mesothelin-specific chimeric antigen receptor (car) for solid tumor cancer immunotherapy

Abstract

The present invention relates to engineered immune cells expressing a chimeric antigen receptor (anti-mesothelin CAR) specific for mesothelin (MLSN) and their use in the treatment of solid tumors, particularly suitable for allogeneic cell immunotherapy.

Description

Technical Field

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

[0002] Chimeric antigen receptors (CARs) are synthetic receptors that target T cells to cell surface antigens and enhance T cell function and persistence. Mesothelin is a cell surface antigen involved in tumor invasion and is highly expressed in mesothelioma, as well as lung, pancreatic, breast, ovarian, and other cancers. Encouragingly, recent clinical trials evaluating active immunization or immune conjugates in patients with pancreatic or mesothelioma have shown reactivity and no toxicity. In conclusion, these findings and preclinical CAR therapy models using systemic or local T cell delivery favorably support mesothelin CAR therapy in a variety of solid tumors.

[0003] Given the potential high efficiency of CAR therapy, it is important to identify appropriate antigens that address solid tumors and thus achieve tumor eradication with minimal or tolerable on-target / off-tumor toxicity to healthy tissues.

[0004] The solid tumor CAR targets in the study are gene products that are altered mainly by genetic mutations or altered splicing (EGFRvIII), altered glycosylation patterns (MUC1), cancer-testis antigen-derived peptides (MAGE), overexpressed differentiation antigens CEA, PSMA, GD2, MUC16, HER2 / ERBB2 and mesothelin (MSLN), or tumor-associated matrix (FAP and VEGFR).

[0005] Although overexpressed antigens are numerous and relatively common, their high sensitivity to low levels of antigen expression (which can be greater than that of monoclonal antibodies) has raised concerns about “target-intervention / de-tumor” side effects. For example, the use of high-dose ERBB2 CAR T cells has led to lethal adverse events, partly attributed to low levels of ERBB2 expression in healthy lung epithelial cells and cardiovascular cells [Morgan, RA et al. (2010) Case report of aserious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther. 18:843–51]. Therefore, the optimal solid tumor antigen target is one whose expression is restricted to tumor cells or occurs only at very low levels in wasting normal tissues.

[0006] Given its low expression on normal mesothelial cells and high expression in a broad spectrum of solid tumors, MSLN has become an attractive target for cancer immunotherapy. Currently reported MSLN-targeted immunotherapies support a good safety profile. MSLN is a potential CAR target in some common solid tumors, such as at least esophageal cancer, breast cancer, gastric cancer, hepatobiliary hepatocellular carcinoma, pancreatic cancer, colon cancer, lung cancer, thymic carcinoma, 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].

[0007] MSLN is a glycoprotein anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) domain. It is initially synthesized as a 69 kDa cell-surface protein. Upon cleavage at the N-terminus by furin, the 40-kDa C-terminal fragment remains attached to the membrane, releasing a soluble 32-kDa N-terminal fragment, termed megakaryocyte-enhancing factor (MPF) [Pastan, I., Hassan, R. (2014) Discovery of mesothelin and exploiting it as a target for immunotherapy. Cancer. Res. 74:2907–12]. A soluble form of MSLN, termed soluble MSLN-associated protein (SMRP), has also been detected in the serum of patients with solid tumors. SMRP is generated through alternative splicing of the mature form of MSLN induced by TNFα-convertase ADAM17 or through proteolytic cleavage.

[0008] Given that MSLN knockout mice exhibit normal development, proliferation, and blood cell counts, the biological function of MSLN appears not to be essential in normal tissues. Instead, preclinical and clinical studies increasingly demonstrate that aberrant MSLN expression plays an active role in both tumor malignant transformation and tumor invasiveness by promoting cancer cell proliferation, thereby promoting local invasion and metastasis and conferring resistance to apoptosis induced by cytotoxic agents. MSLN can function bidirectionally by directly activating intracellular pathways via its GPI domain or through interaction with its receptor CA125 / MUC16. MSLN overexpression alone is sufficient to constitutively activate the intracellular pathways of NFκB, MAPK, and PI3K, thereby promoting cell proliferation and resistance to apoptosis.

[0009] Physiologically, MSLN is expressed on the mesothelial cells of the peritoneum, pleural cavity, and pericardium; it is expressed at a minimal level on the surface of epithelial cells of the trachea, ovary, reticulum testis, tonsils, and fallopian tubes. Initially, MSLN overexpression was observed in mesothelioma and ovarian cancer, and subsequently in lung cancer, esophageal cancer, pancreatic cancer, gastric cancer, biliary cancer, endometrial cancer, thymic cancer, colon cancer, and breast cancer. Therefore, MSLN overexpression alone has an estimated incidence rate of 340,000 patients and a prevalence rate of 2 million patients annually in the United States alone.

[0010] CARs typically consist of an extracellular domain, hinge, transmembrane domain, and intracellular domain (usually containing signal transduction domains derived from CD3ζ and co-stimulatory receptors) derived from a single-stranded variable fragment (scFv). Second-generation CARs further enhance T cell function and persistence by introducing signal transduction domains that rescue and amplify activation signals delivered via the CD3ζ cytoplasmic domain. Dual signal transduction prevents T cell dysfunction and improves persistence and function by enhancing T cell proliferation and cytokine production (IFNγ and IL2) and reducing activation-induced cell death through recruitment of PI3K, TRAF, and / or other pathways. Third-generation CARs contain three signal transduction domains, typically those encompassing CD3ζ, and two co-stimulatory domains, such as CD28 and 4-1BB or CD28 and OX40. Compared to second-generation CARs, third-generation CARs exhibit inconsistent in vivo antitumor activity. Selecting appropriate co-stimulatory domains is essential for maintaining CAR T cell activity and calibrating T cell persistence. However, the ideal co-stimulatory domain can be environment-based, as CAR function depends on a variety of external factors, such as antigen density, CAR chemometrics, CAR affinity, and the immunological properties of the tumor microenvironment.

[0011] For effective initiation of T cell signaling, the spatial distance between the CAR and its target antigen can be equally important, but it depends on a completely different set of structural elements associated with the epitope position on the target molecule and the spacer domain between the scFv and the T cell membrane. Some studies have demonstrated that the same epitope can activate CAR-T cells more efficiently when expressed at a membrane-proximal position than at a membrane-distal position. For example, Hombach et al. demonstrated that CAR T cells recognizing a membrane-distal “N” epitope of carcinoembryonic antigen (CEA) were only moderately activated; however, when they engineered recombinant CEA protein to express the N epitope at a membrane-proximal position, the same CAR T cells were activated more effectively [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]. This suggests that targeting certain distal membrane epitopes on tumor cells can allow large phosphatases, such as CD45 and CD148, to enter the synapse and inhibit phosphorylation events initiated via CAR binding. Modulating the extracellular spacer sequence between the T cell membrane and the ligand-binding scFv to promote synapse formation could help overcome the stereochemical limitations imposed by the location of the target epitope.

[0012] The specific concern regarding MSLN CARs lies in interference from soluble MSLNs, which in principle can occupy and block scFv portions. However, MSLN CAR T cell activation (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].

[0013] The principle that genetically modified T cells expressing novel synthetic CARs can effectively treat advanced refractory cancers has been established, but many questions remain regarding the full potential of this novel therapeutic approach. Engineering safer and more effective CARs for solid tumor cancer therapy requires going beyond conventional empirical approaches to receptor design and cell engineering; ideally, it should be guided by our knowledge of TCR signaling, T cell biology, and the manipulation of the tumor microenvironment. It is now evident that the binding affinity between CARs and target cells, the Kon / Koff ratio, and spatial constraints can influence the ability of CARs to optimally activate for tumor recognition, particularly for T cells in 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].

[0014] Considering the above, screening scFvs solely for their affinity for MSLN antigens or their ability to induce T cell activation and proliferation in vitro does not seem sufficient to engineer the most suitable CAR T cells. Current data indicate that, to date, the optimal CAR affinity cannot be deductively determined for a single target molecule, and there is no unbiased method to identify the ideal affinity range that would allow for the best in vivo results [Srivastava, S. and Ridell, RS (2015) Engineering CAR-TCells: Design Concepts. Trends Immunol. 36(8):494–502].

[0015] Furthermore, the solid tumor microenvironment presents several barriers to MSLN CAR-T cells, which can limit their antitumor efficacy. To optimize CAR T cell efficiency, various approaches are being evaluated to control the host tumor microenvironment or to generate “armored” CAR T cells capable of overcoming immune barriers. These strategies include (i) promoting CAR T cell infiltration, (ii) enhancing the functional durability of CAR T cells, (iii) improving CAR T cells to overcome inhibitory signals encountered in the tumor microenvironment, and (iv) improving safety by preventing on-target / detumescent toxicity. Among 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 diacylglycerolkinases. Cancer Res. 73:3566–77] demonstrated, for example, that deletion of the DGKζ gene significantly enhanced the antitumor activity of MSLN CAR T cells, as demonstrated by enhanced secretion of effector cytokines, expression of FASL and TRAIL, and in vitro cytotoxicity.

[0016] On the other hand, different strategies have been developed to address the risks of CAR T cell on-target / detumescent toxicity and improve safety.

[0017] One approach involves transfecting mRNA encoding MSLN CAR, resulting in transient CAR expression over just a few days. This approach has shown promise in preclinical models; multiple infusions of mRNA CAR T cells produced robust in vivo antitumor activity [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]. However, transient CAR expression can limit the long-term efficacy of the therapy. A clinical trial at the University of Pennsylvania using autologous T cells electroporated with mRNA encoding a second-generation MSLN CAR (SS1-4-1BB CAR) resulted in a moderate clinical response and a transient increase in serum inflammatory cytokines, including IL12, IL6, G-CSF, MIP1β, MCP1, IL1RA, and RANTES.

[0018] If adverse events occur, another way to improve T-cell safety is to use suicide genes to eliminate T cells. In this case, CAR T cells can be eliminated by drug-induced activation of suicide genes, such as herpes simplex thymidine kinase (HSV-TK), gene-inducible caspase-9, or EGFRΔ gene.

[0019] In a previous patent application, WO2016120216, the applicant developed an alternative system for inducing gene suicide, which involves inserting a foreign epitope into the CAR structure recognized by clinically approved antibodies, such as rituximab. This allows for the partial or complete depletion of CAR-positive immune cells infused into a patient as needed. One advantage of this approach is that, in addition to the CAR, no suicide gene must be co-expressed in the cells. However, this epitope insertion can influence the overall CAR structure and can alter how scFv interacts with its homoantigens.

[0020] In terms of its use in allogeneic therapy strategies, this invention aims to address all or part of the above limitations by providing safer engineered immune CAR-positive cells to target MSLN-expressing cells in vivo, such as solid tumors. Summary of the Invention

[0021] The present invention relates primarily to mesothelin-specific chimeric antigen receptors (CARs) expressed in immune cells, preferably T cells, for their therapeutic use against malignant cells or tissues expressing mesothelin.

[0022] These types of CARs typically include the following structure:

[0023] -Extracellular ligand-binding domains, containing VH and VL from monoclonal anti-mesothelin antibodies;

[0024] - Transmembrane domains; and

[0025] -Cytoplasmic domains, including CD3ξ signal transduction domains and co-stimulatory domains.

[0026] The extracellular ligand-binding domain of the CAR according to the invention preferably comprises one or more scFv segments from an antibody called meso1, and more specifically includes CDRs from thereof of SEQ ID NO: 3, 4, 5, 6, 7 and / or 8.

[0027] According to a preferred aspect, the extracellular ligand-binding domain of the CAR comprises:

[0028] - A variable-heavy VH chain comprising a CDR from antibody meso1, wherein the CDR has 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.

[0029] - A variable weight VL chain containing a CDR from antibody Meso1, which has 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.

[0030] The anti-mesothelin CARs of the present invention form exogenous polypeptide sequences that are expressed by immune cells for their exposure on the cell surface. These are encoded by exogenous (relative to the original genome of the immune cell) polynucleotide sequences, preferably inserted into specific genomic loci, such as the TCR, B2m, or PD1 loci, using a rare endonuclease.

[0031] According to some embodiments, the CAR also includes other exogenous polypeptide sequences comprising epitopes that can be targeted by clinically approved antibodies for in vivo depletion or by other ligands for their in vivo or in vitro detection or purification. These other exogenous polypeptide segments can be specifically recognized by rituximab, such as the segment referred to as "R2" in this specification.

[0032] More specifically, this invention relates to immune cells or populations of immune cells transformed with an anti-mesothelin CAR polynucleotide sequence comprising the said polynucleotide sequence and / or an anti-mesothelin CAR sequence expressing a polypeptide.

[0033] According to the present invention, these engineered immune cells or cell populations can be further genetically engineered, mutated, or genetically edited to improve their therapeutic suitability or efficacy, such as improving their persistence or lifespan. According to a preferred aspect, the engineered immune cells according to the present invention combine the expression of the anti-mesothelin CAR sequence with other gene modifications that reduce the expression of their endogenous genes such as TCR, HLA, and / or B2m genes. TGFβ receptor

[0034] According to other preferred aspects, engineered immune cells can be mutated to improve their CAR-dependent immune activation, particularly by reducing or inhibiting the expression of immune checkpoint proteins and / or their receptors such as PD1 / PDL1.

[0035] According to other preferred aspects, engineered immune cells can be mutated to improve their CAR-dependent immune activation, particularly by reducing or inhibiting the TGFβ signaling pathway.

[0036] According to other preferred aspects, other exogenous gene sequences may also be inserted, co-transfected or co-expressed with the anti-mesothelin CAR of the present invention, specifically with an inhibitor or decoy of the TGFβ receptor, such as an inhibitor of the dominant inactivated TGFβ receptor (dnTGFβRII).

[0037] Other examples of exogenous gene sequences whose expression can be combined with anti-mesothelin CAR expression to improve the therapeutic efficacy of immune cells are provided, specifically the following:

[0038] -NK cell inhibitors, such as HLAG, HLAE or ULBP1;

[0039] -CRS inhibitors, such as mutant IL6Ra, sGP130, or IL18-BP; or

[0040] - Cytochrome P450, CYP2D6-1, CYP2D6-2, CYP2C9, CYP3A4, CYP2C19, or CYP1A2, conferring hypersensitivity of the immune cells to drugs such as cyclophosphamide and / or isophosphamide.

[0041] - Dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin or methylguanine transferase (MGMT), mTORmut or Lckmut, confer drug resistance

[0042] - Chemokines or cytokines, such as IL-2, IL-12 and IL-15.

[0043] - Inhibitors of tumor-associated macrophage (TAM) secretion, such as CCR2 / CCL2 neutralizers, to enhance the therapeutic activity of immune cells;

[0044] The engineered immune cells according to the invention are particularly suitable for treating diseases characterized by cells expressing mesothelin, specifically solid tumors, such as esophageal cancer, breast cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, colon cancer, lung cancer, thymic cancer, mesothelioma, ovarian cancer and / or endometrial cancer.

[0045] Therefore, the present invention covers methods for producing engineered cells, the resulting therapeutic cells, cell populations containing these cells and therapeutic compositions containing them, as well as treatment methods capable of addressing pathologies induced by cells expressing mesothelin.

[0046] Explanation of the attached figures and tables

[0047] Figure 1A: A schematic diagram of the preferred form of the anti-mesothelin CAR according to the present invention. A: CAR, comprising: V1 and V2, which represent sequences containing ScFv-specifically binding mesothelin, such as VH and VL or VL and VH from meso1 antibody; L: adapter; R1 and R2, representing exogenous epitopes, such as CD20 epitopes recognized by human-approved monoclonal anti-CD20 antibodies (e.g., rituximab…); TM: transmembrane domain; CO-STIM: co-stimulatory domain; ITAM: stimulatory domain, comprising ITAM (immunoreceptor tyrosine-based activation motif (ITAM)). This CAR typically has at least 80% polypeptide sequence identity with SEQ ID NO: 21. B: CAR without exogenous epitopes, comprising: VH and VL or VL and VH from meso1 antibody; (G4S)3 adapter; CD8a hinge domain; CD8a transmembrane domain; 4-1BB co-stimulatory domain; and CD3z signal transduction domain.

[0048] Figure 2 : A schematic diagram showing immune cells expressing anti-MSLN CAR according to the present invention, wherein optional genetic properties have been further introduced. A. Co-expression with an inactive variant of the TGFβ receptor (e.g., dnTGFβRII) and / or genetic reduction or inactivation of TGFβ receptor expression to resist tumor-induced immunosuppression. Reduction or inactivation of TCR expression (e.g., TCRα) to reduce allogeneic reactivity of immune T cells, leading to GvHD. B. Genetic reduction or inactivation of TGFβ receptor, TCR, and / or CD52 expression via gene editing tools (e.g., TALEN).

[0049] Figure 3 Mesothelin protein expression was observed 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 an APC-conjugated goat anti-mouse polyclonal antibody as the secondary antibody.

[0050] Figure 4 Quantitative expression of mesothelin protein on the surface of HeLa and HPAC cells. MSLN expression levels were analyzed by flow cytometry using a fluorescence-based QIFIKIT.

[0051] Figure 5 : This figure represents a series of killing assays performed in vitro to evaluate the activation of anti-mesothelin CAR-positive cells.

[0052] Figure 6 Original [TCRα] negCAR expression on the surface of T cells (UCART cells). Cryopreserved UCART cells derived from a single donor were stained with histidine-tagged recombinant human cortisol and PE-conjugated antihistamines or biotinylated protein L and Vioblue-bound streptavidin and analyzed by flow cytometry.

[0053] Figure 7 : Expression of CD4 and CD8 in the CAR+ portion of UCART cells. Cryopreserved UCART cells derived from a single donor were stained with FITC-bound anti-CD4 and BV510-bound anti-CD8 antibodies and analyzed by flow cytometry.

[0054] Figure 8 : Production of IFNg in UCART cells. Fresh UCART cells 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.

[0055] Figure 9 : Displayed from the original [TCRα] neg The graph showing the percentage of cytolysis produced by a continuous killing assay of HPAC cells from T cells (UCART cells) is shown in the figure below. Figure 5 As shown, cryopreserved UCART cells from a donor were co-cultured with HPAC cells at an E:T ratio of 1:2 (A) or 1:8 (B) for 15 days.

[0056] Figure 10 TCRαβ expression on the surface of UCART cells. Cryopreserved UCART cells derived from a single donor were stained with an anti-TCRαβ antibody bound to PEvio770 and analyzed by flow cytometry.

[0057] Figure 11 Unengineered T cells, TRAC gene knockout T cells, and TRAC gene knockout T cells with TCRαβ + Flow cytometry analysis of TCRαβ receptor expression on the surface of depleted T cells.

[0058] Figure 12 Flow cytometry analysis of CD25 expression on the surface of unengineered T cells, TRAC knockout T cells, and TRAC knockout T cells depleted in TCRαβ+ cells when exposed 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), respectively.

[0059] Figure 13 Measurement of UCART cell depletion in CDC mediated by rituximab via exogenous epitope peptide R2 included in CAR P4-R2, Meso1-R2 and MESO2-R2.

[0060] Figure 14 : A graph showing the measurement of SMAD2-3 phosphorylation when exposed to TGFβ from CART cells derived from two different donors.

[0061] Figure 15 : Using 3 doses (1×10 6 3×10 6 and 10×10 6 Mean tumor volume (HPAC MSLN+ cells) in mice injected with UCARTmeso (TCR-negative anti-mesothelin P4-R2 CAR-positive cells) per CAR+ cell / mouse.

[0062] Figure 16 : Using 3 doses (1×10 6 3×10 6 and 10×10 6 The average tumor volume in mice injected with UCARTmeso cells (TCR-negative anti-mesothelin Meso2-R2 CAR-positive cells) (1 CAR+ cells / mouse).

[0063] Figure 17 : Using 3 doses (1×10 6 3×10 6 and 10×10 6 The average tumor volume in mice injected with UCARTmeso cells (TCR-negative anti-mesothelin Meso1-R2 CAR-positive cells) (1 CAR+ cells / mouse).

[0064] Figure 18 : Using 3 doses (A: 1×10 6 B: 3×10 6 And C: 10×10 6 Mean tumor volume + / - standard deviation in mice injected with UCARTmeso cells (CAR+ cells / mouse). Comparison of different doses of Meso1-R2, P4-R2, and MESO2-R2.

[0065] Figure 19 : with two doses (3×10 6 and 10×10 6The average tumor volume in mice injected with UCARTmeso cells that also express dnTGFBRII (1 CAR+ cells / mouse) was measured in 1000 cells.

[0066] Figure 20 A. Comparison of CAR and dnTGFBRII detection in UCARTMeso cells expressing P4 or MESO1 constructs with dnTGFBRII. B. Percentage of CD4+ and CD8+ in the CAR-positive fraction of UCARTMeso cells generated in Example 5.

[0067] Figure 21 The percentage of Temra, Tem, Tcm; and Tn / scm cells observed in the CAR+CD4+ fraction (A.) or CAR+CD8+ fraction (B.) of UCARTMeso cells generated in Example 5.

[0068] Figure 22 The percentage of H226 cell killing by different UCARTMeso cells with or without TGFBRII pathway inactivation or non-inactivation, achieved by knockout (KO) or dominant inactivation of TGFBRII (dnTGFBRII) expression.

[0069] Figure 23 Production of IFNg from UCARTMeso cells generated in Example 5 and exposed to (A.) or not exposed to (B.) recombinant mesothelin.

[0070] Figure 24 Evaluation of the sensitivity of UCARTmeso cells to TGFb. A. Percentage of pSMAD2 / 3 positive (gray) or negative (black) cells in the CAR-positive fraction of UCARTmeso produced in Example 5 when treated with TGFb. B. Percentage of proliferation inhibition of different UCARTmeso cells in the presence of TGFb and recombinant mesothelin.

[0071] Table 1: Amino acid sequences of different domains of the P4, Meso1, and MESO2 scFv constituting the CARs shown in the examples.

[0072] Table 2: Amino acid sequences, excluding scFv, that constitute different domains of the MSLN CAR according to the present invention.

[0073] Table 3: Examples of mAb-specific epitopes (and their corresponding mAbs) that can be used in the extracellular binding domain of the CAR of the present invention for engineered cell sorting and depletion.

[0074] Table 4: Amino acid sequences of P4-R2, Meso1-R2, Meso1 and MESO2-R2 CARs.

[0075] 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.

[0076] Table 6: TALE nuclease target sequences of the TGFβRII gene.

[0077] Table 7: CRISPR target sequences of the TGFβRII gene.

[0078] Table 8: Genomic sequences targeted by TALE nuclease (TALEN) to inactivate TCR and CD52.

[0079] Table 9: Characteristics of the engineered T cell populations used in the examples.

[0080] Table 10: Description of the six types of genetically modified T cells produced in the study provided in Example 5. Detailed Implementation

[0081] Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the fields of gene therapy, biochemistry, genetics and molecular biology.

[0082] All methods and materials similar to or equivalent to those described herein, wherein suitable methods and materials are described herein, may be used to practice or test the invention. All patent publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification, including definitions, shall prevail. Furthermore, unless otherwise stated, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0083] Unless otherwise stated, the practice of this invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the scope of the art. These techniques are well described in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, 3rd 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 & S.J. Higgins, ed., 1984); Transcription And Translation (BD Harmes & S.J. Higgins, ed., 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); the series, Methods in Enzymology (edited by J. Abelson and M. Simon, Academic Press, Inc., New York), specifically, Volumes 154 and 155 (edited by Wu et al.) and Volume 185, "Gene Expression Technology" (edited by D. Goeddel); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and MPCalos (Editor-in-Chief, 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Editor-in-chief Mayer and Walker, Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Editor-in-Chief DMWeir and CC Blackwell, 1986); and Manipulating the Mouse Embryo (Editor-in-Chief, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0084] This invention relates to a general method of treating solid tumors by adoptive immune cells targeting the transmembrane protein MSLN, specifically covering a specific epitope region of mesothelin in the polypeptide sequence SEQ ID NO: 25, and more specifically by using allogeneic CAR-T cells targeting this epitope. This method has proven particularly effective.

