors (episome vectors) and / or vectors capable of expressing nucleic acids to which they are ligated (expression vectors). Numerous suitable vectors are known to those skilled in the art and are commercially available. Examples of viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated virus (AAV)), coronaviruses, negative-strand RNA viruses (e.g., orthomyxovirus (e.g., influenza virus)), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses (e.g., picornavirus and alphavirus), as well as double-strand DNA viruses (e.g., adenovirus, herpesvirus (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus)), and poxviruses (e.g., vaccinia, fowlpox, and canary poxvirus). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukemia sarcoma virus, mammalian type C, B, and D viruses, HTLV-BLV group, lentivirus, and spumavirus (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0073] Specifically, the present invention provides, in particular, expression vectors in the form of lentiviral vectors or AAV vectors comprising a polynucleotide sequence encoding a CAR as described herein.

[0074] Such lentiviral vectors may contain a polynucleotide sequence encoding the CAR of the present invention, functionally ligated to a promoter (such as a splenic focus-forming virus promoter (SFFV)). "Functionally ligated" means that the components described are juxtaposed in a relationship that allows them to function as intended. When a gene (such as a polynucleotide sequence encoding the CAR) is "functionally ligated" to a promoter, and its transcription is under the control of the promoter, the product encoded by the gene is produced as a result of this transcription.

[0075] The lentiviral vector of the present invention typically includes regulatory elements such as 5' and 3' terminal repeat sequences (LTRs), but may also include other structural and functional genetic elements originally derived from lentiviruses. Such structural and functional genetic elements are well known in the art. The lentiviral vector may include, for example, the genes gag, pol, and env. However, preferably, the lentiviral vector of the present invention does not include the genes gag, pol, and env. As further regulatory elements, the lentiviral vector may include one or more (e.g., two or more) packaging signals (e.g., packaging signal ψ), primer binding sites, transactivation response regions (TARs), and rev response regions (RREs).

[0076] Typically, the 5' and 3' end repeat sequences (LTRs) on both ends of the lentiviral genome possess promoter / enhancer activity and are essential for the correct expression of the full-length lentiviral vector transcript. LTRs usually contain a repeat sequence U3RU5 present at both the 5' and 3' ends of a double-stranded DNA molecule, which is a combination of a 5'R-U5 segment and a 3'U3-R segment of single-stranded RNA, where the repeat R occurs at both ends of the RNA, while 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. Lentiviral vectors can be made safer by removing the U3 sequence, resulting in a "self-activating" vector where the viral promoter and enhancer sequences originally present in the LTR are completely absent. Therefore, the vector can infect and integrate into the host genome only once, but not again, thus increasing its safety 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 includes a 3'LTR, where the 3'LTR enhancer-promoter sequence (i.e., U3 sequence) is modified (e.g., deleted).

[0078] In some embodiments, the lentiviral vector has the following elements in the order from 5' to 3': • 5' terminal repeat sequence (5'LTR); • Promoter (e.g., EF1-alpha promoter); • A polynucleotide sequence encoding the chimeric antigen receptor of the present invention; and / or • 3'-terminal repeat sequence (3'LTR), preferably 3'-autoinactivated LTR Contains a polynucleotide sequence that includes one or more of the following.

[0079] In certain embodiments, lentiviral vectors have the following elements arranged in the order from 5' to 3': • 5' terminal repeat sequence (5'LTR); • Promoter (e.g., EF1-alpha promoter); The present invention may include safety switches such as R2, • A polynucleotide sequence encoding the 2A peptide; • One or any additional polypeptide to be co-expressed with the CAR, e.g., a polynucleotide sequence encoding dnTGFβR; ; and / or • 3'-terminal repeat sequence (3'LTR), preferably 3'-autoinactivated LTR It may further include a polynucleotide sequence containing at least one of the following:

[0080] Alternatively, lentiviral vectors have the following elements in the order from 5' to 3': • 5' terminal repeat sequence (5'LTR); • Promoter (e.g., EF1-alpha promoter); • One or any additional polypeptide to be co-expressed with the CAR, e.g., a polynucleotide sequence encoding dnTGFβR; • A polynucleotide sequence encoding the 2A peptide; The present invention may include safety switches such as R2, ; and / or • 3'-terminal repeat sequence (3'LTR), preferably 3'-autoinactivated LTR It may contain at least one of the following.

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

[0082] AAV vectors, particularly those derived 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 especially convenient for introducing the MSLN-CAR of the present invention into the genome via site-specific homologous recombination. Generally, site-specific homologous recombination is induced into immune cells by the expression of low-frequency cleavage endonucleases such as TALENs, as has already been taught for other CARs treating hematological cancers in EP3276000 and WO2018073391. Site-specific incorporation of CARs can offer several advantages, including more stable incorporation, incorporation that places the transgene under transcriptional control of an endogenous promoter at a selected locus, and incorporation that can inactivate the endogenous locus. These latter aspects will be discussed in detail in the following sections on genomic engineering of therapeutic immune cells.

[0083] One object of the present invention is to provide an AAV vector comprising a polynucleotide sequence encoding MSLN-CAR as described above, and another sequence encoding a cis-regulatory element (e.g., a 2A peptide cleavage site) or an intra-sequence ribosome entry site (IRES), which optionally enables the co-expression of a third sequence encoding a product that improves the therapeutic efficacy of modified immune cells. An example of dnTGFβRII overexpression is given herein, which has been shown to reduce SMAD2-3 phosphorylation, thereby reducing TGFβ-induced cell removal in the tumor environment.

[0084] " therapeutic propertiesThe term "」 encompasses various strategies by which such cells can be improved from the perspective of their use in therapeutic treatment. This means conferring therapeutically advantageous merits (i.e., therapeutic efficacy) to the cells by genetic engineering, or promoting their use or production. For example, genetic engineering can result in effector cells with better survival rates, faster growth, shorter cell cycles, improved immune activity, more functional, more differentiated, more specific to target cells, more sensitive or resistant to drugs, and more sensitive to glucose deprivation, oxygen, or amino acid deprivation (i.e., more adaptable to the tumor microenvironment). Progenitor cells can be more productive, more acceptable to the recipient patient, and more likely to produce cells that differentiate into the desired effector cells. These examples of "therapeutic characteristics" are presented as non-limiting examples.

[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 in the immune response. Activated T cells include cytotoxic T cells and helper T cells, which are preferred effector cells for executing the cellular response. The category of effector T cells is broad and includes various types of T cells that respond actively to stimuli such as co-stimulation. This includes helper, killer, regulatory, and other potential types of T cells.

[0087] The term "immune cells" typically refers to hematopoietic-derived cells that are functionally involved in the initiation of natural and / or adaptive immune responses, such as CD3 or CD4-positive cells. The immune cells of the present invention can be T cells selected from the group consisting of dendritic cells, killer dendritic cells, mast cells, NK cells, B cells, or inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes.

[0088] "Primary cells" refer to cells that are directly collected from living tissue (e.g., biopsy material), grown in vitro for a limited time, and then established as a cell line so that the population can double a limited number of times. Primary cells are the opposite of persistently tumorigenic or artificially immortalized cell lines. Non-resolved 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, but are not limited to, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from inflammatory sites, ascites, pleural fluid, splenic tissue, and tumor-infiltrating lymphocytes from tumors. In some embodiments, the immune cells may originate from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with an infection. In other embodiments, the cells are part of a mixed population of immune cells exhibiting different phenotypic features, including CD4, CD8, and CD56-positive cells. Primary immune cells are provided from a donor or patient by various methods known in the art, such as leukocyte removal, 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 those 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 in this 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 induced from human embryonic stem cells by a technique well known in the art that does 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 genetic material into cells, modifying them, and / or removing them from cells. gene editing Genetic engineering refers to 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] "Sequence-specific reagent" means any active molecule that has the ability to specifically recognize a selected polynucleotide sequence called a "target sequence," which is generally at least 9 bp, more preferably at least 10 bp, and even more preferably at least 12 pb in length, at a given genomic locus, in view of modifying the expression of that locus. The expression can be modified by mutation, deletion, or insertion in an encoding or regulatory polynucleotide sequence, by epigenetic changes such as methylation or histone modification, or by interference with the transcription level through interaction with transcription factors or polymerases.

[0094] Examples of sequence-specific reagents include endonucleases, RNA guides, RNAi, methylases, exonucleases, histone deacetylases, terminal processing enzymes such as exonucleases, more specifically cytidine deaminases, which bind to systems like CRISPR / Cas9 to perform base editing (i.e., nucleotide substitution), but without necessarily relying on nuclease-mediated cleavage, as described, for example, by 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 present invention, the sequence-specific reagent is preferably a sequence-specific nuclease reagent, such as an RNA guide bound to an induced endonuclease.

[0096] The present invention aims to improve the therapeutic potential of immune cells through gene editing methods, particularly the insertion of targeted genes.

[0097] "Targeted gene integration" means any known site-specific method that enables the insertion, replacement, or modification of a genome-coding sequence in a living cell.

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

[0099] "DNA target," "DNA target sequence," "target DNA sequence," "nucleic acid target sequence," "target sequence," or "processing site" refers to a polynucleotide sequence that can be targeted and processed by the sequence-specific nuclease reagent of the present invention. These terms refer to a specific DNA location within a cell, preferably a genomic location, but also to a part of the genetic material that may exist independently of the genetic material itself, such as a non-specific example, a plasmid, episome, virus, transposon, or part of an organelle such as a mitochondria. A non-specific example of an RNA-inducible target sequence is a genomic sequence that can hybridize with a guide RNA that directs an RNA-inducible endonuclease to a desired locus.