[0085] This document describes a method for generating engineered immune cells targeting human mesothelin (MSLN_human, known as Q13421 in the Uniprot database), and more specifically, the mesothelin polypeptide region represented by SEQ ID NO: 25, which is present on the surface of malignant cells. As shown in the experimental section of this specification, effective CAR T cells have been generated by targeting the antigen region comprising or consisting of SEQ ID NO: 25, specifically by using an scFv containing the antibody Meso1 comprising SEQ ID NO: 9 and SEQ ID NO: 10.

[0086] The engineered immune cells produced according to the present invention, typically NK or T cells containing CARs comprising SEQ ID NO: 9 and / or SEQ ID NO: 10, have shown higher activation, potency, cytotoxic activity, cytokine release, and in vivo persistence than their counterparts with other prior anti-mesothelin CARs.

[0087] Therefore, the present invention relates to CAR immune cells that target the specific epitope contained in the sequence SEQ ID NO.25 of the MSLN protein, which are present on the surface of malignant cells, and are specifically engineered for the treatment of solid tumors.

[0088] Design of MSLN-CAR for expression in immune cells:

[0089] “ Chimeric antigen receptor "CAR" refers to a recombinant receptor containing a targeting moiety associated with one or more signal transduction domains in a single fusion molecule. Typically, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv) containing a variable light and heavy fragment of the monoclonal antibody linked by a flexible linker. Binding moieties based on receptor or ligand domains have also been successfully used. The signal transduction domains of a CAR are typically derived from the cytoplasmic region of the CD3ζ or Fc receptor γ chain and are generally combined with signal transduction domains from co-stimulatory molecules, including CD28, OX-40 (CD134), ICOS, and 4-1BB (CD137), to enhance cell survival and proliferation. CARs are typically expressed in effector immune cells to redirect their immune activity against antigens expressed on the surface of tumor cells from various malignancies, including lymphomas and solid tumors. Components of a CAR are any functional subunits of the CAR encoded by an exogenous polynucleotide sequence introduced into the cell. For example, this component may aid in interaction with the target antigen, stability, or localization of the CAR within the cell.

[0090] Typically, this type of CAR includes:

[0091] -Extracellular ligand-binding domains, containing VH and VL from monoclonal anti-mesothelin antibodies;

[0092] - Transmembrane domains; and

[0093] - Signal transduction domain, preferably a cytoplasmic domain, comprising a CD3ξ signal transduction domain and a co-stimulatory domain.

[0094] More specifically, the present invention relates to a CAR expressed in immune cells such as NK or T cells, said CAR comprising an antigen-binding domain that specifically binds to SEQ ID NO: 25.

[0095] According to a preferred aspect, the mesothelin-specific chimeric antigen receptor (CAR) of the present invention has an extracellular ligand-binding domain comprising at least one CDR region from a variable-weight VH chain of 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 at least one CDR region from a variable-weight VL chain of said 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).

[0096] Typically, the extracellular ligand binding domain includes:

[0097] - A variable-heavy VH chain comprising a CDR from antibody Meso1, wherein the CDR has 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 / or

[0098] - A variable weight VL chain containing a CDR from antibody Meso1, which has at least 90% identity with SEQ ID NO: 6 (CDRL1-Meso1), SEQ ID NO: 7 (CDRL2-Meso1), and SEQ ID NO: 8 (CDRL3-Meso1), respectively.

[0099] According to a preferred embodiment of the invention, the mesothelin-specific chimeric antigen receptor has an extracellular ligand-binding domain comprising VH and VL chains 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. Typically, framework residues in the framework region can be replaced with corresponding residues from a CDR donor antibody to alter, for example, improve antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interaction between the CDR and framework residues to identify framework residues important for antigen binding and by sequence alignment to identify uncommon 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, the entire contents of which are incorporated herein by reference].

[0100] Various embodiments of the invention are provided based on the features set forth in the claims, drawing upon the conventional practice and knowledge of those skilled in the art. Detailed sequences contained in the CAR according to the invention are described in detail in Tables 1, 2, 3, and 4, wherein each row or column should be considered an independent embodiment of the invention.

[0101] Table 1: Amino acid sequences constituting different domains of P4, Meso1, and MESO2 scFv

[0102]

[0103]

[0104] Table 2: Amino acid sequences, excluding scFv, constituting different domains of the MSLN CAR according to the present invention

[0105]

[0106] Table 3: Amino acid sequences of P4-R2, Meso1-R2, Meso1, and MESO2-R2 CARs

[0107]

[0108] Table 4: Complete polypeptide sequences of MSLN CAR, dnTGFβRII, and MSLN epitope regions

[0109]

[0110]

[0111]

[0112]

[0113] The signal transduction domain or intracellular signal transduction domain of the CAR according to the present invention is responsible for intracellular signal transduction following the binding of the extracellular ligand-binding domain to the target, which leads to the activation of immune cells and immune responses. In other words, the signal transduction domain is responsible for the activation of at least one of the normal effector functions of immune cells expressing CAR. For example, the effector functions of T cells can be cytolytic activity or helper activity, including cytokine secretion. Therefore, the term "signal transduction domain" refers to a protein portion that transduces effector signaling and directs the cell to perform specialized functions.

[0114] Preferred examples of signal transduction domains for CARs may be cytoplasmic sequences of T-cell receptors and co-receptors that function consistently to induce signal transduction following antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities. The signal transduction domain comprises two distinct classes of cytoplasmic signal transduction sequences: those that induce antigen-dependent primary activation and those that function independently of the antigen to provide secondary or co-stimulatory signals. Primary cytoplasmic signal transduction sequences may comprise signal transduction motifs known as immune receptor tyrosine-based activation motifs (ITAMs). ITAMs are well-defined signal transduction motifs present in the cytoplasmic tails of various receptors that act as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAMs used in this invention may include, as non-limiting examples, those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the CAR signal transduction domain may include a CD3ζ signal transduction domain having an amino acid sequence that has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity with the amino acid sequence selected from the group consisting of (SEQ ID NO: 9).

[0115] In a specific embodiment, the signal transduction domain of the CAR of the present invention includes a co-stimulatory signaling molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand, which is required for an effective immune response. A "co-stimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a homologous co-stimulatory molecule on a T cell, thereby providing a signal in addition to the primary signal provided by, for example, the binding of the TCR / CD3 complex to an MHC molecule carrying a loaded peptide, which mediates T cell responses, including but not limited to proliferation activation and differentiation. Co-stimulatory ligands may 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), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. In particular, costimulatory ligands also include antibodies that specifically bind to costimulatory molecules presented on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0116] In a preferred embodiment, the signal transduction domain of the CAR of the present invention comprises a portion of a co-stimulatory signaling molecule selected from the group consisting of 4-1BB (GenBank: AAA53133.) and CD28 (NP_006130.1). Specifically, the signal transduction domain of the CAR of the present invention comprises 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. Therefore, the mesothelin-specific chimeric antigen receptor according to the present invention preferably comprises a CD3ξ signal transduction domain having at least 80% identity with SEQ ID NO. 19, and generally comprises a co-stimulatory domain having at least 80% identity with SEQ ID NO. 18. (4-1BB).

[0117] The CAR according to the invention is typically expressed on the surface membrane of a cell. Therefore, the CAR also includes a transmembrane domain. A key characteristic of a suitable transmembrane domain includes the ability to be expressed on the cell surface, preferably an immune cell, specifically a lymphocyte or natural killer (NK) cell, and the ability to interact with each other to direct a cellular response of the immune cell against a predetermined target cell. The transmembrane domain can be derived from a natural or synthetic source. The transmembrane domain can be derived from any membrane-binding protein or transmembrane protein. As a non-limiting example, a transmembrane polypeptide can be a subunit of a T cell receptor, such as α, β, γ, or ζ; a polypeptide constituting the CD3 complex; an IL2 receptor p55 (α chain), p75 (β chain), or γ chain; a subunit chain of an Fc receptor, specifically an Fcγ receptor III or CD protein. Alternatively, the transmembrane domain can be synthetic and can primarily contain hydrophobic residues such as leucine and valine. In a preferred embodiment, the transmembrane domain is derived from a human CD8α 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" generally refers to any oligopeptide or polypeptide used to link the transmembrane domain to the extracellular ligand domain. Specifically, the hinge region provides greater flexibility and accessibility to the extracellular ligand-binding domain. 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 entirely or partially from naturally occurring molecules, such as from the extracellular regions of CD8, CD4, or CD28, or from the antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or it may be a fully synthetic hinge sequence. In a preferred embodiment, the hinge domain comprises a portion of the human CD8α chain, FcγRIIIα receptor, or IgG1, or preferably a hinge polypeptide exhibiting at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity with these polypeptides.

[0118] Therefore, the mesothelin-specific chimeric antigen receptor (CAR) according to the invention comprises a hinge located between an extracellular ligand-binding domain and a transmembrane domain, said hinge being typically selected from CD8α hinges, IgG1 hinges, and FcγRIIIα hinges, or polypeptides sharing at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with these polypeptides, specifically with SEQ ID NO: 16 (CD8α).

[0119] The CAR according to the invention generally also includes a transmembrane domain (TM), which is preferably selected from CD8α and 4-1BB, more preferably selected from CD8α-TM or a polypeptide showing at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity with SEQ ID NO.17 (CD8αTM).

[0120] According to other embodiments, the mesothelin-specific CAR according to the present invention includes a safety switch for classifying, purifying, and / or depleting engineered immune cells. Although the method of implementing the present invention is designed for in vitro implementation, in vivo depletion can be performed to control the expansion of immune cells in a patient and potentially terminate therapeutic effects using antibodies approved by regulatory authorities for human therapeutic use. Table 5 lists examples of mAb-specific epitopes (and their corresponding mAbs) that can be integrated into the extracellular binding domain of the CAR of the present invention.

[0121] 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.

[0122]

[0123]

[0124] Therefore, the mesothelin-specific CAR according to the present invention preferably includes a safety switch containing at least one exogenous mAb epitope listed in Table 5. Preferably, the mesothelin-specific CAR according to the present invention preferably includes a safety switch containing the rituximab-specifically binding epitope CPYSNPSLC (SEQ ID NO: 26). More preferably, the mesothelin-specific CAR includes a safety switch referred to as "R2", which has at least 90% identity with SEQ ID NO: 15.

[0125] The mesothelin-specific CARs according to the present invention typically also include a signal peptide to aid in their expression on the surface of engineered cells. Chimeric antigen receptors (CARs) are typically formed as single-chain polypeptides, but can also be produced in multi-chain forms, as described, for example, in WO2014039523.

[0126] As shown in the examples, the preferred CAR according to the invention is MSLN-CAR-Meso1-R2 or MSLN-CAR-Meso1, which has 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.

[0127] Figure 1The structure of the preferred polypeptide structure of the MSLN-CAR of the present invention is shown.

[0128] More generally, the CAR of the present invention is generated by assembling different polynucleotide sequences encoding consecutive fragments of a CAR polypeptide into vectors for transfection and expression in immune cells described in the art and, for example, reviewed by [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].

[0129] This invention relates to polynucleotides and carriers, as well as any intermediate steps in the production process of the immune cells mentioned herein.

[0130] - "Vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. In this invention, "vector" includes, but is not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules composed of chromosomal, non-chromosomal, semi-synthetic, or synthetic nucleic acids. Preferred vectors are those capable of autonomous replication (attachment vectors) and / or expression (expression vectors) of the nucleic acid attached to them. A large number of suitable vectors are known to those skilled in the art and are commercially available. Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated virus (AAV), coronaviruses), negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses such as picornaviruses and alpha viruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., cowpox, fowlpox, and canarypox). Other viruses include, for example, norovirus, enveloped viruses, flaviviruses, reoviruses, papillomaviruses, hepatotropic DNA viruses, and hepatitis viruses. Examples of retroviruses include: avian leukosis sarcoma, mammalian C, B, and D viruses, HTLV-BLV groups, lentiviruses, and foam viruses (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, 3rd edition, edited by BN Fields et al., Lippincott-Raven Publishers, Philadelphia, 1996.

[0131] Specifically, the present invention provides an expression vector in the form of a lentiviral vector or an AAV vector containing a multinucleotide sequence encoding the CAR described herein.

[0132] The lentiviral vector may contain a polynucleotide sequence encoding a CAR according to the invention, operably linked to a promoter (such as the spleen focal formation virus promoter (SFFV)). "Operably linked" means that the components are juxtaposed in a relationship that enables them to function in their intended manner. A gene (such as a CAR-encoded polynucleotide sequence) is "operably linked" to a promoter when its transcription is controlled by the promoter and such transcription results in the production of a product encoded by the gene.

[0133] The lentiviral vectors of the present invention typically contain regulatory elements, such as 5' and 3' long terminal repeat (LTR) sequences, but may also contain other structural and functional genetic elements primarily derived from lentiviruses. These structural and functional genetic elements are well known in the art. The lentiviral vectors may, for example, contain the genes gag, pol, and env. However, preferably, the lentiviral vectors of the present invention do not contain the genes gag, pol, and env. As other regulatory elements, the lentiviral vectors may contain one or more (e.g., two or more) packaging signals (e.g., packaging signal ψ), promoter binding sites, transactivation response regions (TARs), and rev response elements (RREs).

[0134] Typically, the 5' and 3' long terminal repeat (LTR) sequences flanking the lentiviral genome possess promoter / enhancer activity and are essential for the proper expression of full-length lentiviral vector transcripts. The LTR usually comprises the repeat sequence U3RU5, present at the 5' and 3' ends of a double-stranded DNA molecule. It is a combination of the 5'R-U5 and 3'U3-R segments of a single-stranded RNA, where the repeat R is present at both ends of the RNA, while U5 (unique sequence 5) is present only at the 5' end of the RNA and U3 (unique sequence 3) is present only at the 3' end. The safety of lentiviral vectors can be improved by removing the U3 sequence, resulting in a "self-inactivating" vector completely lacking the viral promoter and enhancer sequences originally present within the LTR. Therefore, the vector can only infect and integrate into the host genome once and cannot be further delivered, thus improving the safety of the vector as a gene delivery vector.

[0135] According to some embodiments, the lentiviral vector is a self-inactivating (SIN) lentiviral vector. According to a specific embodiment, the lentiviral vector contains a 3'LTR, wherein the 3'LTR enhancer-promoter sequence (i.e., the U3 sequence) has been modified (e.g., deleted).

[0136] According to some implementations, the lentiviral vector contains a polynucleotide sequence that includes one or more of the following elements in a 5' to 3' sequence:

[0137] -5' long terminal repeat (5'LTR);

[0138] - Promoters (such as the EF1-α promoter);

[0139] - The multinucleotide sequence encoding a chimeric antigen receptor according to the present invention; and / or

[0140] -3' long terminal repeat (3'LTR), preferably 3' self-inactivating LTR.

[0141] According to a specific embodiment, the lentiviral vector may also contain a polynucleotide sequence comprising at least one of the following elements in a 5' to 3' sequence:

[0142] -5' long terminal repeat (5'LTR);

[0143] - Promoters (such as the EF1-α promoter);

[0144] - CAR according to the invention optionally including a safety switch such as R2,

[0145] - A polynucleotide sequence encoding peptide 2A;

[0146] -A polynucleotide sequence encoding any other polypeptide co-expressed with CAR, such as dnTGFβR;

[0147] and / or

[0148] -3' long terminal repeat (3'LTR), preferably 3' self-inactivating LTR.

[0149] Alternatively, the lentiviral vector may contain at least one of the following elements in a 5' to 3' sequence:

[0150] -5' long terminal repeat (5'LTR);

[0151] - Promoters (such as the EF1-α promoter);

[0152] -A polynucleotide sequence encoding any other polypeptide co-expressed with CAR, such as dnTGFβR;

[0153] - A polynucleotide sequence encoding peptide 2A;

[0154] - CAR according to the invention optionally including a safety switch such as R2,

[0155] and / or

[0156] -3' long terminal repeat (3'LTR), preferably 3' self-inactivating LTR.

[0157] Typically, the resulting vector forms a single transcriptional unit operatively linked to the promoter of item (b) and is transcribed under the control of said promoter.

[0158] AAV vectors, particularly those from the AAV6 family [Wang, J. et al. (2015) Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat Biotechnol 33, 1256–1263], are particularly useful for introducing the MSLN-CAR according to the invention into the genome using site-specific homologous recombination. Typically, as taught in EP3276000 and WO2018073391 relative to other CARs used for the treatment of hematologic malignancies, site-specific homologous recombination is induced in immune cells by expressing a rare endonuclease such as TALEN. Site-specific integration of the CAR can have several benefits, such as more stable integration, integration that places the transgene under transcriptional control of an endogenous promoter at the selected locus, and integration that can inactivate the endogenous locus. These latter aspects are described in detail in the following section on the genome engineering of therapeutic immune cells.

[0159] As an objective of this invention, an AAV vector is provided comprising a multinucleotide sequence encoding an MSLN-CAR as previously described and another sequence optionally encoding a cis-regulatory element (e.g., a 2A peptide cleavage site) or an internal ribosome entry site (IRES), thereby allowing co-expression of a third sequence encoding a product that improves the efficacy of engineered immunotherapy. An example of overexpression of dnTGFβRII is provided herein, which has been found to reduce SMAD2-3 phosphorylation, while simultaneously reducing TGFβ-induced cell exhaustion in the tumor environment.

[0160] the term" Therapeutic natureThis encompasses various ways in which these cells can be improved from the perspective of their use in therapeutic treatment. This refers to genetically engineering cells to endow them with therapeutic advantages (i.e., therapeutic efficacy) or to facilitate their use or their production. For example, genetic engineering can simultaneously provide effector cells with better survival, faster growth, shorter cell cycles, improved immune activity, greater functionality, greater differentiation, greater specificity to their target cells, greater sensitivity or tolerance to drugs, and less sensitivity to glucose deprivation, oxygen or amino acid depletion (i.e., resilience to the tumor microenvironment). Progenitor cells can be more productive, better tolerated by recipient patients, and more likely to generate cells that will differentiate into the desired effector cells. These examples of "therapeutic properties" are provided as examples, but not as limiting.

[0161] MSLN for Cell Therapy Genome engineering of CAR immune cells

[0162] More specifically, this invention relates to cells and reagents having the following properties:

[0163] Effector cells: Effector cells are relatively short-lived, activated cells that protect the body during the immune response. Activated T cells (including cytotoxic T cells and helper T cells) are the preferred effector cells for carrying out cell-mediated responses. The category of effector T cells is broad, encompassing various T cell types that actively respond to stimuli such as co-stimuli. This includes helper T cells, cytotoxic T cells, regulatory T cells, and potentially other T cell types.

[0164] "Immune cells" refer to hematopoietic cells that functionally participate in the initiation and / or execution of innate and / or adaptive immune responses, such as typically CD3 or CD4 positive cells. According to the invention, immune cells may be dendritic cells, cytotoxic dendritic cells, mast cells, NK cells, B cells, or T cells selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes.

[0165] "Primary cells" or "multiple primary cells" are cells directly collected from living tissue (e.g., biopsy material) and established for a limited time of in vitro growth, meaning they can undergo a limited number of population doublings. Primary cells are the opposite of continuously tumorigenic or artificially immortalized cell lines. Non-limiting examples of these 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 Molt 4 cells.

[0166] Primary immune cells can be obtained from a wide range of non-limiting sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumor-derived lymphocytes such as tumor-infiltrating lymphocytes. In some embodiments, the immune cells may be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with an infection. In another embodiment, the cells are part of a mixed population of immune cells exhibiting different phenotypic characteristics, such as cells containing CD4, CD8, and CD56 positive cells. Primary immune cells can be obtained from donors or patients using various methods known in the art, such as leukocyte removal techniques reviewed 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).

[0167] Immune cells derived from stem cells are also considered to be primary immune cells according to the present invention, 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]. 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].

[0168] According to a preferred embodiment of the present invention, immune cells are derived from human embryonic stem cells using techniques well known in the art that do not involve the destruction of human embryos [Chung et al. (2008) Human Embryonic Stem Cell lines generated without embryo destruction, Cell Stem Cell 2(2):113-117].

[0169] "Genetic engineering" refers to any method aimed at introducing genetic material into cells, modifying genetic material, and / or removing genetic material from cells. Gene editing Gene editing refers to genetic engineering that allows the addition, removal, or alteration of genetic material at specific locations (locus) in the genome, including punctual mutations. Gene editing typically involves sequence-specific reagents.

[0170] "Sequence-specific reagents" refer to any active molecule capable of specifically recognizing selected polynucleotide sequences (referred to as "target sequences") at genomic loci, intended to modify the expression of said genomic loci, typically at least 9 bp, more preferably at least 10 bp, and even more preferably at least 12 pb in length. The expression can be altered at the transcriptional level by mutation, deletion, or insertion into the coding or regulatory polynucleotide sequence, by epigenetic changes such as methylation or histone modifications, or by interference at the transcriptional level through interaction with transcription factors or polymerases.

[0171] Examples of sequence-specific reagents include endonucleases, RNA guides, RNAi, methylases, exonucleases, histone deacetylases, endonucleases, end-processing enzymes such as exonucleases, and more specifically cytidine deaminases, such as those coupled to the CRISPR / Cas9 system to perform base editing (i.e., nucleotide substitution) without requiring cleavage by nucleases, as described, for example, by Hess GT et al. [Methods and applications of CRISPR-mediated baseediting in eukaryotic genomes (2017) Mol Cell. 68(1): 26–43].

[0172] According to a preferred aspect of the invention, the sequence-specific reagent is preferably a sequence-specific nuclease reagent, such as an RNA guide coupled with a guide endonuclease.

[0173] This invention aims to improve the therapeutic potential of immune cells through gene editing technology, particularly through gene-targeted integration.

[0174] "Gene-targeted integration" refers to any known site-specific method that allows the insertion, replacement, or modification of genome coding sequences into living cells.

[0175] According to a preferred aspect of the invention, the gene-targeted integration includes homologous gene recombination at a locus of a target gene to result in the insertion or substitution of at least one exogenous nucleotide, preferably a sequence of multiple nucleotides (i.e., a polynucleotide), and more preferably a coding sequence.

[0176] "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 according to the present invention. These terms refer to a specific DNA location, preferably a genomic location in a cell, but also to a portion of genetic material that can exist independently of the main genetic material, such as plasmids, episomes, viruses, transposons, or organelles such as mitochondria as non-limiting examples. Non-limiting examples of RNA-guided target sequences are those genomic sequences that can hybridize with guide RNA, directing the RNA-guided nuclease to the desired locus.

[0177] “Sparse-cut endonucleases” are selected sequence-specific endonuclease reagents that recognize sequences typically ranging from 10 to 50 consecutive base pairs, preferably from 12 to 30 bp and more preferably from 14 to 20 bp.

[0178] According to a preferred aspect of the invention, the endonuclease reagent is a nucleic acid encoding an "engineered" or "programmable" rare-cleaving endonuclease, such as, for example, the homing endonuclease of Arnould S. et al. [WO2004067736], such as, for example, the zinc finger nuclease (ZFN) described by Urnov F. et al. [Highly efficient endogenous human gene correction using designed zinc-finger nucleases (2005) Nature 435:646-651], such as, for example, the TALE nuclease nuclease described by 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 such as, for example, the rare-cleaving nuclease architecture for therapeutic genome engineering (2013) Nucleic Acids Research by Boissel et al. [MegaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering (2013) Nucleic Acids Research] The MegaTAL nuclease described in 42(4):2591-2601.