[0100] "Low-frequency cleavage endonucleases" are freely selectable sequence-specific endonuclease reagents, as long as their recognition sequences are generally in the range of 10 to 50 consecutive base pairs, preferably 12 to 30 bp, and more preferably 14 to 20 bp.

[0101] In preferred aspects of the present invention, the endonuclease reagent is, for example, a homing endonuclease as described in Arnould S., et al. [WO2004067736], a zinc finger nuclease (ZFN) 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 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, for example, Boissel et al. [MegaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering (2013) Nucleic Acids Research]. These are nucleic acids that encode "modified" or "programmable" low-frequency cleavage endonucleases, such as MegaTAL nucleases, as described in [42(4):2591-2601].

[0102] In another embodiment, the endonuclease reagent is an RNA guide used in conjunction with an RNA-inducing endonuclease such as Cas9 or Cpf1, in particular, as taught by 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 present invention, the endonuclease reagent is transiently expressed in cells, meaning that the reagent is not integrated into the genome or persists for a long period of time, as is the case with RNA, more specifically mRNA, proteins, or mixed complexes of proteins and nucleic acids (e.g., ribonucleoproteins).

[0104] To enhance stability, mRNA-based endonucleases are preferably synthesized with a cap using techniques well 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] Generally, the electroporation process used to introduce genes into 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 generates a substantially uniform pulsed electric field through the processing volume between the parallel plate electrodes, with a voltage greater than 100 volts / cm and less than 5000 volts / cm. One such electroporation chamber is preferably such that the quotient of the square of the electrode gap (cm²) is equal to the chamber volume (cm²). 3 The shape factor (cm) is determined by dividing by ). -1 ) has a shape factor of 0.1 cm -1 The following describes the method, where the cell suspension with the sequence-specific reagent is in a culture 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, as reported in Mussolino et al. [TALEN facilitate targeted genome editing in human cells with high specificity and low cytotoxicity (2014) Nucl. Acids Res. 42(10): 6762-6773], TALE nucleases are particularly suitable sequence-specific nuclease reagents for therapeutic use, especially when acting in the form of heterodimers, i.e., pairs of a "right" (also called "5'" or "forward") monomer and a "left" (also called "3'" or "reverse") monomer, due to their relatively high specificity.

[0107] As described above, sequence-specific reagents are preferably in the form of nucleic acids, such as in the form of low-frequency cleavage endonucleases, DNA or RNA encoding subunits thereof, but may also be part of conjugates containing polynucleotides and polypeptides such as so-called "ribonucleoprotein". Such conjugates can be formed with reagents together with, for example, 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 RNA or DNA guides that can be complexed with their respective nucleases.

[0108] An "exogenous sequence" refers to any nucleotide or nucleic acid sequence that was not initially present at the selected locus. This sequence may be homologous to or a copy thereof of the genomic sequence, or it may be an exogenous sequence introduced into the cell. Conversely, an "endogenous sequence" refers to the genomic sequence of the cell that is initially present at the locus. The exogenous sequence preferably encodes a polypeptide that, when expressed, provides therapeutic advantages compared to sister cells in which the exogenous sequence is not incorporated at the locus. Endogenous sequences that have been gene-edited by nucleotide or polynucleotide insertion according to the method of the present invention in order to express a different polypeptide are broadly referred to as exogenous coding sequences.

[0109] By using the reagents and techniques described above, the present invention provides: • The process of preparing immune cells, preferably primary cells, derived from a donor or patient; • The process of expressing MSLN-CAR in such cells, generally by introducing the MSLN-CAR coding sequence into the cell's genome via a viral vector, as described above; - A step of introducing a sequence-specific reagent, such as a low-frequency cleavage endonuclease, into such immune cells in order to induce modification (mutation or coding sequence insertion) at an endogenous gene locus; and / or - A step of introducing an exogenous coding sequence into the cells in order to improve the therapeutic efficacy of the cells, specifically their immune properties. By performing one or more of these steps, we will develop a method for producing therapeutic cells.

[0110] In some aspects of the present invention, the immune cells are derived from a patient or a suitable donor and express MSLN CAR within the cells in view of the implementation of so-called "autologous" injection of modified immune cells. The immune cells may also be derived from stem cells such as iPS cells from such a patient or suitable donor, or from tumor-infiltrating lymphocytes (TILLs).

[0111] In some aspects of the present invention, the method aims to provide a “ready-made” composition of immune cells, said immune cells, which are modified for allogeneic therapeutic treatment.

[0112] "Allogeneic" means that the cells originate from a donor and are produced or differentiated from stem cells, given that they are injected into patients with different haplotypes.

[0113] Such immune cells are generally modified to be less allogeneic and / or more viable in relation to the patient host. More specifically, the method involves reducing or inactivating TCR expression within T cells or T cell-induced stem cells. This can be achieved using various sequence-specific reagents, for example, by gene silencing or gene editing methods (nucleases, base editing, RNAi, etc.).

[0114] The applicant previously made available a robust protocol and gene editing strategy for producing allogeneic therapeutic-grade T cells from PBMCs, particularly by providing a highly safe and specific endonuclease reagent in the form of TALE nuclease (TALEN®). Donor-derived [TCR] negThe production of so-called "universal T cells," which are T cells, has been achieved and successfully injected into patients, resulting in milder 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)]. Furthermore, as described in WO2014184744, for example, inactivation of the TCR or β2m component in primary T cells can be combined with inactivation of further genes encoding checkpoint inhibitor proteins.

[0115] In a preferred embodiment, the present invention provides a method for modifying immune cells, wherein at least one gene encoding TCR alpha or TCR beta is inactivated in the immune cells, preferably by expression of a low-frequency cleavage endonuclease, while an exogenous polynucleotide encoding MSLN-CAR is introduced into the genome of the cells and stably expressed. Preferably, the exogenous sequence is incorporated into the locus encoding TCR alpha or TCR beta, more preferably under transcriptional control of the endogenous promoter of TCR alpha or TCR beta.

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

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

[0118] The present invention also provides modified lymphocytes equipped with chimeric antigen receptors directed to solid tumors, particularly against mesothelin-positive cells, which are used in conjunction with or after a lymphocyte depletion treatment step for the treatment of solid tumor cancer.

[0119] Such lymphocyte depletion regimens may include anti-CD52 reagents, such as alemtuzumab or purine analogs used to treat hematological malignancies.

[0120] In a preferred embodiment, modified lymphocytes comprising the MSLN-CAR described herein are made resistant to such lymphocyte depletion regimens by inhibiting or interfering with the expression of molecules targeted by the lymphocyte depletion reagent, such as the antigen CD52 in the case of alemtuzumab.

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

[0122] As described above, treating solid tumors with genetically modified lymphocytes possessing chimeric antigen receptors resistant to chemotherapy or lymphocyte depletion regimens is a key aspect of the present invention. Such regimens may include not only antibodies targeting antigens such as CD52, CD3, CD4, CD8, and CD45, or other specific markers present on the surface of immune cells, but also less specific drugs such as purine analogs (e.g., fludarabine and / or chlorofarabine) and glucocorticoids. One aspect of the present invention is to make modified lymphocytes resistant to such regimens by inactivating or reducing the expression of a gene encoding at least one molecular target of these lymphocyte depletion reagents, for example, the DCK gene that metabolizes purine analogs, or a gene encoding the glucocorticoid receptor (GR).

[0123] Therefore, the present invention more specifically focuses on CAR-positive cells and reduces their allogeneic reactivity and makes them resistant to lymphocyte depletion regimens by reducing, inactivating, or impairing the expression of their TCR, CD52, and / or DCK and / or GR, in order to enable allogeneic use of these cells in the treatment of solid tumors.

[0124] In a further embodiment, modified immune cells may be further modified to improve their persistence or lifespan in the patient's body, 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, modified immune cells are mutated to improve their CAR-dependent immune activation, specifically by reducing or suppressing the expression of immune checkpoint proteins and / or their receptors, e.g., PD1 or CTLA4 as described in WO2014184744.

[0126] In a further embodiment, the modified immune cell may be further modified to obtain the co-expression of another exogenous gene sequence within the cell, the 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 cause hypersensitivity of the immune cells to drugs such as cyclophosphamide and / or isophosphamide; • Drug-inducing agents that induce drug resistance: dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin, or methylguanine transferase (MGMT), mTORmut, or Lckmut; Chemokines or cytokines such as IL-2, IL-12, and IL-15; Chemokine receptors such as CCR2, CXCR2, or CXCR4; and / or • Inhibitors of tumor-associated macrophage (TAM) secretion, such as CCR2 / CCL2 antagonists, that enhance the therapeutic activity of immune cells. It is selected from those that code.

[0127] This application claims to provide modified immune cells that co-express at least one exogenous sequence encoding an MSLN-CAR described herein and another exogenous sequence encoding a human polypeptide selected from the above list, in order to produce a therapeutic composition for solid tumors.

[0128] Combination of MSLN-CAR expression and TGFbRII signaling pathway disruption in therapeutically modified immune cells More specifically, the present invention combines the expression of the aforementioned exogenous sequence encoding MSLN-CAR with another exogenous sequence encoding a TGF-beta receptor inhibitor, particularly a TGFβRII inhibitor (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 TGFβ in regulation of the tumor microenvironment and drug delivery (Review). International Journal of Oncology, 46, 933-943].

[0130] While the precise role of TGF-beta receptors in tumorigenesis remains debatable, we have found that co-expressing a mesothelin-specific chimeric antigen receptor (CAR) with another exogenous gene sequence encoding an inhibitor of TGF-BRII signaling, and / or inactivating or reducing TGF-beta receptor signaling using sequence-specific reagents, leads to improved therapeutic efficacy of modified immune cells. Specifically, we employed two different approaches to inhibit the TGF-βRII signaling pathway, which may be used in combination: Expression of an inactive ligand of TGFβRII, such as dominant-negative TGFβRII (SEQ ID NO:26), or a similar inactive form of TGFβRII having at least 80%, preferably at least 90%, more preferably at least 95%, identity with the polypeptide sequence SEQ ID NO:26, as described in Hiramatsu, K., et al. [Expression of dominant-negative TGF-β receptors inhibits cartilage formation in conditional transgenic mouse (2011) J. Bone. Miner. Metab. 29: 493]. and / or Specifically, this involves inactivating the endogenous gene sequence of TGFβRII by using low-frequency cleavage endonucleases such as TALE nucleases or RNA-induced 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 CAR of the present invention to inhibit TGF-beta receptor signaling.

[0132] This application discloses a set of TALE nucleases that are particularly specific to a set of target sequences within the TGFβRII gene. These TALE nucleases exhibited the highest TGFβRII knockout efficiency with very little off-target cleavage and generated a large population of modified living 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 related to the TGFβRII gene TIFF0007830327000007.tif72158

[0134] An RNA guide was designed to inactivate the TGFβRII gene using a Cas9 nuclease reagent. The corresponding target sequences are disclosed in Table 7.

[0135] (Table 7) CRISPR target sequences related to the TGFβRII gene TIFF0007830327000008.tif106161TIFF0007830327000009.tif243161TIFF0007830327000010.tif243161TIFF0007830327000011.tif22161

[0136] The present invention therefore encompasses the use of a TALE nuclease or RNA-inducing endonuclease designed to bind to any of the target sequences SEQ ID NO:X~Y listed in Table 5 or 6 to inactivate or reduce the expression of TGFβRII for the production of therapeutic immune cells as taught herein.

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

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

[0139] Accordingly, the present application also claims a vector, in particular a viral vector, such as a lentiviral vector 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, particularly vectors of the AAV6 family, or the chimeric vector AAV2 / 6 already 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 invention is thus the transduction of an AAV vector comprising the MSLN-CAR coding sequence in primary human immune cells, in association 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 invention, the sequence-specific endonuclease reagent can be introduced into the cells by transfection, more preferably by electroporation of the mRNA encoding the sequence-specific endonuclease reagent.

[0143] As a result of the insertion of the foreign nucleic acid sequence, the introduction of genetic material, the modification or replacement of an endogenous sequence can more preferably occur "in-frame" with respect to the endogenous gene sequence at that locus.

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

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

[0146] One object of the present invention is that the AAV vector used in the method may include an exogenous coding sequence that does not include a promoter, and the coding sequence is any of the sequences referred to herein.

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

[0148] Figure 2 maps the main cellular functions that can be modified by gene editing according to the present invention to improve the efficiency of modified immune cells. Any of the gene inactivations described under each function can be combined to obtain a synergistic effect on the overall therapeutic efficacy of immune cells.

[0149] More specifically, this invention provides combinations of genetic modifications (genotypes) in immune cells, which are used in 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]<U+ - [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] - Immediately improve the efficacy of immune cells against MSLN-positive malignant cells such as

[0150] As described above, the present invention also focuses in particular on the use of CAR-positive cells in the treatment of solid tumors, and since these cells are resistant to lymphocyte depletion agents, they can be used in an allogeneic setting, in combination with a lymphocyte depletion regimen, or afterward. 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 immunity to purine analogs: • [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 improvement of therapeutic immune cells through the expression of transgenes at inactivation loci. The preferred genotypes described above can be obtained by target gene integration, preferably at the PD1, TCR (TCR alpha and / or TCR beta), or TGFβRII loci, but also at further select loci described later.

[0152] "Targeted gene integration" means any known site-specific method that enables the insertion, replacement, or modification of a genomic sequence in a living cell. Targeted gene integration typically involves homologous recombination or NHEJ (non-homologous end joining) mechanisms, which are enhanced by endonuclease sequence-specific reagents to result in the insertion or substitution 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 can 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 present invention is a) Polynucleotide sequences whose expression is involved in the reduction of 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) Polynucleotide sequences such as IL27RA, STAT1, STAT3, whose expression upregulates immune checkpoint proteins (e.g., TIM3, CEACAM, LAG3, TIGIT); and / or c) Polynucleotide sequences such as ILT2 or ILT4 whose expression mediates interaction with HLA-G; and / or d) Polynucleotide sequences whose expression is involved in the downregulation of T cell proliferation, such as SEMA7A and SHARPIN which reduce Treg proliferation, STAT1 which reduces apoptosis, PEA15 which increases IL-2 secretion, and RICTOR which supports the differentiation of CD8 memory; and / or e) Polynucleotide sequences, such as mir21, whose expression is involved in the downregulation of T cell activation; and / or f) Polynucleotide sequences, such as JAK2 and AURKA, whose expression is involved in signaling pathways that respond to cytokines; and / or g) Polynucleotide sequences such as DNMT3, miRNA31, MT1A, MT2A, and PTGER2, whose expression is involved in T cell elimination. The process includes introducing a selected mutation or polynucleotide coding sequence into an endogenous locus of an immune cell, preferably by expressing a sequence-specific reagent in the cell that specifically targets the selected endogenous locus.

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

[0156] Targeting one of the aforementioned loci by performing gene integration is beneficial for further improving 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 selective locus are listed below.

[0158] Expression of transgenes that confer resistance to drugs or immunosuppressants In one aspect of this method, the exogenous sequence incorporated into the genomic locus of an immune cell encodes a molecule that confers drug resistance to the immune cell.

[0159] Examples of preferred exogenous sequences include dihydrofolate reductase (DHFR) variants that confer resistance to folate analogs such as methotrexate; inosine monophosphate dehydrogenase 2 (IMPDH2) variants that confer resistance to IMPDH inhibitors such as mycophenolic acid (MPA) or its prodrug mycophenolic acid mofetil (MMF); calcineurin or methylguanine transferase (MGMT) variants that confer resistance to calcineurin inhibitors such as FK506 and / or CsA; mTOR variants such as mTORmut that confer resistance to rapamycin; and Lck variants 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 commonly used to interact with cancer cells and thereby reduce their proliferation or viability. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, ifosamide), antimetabolites (e.g., purine nucleoside antimetabolites, e.g., clofarabine, fludarabine, or 2'-deoxyadenosine, methotrexate (MTX), 5-fluorouracil, or their derivatives), antitumor antibiotics (e.g., mitomycin, adriamycin), plant-derived antitumor agents (e.g., vincristine, vindesine, taxol), cisplatin, carboplatin, etoposide, etc. Examples of such agents include, but are not limited to, the anticancer drugs TRIMETHOTRIXATE® (TMTX), temozolomide®, RALTRITREXED®, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzyguanidine (6-BG), bis-chloronitrosourea (BCNU), and camptothecin®, or any of these therapeutic derivatives.

[0161] As used herein, immune cells are made “resistant or tolerant” to the drug when the cells or cell population are modified, and as a result can grow at least in vitro in a culture medium containing the drug at a 50% maximum inhibitory concentration (IC50) (the IC50 is determined for unmodified cells or cell populations).

[0162] In certain embodiments, the drug resistance may be conferred to immune cells by the expression of at least one "drug resistance coding sequence." The drug resistance coding sequence relates to a nucleic acid sequence that confers "resistance" to a certain agent, for example, one of the chemotherapeutic agents described above. The drug resistance coding sequences of the present invention can encode resistance to antimetabolites, methotrexate, vinblastine, cisplatin, alkylating agents, anthracyclines, cytotoxic antibiotics, antiimmunophilins, and their analogs or derivatives (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-resistant exogenous sequences in immune cells according to the present invention enables the use of such immune cells in a cell therapy treatment scheme that combines cell therapy with chemotherapy, or in patients who have received such drug treatments.