[0179] According to another embodiment, the endonuclease reagent is a guide RNA used to bind RNA-guided endonucleases such as Cas9 or Cpf1, especially as taught by Doudna, J. and Chapentier, E. [The new frontier of genome engineering with CRISPR-Cas9 (2014) Science 346 (6213): 1077], which are incorporated herein by reference.

[0180] According to a preferred aspect of the invention, the nuclease reagent is transiently expressed into the cell, which means that the integration of the reagent into the genome or the long-term persistence of the reagent, such as RNA, more specifically mRNA, protein, or a complex of mixed proteins and nucleic acids (e.g., ribonucleoprotein), is not considered.

[0181] Preferably, the stability of the endonuclease in mRNA form with a cap is improved by means of techniques known in the art, such as those described by 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].

[0182] Typically, the electroporation step for transfecting primary immune cells such as PBMCs is performed in a closed chamber containing parallel plate electrodes, which generate a pulsed electric field, greater than 100 V / cm and less than 5,000 V / cm, substantially uniform throughout the processing volume, as described in WO2004083379 (which is incorporated herein by reference), specifically from page 23, line 25 to page 29, line 11. Such an electroporation chamber preferably has a cross-sectional area (cm) of the square of the electrode spacing. 2 Divide by the room volume (cm³) 3 The geometric factor (cm) defined by the quotient of ) -1 ), where the geometric factor is less than or equal to 0.1 cm -1 The cell suspension and sequence-specific reagents are contained in a medium, the conductivity of which is adjusted to be in the range of 0.01 to 1.0 millisiemens. Typically, the cell suspension is subjected to one or more pulsed electric fields. This method allows for scalable treatment volumes of the suspension and substantially uniform cell treatment times within the chamber.

[0183] Due to their high specificity, TALE nucleases have proven to be suitable sequence-specific nuclease reagents for therapeutic applications, especially in heterodimeric form – i.e., by working with pairs having a “right” monomer (also known as “5’ or “forward”) and a “left” monomer (also known as “3’ or “reverse”), as reported, for example, by 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].

[0184] As described above, sequence-specific reagents are preferably in nucleic acid form, such as in DNA or RNA encoding a rare nuclease and its subunits, but they can also be part of conjugates comprising polynucleotides and polypeptides (such as so-called "ribonucleases"). Such conjugates can be formed with reagents such as Cas9 or Cpf1 (RNA-guided endonucleases), as described by 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 genomeediting using the Natronobacterium gregoryi Argonaute (2016) Nature Biotech], which include RNA or DNA guides that can complex with their respective nucleases.

[0185] "Exogenous sequence" refers to any nucleotide or nucleic acid sequence that is not initially present at the selected locus. This sequence may be homologous to a genomic sequence, a copy of it, or it may be an exogenous sequence introduced into the cell. Conversely, "endogenous sequence" refers to a cellular genomic sequence that is initially present at the locus. The exogenous sequence preferably encodes a polypeptide that confers a therapeutic advantage over sister cells that do not integrate the exogenous sequence at the locus. According to the method of the present invention, the endogenous sequence used for gene editing to express different polypeptides via the insertion of nucleotides or polynucleotides is broadly referred to as an exogenous coding sequence.

[0186] By using the above reagents and techniques, this invention also develops a method for generating therapeutic cells by performing one or more of the following steps:

[0187] - Provide immune cells from a donor or patient, preferably primary cells;

[0188] - The MSLN-CAR as described above is expressed into these cells, usually by introducing the MSLN-CAR coding sequence into the cell genome using a viral vector;

[0189] - Introducing sequence-specific reagents, such as rare-cut endonucleases, into these immune cells to induce modifications (mutations or coding sequence insertions) at loci of endogenous genes; and / or

[0190] - Introducing exogenous coding sequences into the cells to improve their therapeutic efficacy, specifically their immune properties.

[0191] According to some aspects of the invention, immune cells are derived from a patient or compatible donor, wherein MSLN CAR is expressed for so-called "autologous" infusion of engineered immune cells. They may also be derived from stem cells, such as iPS cells, derived from the patient or compatible donor or from tumor-infiltrating lymphocytes (TILL).

[0192] According to some aspects of the invention, the method is designed to provide an “off-the-shelf” composition of immune cells, which are then engineered for use in allogeneic therapeutic treatments.

[0193] "Allogeneic" refers to cells that originate from a donor, are generated or differentiated from stem cells, and are then infused into patients with different haplotypes.

[0194] These immune cells are typically engineered to exhibit lower alloreactivity and / or greater durability relative to their patient host. More specifically, this approach involves steps to reduce or inactivate TCR expression in T cells or stem cells that will be derived into T cells. This can be achieved using various sequence-specific reagents, such as through gene silencing or gene editing technologies (nucleases, base editing, RNAi, etc.).

[0195] This invention has previously made robust procedures and gene-editing strategies available for the generation of allogeneic therapeutic-grade T cells from PBMCs, specifically by providing TALE nucleases. A highly safe and specific form of endonuclease reagent. This enables the development of so-called "universal T-cells" (which are derived from donor [TCRs]). neg The generation of T cells and their successful injection into patients with reduced 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)]. Simultaneously, inactivation of the TCR or β2m component in primary T cells can be combined with inactivation of other genes encoding checkpoint inhibitor proteins, as described, for example, in WO2014184744.

[0196] In a preferred embodiment, the present invention provides a method for engineering immune cells, wherein at least one gene encoding TCRα or TCRβ is inactivated in the immune cells, preferably by expression of a rare endonuclease, and a foreign polynucleotide encoding MSLN-CAR is introduced into the genome of the cell for stable expression. Preferably, the foreign sequence is integrated at the locus encoding TCRα or TCRβ, more preferably under the transcriptional control of the endogenous promoter of TCRα or TCRβ.

[0197] In other embodiments, engineered immune cells may be further modified to confer resistance to at least one immunosuppressive drug, such as by inactivating CD52, a target of anti-CD52 antibodies (e.g., alemtuzumab), which has previously been described with reference to the treatment of blood cancers, for example in WO2013176915.

[0198] Although the use of anti-CD52 lymphocyte scavengers has been limited to liquid tumor cancers to date [Quasim W. et al. (2019) Allogeneic CAR T cell therapies for leukemia Am J Hematol. 94: S50–S54.], a major aspect of the present invention is the use of genetically engineered lymphocytes resistant to lymphocyte scavenging protocols for the treatment of solid tumors.

[0199] The present invention also provides engineered lymphocytes having chimeric antigen receptors for solid tumors, particularly for mesothelin-positive cells, which are used in conjunction with or after a lymphocyte clearance treatment step in the treatment of solid tumor cancer.

[0200] This lymphocyte clearance regimen may include anti-CD52 agents, such as alenzusmab, or purine analogs, such as those used to treat blood cancers.

[0201] In a preferred embodiment, the engineered lymphocytes with MSLN-CAR described herein are made resistant to this lymphocyte clearance regimen by inhibiting or disrupting the expression of molecules targeted by the lymphocyte scavenger, such as, for example, the antigen CD52 in the case of alenzuszumab.

[0202] In other embodiments, engineered immune cells can be further modified to confer resistance to chemotherapy drugs, specifically purine analogues, for example by inactivating DCK as described in WO201575195.

[0203] As previously stated, a key aspect of this invention is the treatment of solid tumor cancers with genetically engineered lymphocytes possessing chimeric antigen receptors, which are resistant to chemotherapy or lymphocyte clearance regimens. Such regimens may comprise antibodies targeting antigens present on the surface of immune cells, such as CD52, CD3, CD4, CD8, CD45, or other specific markers, as well as less specific drugs, such as purine analogs (e.g., fludarabine and / or clofarabine) and glucocorticoids. One aspect of this invention involves making the engineered lymphocytes resistant to such regimens by inactivating or reducing the expression of genes encoding at least one molecular target of these lymphocyte clearance agents, such as the gene DCK that metabolizes purine analogs or the gene encoding the glucocorticoid receptor (GR).

[0204] Therefore, the present invention focuses more specifically on CAR-positive cells for allogeneic use in the treatment of solid cancer, which have reduced, inactivated or defective expression of TCR, CD52 and / or DCK and / or GR, making them less responsive to and tolerant of allogeneic lymphocyte clearance protocols.

[0205] In other embodiments, engineered immune cells may be further modified to improve their persistence or lifespan in patients, specifically by inactivating genes encoding MHC-I components such as HLA or β2m, as described in WO2015136001 or by Liu, X. et al. [CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells (2017) Cell Res27:154–157].

[0206] According to a preferred aspect of the invention, engineered immune cells are mutated to improve their CAR-dependent immune activation, specifically by reducing or inhibiting the expression of immune checkpoint proteins and / or their receptors such as PD1 or CTLA4, as described in WO2014184744.

[0207] In other embodiments, engineered immune cells may be further modified to obtain co-expression of another exogenous gene sequence selected from those encoding one of the following sequences in the cells:

[0208] -NK cell inhibitors, such as HLAG, HLAE or ULBP1;

[0209] -CRS inhibitors, such as mutant IL6Ra, sGP130 or IL18-BP;

[0210] - Cytochrome P450, CYP2D6-1, CYP2D6-2, CYP2C9, CYP3A4, CYP2C19 or CYP1A2, which confer hypersensitivity to drugs such as cyclophosphamide and / or isophosphamide.

[0211] - Dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin or methylguanine transferase (MGMT), mTORmut or Lckmut, confer drug resistance;

[0212] - Chemokines or cytokines, such as IL-2, IL-12 and IL-15;

[0213] - Chemokinin receptors, such as CCR2, CXCR2, or CXCR4; and / or

[0214] - Inhibitors of tumor-associated macrophage (TAM) secretion, such as CCR2 / CCL2 neutralizers, to enhance the therapeutic activity of immune cells;

[0215] This application claims, for the purpose of generating therapeutic compositions targeting solid tumors, the use of engineered immune cells co-expressing at least one exogenous sequence encoding MSLN-CAR as described herein with another exogenous sequence encoding a human polypeptide selected from the above list.

[0216] The combination of MSLN-CAR expression in therapeutically engineered immune cells and disruption of the TGFbRII signaling pathway

[0217] More specifically, this invention combines the expression of the exogenous sequence encoding MSLN-CAR as described above with another exogenous sequence encoding a TGFβ receptor inhibitor, particularly a TGFβRII inhibitor (Uniprot–P37173).

[0218] TGFβ receptor has been described as having a dominant role in the tumor microenvironment [Papageorgis, P. et al. (2015). Role of TGFβin regulation of the tumor microenvironment and drug delivery (Review). International Journal of Oncology, 46, 933-943].

[0219] Although the exact role of the TGFβ receptor in tumorigenesis remains controversial, the inventors have discovered that co-expression of the mesothelin-specific chimeric antigen receptor (CAR) with another exogenous gene sequence encoding an inhibitor of TGFBRII signaling and / or inactivation or reduction of TGFβ receptor signaling through the use of sequence-specific reagents leads to improved therapeutic efficacy of engineered immune cells. Specifically, the inventors have used two different methods to disrupt the TGFβRII signaling pathway, and these two methods can be combined:

[0220] Expression of inactive ligands of TGFβRII, such as dominantly inactive TGFβRII (SEQ ID NO: 26), as described in Hiramatsu, K. et al. [Expression of dominant negative TGF-βreceptors inhibits scartilage formation in conditional transgenic mice (2011) J. Bone. Miner. Metab. 29:493], or expression of similar inactive forms of TGFβRII having at least 80%, preferably at least 90%, more preferably at least 95% identity with the polypeptide sequence SEQ ID NO: 26.

[0221] and / or

[0222] Inactivation of the endogenous gene sequence of -TGFβRII is achieved specifically by using a rare nuclease, such as TALE nuclease, or an RNA-directed nuclease (e.g., Cas9 or Cpf1).

[0223] Anti-TGFβ receptor RII IgG1 monoclonal antibodies, such as LY3022859 [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], which inhibit receptor-mediated signal transduction activation, can also be combined with the CAR according to the present invention for inhibiting TGFβ receptor signal transduction.

[0224] In this application, we disclose the selection of TALE nucleases that are particularly specific for targeting sequences within the TGFβRII gene. These TALE nucleases have demonstrated the highest TGFβRII knockout efficacy with minimal off-target cleavage, resulting in large populations of viable engineered cells sufficient for dose administration to several patients. Table 6 lists these preferred TALE nucleases and their corresponding target sequences.

[0225] Table 6: TALE nuclease target sequences of the TGFβRII gene

[0226]

[0227] An RNA guide avec was also designed to inactivate the TGFβRII gene using a Cas9 nuclease reagent. Their respective target sequences are disclosed in Table 7.

[0228] Table 7: CRISPR target sequences of the TGFβRII gene.

[0229]

[0230]

[0231]

[0232]

[0233] Therefore, this invention covers the use of TALE nucleases or RNA-directed endonucleases designed to combine with any target sequence SEQ ID NO: X to Y mentioned in Table 5 or 6 for inactivating or reducing the expression of TGFβRII for the production of therapeutic immune cells within the teachings of this specification.

[0234] The present invention also relates to engineered immune cells comprising exogenous polynucleotides encoding nucleases, such as one polynucleotide previously mentioned, to inactivate or reduce the expression of the endogenous TGFβRII gene.

[0235] Therefore, this invention application reports engineered immune cells, particularly CAR immune cells, in which exogenous sequences encoding TGFβ receptor inhibitors have been introduced, more specifically sequences encoding dominantly inactivated TGFβ receptors. These cells are more specifically designed for the treatment of solid tumors, particularly MSLN-positive tumors.

[0236] Therefore, this application also claims protection for vectors, particularly viral vectors, such as lentiviral vectors or AAV vectors as described in the art, which at least comprise a polynucleotide sequence encoding a dominant inactivating TGFβRII and optionally a mesothelin-specific chimeric antigen receptor. In a preferred embodiment, the vector comprises a first polynucleotide sequence encoding the dominant inactivating TGFβRII, a second polynucleotide sequence encoding a 2A self-cleaving peptide, and a third polynucleotide sequence encoding the mesothelin-specific chimeric antigen receptor.

[0237] Targeted insertion into immune cells can be significantly improved by using AAV vectors, particularly vectors from the AAV6 family or the chimeric vector AAV2 / 6, as previously described by Sharma A. et al. [Transduction efficiency of AAV2 / 6, 2 / 8 and 2 / 9 vectors for delivering genes in human corneal fibroblasts. (2010) Brain Research Bulletin. 81(2–3): 273-278].

[0238] Therefore, one aspect of the present invention is the transduction of an AAV vector containing an MSLN-CAR coding sequence in human primary immune cells, combined with the expression of a sequence-specific endonuclease reagent such as TALE endonuclease, to enhance gene integration at previously cited loci.

[0239] According to a preferred aspect of the invention, the sequence-specific endonuclease reagent can be introduced into cells by transfection, more preferably by electroporation of mRNA encoding the sequence-specific endonuclease reagent.

[0240] The insertion of the obtained exogenous nucleic acid sequence can lead to the introduction of genetic material, the modification or replacement of endogenous sequences, more preferably "in frame" relative to the endogenous gene sequence located at the locus.

[0241] According to another aspect of the invention, each cell transduces 10 5 Up to 10 7 Preferably 10 6 Up to 10 7 More preferably about 5.10 6 One viral genome.

[0242] According to another aspect of the invention, cells can be treated with proteasome inhibitors such as bortezomib or HDAC inhibitors to further facilitate homologous recombination.

[0243] As an objective of this invention, the AAV vector used in this method may contain a promoterless exogenous coding sequence, which is any of those mentioned in this specification.

[0244] This invention also provides an efficient method for obtaining primary immune cells, which allows for gene editing at multiple loci, more specifically at loci involved in host-transplant interactions and recognition. Other loci can also be edited to improve the activity, survival, or lifespan of engineered primary cells, particularly primary T cells.

[0245] Figure 2 The main cellular functions that can be modified by gene editing according to the present invention to improve the efficiency of engineered immune cells are mapped. Inactivation of any gene listed under each function can be combined with another to achieve a synergistic effect on the overall therapeutic efficacy of the immune cells.

[0246] More specifically, the present invention provides a combination of gene modifications (genotypes) in immune cells that promote improved efficacy of immune cells against solid tumors, particularly against MSLN-positive malignant cells, for example:

[0247] -[MSLN-CAR] + ,

[0248] -[MSLN-CAR] + [dnTGFβRII] + ,

[0249] -[MSLN-CAR] + [dnTGFβRII] + [TCR] - ,

[0250] -[MSLN-CAR] + [dnTGFβRII] + [TGFβRII] - [TCR] - ,

[0251] -[MSLN-CAR] + [TGFβRII] - ,

[0252] -[MSLN-CAR] + [TGFβRII] - [TCR] - ,

[0253] -[MSLN-CAR] + [β2m] - ,

[0254] -[MSLN-CAR] + [dnTGFβRII] + [β2m] - ,

[0255] -[MSLN-CAR] + [TGFβRII] - [β2m] - ,

[0256] -[MSLN-CAR] + [dnTGFβRII] + [β2m] - [TCR] - ,

[0257] -[MSLN-CAR] + [TGFβRII] - [β2m] - [TCR] - ,

[0258] -[MSLN-CAR] + [dnTGFβRII] + [TGFβRII] - [β2m] - [TCR] - ,

[0259] -[MSLN-CAR] + [PD1] - ,

[0260] -[MSLN-CAR] + [TGFβRII] - [PD1] - ,

[0261] -[MSLN-CAR] + [dnTGFβRII] + [PD1] - ,

[0262] -[MSLN-CAR] + [dnTGFβRII] + [TGFβRII] - [PD1] - ,

[0263] -[MSLN-CAR] + [dnTGFβRII] + [β2m] - [PD1] - ,

[0264] -[MSLN-CAR] + [TGFβRII] - [PD1] - [TCR] - ,

[0265] -[MSLN-CAR] + [dnTGFβRII] + [TGFβRII] - [PD1] - [TCR] - ,

[0266] -[MSLN-CAR] + [PD1] - [β2m] - ,

[0267] -[MSLN-CAR] + [TGFβRII] - [PD1] - [β2m] - ,

[0268] -[MSLN-CAR] + [dnTGFβRII] + [PD1] - [β2m] - ,

[0269] -[MSLN-CAR] + [dnTGFβRII] + [TGFβRII] - [PD1] - [β2m] - ,

[0270] -[MSLN-CAR] + [dnTGFβRII] + [β2m] - [PD1] - [TCR] - ,

[0271] -[MSLN-CAR] + [TGFβRII] - [PD1] - [TCR] - [β2m] - ,

[0272] -[MSLN-CAR] + [dnTGFβRII] +[TGFβRII] - [PD1] - [TCR] - [β2m] - .

[0273] As previously mentioned, the present invention also specifically focuses on the use of CAR-positive cells in the treatment of solid cancer, which are resistant to lymphocyte clearance agents, allowing them to be used in combination with or after lymphocyte clearance protocols in an allogeneic setting. These cells preferably exhibit the following genotypes:

[0274] -relative to resistance - CD52 antibody partially or completely tolerated:

[0275] -[MSLN-CAR] + [CD52] - [TCR] - ,

[0276] -[MSLN-CAR] + [CD52] - [TCR] - [β2m] - ,

[0277] -[MSLN-CAR] + [TGFβRII] - [CD52] - [TCR] - ,

[0278] -[MSLN-CAR] + [TGFβRII] - [CD52] - [TCR] - [β2m] - ,

[0279] -[MSLN-CAR] + [dnTGFβRII] + [CD52] - [TCR] - ,

[0280] -[MSLN-CAR] + [dnTGFβRII] + [CD52] - [TCR] - [β2m] - ,

[0281] - Compared to partial or complete tolerance to purine analogs:

[0282] -[MSLN-CAR] + [DCK] - [TCR] - ,

[0283] -[MSLN-CAR] + [DCK] - [TCR] - [β2m] - ,

[0284] -[MSLN-CAR] + [TGFβRII] - [DCK] - [TCR] - ,

[0285] -[MSLN-CAR] + [TGFβRII] - [DCK] - [TCR] - [β2m] - ,

[0286] -[MSLN-CAR] + [dnTGFβRII] + [DCK] - [TCR] - ,

[0287] -[MSLN-CAR] + [dnTGFβRII] + [DCK] - [TCR] - [β2m] - ,

[0288] -Relative to partial or complete tolerance to glucocorticoids:

[0289] -[MSLN-CAR] + [GR] - [TCR] - ,

[0290] -[MSLN-CAR] + [GR] - [TCR] - [β2m] - ,

[0291] -[MSLN-CAR] + [TGFβRII] - [GR] - [TCR] - ,

[0292] -[MSLN-CAR] + [TGFβRII] - [GR] - [TCR] - [β2m] - ,

[0293] -[MSLN-CAR] + [dnTGFβRII] + [GR] - [TCR] - ,

[0294] -[MSLN-CAR] + [dnTGFβRII] + [GR] - [TCR] - [β2m] - ,

[0295] Further improve therapeutic immune cells by expressing transgenes at inactivated loci.

[0296] The preferred genotypes described above can be obtained by targeted integration at the PD1, TCR (TCRα and / or TCRβ) or TGFβRII loci, and at other selected loci as described below.

[0297] "Gene-targeted integration" refers to any known site-specific method that allows the insertion, substitution, or modification of a genomic sequence into living cells. Gene-targeted integration typically involves homologous gene recombination or NHEJ (non-homologous end joining) mechanisms, which are enhanced by endonuclease sequence-specific reagents to produce a sequence of at least one exogenous nucleotide, preferably several nucleotides (i.e., polynucleotides), and more preferably a coding sequence, for insertion or substitution at a predetermined locus.

[0298] The method of the present invention can be combined with other methods involving genetic transformation, such as viral transduction, and can also be combined with other transgenic expression methods that do not necessarily involve integration.

[0299] According to one aspect, the method according to the invention includes the step of introducing a mutated or polynucleotide coding sequence into an endogenous locus of immune cells, the mutated or polynucleotide coding sequence being selected from:

[0300] a) Polynucleotide sequences whose expression involves reduced glycolysis and calcium signaling in response to low glucose conditions, such as SERCA3 which enhances calcium signaling, miR101 and miR26A which enhance glycolysis, and BCAT which mobilizes glycolytic reserves; and / or

[0301] b) Polynucleotide sequences whose expression upregulates immune checkpoint proteins (e.g., TIM3, CEACAM, LAG3, TIGIT), such as IL27RA, STAT1, STAT3; and / or

[0302] c) Polynucleotide sequences whose expression mediates interactions with HLA-G, such as ILT2 or ILT4; and / or

[0303] 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 promotes CD8 memory differentiation; and / or

[0304] e) Polynucleotide sequences whose expression is involved in the downregulation of T cell activation, such as mir21; and / or

[0305] f) Polynucleotide sequences whose expression involves signal transduction pathways in response to cytokines, such as JAK2 and AURKA; and / or

[0306] g) Polynucleotide sequences whose expression is involved in T cell exhaustion, such as DNMT3, miRNA31, MT1A, MT2A, and PTGER2;

[0307] Preferably, this is achieved by expressing a sequence-specific reagent that specifically targets the selected endogenous locus in the cells.

[0308] Preferably, the transgene or exogenous polynucleotide sequence is inserted, thereby placing its expression under the transcriptional control of at least one endogenous promoter present at one of the loci.

[0309] As mentioned above, implementing gene integration targeting a locus will help to further improve the efficacy of the therapeutic immune cells of the present invention.

[0310] The following provides examples of these exogenous sequences or transgenes that can be expressed or overexpressed at selected loci:

[0311] Expression of transgenes conferring resistance to drugs or immune depletion agents

[0312] According to one aspect of the method of the present invention, a foreign sequence integrated into an immune cell genomic locus encodes a molecule that confers resistance to a drug to the immune cell.