[0164] Several drug resistance coding sequences have been identified that may be used to confer the drug resistance of the present invention. An example of a drug resistance coding sequence may be, for example, a variant or modifier of dihydrofolate reductase (DHFR). DHFR is an enzyme involved in regulating the amount of tetrahydrofolate in cells and is essential for DNA synthesis. Folate analogs such as methotrexate (MTX) inhibit DHFR and are therefore used clinically as anti-cancer agents. Various variant forms of DHFR that are resistant to inhibition by folate antimetabolites used in therapy have been described. In certain embodiments, the drug resistance coding sequence of the present invention may be a nucleic acid sequence encoding a variant form of human wild-type DHFR (GenBank: AAH71996.1), which contains at least one mutation that confers resistance to folate antimetabolite therapy such as methotrexate. In certain embodiments, the DHFR variant morphology contains at least one mutant 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 variant morphology contains two mutant amino acids at positions L22 and F31. The amino acid position correspondences described herein are often expressed as the amino acid positions of the wild-type DHFR polypeptide morphology. 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 a folate antimetabolite, preferably 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 resistance coding sequence may be a variant or variant of ionisine-5'-monophosphate dihydrogenase II (IMPDH2), the rate-limiting enzyme for the novel synthesis of guanosine nucleotides. A variant or variant of IMPDH2 is an IMPDH inhibitor resistance gene. The IMPDH inhibitor may be mycophenolic acid (MPA) or its prodrug, mycophenolic acid mofetil (MMF). A variant IMPDH2 may contain at least one, preferably two, mutations in the MAP binding site of wild-type human IMPDH2 (Genebank: NP_000875.2), thus exhibiting significantly increased resistance to IMPDH inhibitors. Mutations in these variants are preferably located 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 resistance coding sequence is a variant form of calcineurin. Calcineurin (PP2B - NCBI: ACX34092.1) is an eccentrically expressed serine / threonine protein phosphatase involved in many biological processes and crucial 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 dephosphates the transcription factor NFAT, allowing it to translocate to the nucleus and other active major target genes such as IL2. FK506 complexed with FKBP12, or cyclosporine A (CsA) complexed with CyPA, interferes with NFAT's access to 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 morphology may include at least one mutant amino acid of the wild-type calcineurin heterodimer at positions V314, Y341, M347, T351, W352, L354, and K360, preferably with double mutations at positions T351 and L354, or V314 and Y341.In certain 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 residue at position 347 may be replaced with a glutamic acid, arginine, or tryptophan residue; the threonine residue 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 residue at position 353 may be replaced with a histidine or asparagine residue; the leucine residue at position 354 may be replaced with an alanine residue; and the lysine residue at position 360 may be replaced with an alanine or phenylalanine residue. In another specific embodiment, the mutant form may include at least one mutant amino acid of wild-type calcineurin heterodimer b at positions V120, N123, L124, or K125, preferably with double mutations at positions L124 and K125. In a specific embodiment, valine at position 120 may be replaced with serine, aspartic acid, phenylalanine, or leucine residue; asparagine at position 123 may be replaced with tryptophan, lysine, phenylalanine, arginine, histidine, or serine; leucine at position 124 may be replaced with a threonine residue; lysine at position 125 may be replaced with alanine, glutamic acid, or tryptophan, or two residues such as leucine-arginine or isoleucine-glutamic acid may be added after lysine at position 125 of the amino acid sequence. The amino acid position correspondences described herein are often expressed as the amino acid positions in the form of 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 nitrosourea and temozolomide (TMZ). 6-benzylguanine (6-BG) is an AGT inhibitor that enhances the toxicity of nitrosourea and, when administered co-administered with TMZ, increases the cytotoxic effects of the drug. Several MGMT variant forms encoding AGT variants are highly resistant to inactivation with 6-BG, yet 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, the AGT variant form may 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 may 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 byproducts across the cell membrane. The P-GP protein exhibits broad specificity to several structurally unrelated chemotherapeutic agents. Therefore, expression of the nucleic acid sequence encoding MDR-1 (Genebank NP_000918) can confer drug resistance to cells.

[0169] Other drug resistance coding sequences, such as those derived from the ble or mcrA genes, may contribute to the production of cytotoxic antibiotics. Ectopic expression of the ble gene or mcrA in immune cells provides 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] Other drug resistance coding sequences may originate from drug target-coding variant genes, such as mTOR mutations (mTOR mut) that confer resistance to rapamycin, as described in 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 specific Lck mutations (Lckmut) that confer resistance to Gleevec, as described in 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 gene modification step of the method may include introducing an exogenous nucleic acid containing at least one sequence encoding a drug resistance coding sequence and a portion of an endogenous gene into a cell, so that homologous recombination occurs between the endogenous gene and the exogenous nucleic acid. In certain embodiments, the endogenous gene may be a wild-type "drug resistance" gene, so that after homologous recombination, the wild-type gene is replaced by a variant form of the gene that confers resistance to the drug.

[0172] Expression of transgenes that enhance the in vivo persistence of immune cells In one aspect of this method, exogenous sequences incorporated into immune cell genomic loci encode molecules that enhance the persistence of immune cells, particularly in vivo persistence in the tumor environment.

[0173] "Enhancing persistence" means extending the lifespan of the immune cells, particularly after they have been injected into a patient. For example, enhanced persistence is achieved when the average lifespan of modified cells is significantly longer than that of unmodified cells, by at least 10%, preferably 20%, more preferably 30%, and even more preferably 50%.

[0174] This is particularly relevant when the immune cells are allogeneic. This can be achieved by creating local immunoprotection by introducing coding sequences that cause immunosuppressive polypeptides to be ectopically expressed and / or secreted at or across the cell membrane. A variety of such polypeptides, specifically immunosuppressive peptides derived from immune checkpoint antagonists, viral envelopes, or NKG2D ligands, can enhance the persistence and / or transplantation 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 number AF414120.1). The ligand polypeptide is preferably an anti-CTLA-4 immunoglobulin, such as CTLA-4a Ig and CTLA-4b Ig, or functional variants thereof.

[0176] In one embodiment, the immunosuppressive polypeptide encoded by the exogenous coding sequence is a PD1 antagonist, such as PD-L1 (also known as CD274, programmed cell death 1 ligand; see UniProt for human polypeptide sequence Q9NZQ7), encoding 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. In this invention, the membrane-bound form of such a PD-L1 ligand is either the original (wild-type) form or a form cleaved by, for example, removal of the intracellular domain or one or more mutations (Wang S et al., 2003, J Exp Med. 2003; 197(9): 1083-1091). Note that PD1 is not considered a membrane-bound form of the PD-L1 ligand in this invention. In another embodiment, the immunosuppressive polypeptide is in a secretory form. Such recombinant secretory PD-L1 (or soluble PD-L1) can be produced by fusing the extracellular domain of PD-L1 to the Fc portion of an 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 terminate PD-1-mediated T cell inhibition. The persistence of both can be further enhanced by co-expressing the PD-L1 ligand with CTLA4 Ig.

[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 exogenous sequence encodes an NKG2D ligand. Some viruses, such as cytomegalovirus, have acquired a mechanism to interfere with the NKG2D pathway by secreting proteins that can evade NK cell-mediated immune surveillance and bind to NKG2D ligands, thereby 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). In tumor cells, mechanisms have evolved to evade the NKG2D response 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 the IL-12 receptor. IL-12 is a well-known activator of immune cells (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 exogenous sequence encodes an antibody directed against an inhibitory peptide or protein. The antibody is preferably secreted in a soluble form by immune cells. In this respect, shark and camel-derived nanobodies 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 fuse more easily with secretory signaling polypeptides and soluble hydrophilic domains.

[0181] The various aspects of enhancing cell persistence described above will be further detailed below, but are particularly favorable when exogenous coding sequences are introduced by interfering with endogenous genes encoding β2m or other MHC components.

[0182] Expression of transgenes that enhance the therapeutic activity of immune cells In one aspect of this method, the exogenous sequence incorporated into the immune cell genome locus encodes a molecule that enhances the therapeutic activity of the immune cell.

[0183] "Enhancing therapeutic activity" means that the immune cells or cell populations modified according to the present invention become more aggressive against a selected type of target cell compared to unmodified cells or cell populations. The target cells consist of a predetermined type of cells or cell population, preferably characterized by common surface markers. Herein, "therapeutic potential" reflects therapeutic activity measured by in vitro experiments. Generally, whether the immune cells are even slightly active against the cells is evaluated using a sensitive cancer cell line such as Daudi cells by measuring cell lysis or decreased proliferation. This can also be evaluated by measuring the degranulation of the immune cells or the levels of chemokine and cytokine production. Experiments can also be performed in mice by injecting tumor cells and observing the resulting tumor growth. Enhancement of activity is considered significant if the number of cells generated 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% by the immune cells.

[0184] In one aspect of the present invention, the exogenous sequence encodes a chemokine or cytokine, such as IL-12. Expression of IL-12 is particularly advantageous because this cytokine promotes the activation of immune cells, as has been widely mentioned in the literature (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 present invention, the exogenous coding sequence encodes or promotes secretion factors that act on other immune cell populations, such as regulatory T cells, to mitigate their inhibitory effects on the 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 transcellular peptide inhibitor of FoxP3, such as 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] "Inhibitors of regulatory T cell activity" refer to molecules secreted by T cells or precursors of such molecules, which allow T cells to escape the downregulatory activity exerted by regulatory T cells on other T cells. Generally, such inhibitors of regulatory T cell activity have the effect of reducing FoxP3 transcriptional activity in the 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 antagonist. Tumor-associated macrophages (TAMs) are important modifiers of the tumor microenvironment. Clinical pathological studies have shown that TAM accumulation in tumors correlates with poor clinical outcomes. Consistent with this evidence, experimental and animal studies support the view that TAMs can provide a microenvironment favorable to promoting tumorigenesis 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 chemotactic protein 1 (MCP1 - NCBI NP_002973.1), is a small cytokine belonging to the CC chemokine family. It is secreted by macrophages and generates chemoattractions in 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 aforementioned locus typically encodes a polypeptide that enhances the therapeutic potential of modified immune cells, but the insertion sequence may also be a nucleic acid capable of directing or blocking the expression of other genes, such as interfering RNA or guide RNA. The polypeptide encoded by the insertion sequence may act directly or indirectly, such as a signaling factor or transcription regulator.

[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 a preferred aspect of the present 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, this population is derived from a patient or donor and is isolated from PBMCs (peripheral blood mononuclear cells) by leukocyte removal.

[0192] The present invention encompasses immune cells containing various exogenous coding sequences and any combination of gene inactivation, each of which has been described independently. Among these combinations, particularly preferred is the combination of CAR expression under transcriptional regulation of an endogenous promoter that is active during immune cell activation, specifically a single promoter located at a single 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 regulation of the hypoxia-inducible factor 1 gene promoter (Uniprot: Q16665).

[0194] The present invention also relates to a pharmaceutical composition comprising the aforementioned modified primary immune cells or immune cell populations with respect to the treatment of infectious diseases or cancer, and a method for treating a patient in need of treatment, wherein the method is • A step of preparing a population of primary immune cells modified by the method of the present invention as described above; - A step of purifying or separating the modified primary immune cells by optional selection; The process of activating the modified primary immune cell population when the cells are injected into the patient, or after injection. Includes.

[0195] T cell activation and proliferation Whether before or after genetic modification, the immune cells of the present invention can be activated or proliferated, even if they can be activated or proliferated independently of the antigen-binding mechanism. In particular, T cells can be activated and proliferated using methods such as those described in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. T cells can be proliferated in vitro or in vivo. Generally, T cells proliferate by generating T cell activation signals through contact with agents that stimulate the CD3 TCR complex and T cell surface co-stimulatory molecules. For example, chemicals such as the calcium ionophore A23187, phorbol 12-myristo 13-acetate (PMA), or mitotic lectins like phytohemagglutinin (PHA) can be used to generate T cell activation signals.

[0196] As a non-established example, T cell populations can be stimulated in vitro, for example, by contact with an anti-CD3 antibody or its antigen-binding fragment immobilized on the surface, or with an anti-CD2 antibody, or by contact with a calcium ionophore along with protein kinase C activator (e.g., bryostatin). Co-stimulation of accessory molecules on the T cell surface is performed using ligands that bind to the accessory molecules. For example, a T cell population can be contacted with anti-CD3 and anti-CD28 antibodies under appropriate conditions to stimulate T cell proliferation. Appropriate conditions for T cell culture include appropriate media (e.g., minimal essential medium, or RPMI Media 1640 or X-vivo 5 (Lonza)) that 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-g, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFp, and TNF, or any other additives for cell proliferation known to those skilled in the art. Other additives for cell proliferation include, but are not limited to, surfactants, plasmamenates, and reducing agents, such as N-acetylcysteine ​​and 2-mercaptoethanol. The culture medium may contain RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, an optimizer, and may be supplemented with amino acids, sodium pyruvate, and vitamins, and may be serum-free or supplemented with an appropriate amount of serum (or plasma), or a set of predetermined hormones, and / or cytokines in sufficient quantities for T cell growth and proliferation. Antibiotics, such as penicillin and streptomycin, are included only in the experimental culture and not in the culture of cells to be injected into the target. Target cells are maintained under conditions necessary to support proliferation, such as appropriate temperature (e.g., 37°C) and air (e.g., air plus 5% CO2). T cells exposed to various stimulation times may exhibit different characteristics.

[0197] In another specific embodiment, the cells may be grown by co-culture with tissue or other cells. The cells may also be grown in vivo, for example, in the blood of a subject after administration of the cells to that subject.

[0198] Therapeutic compositions and uses The method of the present invention described above makes it possible to produce modified primary immune cells within a limited time frame of about 15 to 30 days, preferably 15 to 20 days, and most preferably 18 to 20 days, and therefore they retain the greatest potential for immunotherapy, particularly with respect to cytotoxic activity.

[0199] These cells form cell populations, which are preferably derived from a single donor or patient. These cell populations can be grown in a closed culture recipient, thus complying with the most stringent manufacturing requirements, and can be frozen before injection into the patient, providing a “ready-to-use” or “off-the-shelf” therapeutic composition.

[0200] In this invention, a large number of cells originating from the same leukocyte removal can be obtained, which is important for obtaining a sufficient dose to treat a patient. Although differences may be observed between cell populations originating from various donors, the number of immune cells acquired by leukocyte removal is generally about 10 8 ~10 10 These are individual PBMC cells. PBMCs contain several types of cells, including granulocytes, monocytes, and lymphocytes, with 30-60% being T cells, and primary T cells from a single donor generally number 10. 8 ~10 9 The number is. The method of the present invention is generally about 10 8 Super T cells, more generally about 10 9 Super T cells, and generally about 10 10 Super T cells, normally 10 11 Ultimately, we obtain a population of modified cells that reach the level of super T cells.

[0201] The present invention therefore relates more specifically to a therapeutically effective population of primary immune cells, wherein at least 30%, preferably 50%, and more preferably 80% of the cells in the population are modified according to any one of the methods described herein.

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

[0203] Such compositions or cell populations can therefore be used as pharmaceutical agents, particularly for the treatment of cancer, specifically for the treatment of lymphoma, but also for solid tumors, such as melanoma, neuroblastoma, glioma, or carcinomas, such as lung tumors, breast tumors, rectal tumors, prostate tumors, or ovarian tumors, in patients who require them.

[0204] More specifically, the present invention relates to a population of primary TCR-negative T cells originating from a single donor, wherein at least 20%, preferably 30%, and more preferably 50% of the cells in the population are modified by a sequence-specific reagent at at least two, preferably three different loci.

[0205] In another aspect, the present invention depends on a method for treating a patient in need of treatment, and the said method is (a) A step of determining specific antigen markers present on the surface of a patient's tumor biopsy; (b) A step of preparing a modified primary immune cell population expressing a recombinant receptor, preferably directed to the specific antigen marker, modified by one of the methods of the present invention described above; (c) The step of administering the modified primary immune cell population to the patient. It must include at least one of the following.

[0206] Generally, the aforementioned cell population mainly consists of CD4 and CD8-positive immune cells such as T cells, which can undergo robust in vivo T cell proliferation and can survive for extended periods both in vitro and in vivo.

[0207] Therapies comprising the modified primary immune cells of the present invention may be remittant, curative, or prophylactic. They may be part of autologous immunotherapy or allogeneic immunotherapy.

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

[0209] This includes adult tumors / cancers and pediatric tumors / cancers.

[0210] Therapy using the modified immune cells of the present invention can be used in combination with one or more cancer treatments selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiotherapy.

[0211] In a preferred embodiment of the present invention, the treatment may be administered to a patient receiving immunosuppressive therapy. In fact, the present invention preferably relies on cells or cell populations that have been made resistant to such immunosuppressants thanks to the inactivation of genes encoding receptors for at least one immunosuppressant. In this respect, immunosuppressive therapy should assist in the selection and proliferation of the T cells of the present invention within the patient's body.

[0212] The 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, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullarily, intramuscularly, intravenously or intralymphatically, or intraperitoneally. In one embodiment, the cell composition of the present invention is preferably administered by intravenous injection.

[0213] The administration of cells or cell populations is 10 per kilogram of body weight. 4 ~10 9 Cells, preferably 10 per kilogram of body weight 5 ~10 6 This invention may consist of a cell administration, and includes all integers of the number of cells within these ranges. The present invention therefore originates from a single donor or patient sampling. 6 ~10 8 It may provide doses of more than 10, generally more than 50, more generally more than 100, and usually more than 1000, including gene-edited cells.

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

[0215] In another embodiment, the effective amount of cells or a composition containing such cells is administered parenterally. The administration may be intravenous. The administration may be done directly by tumor injection.

[0216] In certain aspects of the present invention, the cells are administered to the patient in conjunction with any number of relevant therapeutic modalities (e.g., before, simultaneously with, or after) treatment with agents such as antiviral therapy, cidofovir, and 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 may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunosuppressants such as CAMPATH, anti-CD3 antibodies, or other antibody therapies, cytoxin, fludarivine, cyclosporine, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and irradiation methods. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or the p70S6 kinase (rapamycin), which is important for growth factor-induced signaling (Henderson, Naya et al. 1991; Liu, Albers et al. 1992; Bierer, Hollander et al. 1993). In further embodiments, the cell composition of the present invention is administered to a patient together with (e.g., before, simultaneously with, or after) bone marrow transplantation, chemotherapeutic agents 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 composition of the present invention is administered following B-cell depletion therapy, such as rituximab, using agents that react with CD20. For example, in one embodiment, a subject may undergo peripheral blood stem cell transplantation after receiving standard treatment with high-dose chemotherapy. In a specific embodiment, following transplantation, the subject receives an infusion of the proliferated immune cells of the present invention. In an additional embodiment, the proliferated cells are administered before or after surgery.

[0217] The present invention also specifically relates to a general method for treating a patient's solid tumor, the method comprising the steps of immunizing the patient with a lymphocyte depletion regimen and injecting genetically modified lymphocytes that are resistant to the lymphocyte depletion agent used in the lymphocyte depletion regimen and specifically target the solid tumor. Such genetically modified lymphocytes are preferably CAR-positive T cells, and more preferably comprising MSLN-CAR as described herein.

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

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

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

[0221] To this end, the present invention provides a composition for lymphocyte removal in patients affected by solid tumors, comprising a composition containing an anti-CD52 antibody and a population of modified lymphocytes that are insensitive to the antibody and target MSLN, wherein such a population preferably comprises cells that express MSLN-CAR and have impaired CD52 expression. In such modified cells, the allele of the CD52 gene is preferably inactivated by a low-frequency cleavage endonuclease such as the TALE nuclease or RNA-induced endonuclease described above.

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

[0223] "Cytolytic activity," "cytotoxic activity," or "cytotoxicity" refers to the percentage of target cells lysed by immune cells.

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

[0225] For adhesion target cells: 2.10 4 Seed 0.1 ml per well of STA-positive or STA-negative cells into a 96-well plate. The day after seeding, STA-positive and STA-negative cells were labeled with CellTrace CFSE and refracted for 4 hours (4 x 10⁶). 5 The cells are co-cultured with T cells. Then the cells are harvested, stained with a fixable viability-determinating 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 ROR1-positive cells 2 x 10 4 STA-negative cells and 4 x 10⁶ cells in 0.1 ml per well in a 96-well plate. 5The cells are co-cultured with T cells. After incubation for 4 hours, the cells are harvested, stained with a fixable viability-determinating dye (eBioscience), and analyzed using a MACSQuant flow cytometer (Miltenyi).