[0313] Examples of preferred exogenous sequences are variants of dihydrofolate reductase (DHFR) conferring resistance to folic acid analogs such as methotrexate; variants of inosine monophosphate dehydrogenase 2 (IMPDH2) conferring resistance to IMPDH inhibitors such as mycophenolic acid (MPA) or its prodrug mycophenolate mofetil (MMF); variants of calcineurin or methylguanine transferase (MGMT) conferring resistance to calcineurin inhibitors such as FK506 and / or CsA; variants of mTOR conferring resistance to rapamycin such as mTORmut; and variants of Lck conferring resistance to imatinib and glimepiride such as Lckmut.

[0314] The term "drug" is used herein to refer to a compound or derivative thereof that is typically used to interact with cancer cells, thereby reducing the proliferation or survival of the cells, preferably a standard chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, ifosfamide), metabolic antagonists (e.g., purine nucleoside antimetabolites such as clofarabine, fludarabine, or 2'-deoxyadenosine, methotrexate (MTX), 5-fluorouracil, or derivatives thereof), antitumor antibiotics (e.g., mitomycin, adriamycin), plant-derived antitumor agents (e.g., vincristine, vinblastine, taxol), cisplatin, carboplatin, etoposide, etc. These agents may also include (but are not limited to) the anticancer agent TRIMETHOTRIXATE. TM (TMTX), TEMOZOLOMIDE TM RALTRITREXED TM S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanidine (6-BG), dichloronitrosourea (BCNU), and CAMPTOTHECIN TM Or any of its therapeutic derivatives.

[0315] As used herein, when the cells or cell populations are modified such that they can proliferate at least in vitro in a culture medium containing the drug at a half-maximal inhibitory concentration (IC50, which is determined relative to unmodified cells or cell populations), immune cells are made "resistant or tolerant" to the drug.

[0316] In a specific implementation, immune cells can be conferred drug resistance through the expression of at least one "drug resistance coding sequence." The drug resistance coding sequence refers to a nucleic acid sequence that confers "resistance" to a reagent such as one of the chemotherapeutic agents mentioned above. The drug resistance coding sequence of the present invention can encode resistance to metabolites, methotrexate, vincristine, cisplatin, alkylating agents, anthracycline antibiotics, cytotoxic antibiotics, anti-immunogenic avidin, their analogues or derivatives, etc. (Takebe, N., SC Zhao et al. (2001) "Generation of dual resistance to 4-hydroperoxycyclophosphamide and methotrexate by retroviral transfer of the human aldehyde dehydrogenase class 1 gene and a mutated dihydrofolatereductase gene". Mol. Ther. 3(1):88-96), (Zielske, SP, JS Reese et al. (2003) "Invivo 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 antifoliates by retroviral coexpression oftyrosyl DNA phosphodiesterase-I and thymidylate synthase"Cancer ChemotherPharmacol 53(2):107-15), (Bardenheuer, W., K. Lehmberg et al. (2005)."Resistance tocytarabine and gemcitabine and in vitro selection of transduced cells afterretroviral expression of cytidine deaminase in human hematopoietic progenitorcells". Leukemia 19(12):2281-8), (Kushman, ME, SLKabler et al. (2007)"Expression of human glutathione S-transferase P1 confers resistance to benzo[a]pyrene orbenzo[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). .

[0317] More specifically, the expression of these drug-resistant exogenous sequences in the immune cells according to the invention enables the use of the immune cells in cell therapy regimens in which cell therapy is combined with chemotherapy or in patients previously treated with these drugs.

[0318] Several drug resistance coding sequences have been identified that could potentially be used to confer resistance according to the present invention. An example of a drug resistance coding sequence could be a mutant or modified form of, for example, 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 antitumor agents. Different mutant forms of DHFR with enhanced resistance to inhibition by antifolate agents used in therapy have been described. In a specific embodiment, the drug resistance coding sequence according to the present invention could be a nucleic acid sequence encoding a mutant form of human wild-type DHFR (GenBank: AAH71996.1) containing at least one mutation conferring resistance to antifolate treatments such as methotrexate. In a specific embodiment, the mutant form of DHFR 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 US Patent 6,642,043). In a specific embodiment, the mutant form of DHFR contains two mutant amino acids at positions L22 and F31. The amino acid positions described herein are generally indicated relative to the amino acid positions in the wild-type DHFR polypeptide form. In a specific embodiment, the serine residue at position 15 is preferably replaced by a tryptophan residue. In another specific embodiment, the leucine residue at position 22 is preferably replaced by an amino acid that disrupts the binding of the mutant DHFR to an antifolic acid agent, preferably by 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.

[0319] Another example of a resistance-coding sequence is a mutant or modified form of inosine-5'-monophosphate dehydrogenase II (IMPDH2), the rate-limiting enzyme in the de novo synthesis of guanosine nucleotides. Mutants or modified forms of IMPDH2 are IMPDH inhibitor resistance genes. IMPDH inhibitors can be mycophenolic acid (MPA) or its prodrug mycophenolate mofetil (MMF). Mutant IMPDH2 may contain at least one, preferably two, mutations in the MAP binding site of wild-type human IMPDH2 (Genebank: NP_000875.2), resulting in a significant increase in resistance to IMPDH inhibitors. The mutations in these variants are preferably located at position 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).

[0320] Another drug resistance coding sequence is a mutant form of calcineurin. Calcineurin (PP2B-NCBI: ACX34092.1) is a ubiquitously expressed serine / threonine protein phosphatase involved in various biological processes and central to T cell activation. Calcineurin is a heterodimer composed of a catalytic subunit (CnA; 3 isoforms) and a regulatory subunit (CnB; 2 isoforms). Upon T cell receptor binding, calcineurin dephosphorylates the transcription factor NFAT, thereby translocating it to the nucleus and activating key target genes such as IL2. FK506, conjugated with FKBP12, or cyclosporine A (CsA), conjugated with CyPA, blocks NFAT's access to the active site of calcineurin, thereby preventing its dephosphorylation and 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 specific embodiments, the mutant form may contain at least one mutant amino acid of wild-type calcineurin heterodimer a at the following positions: V314, Y341, M347, T351, W352, L354, K360, preferably two mutations at the following positions: T351 and L354 or V314 and Y341. In a specific embodiment, the valine residue at position 341 can be replaced by a lysine or arginine residue; the tyrosine residue at position 341 can be replaced by a phenylalanine residue; the methionine residue at position 347 can be replaced by a glutamic acid, arginine, or tryptophan residue; the threonine residue at position 351 can be replaced by a glutamic acid residue; the tryptophan residue at position 352 can be replaced by a cysteine, glutamic acid, or alanine residue; the serine residue at position 353 can be replaced by a histidine or asparagine residue; the leucine residue at position 354 can be replaced by an alanine residue; and the lysine residue at position 360 can be replaced by an alanine or phenylalanine residue. In another specific embodiment, the mutant form may contain at least one mutant amino acid of the wild-type calcineurin heterodimer b located at the following positions: V120, N123, L124, or K125, preferably two mutations at positions L124 and K125.In specific embodiments, valine at position 120 can be replaced by serine, aspartic acid, phenylalanine, or leucine residues; asparagine at position 123 can be replaced by tryptophan, lysine, phenylalanine, arginine, histidine, or serine; leucine at position 124 can be replaced by threonine residues; and lysine at position 125 can be replaced by alanine, glutamic acid, or tryptophan, or two residues such as leucine-arginine or isoleucine-glutamic acid can be added after lysine at position 125 in the amino acid sequence. The amino acid positions described herein are typically indicated by the amino acid positions in the form of wild-type human calcineurin heterodimer b polypeptide (NCBI: ACX34095.1).

[0321] Another resistance-encoding sequence is O(6)-methylguanine methyltransferase (MGMT-UniProtKB: P16455), which encodes human alkylguanine transferase (hAGT). AGT is a DNA repair protein that confers resistance to the cytotoxic effects of alkylating agents such as nitrosoureas and temozolomide (TMZ). 6-Benzylguanine (6-BG) is an inhibitor of AGT that enhances the toxicity of nitrosoureas and, when co-administered with TMZ, amplifies the cytotoxic effects of that agent. Several mutant forms of MGMT encoding AGT are highly resistant to inactivation by 6-BG but retain their 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 specific embodiments, the AGT mutant form may contain a mutant amino acid at position P140 of the wild-type AGT. In a preferred embodiment, the proline at position 140 is replaced by a lysine residue.

[0322] Another drug resistance coding sequence could be the multidrug resistance protein (MDR1) gene. This gene encodes a membrane glycoprotein called P-glycoprotein (P-GP), which is involved in the transport of metabolic byproducts across the cell membrane. P-GP proteins exhibit broad specificity for some structurally unrelated chemotherapeutic agents. Therefore, cell resistance can be conferred by expressing the nucleic acid sequence encoding MDR-1 (Genebank NP_000918).

[0323] Another resistance-coding sequence can promote the production of cytotoxic antibiotics, such as those from the ble or mcrA genes. Ectopic expression of the ble or mcrA gene in immune cells provides a selective advantage when exposed to their respective chemotherapeutic agents, bleomycin and mitomycin C (Belcourt, MF (1999) "Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA" PNAS. 96(18):10489-94).

[0324] Another resistance-coding sequence can come from a mutated form of the gene encoding the drug target, such as a mutant variant of mTOR (mTOR mut) that confers 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 certain mutant variants of Lck (Lckmut) that confer resistance to imatinib, 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.

[0325] As described above, the gene modification step of this method may include introducing a foreign nucleic acid containing at least a sequence encoding drug resistance and a portion of an endogenous gene into the cell, thereby causing homologous recombination between the endogenous gene and the foreign nucleic acid. In a specific embodiment, the endogenous gene may be a wild-type "drug-resistant" gene, so that after homologous recombination, the wild-type gene is replaced by a mutant form of the drug-resistant gene.

[0326] Enhancing the persistent expression of transgenes in immune cells

[0327] According to one aspect of this method, exogenous sequences integrated into immune cell genomic loci encode molecules that enhance immune cell persistence, particularly in vivo persistence in the tumor setting.

[0328] "Improved durability" refers to extending the survival of immune cells in terms of lifespan, especially once the engineered immune cells are injected into a patient. For example, durability is improved if the average survival 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%.

[0329] This is particularly relevant when the immune cells are allogeneic. This can be achieved by introducing coding sequences for immunosuppressive peptides expressed and / or secreted ectopically on or through the cell membrane, thereby generating local immune protection. Multiple groups of these peptides, specifically immune checkpoint antagonists, immunosuppressive peptides derived from viral envelopes, or NKG2D ligands, can enhance persistence and / or allogeneic immune cell transplantation into patients.

[0330] According to 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.

[0331] According to one embodiment, the immunosuppressive polypeptide encoded by the exogenous coding sequence is an antagonist of PD1, such as PD-L1 (other names: CD274, programmed cell death 1 ligand; UniProt number for human polypeptide sequence: Q9NZQ7), which encodes a 290-amino acid type I transmembrane protein consisting of an IgV-like domain, an IgC-like domain, a hydrophobic transmembrane domain, and a 30-amino acid cytoplasmic tail. This membrane-bound form of the PD-L1 ligand is referred to in this invention as either in its natural (wild-type) form or in a truncated form, such as by removing the intracellular domain, or having one or more mutations (Wang S et al., 2003, J Exp Med. 2003; 197(9):1083–1091). It is noteworthy that PD1 is not considered to be the membrane-bound form of the PD-L1 ligand according to the invention. According to another embodiment, the immunosuppressive polypeptide is in a secretory form. This recombinant secreted PD-L1 (or soluble PD-L1) can be generated by fusing the extracellular domain of PD-L1 to the Fc region 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 suppression. PD-L1 ligands can be co-expressed with CTLA4 Ig for further enhanced persistence of both.

[0332] According to 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 β2microglobulin / CD3ζpolypeptides 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.

[0333] According to one implementation, the exogenous sequence encodes the NKG2D ligand. Some viruses, such as cytomegalovirus, have acquired mechanisms to avoid NK cell-mediated immune surveillance by secreting proteins that can bind to the NKG2D ligand and prevent them from being expressed on their surface (Welte, SA et al. (2003) "Selective intracellular retention of virally induced NKG2D ligands by the human cytomegalovirus UL16glycoprotein". Eur. J. Immunol., 33, 194–203). In tumor cells, several 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).

[0334] According to one implementation, the exogenous sequence encodes a cytokine receptor such as the IL-12 receptor. IL-12 is a well-known activator of immune cell activation (Curtis JH (2008) "IL-12 Produced by Dendritic Cells Augments CD8+T Cell Activation through the Production of the Chemokines CCL1 and CCL171". The Journal of Immunology. 181(12): 8576-8584).

[0335] According to one embodiment, the exogenous sequence encodes an antibody against an inhibitory peptide or protein. The antibody is preferably secreted by immune cells in a soluble form. Nanobodies derived from sharks and camels are advantageous in this regard 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 considered to be more readily fused with secretory signaling peptides and with soluble hydrophilic domains.

[0336] The various aspects of improving cell durability developed above are particularly preferred when an exogenous coding sequence is introduced by disrupting an endogenous gene encoding β2m or another MHC component, as detailed in further detail.

[0337] Expression of transgenes that enhance the therapeutic activity of immune cells

[0338] According to one aspect of this method, exogenous sequences integrated into immune cell genomic loci encode molecules that enhance the therapeutic activity of immune cells.

[0339] "Enhanced therapeutic activity" means that, relative to the selected target cell type, the engineered immune cells or cell populations according to the present invention become more invasive than unengineered cells or cell populations. The target cells consist of a defined type of cells or cell populations, preferably characterized by common surface markers. In this specification, "therapeutic potential" reflects therapeutic activity, as measured by in vitro experiments. Typically, sensitive cancer cell lines such as Daudi cells are used to evaluate whether immune cells are more or less active against said cells by performing cell lysis or growth reduction measurements. This can also be evaluated by measuring the level of immune cell degranulation or the production of chemokines and cytokines. Experiments can also be performed in mice injected with tumor cells by monitoring the resulting tumor enlargement. Significantly enhanced activity is considered when immune cells reduce the number of cells appearing in these experiments by more than 10%, preferably more than 20%, more preferably more than 30%, and even more preferably more than 50%.

[0340] According to one aspect of the invention, the exogenous sequence encodes a chemokine or cytokine, such as IL-12. Expression of IL-12 is particularly advantageous because this cytokine is widely mentioned in the literature as promoting the activation of immune cells (Colombo M.P. et al. (2002) "Interleukin-12 in anti-tumor immunity and immunotherapy" Cytokine Growth Factor Rev. 13(2):155-68).

[0341] According to a preferred aspect of the invention, the exogenous coding sequence encodes or promotes the secretion of factors that act on other immune cells, such as T-regulatory cell populations, to alleviate their inhibitory effects on said immune cells.

[0342] According to one aspect of the invention, the exogenous sequence encodes an inhibitor of regulatory T cell activity, which is a peptide inhibitor of forkhead / winged helix transcription factor 3 (FoxP3), and more preferably an inhibitor of a cell-penetrating peptide of FoxP3, such as an inhibitor called 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).

[0343] "Inhibitors of regulatory T cell activity" refer to molecules or precursors of such molecules secreted by T cells that allow T cells to escape downregulated activity by regulatory T cells. Typically, such inhibitors of regulatory T cell activity reduce FoxP3 transcriptional activity in these cells.

[0344] According to one aspect of the invention, the exogenous sequence encodes a secretion inhibitor of tumor-associated macrophages (TAMs), such as a CCR2 / CCL2 neutralizer. Tumor-associated macrophages (TAMs) are major regulators of the tumor microenvironment. Clinicopathological studies have shown that the accumulation of TAMs in tumors is associated with poor clinical outcomes. Consistent with this evidence, experimental and animal studies support the idea that TAMs can provide a favorable microenvironment to promote tumor development and progression. (Theerawut C. et al. (2014) "Tumor-Associated Macrophages as Major Players in the Tumor Microenvironment" Cancers (Basel) 6(3):1670–1690). Chemokinase ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP1-NCBI NP_002973.1), is a small cytokine belonging to the CC chemokine family, secreted by macrophages, and produces chemical attraction on monocytes, lymphocytes, and basophils. CCR2 (CC chemokine receptor type 2-NCBINP_001116513.2) is the receptor for CCL2.

[0345] Although the coding sequence inserted at the locus typically encodes a polypeptide that improves the therapeutic potential of engineered immune cells, the inserted sequence can also be a nucleic acid capable of directing or inhibiting the expression of other genes, such as interfering RNA or guide RNA. The polypeptide encoded by the inserted sequence can act directly or indirectly, such as a signal transduction protein or transcription regulator.

[0346] Engineered immune cells and immune cell populations

[0347] The present invention also relates to a variety of engineered immune cells that can be obtained according to one of the methods described herein, either in isolated form or as part of a cell population.

[0348] According to a preferred aspect of the invention, the engineered cells are primary immune cells, such as NK cells or T cells, which are typically part of a cell population that may include different types of cells. Generally, the population derived from a patient or donor is isolated from PBMCs (peripheral blood mononuclear cells) via leukocyte ablation.

[0349] This invention covers immune cells comprising any combination of different exogenous coding sequences and gene inactivation, which have been described independently above. Among these combinations, endogenous promoters that are active during immune cell activation are particularly preferred, specifically those promoters located at a TCR locus, specifically those combining CAR expression under the transcriptional control of the TCRα promoter.

[0350] Another preferred combination is to insert a foreign sequence encoding a CAR or one of its components under the transcriptional control of the hypoxia-inducible factor 1 gene promoter (Uniprot: Q16665).

[0351] The present invention also relates to pharmaceutical compositions for treating infections or cancer, comprising engineered primary immune cells or populations of immune cells as described above, and to methods for treating patients in need of such treatment, wherein the methods include:

[0352] - Prepare engineered primary immune cell populations according to the method of the present invention as described above;

[0353] -Optionally, the engineered primary immune cells are purified or sorted;

[0354] - Activate the engineered primary immune cell population during or after infusion of the cells into the patient.

[0355] T cell activation and expansion

[0356] Whether before or after genetic modification, the immune cells according to the present invention can be activated or expanded, even if they can be activated or proliferated independently of antigen-binding mechanisms. Specifically, T cells can be activated and expanded using, for example, methods described in U.S. Patents 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 expanded in vitro or in vivo. T cells are typically expanded by contacting reagents that stimulate the CD3 TCR complex and co-stimulatory molecules on the surface of T cells to generate activation signals for T cells. For example, chemicals such as calcium ion carrier A23187, phorbol 12-myristate 13-acetate (PMA), or mitotic lectins such as phytohemagglutinin (PHA) can be used to generate activation signals for T cells.

[0357] As a non-limiting example, T cell populations can be stimulated in vitro, such as by contacting anti-CD3 antibodies or their antigen-binding fragments or anti-CD2 antibodies immobilized on a surface, or by contacting calcium ionocarriers bound to protein kinase C activators (e.g., lichenin). To co-stimulate helper molecules on the T cell surface, ligands binding helper molecules are used. For example, T cell populations can be contacted with anti-CD3 and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. Suitable conditions for T cell culture include appropriate culture media (e.g., minimum essential medium or RPMI 1640 or X-vivo 5 (Lonza)) that may contain factors necessary for proliferation and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGF-β, and TNF—or any other additives known to the art for cell growth. Other additives used for cell growth include, but are not limited to, surfactants, human plasma protein powder, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Culture media may include RPMI 1640, A1M-V, DMEM, MEM, α-MEM, F-12, X-Vivo 1, and X-Vivo 20; an optimizer containing added amino acids, sodium pyruvate, and vitamins; serum-free or supplemented with adequate amounts of serum (or plasma) or a defined group of hormones; and / or sufficient amounts of cytokines to promote T cell growth and expansion. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures and not in cell cultures to be infused into subjects. Target cells are maintained under conditions necessary to support growth, such as appropriate temperature (e.g., 37°C) and atmosphere (e.g., air with 5% CO2). T cells exposed to different stimulation times may exhibit different characteristics.

[0358] In another specific embodiment, the cells can be expanded by co-culturing with tissues or cells. The cells can also be expanded in vivo, for example, in the blood of a subject after the cells have been administered to that subject.

[0359] Therapeutic Compositions and Applications

[0360] The method of the present invention described above allows for the generation of engineered primary immune cells within a limited timeframe of about 15 to 30 days, preferably 15 to 20 days, and most preferably 18 to 20 days, so that they retain their full immunotherapeutic potential, particularly in terms of cytotoxic activity.

[0361] These cells form cell populations, which are preferably derived from a single donor or patient. These cell populations can be expanded under closed culture receivers to meet the highest production practice requirements and can be frozen before infusion into a patient, thereby providing “off-the-shelf” or “ready-to-use” therapeutic compositions.

[0362] According to the present invention, a large number of cells derived from the same leukocyte ablation technique can be obtained, which is crucial for obtaining a sufficient dose to treat patients. Although differences can be observed between cell populations derived from different donors, the number of immune cells obtained by leukocyte ablation is typically around 10. 8 Up to 10 10 PBMCs contain several cell types: granulocytes, monocytes, and lymphocytes, with 30 to 60% being T cells, which typically represent 10 cells from a single donor. 8 Up to 10 9 Primary T cells. The method of this invention typically concludes with an engineered cell population, generally exceeding approximately 10n. 8 T cells, more often than about 10 9 T cells, or even more than about 10 T cells in general. 10 10 T cells, and often more than 10 11 T cells.

[0363] Therefore, the present invention relates more specifically to therapeutically effective primary immune cell populations, wherein at least 30%, preferably 50%, more preferably 80% of the cells in the population have been modified according to any of the methods described herein.

[0364] According to a preferred aspect of the invention, more than 50% of the population comprises TCR-negative T cells. According to a more preferred aspect of the invention, more than 50% of the population comprises CAR-positive T cells. Engineered immune cells, cell populations, therapeutic compositions, and uses are also described.

[0365] Therefore, this composition or population of cells can be used as a medicine; especially for the treatment of cancer in patients in need, particularly for the treatment of lymphoma, but also for the treatment of solid tumors such as melanoma, neuroblastoma, glioma, or cancers such as lung cancer, breast cancer, colon cancer, prostate cancer, or ovarian cancer.

[0366] More specifically, the present invention relates to a population of primary TCR-negative T cells derived from a single donor, wherein at least 20%, preferably 30%, more preferably 50% of the cells in the population have been modified at at least two (preferably three) different loci using sequence-specific reagents.

[0367] In another respect, the present invention relates to a method for treating a patient in need of treatment, the method comprising at least one of the following steps:

[0368] (a) Identify specific antigenic markers present on the surface of a patient’s tumor biopsy;

[0369] (b) Providing an engineered primary immune cell population engineered by one of the methods of the present invention as described above, which preferably expresses a recombinant receptor against the specific antigen marker;

[0370] (c) The engineered population of the engineered primary immune cells is given to the patient.

[0371] Typically, the cell population mainly comprises CD4 and CD8 positive immune cells such as T cells, which can undergo robust in vivo T cell expansion and can sustain prolonged levels both in vitro and in vivo.

[0372] Treatment involving engineered primary immune cells according to the present invention can be ameliorative, curative, or preventative. It can be part of autologous immunotherapy or allogeneic immunotherapy.

[0373] In another embodiment, the isolated cells according to the invention or the 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, hepatobiliary cancer, pancreatic cancer, colon cancer, lung cancer, thymic cancer, mesothelioma, ovarian cancer and / or endometrial cancer.

[0374] It also includes adult tumors / cancers and childhood tumors / cancers.

[0375] Treatment using engineered immune cells according to the present invention can be combined with one or more anticancer therapies selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser therapy, and radiotherapy.

[0376] According to a preferred embodiment of the invention, the treatment can be administered to a patient who has undergone immunosuppressive therapy. Indeed, the invention preferably relies on cells or cell populations that have developed resistance to at least one immunosuppressant due to the inactivation of genes encoding receptors for such immunosuppressants. In this respect, immunosuppressive therapy should facilitate the selection and expansion of T cells according to the invention within the patient.

[0377] The administration of cells or cell populations according to the invention can be performed in any convenient manner, including via aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered to patients via subcutaneous, intradermal, intratumoral, intranodular, intramedullary, intramuscular, intravenous, or intralymphatic injection, or intraperitoneal injection. In one embodiment, the cell compositions of the invention are preferably administered via intravenous injection.

[0378] Cells or cell populations can be administered at a dose of 10 per kg of body weight. 4 -10 9 Composed of 10 cells, preferably 10 5 Up to 10 6 Cells / kg body weight, including integer values ​​for all cell numbers within these ranges. Therefore, the present invention can provide doses of more than 10, typically more than 50, more usually more than 100, and often more than 1000, comprising 10 cells / kg body weight from a single donor or patient sample. 6 -10 8 A gene-edited cell.

[0379] Cells or cell populations may be administered in one or more doses. In another embodiment, the effective amount of cells is administered in a single dose. In another embodiment, the effective amount of cells is administered in more than one dose over a period of time. The timing of administration is within the judgment of the administering physician and depends on the patient's clinical condition. Cells or cell populations can be obtained from any source, such as blood banks or donors. Although individual needs vary, the determination of the optimal range of effective amounts for a given cell type for a particular disease or condition is within the scope of those skilled in the art. An effective amount is the amount that provides therapeutic or preventative benefit. The dose administered will depend on the recipient's age, health, and weight, the type of concurrent treatments (if present), the frequency of treatment, and the nature of the desired effect.

[0380] In another embodiment, the effective amount of cells or a composition containing those cells is administered parenterally. The administration may be intravenous. The administration may also be performed directly by intratumoral injection.

[0381] In some embodiments of the invention, the cells are administered to the patient in combination with any number of relevant treatment modalities (e.g., before, during, or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or nastatinumab for MS patients, efariziumab for psoriasis patients, or other treatments for PML patients. In other embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506), antibodies or other immunoablation agents (such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate mofetil, steroids, FR901228, cytokines, and radiation). These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for signal transduction induced by growth factors (Henderson, Naya et al., 1991; Liu, Albers et al., 1992; Bierer, Hollander et al., 1993). In other embodiments, the cell composition of the present invention is administered to the patient in combination with bone marrow transplantation, T-cell ablation therapy using chemotherapeutic agents (such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide), or antibodies (such as OKT3 or CAMPATH) (e.g., before, simultaneously with, or after). In another embodiment, the cell composition of the present invention is administered after B-cell ablation therapy (such as agents that react with CD20, such as rituximab). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an infusion of expanded immune cells of the present invention after transplantation. In other embodiments, the expanded cells are administered before or after surgery.

[0382] The present invention also specifically relates to a general method for treating solid tumors in patients, comprising the steps of immune exhaustion of the patient by a lymphocyte depletion protocol and infusion of genetically engineered lymphocytes that are tolerant to the lymphocyte depletion reagents used in the lymphocyte depletion protocol and specifically target the solid tumor. These genetically engineered lymphocytes are preferably CAR-positive T cells, more preferably having MSLN-CAR as described herein.

[0383] The lymphocyte clearance protocol preferably includes antibodies against antigens present on the surface of immune cells, such as CD52, CD3, CD4, CD8, CD45, or other specific markers or drugs, such as purine analogs (e.g., fludarabine and / or clofarabine) and glucocorticoids.

[0384] According to a preferred embodiment of the invention, the method includes subjecting the patient to a lymphocyte clearance regimen comprising an anti-CD52 antibody and administering engineered CAR T cells with MSLN-CAR, wherein CD52 expression is reduced, insufficient, or inactivated.

[0385] In a preferred embodiment of the invention, lymphocyte depletion therapy may contain an anti-CD52 antibody, such as alenzumab, alone or in combination. For example, a lymphocyte depletion regimen may combine cyclophosphamide (typically 1 to 3 days), fludarabine (1 to 5 days), and alenzumab (1 to 5 days). Typically, a lymphocyte depletion regimen may contain cyclophosphamide at 50 to 70 mg / kg / day, fludarabine at 20 to 40 mg / m² / day, and alenzumab at 0.1 to 0.5 mg / kg / day, alone or in combination.

[0386] To this end, the present invention provides a combination of compositions for lymphocyte clearance in patients affected by solid tumors, the compositions comprising an anti-CD52 antibody and an engineered lymphocyte population targeting MSLNs that is insensitive to said antibody, the population preferably comprising MSLN-CARs expressing MSLN-CARs and having impaired CD52 expression. In these engineered cells, the CD52 gene allele has preferably been inactivated by a dilute endonuclease, such as the previously described TALE nuclease or an RNA-directed endonuclease.

[0387] The present invention also provides a medical kit for use in the treatment of solid tumor cancer, comprising the lymphocyte scavenging composition and the engineered cell population to which it is resistant.

[0388] "Cytolytic activity" or "cytotoxic activity" refers to the percentage of cell lysis in target cells imparted by immune cells.

[0389] The methods used to determine cytotoxicity are as follows:

[0390] For adherent target cells: 2.10 4 0.1 ml of STA-positive or STA-negative cells were seeded into each well of a 96-well plate. The day after seeding, STA-positive and STA-negative cells were labeled with CellTrace CFSE and seeded at 4 × 10⁻⁶ cells per well. 5 T cells were co-cultured for 4 hours. Then, the cells were harvested, stained with a fixable viability dye (eBioscience), and analyzed using a MACSQuant flow cytometer (Miltenyi).

[0391] For suspension target cells: STA-positive and STA-negative cells were labeled with CellTrace CFSE and CellTrace Violet, respectively. Approximately 2 × 10⁶ cells were used. 4 ROR1 positive cells and 2×10 4 STA-negative cells and 4 × 10 5 T cells were co-cultured in 96-well plates at 0.1 ml per well. After 4 hours of culture, cells were harvested, stained with a fixable viability dye (eBioscience), and analyzed using a MACSQuant flow cytometer (Miltenyi).

[0392] The percentage of specific cell lysis can be calculated using the following formula:

[0393]

[0394] "Enhanced cytotoxicity" means that the percentage of cell lysis of target cells conferred by engineered immune cells is increased by at least 10%, such as 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 cell lysis of target cells conferred by unengineered immune cells.

[0395] "Identity" refers to the sequence similarity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing positions in each sequence, which can be aligned for comparative purposes. When positions in the compared sequences are occupied by the same base, the molecules are identical at that position. The degree of similarity or identity between nucleic acid or amino acid sequences depends on the number of identical or matching nucleotides at common positions in the nucleic acid sequences. Various alignment algorithms and / or procedures can be used to calculate the identity between two sequences, including FASTA or BLAST, which are available as part of the GCG Sequence Analysis Package (University of Wisconsin, Madison, Wis.) and can be used, for example, with default settings. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98%, or 99% identity with the specific polypeptides described herein and preferably exhibiting substantially the same function, as well as the polynucleotides encoding these polypeptides, are considered.

[0396] -As used herein, the terms “subject” or “patient” generally refer to mammals, preferably primates, and more preferably humans.

[0397] The foregoing written description of the invention provides ways and means of making and using the invention, thereby enabling any person skilled in the art to make and use the invention. This enabling capability is specifically provided for by the subject matter of the appended claims, which form part of the original description.

[0398] When describing numerical limits or ranges in this document, the endpoint is included. Furthermore, all values ​​and subranges within a numerical limit or range are explicitly included as if explicitly stated.

[0399] The invention has been generally described, and further understanding can be obtained by referring to certain specific embodiments, which are provided for illustrative purposes only and are not intended to limit the scope of the claimed invention.

[0400] Example

[0401] Mesothelin (MSLN) is a glycophosphatidylinositol (GPI)-linked cell surface protein that is typically expressed in mesothelioma cells that fill the pleura, peritoneum, and pericardium. The MSLN gene encodes a 71 kDa precursor protein, which is processed into a 31 kDa shed protein called MPF (megakaryocyte strengthening factor) and a 40 kDa membrane-bound protein called mesothelin.

[0402] Mesothelin has been reported to be highly expressed in several types of malignant tumors, such as malignant mesothelioma, ovarian cancer, pancreatic cancer, and lung adenocarcinoma (Morello et al., 2016; O'Hara et al., 2016). In some cases, mesothelin expression has been associated with increased tumor invasiveness and poor clinical outcomes.

[0403] Description of MSLN-specific CARs used in the study

[0404] Three second-generation CARs were generated, each consisting of scFvs P4, meso1, and MESO2, containing a CD8α hinge / transmembrane domain and 4-1BB and CD3ζ activation domains, and were transmitted through the primary mesoCAR. + The activity of T cells against target cell lines expressing different levels of mesothelin (MSLN) was screened for chimeric antigen receptor (CAR) expression and in vitro antitumor activity. In some forms, a self-destructing switch “R2” is introduced into the CAR structure. The “R2” polypeptide includes two CD20 mimotopes located between the scFv and the hinge to confer sensitivity to anti-CD20 therapeutic antibodies such as rituximab, as previously described in WO2016120216.

[0405] Figure 1 A schematic diagram of the CAR structure is provided.

[0406] The different sequences contained in each CAR are described in detail in Tables 1, 2 and 3, and Table 4 shows its complete amino acid sequence.

[0407] 1-In vitro assay

[0408] CAR expression was screened in primary T cells derived from PBMCs, and their antitumor activity against three target cell lines expressing different levels of MSLN was determined:

[0409] -HeLa( CCL-2), epithelial cell cervical adenocarcinoma,

[0410] -HPAC( CRL-2119), epithelial pancreatic adenocarcinoma, and

[0411] -293H or A2058 cells are mesothelin-negative cells.

[0412] like Figure 3 and 4 As shown, the expression of mesothelin on the surface of these cells was evaluated by flow cytometry.

[0413] 2-Gene-edited MSLN-UCART cell generation

[0414] - On day 0, frozen human peripheral blood mononuclear cells (PBMCs) from Hemacare (Northridge, CA 91325, USA) were thawed, washed, counted, and resuspended in X-vivo 15 medium supplemented with 5% AB serum. The cells were then transferred to an incubator set at 37°C and 5% CO2.

[0415] - On day 1, PBMCs were counted, and CD3+ cell percentage was evaluated by flow cytometry. Cells were centrifuged and resuspended in X-vivo15 medium supplemented with 5% AB serum, 350 UI / ml IL2, and MACS GMP T Cell TransAct (60 μl per million CD3+ cells). Cells were then transferred to an incubator set at 37°C and 5% CO2.

[0416] - On day 4, T cells and the rLV vector with the polynucleotide sequence encoding the anti-MSLN CAR were resuspended in X-vivo 15 medium supplemented with 5% AB serum and 350 UI / ml IL2, and seeded onto plates coated with retronectin. The plates were then transferred to an incubator set to 37°C and 5% CO2.

[0417] - On day 5, T cells were washed and resuspended in X-vivo 15 medium supplemented with 5% AB serum and 350 IU / ml IL2. The cells were then transferred to an incubator set at 37°C and 5% CO2.

[0418] - As previously reported, on day 6, T cells were co-electroplated with mRNAs encoding the right and left arms of TRAC TALEN and CD52 TALEN, respectively [Poirot et al. (2013) Blood. 122(21): 1661] to effectively inactivate the TCRα and CD52 genes and prevent TCRαβ expression on the surface of primary T cells. TALEN is the registered name of TALE nucleases designed by Cellectis (8, rue de la Croix Jarry, 75013 Paris, France). The genomic target sequences of these TALE nucleases are indicated in Table 8 below. Transfection was performed using the AgilePulse technique. Cells were then transferred to an incubator set to 37°C and 5% CO2.

[0419] - On day 7, T cells were washed and resuspended in X-vivo 15 medium supplemented with 5% AB serum and 350 IU / ml IL2. The cells were then transferred to an incubator set at 37°C and 5% CO2.

[0420] - T cells were expanded in the GRex apparatus between days 7 / 8 and 18. When using GRex 6-well cell culture plates, half of the culture medium was removed on days 11 and 15 and replaced with fresh medium containing IL2, with fresh IL2 added on day 13. When using GRex 100M, fresh IL2 was added on days 11, 13, and 15 without any medium replacement. During expansion, the cell cultures were incubated at 37°C and 5% CO2.

[0421] - On day 18, all UCART cells were cryopreserved for use in subsequent in vitro and in vivo assays.

[0422] Table 8: Genomic sequences targeted by TALE nuclease (TALEN)

[0423]

[0424] 2.1 MSLN-CAR expression analysis

[0425] P4-R2, Meso1-R2, and MESO2-R2 CARs were used to generate three MSLN-specific UCART cell products. The different UCART cell products were then evaluated in vitro.

[0426] The first in vitro study of four UCART cell products on day 18 aimed to determine the UCART cell phenotype. For this purpose, 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+ portion of UCART cells. CAR surface expression was evaluated using His-tagged recombinant human cortisol or biotinylated protein L, which both recognize the scFv portion of CAR, or biotinylated rituximab, which recognizes the R2 self-destructing switch portion of CAR. TCRαβ receptor surface expression was assessed using a PE-vio770-conjugated anti-TCRαβ antibody. CD4 and CD8 surface expression were assessed using FITC-conjugated CD4 and BV510-conjugated CD8 antibodies.

[0427] CAR surface expression was assessed by flow cytometry using a His-tagged recombinant human cortisol protein that recognizes the scFv portion of the CAR, or a biotinylated rituximab that recognizes the R2 self-destructing switch portion of the CAR.

[0428] like Figure 6 As shown, at least 50% of UCART cells modified with P4-R2, Meso1-R2, or MESO2-R2 were CAR-positive. However, P4-R2 CAR showed a higher expression level than Meso1-R2 and MESO2-R2 CAR.

[0429] like Figure 7 As shown, at least 51% of the CAR+ portion of UCART cells modified with P4-R2, Meso1-R2, and MESO2-R2 CARs is CD4+.

[0430] 2.2 IFNg production and killing activity

[0431] A second in vitro study of three UCART cell products aimed to analyze UCART cell function. The ability of UCART cells to produce cytokines was assessed by co-culturing with HPAC (MSLN+) or 293H (MSLN-) cells for 24 hours and quantifying IFNg in the cell culture supernatant using a standard ELISA procedure.

[0432] like Figure 8As shown, UCART cells modified with P4-R2 and Meso1-R2 CARs, when co-cultured with MSLN+ cell lines, produced IFNg levels exceeding 40,000 pg / ml and 50,000 pg / ml, respectively, while those co-cultured with MSLN- cell lines produced IFNg levels less than 1,500 and 200 pg / ml, respectively. Although UCART cells modified with Meso1-R2 CARs produced similar levels of IFNg as those modified with P4-R2 CARs when co-cultured with MSLN+ cell lines, cells with Meso1-R2 CARs produced extremely low levels of IFNg when cultured with MSLN- cell lines. UCART cells modified with MESO2-R2 CARs, when co-cultured with MSLN+ cell lines, produced IFNg levels not exceeding 15,000 pg / ml.

[0433] Then, the ability of UCART cells to carry out continuous killing of HPAC cells was evaluated over a 15-day period, which included six rounds of exposure to MSLN+ (HPAC) cells at ratios of 1:2 and 1:8.

[0434] like Figure 9 As shown, after a single round of exposure to HPAC cells, CAR cells modified with P4-R2, Meso1-R2, and MESO2-R2 CARs exhibited similar levels of cytotoxic activity. However, after several rounds of exposure, T cells with Meso1-R2 and P4-R2 showed significantly higher sustained cytotoxic activity than CAR cells with MESO2-R2 CARs, while CAR cells modified with Meso1-R2 showed more sustained activity than those modified with P4-R2.

[0435] Importantly, none of these CART cell products showed significant killing activity against mesothelin-negative A2058 and 293H cells.

[0436] 3. UCART Functional validation of the genetic attributes of MSLN cells

[0437] 3.1 TRAC and / or CD52 gene knockout

[0438] like Figure 10 As shown, less than 20% of UCART cells modified with P4-R2, Meso1-R2, and MESO2-R2 CARs maintained TCRαβ+, indicating that the TALEN-mediated inactivation level of the TCRα gene is highly effective in the cell population.

[0439] In addition, according to Figure 11 The provided flow cytometry data indicate that TCRαβ+ depletion is the result of removing unengineered cells and selecting [CAR].+ [TCR] - Effective steps for UCART cells: 84% of unengineered T cells were TCRαβ+, compared to 8% of TRAC knockout T cells and 0.2% of TRAC knockout T cells depleted in TCRαβ+ cells.

[0440] To fully confirm that the absence of TCRαβ receptor detection on the cell surface of TRAC gene knockout and depleted in TCRαβ+ cells is associated with the absence of functional TCRαβ receptor expression, unengineered T cells, TRAC gene knockout T cells, and TRAC gene knockout and depleted in TCRαβ+ cells were exposed to phytohemagglutinin (PHA) for 24 hours and the expression of the activation marker CD25 was monitored by flow cytometry.

[0441] like Figure 12 As shown, upon exposure to PHA, only unengineered T cells expressed significant levels of CD25 on their surface. These data clearly demonstrate that knockout of the TRAC gene in T cells prevents the expression of the functional TCRαβ receptor on the cell surface.

[0442] After culturing for 7 days in 50 μg / ml anti-CD52 monoclonal antibody (with or without 30% rabbit complement (Cedarlane) or rat IgG as a control), CD52 gene inactivation was evaluated by culturing engineered cells. After culturing at 37°C for 2 hours, cells were labeled with fluorescently conjugated anti-CD52 antibody and a fluorescent viability dye (eBioscience), and the frequency of CD52-positive and CD52-negative cells in live cells was measured by flow cytometry. Alternatively, cells were co-cultured with the antibody to select for resistance.

[0443] 3.2 UCART cell depletion using rituximab targeting the R2 peptide.

[0444] Another in vitro study of UCART cell products aimed to evaluate the ability of UCART cells to be depleted when treated with rituximab.

[0445] UCART cells modified with P4-R2, Meso1-R2, and MESO2-R2 CARs were co-cultured with HPAC cells for 2 days, then exposed to culture medium, rituximab (RTX), rabbit complement (BRC), or a mixture of rituximab and rabbit complement for 2 hours. The percentage of CAR+ cells was monitored by flow cytometry. Figure 13As shown, UCART cells modified with P4-R2, Meso1-R2, and MESO2-R2 were effectively depleted when treated with rituximab and complement from infant rabbits. In contrast, UCART cells modified with naked P4 (MSLN CAR with P4 structure but no R2 sequence) were not depleted.

[0446] 3.3 Inactivation of the TGFβ signaling pathway in CAR T cells

[0447] Another property offered to MesoCAR T cells is tolerance to the tumor microenvironment by inactivating the TGFb signaling pathway. Two different strategies were investigated: inactivation of the TGFbRII gene expression by knockout or by overexpression of the dominant inactivated form of the TGFbRII gene (dnTGFbRII).

[0448] 3.3.1. Inactivation of TGFbRII via KO

[0449] T cells were electroporated with 10 μg of mRNA encoding two TGFbRII genes (pCLS32939 and SEQ ID NO: 156 (pCLS32940) or SEQ ID NO: 157 (pCLS32967) and SEQ ID NO: 158 (pCLS32968)) from the right and left arms of SEQ ID NO: 155. Three days post-transfection, T cells were harvested, gDNA was extracted, and PCR was performed to amplify the amplicon. Analysis of the PCR products by deep sequencing showed that transfection with TALENs encoded by pCLS32939 and pCLS32940, or TALENs encoded by pCLS32967 and pCLS32968, demonstrated 96.62% and 97.28% gene editing (i.e., insertion and / or deletion), respectively, confirming the high efficiency of TGFbRII KO.

[0450] 3.3.2. Inactivation of TGFbRII via overexpression of dnTGFbRII

[0451] MSLN-CAR T cells were generated as described in Example 2 using two different donors and rLV vectors encoding different MSLN CARs with or without dnTGFbRII (SEQ ID NO: 24) separated by a 2A cleavage peptide. Following the generation process, the different MSLN-CAR T cells were thawed and seeded at 3 million cells / ml with 70 IU / ml IL-2. One day after thawing, the cells were exposed to 5 ng / ml TGFb (R&D systems). One hour later, the cells were stained for CAR expression by staining the cell surface with biotinylated recombinant mesothelin (LakePharma) and Brilliant Violet 421 antibiotic streptavidin (BD). Additionally, intracellular staining of MSLN-CAR T cells was performed using PE-conjugated antiphospho-SMAD2 / 3 (BD) according to the supplier's instructions. Cells were analyzed by flow cytometry to determine the phosphorylated SMAD2 / 3 status in CAR-positive or CAR-negative subsets. Figure 14 The results confirmed that in the absence of dnTGFbRII, cells were positive for phosphorylation of SMAD2 / 3. Figure 14 (Left figure), while in the presence of dnTGFbRII, only CAR-positive cells showed reduced SMAD2 / 3 phosphorylation ( Figure 14 (See right figure). These results suggest that TGFb signaling may be impaired in MSLN-CART cells expressing dnTGFbRII.

[0452] 4-In vivo experiments

[0453] Preliminary in vivo studies were conducted to define and validate the animal / tumor model and the administration route of CAR T cells. HPAC tumor cells injected subcutaneously (SC) into NSG mice were selected as the animal / tumor model for evaluating the in vivo antitumor activity of T cells expressing three selected mesoCAR constructs. Although meso1-R2 was lower in terms of cell surface expression levels, cytotoxicity, and IFNγ secretion, this was preserved in the study and compared with P4-R2 and MESO2-R2 CARs.

[0454] Then, the in vivo antitumor activity of human T cells expressing mesoCAR candidate and P4-CAR was evaluated. In short, MSLN was implanted into NSG mice. + Cell line (HPAC cells, SC injection), then mesoCAR + T-cell treatment (IV injection, 3 doses). Evaluation of mesoCAR by monitoring tumor growth. + T cell activity.

[0455] The three mesoCARs evaluated + T cells exhibited in vivo antitumor activity against HPAC tumor cells at varying activity levels. This was compared with other mesoCARs evaluated. + Compared to T cells, T cells expressing MESO2-R2 CAR showed lower activity.

[0456] 4.1 Basis for animal model selection

[0457] Due to mesoCAR + Human-specific T-cell activity is not suitable for studies in standard immunologically active animal models due to the rapid targeting and depletion of human T-cells via xenogeneic immune responses. The selected animal model was a highly immunodeficient NSG mouse strain (from Jackson Laboratory's NOD.Cg-Prkdc). scid Il2rg tm1Wjl / SzJ strain), because it allows human MSLN + Transplantation of both tumor cells and human CAR T cells.

[0458] 4.2 Establishment of animal / tumor models

[0459] 4.2.1 Hela and HPAC transplantation

[0460] Two mesothelin-expressing tumor cell lines were used in this study: HeLa ( CCL-2), epithelial cervical adenocarcinoma, and HPAC ( CRL-2119), epithelial pancreatic adenocarcinoma. The aim of this first study was to evaluate tumor take and tumor growth parameters of HeLa and HPAC cells following subcutaneous (SC) injection in NSG mice (6-8 weeks old).