[0227] The percentage of specific dissolution can be calculated using the following formula. TIFF0007830327000012.tif25128

[0228] "Increased cytotoxicity" means that the percentage of target cell lysis delivered by modified immune cells is increased by at least 10%, for example, 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 percentage of target cell lysis delivered by unmodified immune cells.

[0229] "Identity" refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing their positions within each sequence, and they may be aligned for comparison. If the positions within the compared sequences are occupied by the same bases, then those 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 consistent nucleotides at the same positions common to the nucleic acid sequences. Identity between two sequences can be calculated using various alignment algorithms and / or programs, 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 that have at least 70%, 85%, 90%, 95%, 98%, or 99% identity with a particular polypeptide described herein, and preferably exhibit substantially the same function, as well as polynucleotides encoding such polypeptides, are assumed.

[0230] When used herein, the terms “subject” or “patient” generally refer to mammals, preferably primates, and more preferably humans.

[0231] The above-described specification of the present invention provides modes and methods for carrying out and using the present invention, enabling any person skilled in the art to carry out and use the present invention, which is provided specifically with respect to the subject matter of the appended claims, which constitute a part of this specification.

[0232] Where numerical limits or ranges are specified herein, the endpoints are also included. Furthermore, all values ​​and subranges within numerical limits or ranges are specifically included as if they were explicitly stated.

[0233] While the present invention has been described in broad terms, further understanding can be gained by referring to specific examples. Such specific examples are provided herein for illustrative purposes only and do not limit the scope of the claimed invention. [Examples]

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

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

[0236] Description of MSLN-specific CAR used in this study Three second-generation CARs were constructed, each consisting of scFv P4, meso1, and MESO2, and containing a CD8α hinge / transmembrane domain, as well as 4-1BB and CD3ζ activation domains, to create primary mesoCARs for target cell lines expressing different levels of mesothelin (MSLN). + We screened for in vitro chimeric antigen receptor (CAR) expression and antitumor activity through T cell activity. In specific versions, a suicide switch "R2" was introduced into the CAR structure. As already described in WO2016120216, the "R2" polypeptide containing two CD20 mimotopes was positioned between the scFv and the 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 We selected CARs for expression in primary T cells derived from PBMCs and identified three target cell lines expressing different levels of MSLN. ·Epithelial cervical adenocarcinoma HeLa (ATCC (registered trademark) CCL-2), • Epithelial splenic adenocarcinoma (HPAC) (ATCC(registered trademark) CRL-2119), and • 293H or A2058 cells, mesothelin-negative cells The antitumor activity of each of them was assayed.

[0240] The expression of mesothelin on the surface of these cells was evaluated 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, California 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 to 37°C and 5% CO2. On day 1, PBMCs were counted, analyzed by flow cytometry to assess the percentage of 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 / 1 million CD3+ cells). The cells were then transferred to an incubator set to 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 to 37°C and 5% CO2. On day 5, the T cells were washed and resuspended in X-vivo 15 medium supplemented with 5% AB serum and 350 UI / ml IL2. The cells were then transferred to an incubator set to 37°C and 5% CO2. On day 6, as previously reported [Poirot et al. (2013) Blood. 122 (21): 1661], mRNA encoding the right and left arms of TRAC TALEN and CD52 TALEN, respectively, was introduced into T cells by simultaneous electroporation to efficiently inactivate the TCRα and CD52 genes and prevent the expression of TCRαβ on the surface of primary T cells. TALEN is the registered name for TALE nucleases designed by Cellectis (75013 Paris, France, rue de la Croix Jarry 8). The genomic target sequences of these TALE nucleases are shown in Table 8 below. Gene transfer was performed using the AgilePulse method. The cells were then transferred to an incubator set to 37°C and 5% CO2. On day 7, the T cells were washed and resuspended in X-vivo 15 medium supplemented with 5% AB serum and 350 UI / ml IL2. The cells were then transferred to an incubator set to 37°C and 5% CO2. T cells were grown in a GRex device between days 7 / 8 and 18. When using a 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 a GRex 100M, fresh IL2 was added on days 11, 13, and 15 without changing the medium. Cell cultures were incubated at 37°C under 5% CO2 during the growth period. On day 18, all UCART cells were cryopreserved for later use in in vitro and in vivo assays.

[0242] (Table 8) Genome sequences targeted by TALE nucleases (TALENs) TIFF0007830327000013.tif58156

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

[0244] The initial in vitro studies conducted on four UCART cell products aimed to determine the phenotype of UCART cells on day 18. To this end, flow cytometry was used to analyze 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. CAR surface expression was assessed using either His-tagged recombinant human mesothelin protein or biotinylated protein L, both of which recognize biotinylated rituximab that recognizes either the scFv portion of CAR or the R2 suicide switch portion of CAR. TCRαβ receptor surface expression was assessed using an anti-TCRαβ antibody conjugated with PE-vio770. CD4 and CD8 surface expression was assessed using a CD4 antibody conjugated with FITC and a CD8 antibody conjugated with BV510.

[0245] CAR surface expression was evaluated by flow cytometry using either His-tagged recombinant human mesothelin protein, which recognizes the scFv region of CAR, or biotinylated rituximab, which recognizes the R2 suicide switch region 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, P4-R2 CAR showed higher expression levels than Meso1-R2 CAR and MESO2-R2 CAR.

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

[0248] 2.2 IFNg production and chemotactic activity The second in vitro study, conducted on three UCART cell products, aimed to analyze the function of UCART cells. The ability of UCART cells to produce cytokines 24 hours after co-culture with HPAC(MSLN+) cells or 293H(MSLN-) cells was evaluated by quantifying IFNg in the cell culture supernatant using a standard ELISA procedure.

[0249] As shown in Figure 8, UCART cells modified with P4-R2 CAR and Meso1-R2 CAR produced IFNg levels exceeding 40,000 pg / ml and 50,000 pg / ml, respectively, after co-culture with MSLN+ cell lines, and below 1,500 pg / ml and 200 pg / ml, respectively, after co-culture with MSLN- cell lines. Although UCART cells modified with Meso1-R2 CAR produced IFNg levels similar to those of UCART cells modified with P4-R2 CAR after co-culture with MSLN+ cell lines, cells with Meso1-R2 CAR produced extremely low levels of IFNg after co-culture with MSLN- cell lines. UCART cells modified with MESO2-R2 CAR produced only IFNg levels below 15,000 pg / ml after co-culture with MSLN+ cell lines.

[0250] Next, the ability of UCART cells to perform sequential killing of HPAC cells was evaluated over 15 days, 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 showed similar levels of killing activity against HPAC cells after one round of exposure. However, after several rounds of exposure, T cells with Meso1-R2 and P4-R2 showed much higher continuous killing activity than CART cells with MESO2-R2 CAR, while CART cells modified with Meso1-R2 showed more sustained activity than CART cells modified with P4-R2.

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

[0253] 3. Functional verification of the genetic characteristics of UCART MSLN cells 3.1 TRAC and / or CD52 gene knockout 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 TALEN-mediated inactivation of the TCRα gene was highly efficient in the cell population.

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

[0255] To fully demonstrate that the absence of TCRαβ receptor detection on the surface of TRAC gene knockout and TCRαβ+ cell-depleted cells is associated with the absence of functional TCRαβ receptor expression, 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 evaluated by culturing modified cells for 7 days in 50 μg / ml anti-CD52 monoclonal antibody (or rat IgG as a control) with or without 30% rabbit complement (Cedarlane), followed by incubation. Two hours after incubation at 37°C, cells were labeled with anti-CD52 antibody conjugated to a fluorescent dye (eBioscience) and analyzed by flow cytometry to determine the frequency of CD52-positive and CD52-negative cells among viable cells. Cells were also cultured with the antibody to select for resistance.

[0258] 3.2 UCART cell removal using rituximab against R2 polypeptide Another in vitro study conducted on UCART cell products aimed to evaluate the ability of UCART cells to have their CAR+ fraction removed after treatment with rituximab.

[0259] UCART cells modified with P4-R2, Meso1-R2, and MESO2-R2 CARs were co-cultured with HPAC cells for 2 days and exposed to culture medium, rituximab (RTX), rabbit complement (BRC), or a mixture of rituximab and rabbit complement for 2 hours. Flow cytometry analysis was then performed to observe the percentage of CAR+ cells. As shown in Figure 13, UCART cells modified with P4-R2, Meso1-R2, and MESO2-R2 were efficiently removed after treatment with rituximab and rabbit complement. In contrast, UCART cells modified with P4-naked (MSLN CARs with a P4 structure but no R2 sequence) were not removed.

[0260] 3.3 Inactivation of the TGFβ signaling pathway in CAR T cells Another characteristic conferred to MesoCAR T cells was resistance to the tumor microenvironment through inactivation of the TGFb signaling pathway. We investigated two different strategies for inactivating TGFbRII gene expression, either by knockout or by overexpression of the dominant-negative form of the TGFbRII gene (dnTGFbRII).

[0261] 3.3.1. Inactivation of TGFbRII by knockout 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, the T cells were harvested, gDNA was extracted, and the amplicons were amplified by PCR. Analysis of PCR products by deep sequencing revealed that gene transfer of TALENs encoded by pCLS32939 and pCLS32940, or TALENs encoded by pCLS32967 and pCLS32968, resulted in 96.62% and 97.28% gene editing (i.e., insertions and / or deletions), respectively, demonstrating the high efficiency of TGFbRII knockout.