[0461] In short, on day 0, mice were randomly divided into 4 groups of 6 mice each according to their individual weight and received HeLa (1×10⁻⁶). 6 Or 10×10 6 (cells / mouse) or HPAC cells (2×10⁶ cells / mouse) 6 Or 10×10 6SC injection (cells / mouse). The amount of tumor cells was selected according to the literature [Abate-Daga, D. et al. (2014). A Novel Chimeric Antigen Receptor Against Prostate Stem Cell Antigen Mediates Tumor Destruction in a Humanized Mouse Model of Pancreatic. Cancer. Hum. Gene Ther.; Arjomandnejad et al. (2014) Helacell line xenograft tumor as a suitable cervical cancer model: Growth kinetic characterization and immunohistochemistry array. Arch. Iran. Med.; Kusakawa et al. (2015) Characterization of invivo tumorigenicity tests using severe immunodeficient NOD / Shi-scid IL2Rγ null mice for detection of tumorigenic cellular impurities in human cell-processed therapeutic products. Regen. Ther.].

[0462] Weight, viability, and behavior were monitored daily. Tumor volume was measured three times a week. Surviving mice were terminated on day 61 (end of study). Autopsies (macroscopic examinations) were performed on all animals terminated in this study, and on all euthanized dying or deceased animals, if possible.

[0463] Both HPAC and Hela tumors grew in NSG mice. HPAC tumors grew faster than Hela tumors. Injection of 1×10 6 and 10×10 6 The average tumor volume V (500 mm) of mice with HeLa cells 3 ) respectively 519mm 3 and 498mm 3 The average time to achieve the desired effect was 55 days and 49 days, respectively. Injection 2×10 6 and 10×10 6 The average tumor volume V (500 mm²) in mice with HPAC cells 3 ) are 557mm respectively3 and 500mm 3 The average arrival time is 24 days and 23 days.

[0464] 4.2.2 Mesothelin expression on HeLa and HPAC tumors in NSG mice

[0465] The second study aimed to assess the expression levels of interstitial in HPAC and Hela tumors after subcutaneous injection and growth of tumor cells in NSG mice.

[0466] In short, on day 0, mice were randomly divided into two groups of three mice each based on their individual body weight and received HeLa (10 × 10⁻⁶). 6 (cells / mouse) or HPAC cells (2×10⁶ cells / mouse) 6 Administer SC (cells / mouse) via injection. Monitor body weight, viability, and behavior daily. Measure tumor volume three times a week. When the tumor volume reaches 300-500 mm... 3 Tumors were collected at the designated time. Tumor samples were analyzed by immunohistochemical (IHC) analysis of mesothelin expression. The last mice were sacrificed on day 63 (end of study).

[0467] Both HPAC and HeLa tumors grew in NSG mice. As observed in previous studies, HPAC tumors grew faster than HeLa tumors. Mice injected with HeLa cells or HPAC cells reached 300 mm at 40 days and 28 days, respectively. 3 The average tumor volume.

[0468] In addition, the expression of mesothelin on tumor cells was examined using IHC in tumors collected from mice. Both types of tumors expressed mesothelin.

[0469] Based on this confirmation data, it was decided to use HPAC cells (2 × 10⁻⁶). 6 To evaluate mesoCAR (cells, SC injection) + The anti-tumor activity of T cells.

[0470] 4.2.3 HPAC-luc-GFP tumor cell transplantation

[0471] HPAC cells expressing firefly luciferase and GFP (HPAC-luc-GFP) were generated using Cellectis, and tumorigenesis and tumor growth of HPAC-luc-GFP cells in NSG mice were evaluated as previously described.

[0472] In short, on day 0, three mice received HPAC-luc-GFP cells (2 × 10⁻⁶). 6SC injection (cells / mouse). Monitor body weight, viability, and behavior daily. Measure tumor volume three times a week and perform bioluminescence imaging on days 7, 14, and 24. Monitor viability and behavior daily.

[0473] Because these conditions provide sensitivity for this assay, it was decided to use wild-type HPAC cells (SC injection, 2 × 10⁻⁶). 6 To evaluate mesoCAR (cells / mouse) + T cell activity in vivo.

[0474] 4.3 Evaluation of the antitumor activity of the UCARTmeso candidate against HPAC tumors in NSG mice

[0475] The aim 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 treatment conditions preliminarily defined in this study. The three CARs were evaluated. + T cell doses (1, 3, and 10 × 10⁻⁶) 6 (1 CAR-positive cell / mouse).

[0476] UCARTmeso and control T cells were generated using PBMCs from the same donor. UCARTmeso and control T cells were not purified from TCRαβ-negative cells. The characteristics of the T cells used are shown in Table 9.

[0477] Table 9: Characteristics of the T cells used in this study.

[0478]

[0479] Day 18: Production process ends (before freezing);

[0480] %CAR + On day 18, relative to CD45 + %CD45 + / CAR + (Recombinant MSLN protein was used for P4-R2 CAR and L-protein was used for Meso1-R2 and MESO2-R2 CAR, as measured by flow cytometry)

[0481] %CD4 + On day 18, relative to CD45 + %CD45 + / CAR + / CD4 +

[0482] %CD8 + On day 18, relative to CD45+ %CD45 + / CAR + / CD8 +

[0483] %TCRαβ - On day 18, relative to CD45 + %CD45 + / TCRαβ -

[0484] In short, on day 7, 85 NSG mice received HPAC tumor cells (2 × 10⁻⁶). 6 The mice were injected subcutaneously with 1 cell per mouse. On day 0, 80 mice with tumors were randomly divided into 16 groups of 5 mice each, based on tumor volume.

[0485] Human T cells (UCARTmeso cells and KO TRAC / NT cells from the control group) were injected on day 0 (for groups 1–15) or day 12 (for group 16). For UCARTmeso cells, the total number of cells injected was defined based on the percentage of CAR-positive cells in the batch, to inject a specified number of CAR-positive cells (1, 3, or 10 × 10⁶) per mouse. 6 (1 CAR-positive cell).

[0486] The antitumor activity of UCART meso candidate CARs (P4-R2, Meso1-R2, and MESO2-R2) was assessed by measuring tumor volume. Figure 15 , 16 (and 17). All UCARTmeso cells showed antitumor activity, although the activity levels varied among different CAR T cells.

[0487] like Figure 18 As shown, at high doses (10×10 6 Antitumor activity was observed in all CAR candidates. 3×10 6 MESO2-R2 CAR + Cells cannot control HPAC tumor growth.

[0488] in conclusion

[0489] Animal / tumor models and treatment conditions have been established to allow for the evaluation of the antitumor activity of CAR T cells targeting mesothelin. The activity of three CAR candidates (MESO2-R2, Meso1-R2, and P4-R2) was evaluated in vivo using these animal models, and all CAR T cells showed antitumor activity against HPAC cells.

[0490] However, MESO2-R2 CAR+ T cells have lower activity compared to CAR+ T cells expressing Meso1-R2.

[0491] Among CAR T cells with the selected properties (R2 self-destructing switch and TRAC KO), the Meso1-R2 CAR T cell candidate showed the highest in vivo activity at the three evaluated doses.

[0492] 4.4 Evaluation of the antitumor activity of UCARTmeso candidates expressing dnTGFBRII

[0493] The aim of this study was to compare the antitumor activity of two UCARTmeso candidates (P4-R2 and MESO1-R2) that also express dnTGFBRII in NSG mice. In short, on day 0, 4 to 6 mice in each group received HPAC cells (2 × 10⁻⁶ cells). 6 Administer SC (cells / mouse) via injection. Monitor body weight, viability, and behavior daily. Measure tumor volume three times a week. Monitor viability and behavior daily.

[0494] All UCARTmeso expressing dnTGFBRII were generated using PBMCs from the same donor, and two CARs were evaluated. + T cell dose (3 and 10 × 10) 6 (1 CAR-positive cell / mouse).

[0495] The antitumor activity of UCARTmeso candidates (P4-R2, MESO1-R2) expressing dnTGFBRII was evaluated by measuring tumor volume. Figure 19 Both UCARTmeso cell lines exhibited antitumor activity, albeit at different levels, with the MESO1 construct showing better antitumor activity.

[0496] 5- For UCART targeting mesothelin (UCART) TGFBRII gene inactivation (KO) of MESO TGFBRII) and Comparison of dnTGFBRII gene overexpression methods

[0497] The work presented in this study aims to compare TGFBRII KO and dnTGFBRII gene overexpression methods and select the best method for UCART MESO.

[0498] To conduct this study, six types of genetically modified T cells were generated and tested (as defined in Table 10).

[0499] Table 10: Description of the six types of genetically modified T cells produced in the study.

[0500]

[0501] 5.1 Different UCART The generation of MESO

[0502] - On day 0, frozen human peripheral blood mononuclear cells (PBMCs) from Hemacare (Northridge, CA91325, USA) were thawed, washed, counted, and resuspended in X-vivo 15 medium supplemented with 5% AB serum. The cells were then transferred to an incubator set at 37°C and 5% CO2.

[0503] - On day 1, PBMCs were counted, and CD3+ cell percentage was evaluated by flow cytometry. Cells were centrifuged and resuspended in X-vivo15 medium supplemented with 5% AB serum, 350 UI / ml IL2, and MACS GMP T Cell TransAct (60 μl per million CD3+ cells). Cells were then transferred to an incubator set at 37°C and 5% CO2.

[0504] - On day 4, T cells and rLV vectors with different polynucleotide sequences encoding anti-mesothelin CARs P4-CAR (SEQ ID NO: 161) and MESO1 CAR (SEQ ID NO: 22) and 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 on fibronectin-coated plates. The plates were then transferred to an incubator set at 37°C and 5% CO2.

[0505] - On day 5, T cells were washed and resuspended in X-vivo 15 medium supplemented with 5% AB serum and 350 IU / ml IL2. The cells were then transferred to an incubator set at 37°C and 5% CO2.

[0506] - On day 6, T cells were electroporated with mRNA encoding the right and left arms of TRAC TALEN, with or without mRNA encoding the right and left arms of TGFBRIITALEN (SEQ ID NO: 157 and SEQ ID NO: 158). Transfection was performed using the AgilePulse technique. Cells were then transferred to an incubator set to 37°C, 5% CO2.

[0507] - On day 7, T cells were washed and resuspended in X-vivo15 medium supplemented with 5% AB serum and 350 IU / ml IL2. The cells were then transferred to an incubator set at 37°C and 5% CO2.

[0508] - Between day 7 / 8 and day 18, T cells were expanded in the GRex device. During expansion, the cell culture was incubated at 37°C and 5% CO2 with the culture medium changed periodically.

[0509] - On day 18, all UCART cells were cryopreserved for use in subsequent in vitro and in vivo assays.

[0510] 5.2 Assessment of UCART cell population

[0511] On day 18, UCART was analyzed by flow cytometry. CAR surface expression was assessed using recombinant mesothelin protein that recognizes the scFv moiety of CAR and biotinylated streptavidin conjugated to PE. dnTGFBRII was assessed using anti-TGFBRII (from Abcam) and APC-conjugated anti-mouse IgG antibodies. CD4 and CD8 surface expression were assessed using FITC-conjugated CD4 and BV510-conjugated CD8 antibodies. Additionally, stemness of UCART cells in CAR+CD4+ or CAR+CD8+ positive cells was analyzed using anti-CD62L conjugated to PECy7 and anti-CD45RA conjugated to APC.

[0512] like Figure 20 As shown in Figure A, P4 expression was detected in 58% of UCART cells and MESO1 expression was detected in 37% of UCART cells. Additionally, dnTGFBR2 was detected in 32% and 17% of UCART cells, respectively, when transduced using P4-dnTGFBRII and MESO1-dnTGFBRII constructs. These results reflect the low expression of dnTGFBRII, which is located downstream of the 2A peptide and CAR.

[0513] Interestingly, in CAR-positive cells, the percentage of CD8+ positive cells varied between 27% and 35% when UCART cells expressed the P4 CAR construct. However, when the MESO1 construct was expressed, the percentage of CD8+ cells (in CAR-positive cells) varied between 36% and 51%. Figure 20 B).

[0514] In addition, stemness analysis showed that in CAR+CD4+ cells ( Figure 21 A) Using P4CAR, the percentage of untreated T cells (Tn) and T memory stem cells (Tscm) varied between 1% and 3%, while in T cells expressing MESO1 CAR, this subtype was higher and varied at around 6%. This effect was also observed to a lower extent in CAR+CD8+ cells, where the percentage of this T cell subtype varied between 15% and 19% and between 19% and 22% respectively, using either the P4 or MESO1 construct. Figure 21 B).

[0515] These results confirm that even with lower expression or detection, MESO1 CAR resulted in a higher proportion of CD8+ (i.e., cytotoxic) and a higher proportion of Tn and Tscm isotypes compared to P4CAR. Importantly, overexpression of the dnTGFBRII construct or inactivation of TGFBRII via KO had no effect on any of the analyzed phenotypes.

[0516] 5.3 Assessment of UCART cytotoxicity and IFNγ production

[0517] Sixteen hours after the post-melting recovery phase, the genetically modified T cells shown in Table 9 were mixed with H226-Luc / GFP cells at effector-to-target (E:T) ratios of 1:3, 1:1, 3:1, and 10:1. The co-cultures were then incubated overnight at 37°C, and bioluminescence was measured to quantify H226 cell lysis. Alternatively, after the post-melting recovery phase, these genetically modified T cells were resuspended in culture medium and seeded at a density of 200,000 cells / well in 96-well untreated or previously coated plates with 75 ng / well of His-tagged recombinant mesothelin. After a 24-hour incubation period, the cell supernatant was collected and analyzed by ELISA to quantify IFNg production.

[0518] like Figure 22 As shown, compared with UCART expressing P4, UCART expressing MESO1 can induce higher cytotoxicity at the lowest doses (1:3 and 1:1 ratios).

[0519] Figure 23 A confirmed that recombinant mesothelin protein could induce IFNg secretion in all produced UCAR T cells. This production varied between 40,000 and up to 90,000 pg / ml, and no significant effect was observed from CAR constructs or TGFB pathway inhibition. However, when analyzing IFNg secretion in the absence of recombinant mesothelin ( Figure 23 B) Unexpectedly, the MESO1CAR construct produced less IFNg than the P4 construct. This result indicates that UCART cells expressing the MESO1CAR construct received less stimulation in the absence of the antigen, suggesting that MESO1CAR possesses reduced “self-activation.” This is an important property in the therapeutic context, as “self-activation” often leads to CAR T cell depletion.

[0520] 5.4 Assessment of TGFb sensitivity

[0521] To determine the effect of TGFB pathway inactivation via KO or dnTGFBRII overexpression, T cells with the gene modifications described in Table 9 were exposed to TGFb for 1 hour and analyzed by flow cytometry to assess the fraction of pSMAD2 / 3 positive cells relative to pSMAD2 / 3 negative cells in CAR-positive cells. In another set of experiments, the resulting UCART cells were exposed to recombinant mesothelin protein in the presence or absence of TGFb and counted after 7 days to evaluate proliferation.

[0522] Figure 24 A showed that in the absence of TGFb pathway inhibition, over 95% of the CAR-positive fraction in UCART cells were pSMAD2 / 3 positive. When inhibited by overexpression of dnTGFBRII, 67% and 60% of the CAR-positive fractions in UCART cells expressing the P4 or MESO1 constructs, respectively, were pSMAD2 / 3 negative. Interestingly, when expressing the P4 or MESO1 constructs, inactivation by KO resulted in 85% and 83% of pSMAD2 / 3 negative cells, respectively. These results confirm that TGFBRII KO has a stronger ability to reduce SMAD2 / 3 phosphorylation.

[0523] Figure 24 B showed that TGFb could inhibit antigen-mediated proliferation of UCART expressing either the P4 or MESO1 constructs to the same extent. Importantly, inhibition of the TGFB pathway via KO or dnTGFBRII overexpression reduced or even eliminated this inhibition. sequence list <110> Cellectis <120> Mesothelin-specific chimeric antigen receptor (CAR) for immunotherapy of solid tumors <130> P81905747PCT00 <150> PA201970835 <151> 2019-12-23 <160> 161 <170> PatentIn version 3.5 <210> 1 <211> 127 <212> PRT <213> artificial <220> <223> P4 Heavy Chain Variable Region <400> 1 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Thr Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Val Ser Val Lys Ser Arg Met Ser Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Gly Met Met Thr Tyr Tyr Tyr Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ile Leu Gly 115 120 125 <210> 2 <211> 116 <212> PRT <213> Artificial <220> <223> Variable region of P4 light chain <400> 2 Gln Pro Val Leu Thr Gln Ser Ser Ser Leu Ser Ala Ser Pro Gly Ala 1 5 10 15 Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn Val Gly Pro 20 25 30 Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr 35 40 45 Leu Leu Asn Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Val 65 70 75 80 Leu Leu Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 85 90 95 Met Ile Trp His Ser Ser Ala Ala Val Phe Gly Gly Gly Thr Gln Leu 100 105 110 Thr Val Leu Ser 115 <210> 3 <211> 5 <212> PRT <213> artificial <220> <223> CDRH1‑meso1 <400> 3 Watch Tyr Tyr Trp Watch 1 5 <210> 4 <211> 16 <212> PRT <213> artificial <220> <223> CDRH2‑meso1 <400> 4 Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 5 <211> 8 <212> PRT <213> artificial <220> <223> CDRH3‑meso1 <400> 5 Val Asp Tyr Lys Ala Phe Asp Ile 1 5 <210> 6 <211> 11 <212> PRT <213> artificial <220> <223> CDRL1‑meso1 <400> 6 Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu His 1 5 10 <210> 7 <211> 7 <212> PRT <213> artificial <220> <223> CDRL2‑meso1 <400> 7 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 8 <211> 9 <212> PRT <213> artificial <220> <223> CDRL3‑meso1 <400> 8 Leu Gln His Tyr Ser Tyr Pro Trp Thr 1 5 <210> 9 <211> 116 <212> PRT <213> artificial <220> <223> meso1 heavy chain variable region <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Val Asp Tyr Lys Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser 115 <210> 10 <211> 107 <212> PRT <213> artificial <220> <223> meso1 light chain variable region <400> 10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Tyr Ser Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 11 <211> 121 <212> PRT <213> Artificial <220> <223> meso 2 heavy chain variable region <400> 11 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Met Pro Ser Asp Ser Tyr Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly His Gly Met Tyr Gly Gly Ala Leu Asp Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 12 <211> 111 <212> PRT <213> Artificial <220> <223> meso2 light chain variable region <400> 12 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Ser 20 25 30 Arg Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Lys Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser His Asp Pro 85 90 95 Ser Gly Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 <210> 13 <211> 15 <212> PRT <213> artificially <220> <223> connector <400> 13 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 14 <211> 21 <212> PRT <213> artificially <220> <223> CD8α signal peptide <400> 14 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 15 <211> 40 <212> PRT <213> artificial <220> <223> R2 self-extinguishing switch <400> 15 Ser Asp Pro Gly Ser Gly Gly Gly Gly Ser Cys Pro Tyr Ser Asn Pro 1 5 10 15 Ser Leu Cys Ser Gly Gly Gly Gly Ser Cys Pro Tyr Ser Asn Pro Ser 20 25 30 Leu Cys Ser Gly Gly Gly Gly Ser 35 40 <210> 16 <211> 45 <212> PRT <213> artificial <220> <223> CD8α hinge <400> 16 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 17 <211> twenty four <212> PRT <213> artificial <220> <223> CD8α transmembrane domain <400> 17 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 18 <211> 42 <212> PRT <213> artificial <220> <223> 4-1BB co-stimulatory domain <400> 18 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 19 <211> 112 <212> PRT <213> artificial <220> <223> CD3ζ signal transduction domain <400> 19 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 20 <211> 542 <212> PRT <213> artificially <220> <223> P4‑R2 CAR complete sequence <400> 20 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu 20 25 30 Val Thr Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp 35 40 45 Ser Val Ser Ser Asn Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro 50 55 60 Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp 65 70 75 80 Tyr Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Met Ser Ile Asn Pro 85 90 95 Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro 100 105 110 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Met Met Thr Tyr Tyr 115 120 125 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Gly Ile Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gln Pro Val Leu Thr Gln Ser Ser Ser Leu Ser Ala Ser 165 170 175 Pro Gly Ala Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn 180 185 190 Val Gly Pro Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro 195 200 205 Pro Gln Tyr Leu Leu Asn Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly 210 215 220 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn 225 230 235 240 Ala Gly Val Leu Leu Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp 245 250 255 Tyr Tyr Cys Met Ile Trp His Ser Ser Ala Ala Val Phe Gly Gly Gly 260 265 270 Thr Gln Leu Thr Val Leu Ser Ser Asp Pro Gly Ser Gly Gly Gly Gly 275 280 285 Ser Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser Gly Gly Gly Gly Ser 290 295 300 Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser Gly Gly Gly Gly Ser Thr 305 310 315 320 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 325 330 335 Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 340 345 350 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 355 360 365 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 370 375 380 Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 385 390 395 400 Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 405 410 415 Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 420 425 430 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 435 440 445 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 450 455 460 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 465 470 475 480 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 485 490 495 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 500 505 510 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 515 520 525 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 530 535 540 <210> 21 <211> 522 <212> PRT <213> artificial <220> <223> meso1‑R2 CAR complete sequence <400> 21 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu 20 25 30 Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly 35 40 45 Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys 50 55 60 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr 65 70 75 80 Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys 85 90 95 Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Val Asp Tyr Lys Ala Phe Asp Ile Trp Gly 115 120 125 Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 145 150 155 160 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 165 170 175 Ala Ser Gln Gly Ile Arg Asn Asp Leu His Trp Tyr Gln Gln Lys Pro 180 185 190 Gly Lys Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser 195 200 205 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 210 215 220 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 225 230 235 240 Leu Gln His Tyr Ser Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val 245 250 255 Glu Ile Lys Ser Asp Pro Gly Ser Gly Gly Gly Gly Ser Cys Pro Tyr 260 265 270 Ser Asn Pro Ser Leu Cys Ser Gly Gly Gly Gly Ser Cys Pro Tyr Ser 275 280 285 Asn Pro Ser Leu Cys Ser Gly Gly Gly Gly Ser Thr Thr Thr Pro Ala 290 295 300 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 305 310 315 320 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 325 330 335 Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 340 345 350 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys 355 360 365 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 370 375 380 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 385 390 395 400 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 405 410 415 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 420 425 430 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 435 440 445 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 450 455 460 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 465 470 475 480 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 485 490 495 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 500 505 510 Ala Leu His Met Gln Ala Leu Pro Pro Arg 515 520 <210> 22 <211> 482 <212> PRT <213> artificially <220> <223> meso1 CAR complete sequence <400> 22 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu 20 25 30 Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly 35 40 45 Ser Ile Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys 50 55 60 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr 65 70 75 80 Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys 85 90 95 Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Val Asp Tyr Lys Ala Phe Asp Ile Trp Gly 115 120 125 Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 145 150 155 160 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 165 170 175 Ala Ser Gln Gly Ile Arg Asn Asp Leu His Trp Tyr Gln Gln Lys Pro 180 185 190 Gly Lys Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser 195 200 205 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 210 215 220 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 225 230 235 240 Leu Gln His Tyr Ser Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val 245 250 255 Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 260 265 270 Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 275 280 285 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 290 295 300 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 305 310 315 320 Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu 325 330 335 Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 340 345 350 Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 355 360 365 Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 370 375 380 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 385 390 395 400 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 405 410 415 Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 420 425 430 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 435 440 445 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 450 455 460 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 465 470 475 480 Pro Arg <210> 23 <211> 531 <212> PRT <213> Artificial <220> <223> Complete sequence of meso2-R2 <400> 23 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30 Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr 35 40 45 Ser Phe Thr Asn Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys 50 55 60 Gly Leu Glu Trp Met Gly Val Ile Met Pro Ser Asp Ser Tyr Thr Arg 65 70 75 80 Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser 85 90 95 Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr 100 105 110 Ala Met Tyr Tyr Cys Ala Arg Tyr Gly His Gly Met Tyr Gly Gly Ala 115 120 125 Leu Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val 145 150 155 160 Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala 165 170 175 Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Ser Arg Leu Ala 180 185 190 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly 195 200 205 Ala Ser Lys Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly Ser Gly 210 215 220 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp 225 230 235 240 Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser His Asp Pro Ser Gly Thr 245 250 255 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Ser Asp Pro Gly 260 265 270 Ser Gly Gly Gly Gly Ser Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser 275 280 285 Gly Gly Gly Gly Ser Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser Gly 290 295 300 Gly Gly Gly Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 305 310 315 320 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 325 330 335 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 340 345 350 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 355 360 365 Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu 370 375 380 Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 385 390 395 400 Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 405 410 415 Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 420 425 430 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 435 440 445 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 450 455 460 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 465 470 475 480 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 485 490 495 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 500 505 510 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 515 520 525 Pro Pro Arg 530 <210> 24 <211> 199 <212> PRT <213> Artificial <220> <223> Dominant negative dnTGFβRII <400> 24 Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val 20 25 30 Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 35 40 45 Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 50 55 60 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 65 70 75 80 Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 85 90 95 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 100 105 110 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 115 120 125 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 130 135 140 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu 145 150 155 160 Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu 165 170 175 Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn 180 185 190 Arg Gln Gln Lys Leu Ser Ser 195 <210> 25 <211> 291 <212> PRT <213> Homo sapiens <220> <223> MSLN target antigen region <400> 25 Glu Val Glu Lys Thr Ala Cys Pro Ser Gly Lys Lys Ala Arg Glu Ile 1 5 10 15 Asp Glu Ser Leu Ile Phe Tyr Lys Lys Trp Glu Leu Glu Ala Cys Val 20 25 30 Asp Ala Ala Leu Leu Ala Thr Gln Met Asp Arg Val Asn Ala Ile Pro 35 40 45 Phe Thr Tyr Glu Gln Leu Asp Val Leu Lys His Lys Leu Asp Glu Leu 50 55 60 Tyr Pro Gln Gly Tyr Pro Glu Ser Val Ile Gln His Leu Gly Tyr Leu 65 70 75 80 Phe Leu Lys Met Ser Pro Glu Asp Ile Arg Lys Trp Asn Val Thr Ser 85 90 95 Leu Glu Thr Leu Lys Ala Leu Leu Glu Val Asn Lys Gly His Glu Met 100 105 110 Ser Pro Gln Ala Pro Arg Arg Pro Leu Pro Gln Val Ala Thr Leu Ile 115 120 125 Asp Arg Phe Val Lys Gly Arg Gly Gln Leu Asp Lys Asp Thr Leu Asp 130 135 140 Thr Leu Thr Ala Phe Tyr Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu 145 150 155 160 Glu Leu Ser Ser Val Pro Pro Ser Ser Ile Trp Ala Val Arg Pro Gln 165 170 175 Asp Leu Asp Thr Cys Asp Pro Arg Gln Leu Asp Val Leu Tyr Pro Lys 180 185 190 Ala Arg Leu Ala Phe Gln Asn Met Asn Gly Ser Glu Tyr Phe Val Lys 195 200 205 Ile Gln Ser Phe Leu Gly Gly Ala Pro Thr Glu Asp Leu Lys Ala Leu 210 215 220 Ser Gln Gln Asn Val Ser Met Asp Leu Ala Thr Phe Met Lys Leu Arg 225 230 235 240 Thr Asp Ala Val Leu Pro Leu Thr Val Ala Glu Val Gln Lys Leu Leu 245 250 255 Gly Pro His Val Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val 260 265 270 Arg Asp Trp Ile Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu Gly 275 280 285 Leu Gly Leu 290 <210> 26 <211> 9 <212> PRT <213> Artificial <220> <223> Simulated epitope R2 (Rituximab) <400> 26 Cys Pro Tyr Ser Asn Pro Ser Leu Cys 1 5 <210> 27 <211> twenty four <212> PRT <213> artificial <220> <223> Pallizumab Epitope <400> 27 Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp 1 5 10 15 Gln Lys Lys Leu Met Ser Asn Asn 20 <210> 28 <211> 12 <212> PRT <213> artificial <220> <223> Cetuximab epitope 1 <400> 28 Cys Gln Phe Asp Leu Ser Thr Arg Arg Leu Lys Cys 1 5 10 <210> 29 <211> 12 <212> PRT <213> artificial <220> <223> Cetuximab epitope 2 <400> 29 Cys Gln Tyr Asn Leu Ser Ser Arg Ala Leu Lys Cys 1 5 10 <210> 30 <211> 12 <212> PRT <213> artificial <220> <223> Cetuximab epitope 3 <400> 30 Cys Val Trp Gln Arg Trp Gln Lys Ser Tyr Val Cys 1 5 10 <210> 31 <211> 12 <212> PRT <213> artificial <220> <223> Cetuximab epitope 4 <400> 31 Cys Met Trp Asp Arg Phe Ser Arg Trp Tyr Lys Cys 1 5 10 <210> 32 <211> 25 <212> PRT <213> artificial <220> <223> Nivolumab epitope 1 <400> 32 Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp 1 5 10 15 Lys Leu Ala Ala Phe Pro Glu Asp Arg 20 25 <210> 33 <211> 19 <212> PRT <213> artificial <220> <223> Nivolumab epitope 2 <400> 33 Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln 1 5 10 15 Ile Lys Glu <210> 34 <211> twenty four <212> PRT <213> artificial <220> <223> QBEND-10 Tablet <400> 34 Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser Thr Asn Val Ser 1 5 10 15 Pro Ala Lys Pro Thr Thr Thr Ala 20 <210> 35 <211> 12 <212> PRT <213> artificial <220> <223> Alenduzumab epitope <400> 35 Gly Gln Asn Asp Thr Ser Gln Thr Ser Ser Pro Ser 1 5 10 <210> 36 <211> 49 <212> DNA <213> Homo sapiens <220> <223> T003387 target sequence TGFbetaRII <400> 36 ttttgtttcc ccatcagaat ataacaccag caatcctgac ttgttgcta 49 <210> 37 <211> 49 <212> DNA <213> Homo sapiens <220> <223> T003401 target sequence TGFbetaRII <400> 37 tccctatgag gagtatgcct cttggaagac agagaaggac atcttctca 49 <210> 38 <211> 49 <212> DNA <213> Homo sapiens <220> <223> T003400 target sequence TGFbetaRII <400> 38 tccctatgag gagtatgcct cttggaagac agagaaggac atcttctca 49 <210> 39 <211> 49 <212> DNA <213> Homo sapiens <220> <223> T003405 target sequence TGFbetaRII <400> 39 tgtggggaggc ccaagatgcc catcgtgcac agggacctca agagctcca 49 <210> 40 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 40 acagtgatca cactccatgt ggg 23 <210> 41 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 41 gcagaagctg agttcaacct ggg 23 <210> 42 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 42 aggttaggtc gttcttcacg agg 23 <210> 43 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 43 aaagcgacct ttccccacca ggg 23 <210> 44 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 44 tggatgacct ggctaacagt ggg 23 <210> 45 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 45 cctgggaaac cggcaagacg cgg 23 <210> 46 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 46 acagatatgg caactcccag tgg 23 <210> 47 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 47 gtggaggtga gcaatccccc ggg 23 <210> 48 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 48 acctacagga gtacctgacg cgg 23 <210> 49 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 49 gtgatcacac tccatgtggg agg 23 <210> 50 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 50 gctggtgtta tattctgatg ggg 23 <210> 51 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 51 cacagtgatc acactccatg tgg 23 <210> 52 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 52 cacatggagt gtgatcactg tgg 23 <210> 53 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 53 cagagtaggg tccagacgca ggg 23 <210> 54 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 54 gcttctgctg ccggttaacg cgg 23 <210> 55 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 55 gtggatgacc tggctaacag tgg 23 <210> 56 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 56 gggaaagccc aaagtcacac agg 23 <210> 57 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 57 aatatgacta gcaacaagtc agg 23 <210> 58 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 58 atggagtgtg atcactgtgg agg 23 <210> 59 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 59 atcaccgcct tccacgccaa ggg 23 <210> 60 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 60 aggagcggaa gacggagttg ggg 23 <210> 61 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 61 ccacgccaag ggcaacctac agg 23 <210> 62 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 62 caagatgccc atcgtgcaca ggg 23 <210> 63 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 63 gatatggcaa ctcccagtgg tgg 23 <210> 64 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 64 agcagaagct gagttcaacc tgg 23 <210> 65 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 65 cctgtaggtt gcccttggcg tgg 23 <210> 66 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 66 gtgagcaatc ccccgggcga ggg 23 <210> 67 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 67 acagagtagg gtccagacgc agg 23 <210> 68 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 68 tagcaacaag tcaggattgc tgg 23 <210> 69 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 69 ccaagatgcc catcgtgcac agg 23 <210> 70 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 70 tgtggaggtg agcaatcccc cgg 23 <210> 71 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 71 tgcctcttgg aagacagaga agg 23 <210> 72 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 72 gcccattgag ctggacaccc tgg 23 <210> 73 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 73 ctgagttcaa cctgggaaac cgg 23 <210> 74 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 74 tgggaggacc tgcgcaagct ggg 23 <210> 75 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 75 gtactcctgt aggttgccct tgg 23 <210> 76 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 76 tcttccgctc ctcagccgtc agg 23 <210> 77 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 77 ctgggcagct ccctcgcccg ggg 23 <210> 78 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 78 cattgagctg gacaccctgg tgg 23 <210> 79 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 79 tggcaactcc cagtggtggc agg 23 <210> 80 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 80 caactcccag tggtggcagg agg 23 <210> 81 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 81 cgagcactgt gccatcatcc tgg 23 <210> 82 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 82 gcggtcatct tccaggatga tgg 23 <210> 83 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 83 agagctgctg cccattgagc tgg 23 <210> 84 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 84 accagggtgt ccagctcaat ggg 23 <210> 85 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 85 ctggacaccc tggtggggaa agg 23 <210> 86 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 86 caaagcgacc tttccccacc agg 23 <210> 87 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 87 gacggctgag gagcggaaga cgg 23 <210> 88 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 88 tactctgtct gtggatgacc tgg 23 <210> 89 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 89 cggcaagacg cggaagctca tgg 23 <210> 90 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 90 cccaaagtca cacaggcagc agg 23 <210> 91 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 91 taggttgccc ttggcgtgga agg 23 <210> 92 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 92 tgaggagcgg aagacggagt tgg 23 <210> 93 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 93 cctgtgcacg atgggcatct tgg 23 <210> 94 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 94 gggagctgcc cagcttgcgc agg 23 <210> 95 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 95 ttgaactcag cttctgctgc cgg 23 <210> 96 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 96 ccaagaggca tactcctcat agg 23 <210> 97 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 97 catgagcttc cgcgtcttgc cgg 23 <210> 98 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 98 cggagttggg gaaacaatac tgg 23 <210> 99 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 99 gctcctcagc cgtcaggaac tgg 23 <210> 100 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 100 caccagggtg tccagctcaa tgg 23 <210> 101 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 101 ctagtcatat ttcaagtgac agg 23 <210> 102 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 102 gctgggcagc tccctcgccc ggg 23 <210> 103 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 103 tgtcagagcg gtcatcttcc agg 23 <210> 104 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 104 ctgggaggac ctgcgcaagc tgg 23 <210> 105 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 105 agctgggcag ctccctcgcc cgg 23 <210> 106 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 106 tgtgttgtgg ttgatgttgt tgg 23 <210> 107 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 107 cctgctgcct gtgtgacttt ggg 23 <210> 108 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 108 acctgctgcc tgtgtgactt tgg 23 <210> 109 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 109 gacgcggcat gtcatcagct ggg 23 <210> 110 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 110 ggtcatccac agacagagta ggg 23 <210> 111 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 111 agtcaagatc tttccctatg agg 23 <210> 112 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 112 gaacatactc cagttcctga cgg 23 <210> 113 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 113 acgtggagct gatgtcagag cgg 23 <210> 114 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 114 ggggaaaggt cgctttgctg agg 23 <210> 115 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 115 tctggaccct actctgtctg tgg 23 <210> 116 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 116 tgggcagctc cctcgcccgg ggg 23 <210> 117 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 117 agctgatgac atgccgcgtc agg 23 <210> 118 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 118 gccgcgtcag gtactcctgt agg 23 <210> 119 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 119 caagaggcat actcctcata ggg 23 <210> 120 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 120 ggtgagcaat cccccgggcg agg 23 <210> 121 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 121 ttgctggtgt tatattctga tgg 23 <210> 122 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 122 cctatgagga gtatgcctct tgg 23 <210> 123 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 123 tgctggtgtt atattctgat ggg 23 <210> 124 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 124 cgaggatatt ggagctcttg agg 23 <210> 125 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 125 gttgatgttg ttggcacacg tgg 23 <210> 126 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 126 tgacgcggca tgtcatcagc tgg 23 <210> 127 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 127 ttgagctgga caccctggtg ggg 23 <210> 128 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 128 ttcagagcag tttgagacag tgg 23 <210> 129 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 129 gcggcatgtc atcagctggg agg 23 <210> 130 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 130 aggtcatcca cagacagagt agg 23 <210> 131 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 131 gaagatgatg atgacagata tgg 23 <210> 132 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 132 tgacctggct aacagtgggc agg 23 <210> 133 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 133 tctactgcta ccgcgttaac cgg 23 <210> 134 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 134 gtccttctct gtcttccaag agg 23 <210> 135 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 135 attgagctgg acaccctggt ggg 23 <210> 136 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 136 gtcgttcttc acgaggatat tgg 23 <210> 137 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 137 catcagcctc ctgccaccac tgg 23 <210> 138 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 138 agttcctgac ggctgaggag cgg 23 <210> 139 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 139 actccagttc ctgacggctg agg 23 <210> 140 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 140 cttgaggtcc ctgtgcacga tgg 23 <210> 141 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 141 ttgaggtccc tgtgcacgat ggg 23 <210> 142 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 142 ccgcgtcttg ccggtttccc agg 23 <210> 143 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 143 gttgcccttg gcgtggaagg cgg 23 <210> 144 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 144 ctttgggctt tccctgcgtc tgg 23 <210> 145 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 145 gatcaccgcc ttccacgcca agg 23 <210> 146 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 146 tgaagtgttc tgcttcagct tgg 23 <210> 147 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 147 ctgtgcacga tgggcatctt ggg 23 <210> 148 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 148 ggcatcttgg gcctcccaca tgg 23 <210> 149 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 149 ttacctgccc actgttagcc agg 23 <210> 150 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 150 atcagcctcc tgccaccact ggg 23 <210> 151 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 151 tcgctttgct gaggtctata agg 23 <210> 152 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 152 agtcacacag gcagcaggtt agg 23 <210> 153 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 153 gaggagcgga agacggagtt ggg 23 <210> 154 <211> twenty three <212> DNA <213> Homo sapiens <220> <223> CRISPR target sequences for TGFβ; RII gene <400> 154 tgggcagcag ctctgtgttg tgg 23 <210> 155 <211> 2781 <212> DNA <213> artificial <220> <223> R-TALEN TGFbRII pCLS32939 <400> 155 atgggcgatc ctaaaaagaa acgtaaggtc atcgatatcg ccgatctacg cacgctcggc 60 tacagccagc agcaacagga gaagatcaaa ccgaaggttc gttcgacagt ggcgcagcac 120 cacgaggcac tggtcggcca cgggtttaca cacgcgcaca tcgttgcgtt aagccaacac 180 ccggcagcgt tagggaccgt cgctgtcaag tatcaggaca tgatcgcagc gttgccagag 240 gcgacacacg aagcgatcgt tggcgtcggc aaacagtggt ccggcgcacg cgctctggag 300 gccttgctca cggtggcggg agagttgaga ggtccaccgt tacagttgga cacaggccaa 360 cttctcaaga ttgcaaaacg tggcggcgtg accgcagtgg aggcagtgca tgcatggcgc 420 aatgcactga cgggtgcccc gctcaacttg accccccagc aggtggtggc catcgccagc 480 aatggcggtg gcaagcaggc gctggagacg gtccagcggc tgttgccggt gctgtgccag 540 gcccacggct tgacccccca gcaggtggtg gccatcgcca gcaatggcgg tggcaagcag 600 gcgctggaga cggtccagcg gctgttgccg gtgctgtgcc aggcccacgg cttgaccccc 660 cagcaggtgg tggccatcgc cagcaatggc ggtggcaagc aggcgctgga gacggtccag 720 cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cccagcaggt ggtggccatc 780 gccagcaata atggtggcaa gcaggcgctg gagacggtcc agcggctgtt gccggtgctg 840 tgccaggccc acggcttgac cccccagcag gtggtggcca tcgccagcaa tggcggtggc 900 aagcaggcgc tggagacggt ccagcggctg ttgccggtgc tgtgccaggc ccacggcttg 960 accccccagc aggtggtggc catcgccagc aatggcggtg gcaagcaggc gctggagacg 1020 gtccagcggc tgttgccggt gctgtgccag gcccacggct tgacccccca gcaggtggtg 1080 gccatcgcca gcaatggcgg tggcaagcag gcgctggaga cggtccagcg gctgttgccg 1140 gtgctgtgcc aggcccacgg cttgaccccg gagcaggtgg tggccatcgc cagccacgat 1200 ggcggcaagc aggcgctgga gacggtccag cggctgttgc cggtgctgtg ccaggcccac 1260 ggcttgaccc cggagcaggt ggtggccatc gccagccacg atggcggcaa gcaggcgctg 1320 gagacggtcc agcggctgtt gccggtgctg tgccaggccc acggcttgac cccggagcag 1380 gtggtggcca tcgccagcca cgatggcggc aagcaggcgc tggagacggt ccagcggctg 1440 ttgccggtgc tgtgccaggc ccacggcttg accccggagc aggtggtggc catcgccagc 1500 cacgatggcg gcaagcaggc gctggagacg gtccagcggc tgttgccggt gctgtgccag 1560 gcccacggct tgaccccgga gcaggtggtg gccatcgcca gcaatattgg tggcaagcag 1620 gcgctggaga cggtgcaggc gctgttgccg gtgctgtgcc aggcccacgg cttgaccccc 1680 cagcaggtgg tggccatcgc cagcaatggc ggtggcaagc aggcgctgga gacggtccag 1740 cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cggagcaggt ggtggccatc 1800 gccagccacg atggcggcaa gcaggcgctg gagacggtcc agcggctgtt gccggtgctg 1860. tgccaggccc acggcttgac cccggagcag gtggtggcca tcgccagcaa tattggtggc aagcaggcgc tggagacggt gcaggcgctg ttgccggtgc tgtgccaggc ccacggcttg acccctcagc aggtggtggc catcgccagc aatggcggcg gcaggccggc gctggagagc attgttgccc agttatctcg ccctgatccg gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggggcgtcct gcgctggatg cagtgaaaaa gggattgggg 2160 gatcctatca gccgttccca gctggtgaag tccgagctgg aggagaaga atccgagttg aggcacaagc tgaagtacgt gccccacgag tacatcgagc tgatcgagat cgcccggaac 2280. agcacccagg accgtatcct ggagatgaag gtgatggagt tcttcatgaa ggtgtacggc tacaggggca agcacctggg cggctccagg aagcccgacg gcgccatcta caccgtgggc tcccccatcg actacggcgt gatcgtggac accaaggcct actccggcgg ctacaacctg 2460 cccatcggcc aggccgacga aatgcagagg tacgtggagg agaaccagac caggaacaag cacatcaacc ccaacgagtg gtggaaggtg tacccctcca gcgtgaccga gttcaagttc 2580 ctgttcgtgt ccggccactt caagggcaac tacaaggccc agctgaccag gctgaaccac 2640 atcaccaact gcaacggcgc cgtgctgtcc gtggaggagc tcctgatcgg cggcgagatg 2700 atcaaggccg gcaccctgac cctggaggag gtgaggagga agttcaacaa cggcgagatc 2760 aacttcgcgg ccgactgata a 2781 <210> 156 <211> 2781 <212> DNA <213> Artificial <220> <223> L-TALEN TGFbRII pCLS32940 <400> 156 atgggcgatc ctaaaaagaa acgtaaggtc atcgatatcg ccgatctacg cacgctcggc 60 tacagccagc agcaacagga gaagatcaaa ccgaaggttc gttcgacagt ggcgcagcac 120 cacgaggcac tggtcggcca cgggtttaca cacgcgcaca tcgttgcgtt aagccaacac 180 ccggcagcgt tagggaccgt cgctgtcaag tatcaggaca tgatcgcagc gttgccagag 240 gcgacacacg aagcgatcgt tggcgtcggc aaacagtggt ccggcgcacg cgctctggag 300 gccttgctca cggtggcggg agagttgaga ggtccaccgt tacagttgga cacaggccaa 360 cttctcaaga ttgcaaaacg tggcggcgtg accgcagtgg aggcagtgca tgcatggcgc 420 aatgcactga cgggtgcccc gctcaacttg accccggagc aggtggtggc catcgccagc 480 aatattggtg gcaagcaggc gctggagacg gtgcaggcgc tgttgccggt gctgtgccag 540 gcccacggct tgacccccca gcaggtggtg gccatcgcca gcaataatgg tggcaagcag 600 gcgctggaga cggtccagcg gctgttgccg gtgctgtgcc aggcccacgg cttgaccccg 660 gagcaggtgg tggccatcgc cagccacgat ggcggcaagc aggcgctgga gacggtccag 720 cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cggagcaggt ggtggccatc 780 gccagcaata ttggtggcaa gcaggcgctg gagacggtgc aggcgctgtt gccggtgctg 840 tgccaggccc acggcttgac cccggagcag gtggtggcca tcgccagcaa tattggtggc 900 aagcaggcgc tggagacggt gcaggcgctg ttgccggtgc tgtgccaggc ccacggcttg 960 accccggagc aggtggtggc catcgccagc cacgatggcg gcaagcaggc gctggagacg 1020 gtccagcggc tgttgccggt gctgtgccag gcccacggct tgaccccgga gcaggtggtg 1080 gccatcgcca gcaatattgg tggcaagcag gcgctggaga cggtgcaggc gctgttgccg 1140 gtgctgtgcc aggcccacgg cttgaccccg gagcaggtgg tggccatcgc cagcaatatt 1200 ggtggcaagc aggcgctgga gacggtgcag gcgctgttgc cggtgctgtg ccaggcccac 1260 ggcttgaccc cccagcaggt ggtggccatc gccagcaata atggtggcaa gcaggcgctg 1320 gagacggtcc agcggctgtt gccggtgctg tgccaggccc acggcttgac cccccagcag 1380 gtggtggcca tcgccagcaa tggcggtggc aagcaggcgc tggagacggt ccagcggctg 1440 ttgccggtgc tgtgccaggc ccacggcttg accccggagc aggtggtggc catcgccagc 1500 cacgatggcg gcaagcaggc gctggagacg gtccagcggc tgttgccggt gctgtgccag 1560 gcccacggct tgaccccgga gcaggtggtg gccatcgcca gcaatattgg tggcaagcag 1620 gcgctggaga cggtgcaggc gctgttgccg gtgctgtgcc aggcccacgg cttgaccccc 1680 cagcaggtgg tggccatcgc cagcaataat ggtggcaagc aggcgctgga gacggtccag 1740 cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cccagcaggt ggtggccatc gccagcaata atggtggcaa gcaggcgctg gagacggtcc agcggctgtt gccggtgctg tgccaggccc acggcttgac cccggagcag gtggtggcca tcgccagcaa tattggtggc aagcaggcgc tggagacggt gcaggcgctg ttgccggtgc tgtgccaggc ccacggcttg acccctcagc aggtggtggc catcgccagc aatggcggcg gcaggccggc gctggagagc attgttgccc agttatctcg ccctgatccg gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggggcgtcct gcgctggatg cagtgaaaaa gggattgggg 2160 gatcctatca gccgttccca gctggtgaag tccgagctgg aggagaaga atccgagttg aggcacaagc tgaagtacgt gccccacgag tacatcgagc tgatcgagat cgcccggaac 2280. agcacccagg accgtatcct ggagatgaag gtgatggagt tcttcatgaa ggtgtacggc tacaggggca agcacctggg cggctccagg aagcccgacg gcgccatcta caccgtgggc tcccccatcg actacggcgt gatcgtggac accaaggcct actccggcgg ctacaacctg 2460 cccatcggcc aggccgacga aatgcagagg tacgtggagg agaaccagac caggaacaag 2520 cacatcaacc ccaacgagtg gtggaaggtg tacccctcca gcgtgaccga gttcaagttc 2580 ctgttcgtgt ccggccactt caagggcaac tacaaggccc agctgaccag gctgaaccac 2640 atcaccaact gcaacggcgc cgtgctgtcc gtggaggagc tcctgatcgg cggcgagatg 2700 atcaaggccg gcaccctgac cctggaggag gtgaggagga agttcaacaa cggcgagatc 2760 aacttcgcgg ccgactgata a 2781 <210> 157 <211> 2781 <212> DNA <213> Artificial <220> <223> R-TALEN TGFbRII pCLS32967 <400> 157 atgggcgatc ctaaaaagaa acgtaaggtc atcgatatcg ccgatctacg cacgctcggc 60 tacagccagc agcaacagga gaagatcaaa ccgaaggttc gttcgacagt ggcgcagcac 120 cacgaggcac tggtcggcca cgggtttaca cacgcgcaca tcgttgcgtt aagccaacac 180 ccggcagcgt tagggaccgt cgctgtcaag tatcaggaca tgatcgcagc gttgccagag 240 gcgacacacg aagcgatcgt tggcgtcggc aaacagtggt ccggcgcacg cgctctggag 300 gccttgctca cggtggcggg agagttgaga ggtccaccgt tacagttgga cacaggccaa 360 cttctcaaga ttgcaaaacg tggcggcgtg accgcagtgg aggcagtgca tgcatggcgc 420 aatgcactga cgggtgcccc gctcaacttg accccggagc aggtggtggc catcgccagc 480 cacgatggcg gcaagcaggc gctggagacg gtccagcggc tgttgccggt gctgtgccag 540 gcccacggct tgaccccgga gcaggtggtg gccatcgcca gccacgatgg cggcaagcag 600 gcgctggaga cggtccagcg gctgttgccg gtgctgtgcc aggcccacgg cttgaccccg 660 gagcaggtgg tggccatcgc cagccacgat ggcggcaagc aggcgctgga gacggtccag 720 cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cccagcaggt ggtggccatc 780 gccagcaatg gcggtggcaa gcaggcgctg gagacggtcc agcggctgtt gccggtgctg 840 tgccaggccc acggcttgac cccggagcag gtggtggcca tcgccagcaa tattggtggc 900 aagcaggcgc tggagacggt gcaggcgctg ttgccggtgc tgtgccaggc ccacggcttg 960 accccccagc aggtggtggc catcgccagc aatggcggtg gcaagcaggc gctggagacg 1020 gtccagcggc tgttgccggt gctgtgccag gcccacggct tgacccccca gcaggtggtg 1080 gccatcgcca gcaataatgg tggcaagcag gcgctggaga cggtccagcg gctgttgccg 1140 gtgctgtgcc aggcccacgg cttgaccccg gagcaggtgg tggccatcgc cagcaatatt 1200 ggtggcaagc aggcgctgga gacggtgcag gcgctgttgc cggtgctgtg ccaggcccac 1260 ggcttgaccc cccagcaggt ggtggccatc gccagcaata atggtggcaa gcaggcgctg 1320 gagacggtcc agcggctgtt gccggtgctg tgccaggccc acggcttgac cccccagcag 1380 gtggtggcca tcgccagcaa taatggtggc aagcaggcgc tggagacggt ccagcggctg 1440 ttgccggtgc tgtgccaggc ccacggcttg accccggagc aggtggtggc catcgccagc 1500 aatattggtg gcaagcaggc gctggagacg gtgcaggcgc tgttgccggt gctgtgccag 1560 gcccacggct tgacccccca gcaggtggtg gccatcgcca gcaataatgg tggcaagcag 1620 gcgctggaga cggtccagcg gctgttgccg gtgctgtgcc aggcccacgg cttgaccccc 1680 cagcaggtgg tggccatcgc cagcaatggc ggtggcaagc aggcgctgga gacggtccag 1740. cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cggagcaggt ggtggccatc 1800 gccagcaata ttggtggcaa gcaggcgctg gagacggtgc aggcgctgtt gccggtgctg tgccaggccc acggcttgac cccccagcag gtggtggcca tcgccagcaa tggcggtggc 1920. aagcaggcgc tggagacggt ccagcggctg ttgccggtgc tgtgccaggc ccacggcttg acccctcagc aggtggtggc catcgccagc aatggcggcg gcaggccggc gctggagagc attgttgccc agttatctcg ccctgatccg gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggggcgtcct gcgctggatg cagtgaaaaa gggattgggg 2160 gatcctatca gccgttccca gctggtgaag tccgagctgg aggagaaga atccgagttg aggcacaagc tgaagtacgt gccccacgag tacatcgagc tgatcgagat cgcccggaac 2280. agcacccagg accgtatcct ggagatgaag gtgatggagt tcttcatgaa ggtgtacggc tacaggggca agcacctggg cggctccagg aagcccgacg gcgccatcta caccgtgggc tcccccatcg actacggcgt gatcgtggac accaaggcct actccggcgg ctacaacctg 2460 cccatcggcc aggccgacga aatgcagagg tacgtggagg agaaccagac caggaacaag 2520 cacatcaacc ccaacgagtg gtggaaggtg tacccctcca gcgtgaccga gttcaagttc 2580 ctgttcgtgt ccggccactt caagggcaac tacaaggccc agctgaccag gctgaaccac 2640 atcaccaact gcaacggcgc cgtgctgtcc gtggaggagc tcctgatcgg cggcgagatg 2700 atcaaggccg gcaccctgac cctggaggag gtgaggagga agttcaacaa cggcgagatc 2760 aacttcgcgg ccgactgata a 2781 <210> 158 <211> 2781 <212> DNA <213> Artificial <220> <223> L-TALEN TGFbRII pCLS32968 <400> 158 atgggcgatc ctaaaaagaa acgtaaggtc atcgatatcg ccgatctacg cacgctcggc 60 tacagccagc agcaacagga gaagatcaaa ccgaaggttc gttcgacagt ggcgcagcac 120 cacgaggcac tggtcggcca cgggtttaca cacgcgcaca tcgttgcgtt aagccaacac 180 ccggcagcgt tagggaccgt cgctgtcaag tatcaggaca tgatcgcagc gttgccagag 240 gcgacacacg aagcgatcgt tggcgtcggc aaacagtggt ccggcgcacg cgctctggag 300 gccttgctca cggtggcggg agagttgaga ggtccaccgt tacagttgga cacaggccaa 360 cttctcaaga ttgcaaaacg tggcggcgtg accgcagtgg aggcagtgca tgcatggcgc 420 aatgcactga cgggtgcccc gctcaacttg accccccagc aggtggtggc catcgccagc 480 aataatggtg gcaagcaggc gctggagacg gtccagcggc tgttgccggt gctgtgccag 540 gcccacggct tgaccccgga gcaggtggtg gccatcgcca gcaatattgg tggcaagcag 600 gcgctggaga cggtgcaggc gctgttgccg gtgctgtgcc aggcccacgg cttgaccccc 660 cagcaggtgg tggccatcgc cagcaataat ggtggcaagc aggcgctgga gacggtccag 720 cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cggagcaggt ggtggccatc 780 gccagcaata ttggtggcaa gcaggcgctg gagacggtgc aggcgctgtt gccggtgctg 840 tgccaggccc acggcttgac cccggagcag gtggtggcca tcgccagcaa tattggtggc 900 aagcaggcgc tggagacggt gcaggcgctg ttgccggtgc tgtgccaggc ccacggcttg 960 accccccagc aggtggtggc catcgccagc aataatggtg gcaagcaggc gctggagacg 1020 gtccagcggc tgttgccggt gctgtgccag gcccacggct tgaccccgga gcaggtggtg 1080 gccatcgcca gcaatattgg tggcaagcag gcgctggaga cggtgcaggc gctgttgccg 1140 gtgctgtgcc aggcccacgg cttgaccccc cagcaggtgg tggccatcgc cagcaatggc 1200 ggtggcaagc aggcgctgga gacggtccag cggctgttgc cggtgctgtg ccaggcccac 1260 ggcttgaccc cccagcaggt ggtggccatc gccagcaata atggtggcaa gcaggcgctg 1320 gagacggtcc agcggctgtt gccggtgctg tgccaggccc acggcttgac cccccagcag 1380 gtggtggcca tcgccagcaa tggcggtggc aagcaggcgc tggagacggt ccagcggctg 1440 ttgccggtgc tgtgccaggc ccacggcttg accccggagc aggtggtggc catcgccagc 1500 cacgatggcg gcaagcaggc gctggagacg gtccagcggc tgttgccggt gctgtgccag 1560 gcccacggct tgaccccgga gcaggtggtg gccatcgcca gccacgatgg cggcaagcag 1620 gcgctggaga cggtccagcg gctgttgccg gtgctgtgcc aggcccacgg cttgaccccc 1680 cagcaggtgg tggccatcgc cagcaatggc ggtggcaagc aggcgctgga gacggtccag 1740. cggctgttgc cggtgctgtg ccaggcccac ggcttgaccc cccagcaggt ggtggccatc gccagcaatg gcggtggcaa gcaggcgctg gagacggtcc agcggctgtt gccggtgctg 1860. tgccaggccc acggcttgac cccggagcag gtggtggcca tcgccagcca cgatggcggc 1920 aagcaggcgc tggagacggt ccagcggctg ttgccggtgc tgtgccaggc ccacggcttg acccctcagc aggtggtggc catcgccagc aatggcggcg gcaggccggc gctggagagc attgttgccc agttatctcg ccctgatccg gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggggcgtcct gcgctggatg cagtgaaaaa gggattgggg 2160 gatcctatca gccgttccca gctggtgaag tccgagctgg aggagaaga atccgagttg aggcacaagc tgaagtacgt gccccacgag tacatcgagc tgatcgagat cgcccggaac 2280. agcacccagg accgtatcct ggagatgaag gtgatggagt tcttcatgaa ggtgtacggc tacaggggca agcacctggg cggctccagg aagcccgacg gcgccatcta caccgtgggc 2400 tcccccatcg actacggcgt gatcgtggac accaaggcct actccggcgg ctacaacctg 2460 cccatcggcc aggccgacga aatgcagagg tacgtggagg agaaccagac caggaacaag 2520 cacatcaacc ccaacgagtg gtggaaggtg tacccctcca gcgtgaccga gttcaagttc 2580 ctgttcgtgt ccggccactt caagggcaac tacaaggccc agctgaccag gctgaaccac 2640 atcaccaact gcaacggcgc cgtgctgtcc gtggaggagc tcctgatcgg cggcgagatg 2700 atcaaggccg gcaccctgac cctggaggag gtgaggagga agttcaacaa cggcgagatc 2760 aacttcgcgg ccgactgata a 2781 <210> 159 <211> 49 <212> DNA <213> Artificial <220> <223> TRAC TALEN target sequence <400> 159 ttgtcccaca gatatccaga accctgaccc tgccgtgtac cagctgaga 49 <210> 160 <211> 49 <212> DNA <213> Artificial <220> <223> CD52 TALEN target sequence <400> 160 ttcctcctac tcaccatcag cctcctggtt atggtacagg taagagcaa 49 <210> 161 <211> 502 <212> PRT <213> Artificial <220> <223> Complete sequence of P4 CAR <400> 161 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu 20 25 30 Val Thr Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp 35 40 45 Ser Val Ser Ser Asn Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro 50 55 60 Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp 65 70 75 80 Tyr Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Met Ser Ile Asn Pro 85 90 95 Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro 100 105 110 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Met Met Thr Tyr Tyr 115 120 125 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Gly Ile Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gln Pro Val Leu Thr Gln Ser Ser Ser Leu Ser Ala Ser 165 170 175 Pro Gly Ala Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn 180 185 190 Val Gly Pro Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro 195 200 205 Pro Gln Tyr Leu Leu Asn Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly 210 215 220 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn 225 230 235 240 Ala Gly Val Leu Leu Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp 245 250 255 Tyr Tyr Cys Met Ile Trp His Ser Ser Ala Ala Val Phe Gly Gly Gly 260 265 270 Thr Gln Leu Thr Val Leu Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 275 280 285 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 290 295 300 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 305 310 315 320 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 325 330 335 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 340 345 350 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 355 360 365 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 370 375 380 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 385 390 395 400 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 405 410 415 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 420 425 430 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu 435 440 445 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile 450 455 460 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr 465 470 475 480 Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met 485 490 495 Gln Ala Leu Pro Pro Arg 500