[0262] 3.3.2. Inactivation of TGFbRII by overexpression of dnTGFbRII As described in Example 2, MSLN-CAR T cells with and without dnTGFbRII (SEQ ID NO:24), separated by a 2A cleavage peptide, were produced using two different donors and rLV vectors encoding different MSLN CARs. 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. On day 1 after thawing, the cells were exposed to 5 ng / ml of TGFb (R&D systems). After 1 hour, the cells were stained for CAR expression by performing cell surface staining with 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, as instructed by the supplier. Cells were analyzed by flow cytometry to determine the phosphorylated SMAD2 / 3 status in CAR-positive and CAR-negative subpopulations (Figure 14). The results demonstrated that in the absence of dnTGFbRII, cells were SMAD2 / 3 phosphorylated (Figure 14, left panel), but in the presence of dnTGFbRII, only CAR-positive cells showed decreased SMAD2 / 3 phosphorylation (Figure 14, right panel). These results indicate that TGFb signaling can be impaired in MSLN-CART cells expressing dnTGFbRII.

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

[0264] Subsequently, the in vivo antitumor activity of human T cells expressing mesoCAR candidates and P4-CAR was evaluated. Briefly, MSLN was introduced into NSG mice. + After transplanting the cell line (injecting HPAC cells via SC), mesoCAR + T-cell treatment (IV injection, 3 doses). mesoCAR + T cell activity was assessed by observing tumor growth. Three mesoCARs were evaluated. + T cells showed different levels of in vivo antitumor activity against HPAC tumor cells. T cells expressing MESO2-R2 CAR showed different levels of activity compared to other mesoCARs evaluated. + Compared to T cells, it showed lower activity.

[0265] 4.1 Rationale for selecting animal models mesoCAR + Since T cells are human-specific, standard immunocompetent animal models are unusable because human T cells are rapidly targeted and eliminated by heterologous immune responses. The selected animal model is the hyperimmune-deficient NSG mouse strain (NOD.Cg-Prkdc from Jackson Laboratory). scid Il2rg tm1Wjl (SzJ lineage) is human MSLN + This is to allow for the transplantation of both tumor cells and human CAR T cells.

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

[0267] To put it simply, on day 0, the mice were randomly divided into 4 groups of 6 based on their individual body weight, and HeLa cells (1x10) were administered. 6 or 10x10 6 Cells / mouse) or HPAC cells (2x10) 6 or 10x10 6 The mice received SC injections of cells (or mice). 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 immunohistochemis-try array. Arch. Iran. Med.; Kusakawa et al. (2015) Characterization of invivo tumorigenicity tests using severe immunodeficient NOD / Shi-scid IL2Rγnull mice for detection of tumorigenic cellular impurities in human cell-processed therapeutic products. Regen. Ther.].

[0268] Weight, survival, and behavior were observed daily. Tumor volume was measured three times a week. Surviving mice were euthanized on day 61 (end of the experiment). Autopsies (microscopic examination) were performed on all euthanized test animals, and, where possible, on all euthanized, dying, or dead animals found at the time of discovery.

[0269] Both HPAC tumors and HeLa tumors grew within NSG mice. HPAC tumors grew faster than HeLa tumors. 1x10 6 and 10x10 6 The average tumor volume V (500 mm) of mice injected with HeLa cells 3 ) are 519 mm each 3 and 498 mm 3 The average time to reach this point was 55 days and 49 days, respectively. 2x10 6 and 10x10 6 The average tumor volume V (500 mm) of mice injected with HPAC cells 3 ) are each 557 mm 3 and 500 mm 3 The average time to reach that point was 24 days and 23 days, respectively.

[0270] 4.2.2 Mesothelin expression in Hela tumors and HPAC tumors in NSG mice The objective of the second study was to evaluate the expression levels of mesothelin in both HPAC tumors and HeLa tumors after tumor cells were subcutaneously injected into NSG mice and allowed to grow.

[0271] To put it simply, on day 0, the mice were randomly divided into two groups of three based on their individual body weight, and HeLa cells (10x10) were administered. 6 Cells / mouse) or HPAC cells (2x10) 6 The mice received SC injections of cells (mice). Body weight, survival, and behavior were observed daily. Tumor volume was measured three times a week. Tumor volume was 300-500 mm². 3 The tumors were collected once the required number of cells was reached. Mesothelin expression in the tumor samples was analyzed by immunohistochemistry (IHC). The last cell was killed on day 63 (end of experiment).

[0272] Both HPAC tumors and HeLa tumors grew within NSG mice. As observed in previous studies, HPAC tumors grew faster than HeLa tumors. The average tumor volume of mice injected with HeLa or HPAC cells was 300 mm². 3 These were reached in 40 and 28 days, respectively.

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

[0274] Based on this verification data, mesoCAR + To evaluate the antitumor activity of T cells, HPAC cells (2x10) 6 We decided to use cells (SC injection).

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

[0276] To put it simply, on day 0, three mice received an SC injection of HPAC-luc-GFP cells (2x10). 6 Cells / mouse). Body weight, survival, and behavior were observed daily. Tumor volume was measured three times a week, and bioluminescence imaging was performed on days 7, 14, and 24. Survival and behavior were observed daily.

[0277] These conditions provided sensitivity to this assay, so wild-type HPAC cells (SC injection, 2x10) were used. 6 mesoCAR using cells / mouse + We decided to evaluate T cell activity in vivo.

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

[0279] UCARTmeso and control T cells were produced using PBMCs derived from the same donor. Neither UCARTmeso nor control T cells were 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 TIFF0007830327000014.tif4915318th day: Production process complete (before freezing); % CAR + CD45 on day 18 + CD45 + / CAR + The percentage (%) (For P4-R2 CAR, this was measured using recombinant MSLN protein; for Meso1-R2 CAR and MESO2-R2 CAR, it was measured using L-protein by flow cytometry) % CD4 + CD45 on day 18 + CD45 + / CAR + / CD4 + Percentage (%) % CD8 + CD45 on day 18 + CD45 + / CAR + / CD8 + Percentage (%) % TCRαβ - CD45 on day 18 + CD45 + / TCRαβ - Percentage (%)

[0281] In short, 85 NSG mice were given subcutaneous injections of HPAC tumor cells (2x10) on day minus 7. 6 The mice received cell / mouse therapy. On day 0, 80 mice with tumors 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). In the case of UCARTmeso cells, the total number of cells to be injected was determined according to the percentage of CAR-positive cells in the batch, with a specified number of CAR-positive cells (1, 3, or 10x10) per mouse. 6 They started injecting CAR-positive cells.

[0283] The antitumor activity of candidate UCARTmeso CARs (P4-R2, Meso1-R2, and MESO2-R2) was evaluated by measuring tumor volume (Figures 15, 16, and 17). All UCARTmeso cells showed antitumor activity, but the level of activity differed among different CART T cells.

[0284] As shown in Figure 18, the higher the dose (10x10 6 Antitumor activity was observed in all CAR candidates. 3x10 6 MESO2-R2 CAR + The cells were unable to control HPAC tumor growth.

[0285] conclusion We established an animal / tumor model and therapeutic conditions that enable the evaluation of the antitumor activity of mesothelin-targeting CAR T cells. Using this animal model, we evaluated the in vivo activity of three candidate CARs, MESO2-R2, Meso1-R2, and P4-R2, and found that all CAR T cells exhibited 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 the CAR T cells with selected characteristics (R2 suicide switch and TRAC KO), the Meso1-R2 CAR T cell candidate showed the highest in vivo activity at the three doses evaluated.

[0288] 4.4 - Evaluation of the antitumor activity of dnTGFBRII-expressing UCARTmeso candidate genes. The objective of this study was to compare the antitumor activity of two UCARTmeso candidates (P4-R2, MESO1-R2) that also express dnTGFBRII in NSG mice. Briefly, on day 0, 4-6 mice / group were induced to HPAC cells (2x10⁻¹⁰). 6 The mice received SC injections of cells (mice). Body weight, survival, and behavior were observed daily. Tumor volume was measured three times a week. Survival and behavior were observed daily.

[0289] All dnTGFBRII-expressing UCARTmeso are produced using PBMCs derived from the same donor, and two doses of CAR are used. + T cells were evaluated (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 showed antitumor activity, but the levels of activity differed, with the MESO1 construct showing higher antitumor activity.

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

[0292] To conduct this research, we created and tested six types of genetically modified T cells (defined in Table 10).

[0293] (Table 10) Description of the six types of genetically modified T cells created for this study TIFF0007830327000015.tif75158

[0294] 5.1 Different UCART MESO production On day 0, frozen human peripheral blood mononuclear cells (PBMCs) from Hemacare (Northridge, California 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 to 37°C and 5% CO2. On day 1, PBMCs were counted, analyzed by flow cytometry to assess the percentage of 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 / 1 million CD3+ cells). The cells were then transferred to an incubator set to 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 seeded onto retronectin-coated plates. The plates were then transferred to an incubator set to 37°C and 5% CO2. On day 5, the T cells were washed and resuspended in X-vivo 15 medium supplemented with 5% AB serum and 350 UI / ml IL2. The cells were then transferred to an incubator set to 37°C and 5% CO2. On day 6, T cells were introduced with or without mRNA encoding the right and left arms of TGFBRII TALEN (SEQ ID NO: 157 and SEQ ID NO: 158) by simultaneous electroporation. Gene transfer was performed using the AgilePulse method. The cells were then transferred to an incubator set to 37°C and 5% CO2. On day 7, the T cells were washed and resuspended in X-vivo 15 medium supplemented with 5% AB serum and 350 UI / ml IL2. The cells were then transferred to an incubator set to 37°C and 5% CO2. T cells were grown using a GRex device between days 7 / 8 and 18. During the growth period, the cell cultures were incubated at 37°C under 5% CO2, and the culture medium was changed occasionally. On day 18, all UCART cells were cryopreserved for later use in in vitro and in vivo assays.

[0295] 5.2 Evaluation of the UCART cell population UCART cells were analyzed by flow cytometry on day 18. CAR surface expression was evaluated using biotinylated recombinant mesothelin protein that recognizes the scFv portion of CAR and streptavidin conjugated to PE. dnTGFBRII was evaluated using anti-TGFBRII (Abcam) and anti-mouse IgG antibodies conjugated to APC. CD4 and CD8 surface expression was evaluated using CD4 antibody conjugated to FITC and CD8 antibody conjugated to BV510. In addition, the stem cell properties of UCART cells in CAR+CD4+ or CAR+CD8+ positive cells were analyzed using anti-CD62L conjugated to PECy7 and anti-CD45RA conjugated to 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, dnTGFBR2 was detected in 32% and 17% of UCART cells when transduced with the P4-dnTGFBRII construct and the MESO1-dnTGFBRII construct, respectively. These results may reflect the lower expression of dnTGFBRII, which is located downstream of the 2A peptide and CAR.

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

[0298] In addition, stem cell analysis revealed that in CAR+CD4+ cells (Figure 21A), the proportion of P4CAR naive T cells (Tn) and memory stem T cells (Tscm) ranged from 1% to 3%, but this subset was higher and ranged at about 6% in T cells expressing MESO1 CAR. In CAR+CD8+ cells, this T cell subset also showed a range of 15% to 19% and 19% to 22% depending on whether the P4 construct or MESO1 construct was used, so this effect was observed, albeit less significantly (Figure 21B).

[0299] These results demonstrate that even with low expression or detection, MESO1 CARs yield a higher proportion of CD8+ (i.e., cytotoxicity) and a higher proportion of Tn and Tscm subsets compared to P4CARs. Importantly, inactivation of TGFBRII, whether by overexpression or knockout of the dnTGFBRII construct, did not affect any of the phenotypes analyzed.

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

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

[0303] 5.4 Evaluation of TGFb sensitivity To determine the effects of TGFB pathway inactivation by either KO or dnTGFBRII overexpression, genetically modified T cells listed in Table 9 were exposed to TGFb for 1 hour and analyzed by flow cytometry to evaluate the pSMAD2 / 3-positive vs. pSMAD2 / 3-negative fractions of CAR-positive cells. In a separate experimental set, produced UCART cells were exposed to recombinant mesothelin protein in or without TGFb and their proliferation was assessed by counting after 7 days.

[0304] Figure 24A shows that without inhibition of the TGFb pathway, over 95% of the CAR-positive fraction of UCART cells were pSMAD2 / 3 positive. Inhibition by dnTGFBRII overexpression resulted in 67% and 60% of the CAR-positive fraction of UCART cells expressing the P4 construct or MESO1 construct being pSMAD2 / 3 negative. Interestingly, inactivation by knockout (KO) of cells expressing the P4 construct or MESO1 construct 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 similarly inhibit antigen-mediated proliferation of UCARTs expressing either the P4 construct or the MESO1 construct. Interestingly, inhibition of the TGFB pathway, whether by knockout or dnTGFBRII overexpression, can reduce or even eliminate such inhibition.

Claims

1. - Extracellular ligand-binding domains including VH and VL derived from a monoclonal anti-mesothelin antibody, directed to the MSLN antigen polypeptide region SEQ ID NO:

25. - Transmembrane domain, and - Cytoplasmic domain containing the CD3 zeta signaling domain, and co-stimulatory domain A mesothelin-specific chimeric antigen receptor (CAR) comprising at least the following: The extracellular ligand-binding domain, - A variable heavy chain (VH) containing a CDR derived from antibody Meso1, which has at least 90% sequence identity with SEQ ID NO:9 (Meso1-VH) and has SEQ ID NO:3 (CDRH1-Meso1), SEQ ID NO:4 (CDRH2-Meso1), and SEQ ID NO:5 (CDRH3-Meso1), respectively, and Variable light chains (VLs) containing antibody Meso1-derived CDRs that have at least 90% sequence identity with SEQ ID NO:10 (Meso1-VL) and also have SEQ ID NO:6 (CDRL1-Meso1), SEQ ID NO:7 (CDRL2-Meso1), and SEQ ID NO:8 (CDRL3-Meso1), respectively. including, The aforementioned mesothelin-specific chimeric antigen receptor (CAR).

2. The mesothelin-specific chimeric antigen receptor according to claim 1, wherein the extracellular ligand-binding domain comprises a VH chain and a VL chain having at least 95% sequence identity with SEQ ID NO:9 (Meso1-VH) and SEQ ID NO:10 (Meso1-VL), respectively.

3. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 2, wherein the extracellular ligand-binding domain comprises a VH chain and a VL chain having at least 99% sequence identity with SEQ ID NO:9 (Meso1-VH) and SEQ ID NO:10 (Meso1-VL), respectively.

4. The mesothelin-specific chimeric antigen receptor (CAR) according to any one of claims 1 to 3, wherein the transmembrane domain has at least 90% sequence identity with SEQ ID NO:6 derived from CD8α.

5. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 4, wherein the transmembrane domain has at least 90% sequence identity with SEQ ID NO:6 derived from CD8α.

6. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 5, wherein the transmembrane domain has at least 95% sequence identity with SEQ ID NO:6 derived from CD8α.

7. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 6, wherein the transmembrane domain has at least 99% sequence identity with SEQ ID NO:6 derived from CD8α.

8. A mesothelin-specific chimeric antigen receptor (CAR) according to any one of claims 1 to 7, further comprising a hinge between the extracellular ligand-binding domain and the transmembrane domain, The mesothelin-specific chimeric antigen receptor (CAR) wherein the hinge is selected from the CD8α hinge, the IgG1 hinge, and the FcγRIIIα hinge.

9. A mesothelin-specific chimeric antigen receptor according to any one of claims 1 to 7, comprising a costimulatory domain derived from 4-1BB or CD28.

10. The mesothelin-specific chimeric antigen receptor according to claim 9, comprising a costimulatory domain derived from 4-1BB or CD28 having at least 90% identity with SEQ ID NO:

18.

11. The mesothelin-specific CAR according to any one of claims 1 to 10, wherein the CD3 zeta signaling domain has at least 90% identity with SEQ ID NO:

19.

12. The mesothelin-specific CAR according to any one of claims 1 to 11, wherein the CD3 zeta signaling domain comprises a signal peptide.

13. A mesothelin-specific chimeric antigen receptor (CAR) according to any one of claims 1 to 12, having at least 90% overall amino acid sequence identity with SEQ ID NO:21 (Meso1 CAR) or SEQ ID NO:22 (Meso1-R2 CAR).

14. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 13, having at least 90% overall amino acid sequence identity with SEQ ID NO:21 (Meso1 CAR) or SEQ ID NO:22 (Meso1-R2 CAR).

15. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 14, having at least 95% overall amino acid sequence identity with SEQ ID NO:21 (Meso1 CAR) or SEQ ID NO:22 (Meso1-R2 CAR).

16. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 15, having at least 99% overall amino acid sequence identity with SEQ ID NO:21 (Meso1 CAR) or SEQ ID NO:22 (Meso1-R2 CAR).

17. A polynucleotide encoding a chimeric antigen receptor according to any one of claims 1 to 16.

18. Modified immune cells comprising the polynucleotide described in claim 17.

19. Modified immune cells expressing a mesothelin-specific chimeric antigen receptor according to any one of claims 1 to 16 on their cell surface membrane.

20. A modified immune cell according to claim 19, which is a T lymphocyte.

21. The modified immune cells according to any one of claims 18 to 20, wherein the expression of TCRs is reduced or suppressed in the immune cells.

22. The modified immune cell according to claim 21, wherein the expression of one gene encoding TCR alpha or TCR beta is reduced or suppressed.

23. Modified immune cells according to any one of claims 18 to 22, which are mutated to confer resistance to at least one immunosuppressant.

24. Modified immune cells according to claim 23, which have been mutated to confer resistance to an anti-CD52 antibody.

25. Modified immune cells according to any one of claims 18 to 24, further mutated to confer resistance to at least one chemotherapy agent.

26. Modified immune cells according to claim 25, further mutated to confer resistance to at least purine analog drugs.

27. Modified immune cells according to any one of claims 18 to 26, which are modified by inactivating genes encoding MHCI components.

28. Modified immune cells according to claim 27, which are modified by inactivating a gene encoding HLA or B2m.

29. A modified immune cell according to any one of claims 18 to 28, wherein the CAR is as described in any one of claims 1 to 16.

30. NK cell inhibitors selected from the group consisting of HLAG, HLAE, and ULBP1; A CRS inhibitor selected from the group consisting of mutant IL6Ra, sGP130, or IL18-BP; - Cytochrome P450, CYP2D6-1, CYP2D6-2, CYP2C9, CYP3A4, CYP2C19, or CYP1A2 that cause hypersensitivity of the aforementioned immune cells to the drug; Drug-inducing agents: dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin, or methylguanine transferase (MGMT), mTORmut, or Lckmut; - A chemokine or cytokine selected from the group consisting of IL-2, IL-12, and IL-15; - Chemokine receptors selected from the group consisting of CCR2, CXCR2, and CXCR4; - Inhibitors of tumor-associated macrophage (TAM) secretion that enhance the therapeutic activity of the aforementioned immune cells. The modified immune cell according to any one of claims 18 to 29, which also expresses another exogenous gene sequence selected from those encoding the same gene.

31. The modified immune cell according to claim 30, wherein the TAM is a CCR2 / CCL2 neutralizing agent.

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