Claims

1. A mesothelin-specific chimeric antigen receptor (CAR), comprising at least: - An extracellular ligand-binding domain comprising VH and VL from a monoclonal anti-mesothelin antibody, wherein the VH chain contains three CDRs, the sequence of CDRH1 is shown in SEQ ID NO: 3, the sequence of CDRH2 is shown in SEQ ID NO: 4, and the sequence of CDRH3 is shown in SEQ ID NO: 5, and the VL chain contains three CDRs, the sequence of CDRL1 is shown in SEQ ID NO: 6, the sequence of CDRL2 is shown in SEQ ID NO: 7, and the sequence of CDRL3 is shown in SEQ ID NO: 8; -CD8α transmembrane domain; and - Cytoplasmic domains, including the CD3 zeta signal transduction domain and the 4-1BB costimulatory domain; The extracellular ligand-binding domain is specifically targeted at the MSLN antigen peptide region SEQ ID NO:

25.

2. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 1, wherein the extracellular ligand binding domain comprises VH and VL chains having at least 80% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

3. The mesothelin-specific chimeric antigen receptor according to claim 2, wherein the extracellular ligand binding domain comprises VH and VL chains having at least 90% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

4. The mesothelin-specific chimeric antigen receptor according to claim 3, wherein the extracellular ligand binding domain comprises VH and VL chains having at least 95% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

5. The mesothelin-specific chimeric antigen receptor according to claim 4, wherein the extracellular ligand binding domain comprises VH and VL chains having at least 99% sequence identity with SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

6. The mesothelin-specific chimeric antigen receptor (CAR) according to any one of claims 1 to 5, wherein the amino acid sequence of the transmembrane domain shares at least 80% sequence identity with SEQ ID NO. 17 from CD8α.

7. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 6, wherein the amino acid sequence of the transmembrane domain shares at least 90% sequence identity with SEQ ID NO. 17 from CD8α.

8. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 7, wherein the amino acid sequence of the transmembrane domain shares at least 95% sequence identity with SEQ ID NO. 17 from CD8α.

9. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 8, wherein the amino acid sequence of the transmembrane domain shares at least 99% sequence identity with SEQ ID NO. 17 from CD8α.

10. The mesothelin-specific chimeric antigen receptor (CAR) according to any one of claims 1 to 5, further comprising a CD8α hinge located between the extracellular ligand-binding domain and the transmembrane domain.

11. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 10, wherein the amino acid sequence of the hinge shares at least 80% sequence identity with SEQ ID NO.

16.

12. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 11, wherein the amino acid sequence of the hinge shares at least 90% sequence identity with SEQ ID NO.

16.

13. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 12, wherein the amino acid sequence of the hinge shares at least 95% sequence identity with SEQ ID NO.

16.

14. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 13, wherein the amino acid sequence of the hinge shares at least 99% sequence identity with SEQ ID NO.

16.

15. The mesothelin-specific CAR according to any one of claims 1 to 5, wherein the CAR has 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.

16. The mesothelin-specific CAR according to any one of claims 1 to 5, further comprising a safety switch, said safety switch comprising an epitope CPYSNPSLC (SEQ ID NO: 26) specifically bound by rituximab.

17. The mesothelin-specific CAR of claim 16, wherein the CAR comprises a safety switch R2 having at least 90% identity with SEQ ID NO:

15.

18. The mesothelin-specific CAR according to any one of claims 1 to 5, wherein the amino acid sequence of the co-stimulatory domain has at least 80% identity with SEQ ID NO:

18.

19. The mesothelin-specific CAR according to any one of claims 1 to 5, wherein the amino acid sequence of the CD3 zeta signal transduction domain has at least 80% identity with SEQ ID NO:

19.

20. The mesothelin-specific CAR according to any one of claims 1 to 5, further comprising a signal peptide.

21. The mesothelin-specific chimeric antigen receptor (CAR) according to any one of claims 1 to 5, wherein the CAR is a single-chain polypeptide.

22. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 21, wherein the CAR has at least 80% overall amino acid sequence identity with SEQ ID NO: 21 or SEQ ID NO:

22.

23. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 22, wherein the CAR has at least 90% overall amino acid sequence identity with SEQ ID NO: 21 or SEQ ID NO:

22.

24. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 23, wherein the CAR has at least 95% overall amino acid sequence identity with SEQ ID NO: 21 or SEQ ID NO:

22.

25. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 24, wherein the CAR has at least 99% overall amino acid sequence identity with SEQ ID NO: 21 or SEQ ID NO:

22.

26. The mesothelin-specific chimeric antigen receptor (CAR) according to claim 25, wherein the amino acid sequence of the CAR is SEQ ID NO: 21 or SEQ ID NO:

22.

27. A polynucleotide encoding a chimeric antigen receptor according to any one of claims 1 to 26.

28. An expression vector comprising the polynucleotide of claim 27.

29. An engineered immune cell comprising the polynucleotide of claim 27 or the expression vector of claim 28.

30. An engineered immune cell expressing, at its cell surface membrane, a mesothelin-specific chimeric antigen receptor according to any one of claims 1 to 26.

31. The engineered immune cell according to claim 29, wherein the immune cell is a T-lymphocyte.

32. The engineered immune cells according to claim 31, wherein the T-lymphocytes are derived from primary cells or differentiated from stem cells.

33. The engineered immune cells according to claim 32, wherein the T-lymphocytes are differentiated from iPS cells.

34. The engineered immune cells according to claim 31, wherein the engineered immune cells are derived from inflammatory T lymphocytes, cytotoxic T lymphocytes, or helper T lymphocytes.

35. The engineered immune cell of claim 29, wherein the expression of TCR in the immune cell is reduced or inhibited.

36. The engineered immune cell of claim 35, wherein at least one gene encoding TCRα or TCRβ is inactivated in the cell.

37. The engineered immune cell of claim 36, wherein at least one gene encoding TCRα or TCRβ is cleaved by a rare endonuclease.

38. The engineered immune cell of claim 36, wherein the polynucleotide encoding a mesothelin-specific CAR is integrated into an endogenous locus transcribed under the control of an endogenous promoter.

39. The engineered immune cell of claim 38, wherein the polynucleotide encoding the mesothelin-specific CAR is integrated into the TCRα or TCRβ locus.

40. The engineered immune cells according to claim 38 are derived from a donor used for allogeneic transplantation.

41. The engineered immune cell according to any one of claims 29 to 40, wherein the cell is mutated to confer resistance to at least one immunosuppressive drug.

42. The engineered immune cell of claim 41, wherein the cell is mutated to confer resistance to the anti-CD52 antibody.

43. The engineered immune cell according to any one of claims 29 to 40, wherein the cell is further mutated to confer resistance to at least one chemotherapeutic agent.

44. The engineered immune cell according to any one of claims 29 to 40, wherein the cell is mutated in a gene encoding HLA or B2m.

45. The engineered immune cell according to any one of claims 29 to 40, wherein the cell is mutated to reduce or inhibit the expression of immune checkpoint proteins and / or their receptors, thereby improving its CAR-dependent immune activation.

46. ​​The engineered immune cell according to any one of claims 29 to 40, wherein the mesothelin-specific chimeric antigen receptor (CAR) is co-expressed in the cell with another exogenous gene sequence encoding a TGFβ receptor inhibitor or a decoy.

47. The engineered immune cell of claim 46, wherein the decoy of the TGFβ receptor is a dominantly inactivated TGFβ receptor having at least 80% polypeptide sequence identity with SEQ ID NO.

24.

48. The engineered immune cell of claim 47, wherein the cell comprises an exogenous polynucleotide, the exogenous polynucleotide comprising a first polynucleotide sequence encoding the mesothelin-specific CAR, a second polynucleotide encoding a 2A self-cleaving peptide, and a third polynucleotide encoding the dominant-inactivated TGFβ receptor.

49. The engineered immune cell according to any one of claims 29 to 40, wherein the cell reduces or inactivates the expression of at least one TGFβ receptor gene.

50. The engineered immune cell according to claim 49, wherein the TGFβ receptor gene is TGFβRII.

51. The engineered immune cell according to any one of claims 29 to 40, wherein the mesothelin-specific chimeric antigen receptor (CAR) is co-expressed in the cell with another exogenous gene sequence selected from sequences encoding: -NK cell inhibitors; -CRS inhibitor; - Cytochrome P450, CYP2D6-1, CYP2D6-2, CYP2C9, CYP3A4, CYP2C19, or CYP1A2, conferring hypersensitivity of the immune cells to the drug. - Dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin or methylguanine transferase (MGMT), mTORmut or Lckmut, confer drug resistance; -Chemokines or cytokines; -Chemokinin receptors; and - Inhibitors of tumor-associated macrophage (TAM) secretion to enhance the therapeutic activity of these immune cells.

52. The engineered immune cells according to claim 51, wherein the NK cell inhibitor is HLAG, HLAE, or ULBP1.

53. The engineered immune cells of claim 51, wherein the CRS inhibitor is a mutated IL6Ra, sGP130, or IL18-BP.

54. The engineered immune cells of claim 51, wherein the chemokine or cytokine is IL-2, IL-12, and IL-15.

55. The engineered immune cell according to claim 51, wherein the chemokine receptor is CCR2, CXCR2, or CXCR4.

56. The engineered immune cells of claim 51, wherein TAM is a CCR2 / CCL2 neutralizer.

57. The engineered immune cells of claim 43, wherein the chemotherapeutic agent is a purine analogue.

58. Use of engineered immune cells according to any one of claims 29 to 57 in the preparation of an agent for treating a pre- or malignant cancerous condition characterized by mesothelin-expressing cells, wherein the pre- or malignant cancerous condition is selected from esophageal cancer, breast cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, colon cancer, lung cancer, thymic cancer, mesothelioma, ovarian cancer, and endometrial cancer.

59. Use of CAR-positive engineered immune cells in the preparation of a medicament for treating patients with a condition characterized by mesothelin-expressing cells, said condition being selected from esophageal cancer, breast cancer, gastric cancer, hepatobiliary cancer, pancreatic cancer, colon cancer, lung cancer, thymic carcinoma, mesothelioma, ovarian cancer, and endometrial cancer, wherein said treatment comprises the following steps: - Engineer immune cells from a donor to express mesothelin-specific chimeric antigen receptor (CAR) according to any one of claims 1 to 26. - The CAR-positive engineered immune cells were administered to the patient to eliminate cells expressing mesothelin.

60. The use according to claim 59, wherein the treatment includes further treatment steps in which the patient's lymphocytes are cleared.

61. The use according to claim 60, wherein the CAR-positive engineered immune cell mutation that eliminates cells expressing mesothelin is used to confer resistance to lymphocyte clearance therapy.

62. The use according to claim 61, wherein the CAR-positive engineered immune cells that eliminate cells expressing mesothelin are mutated in their CD52 gene.

Images