Novel anti-MUC1 CARs and gene-edited immune cells for cancer immunotherapy of solid tumors

Abstract

The present invention relates to genetically modified immune cells expressing a novel anti-MUC1 chimeric antigen receptor and their use in the treatment of solid tumors, particularly suitable for allogeneic cellular immunotherapy.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the field of cellular immunotherapy, and more specifically to engineered immune cells expressing anti-MUC1 chimeric antigen receptors (CARs), useful in the treatment of solid tumors. [Background technology]

[0002] 2. Background of the Invention CAR immune cell therapy, primarily T cells and NK cells, has revolutionized the treatment of blood cancers over the past decade. However, to date, the same types of therapy have not been an effective means of treating solid tumors [June et al., CAR T cell immunotherapy for human cancer (2018) Science 359:1361-1365].

[0003] Indeed, challenges in treating solid tumors arise from two major areas: 1) off-tumor toxicity due to the lack of exclusive tumor specificity, and 2) the complex immunosuppressive biology of solid tumors [Marofi, F., et al. (2021) CAR T cells in solid tumors: challenges and opportunities. Stem Cell Res Ther. 18(4):843-851 (Non-Patent Document 2)].

[0004] The specificity of CAR immune cells remains a key challenge due to on-target off-tumor activity resulting in damage to healthy tissues. In this regard, the efficiency of therapeutic antibodies in immunotherapy has proven to be of low predictive value for the specificity / activity of CAR-T cells using the same ScFv as the previously developed antibody. The reverse is also true, as anti-CD19 CARs have proven to be highly efficient in cases where CD19 antibodies could not be developed for hematological cancers.

[0005] In hematological tumors, off-target damage is associated with a lower risk. In general, the associated toxicity is well tolerated or effectively managed. This is in part due to the antigen being restricted to a specific cell lineage of the hematological compartment, such as CD19 on B cells. Loss of healthy B cells due to CD19 CAR-T activity may result in B cell hypoplasia, which is associated with a higher risk of infection but is otherwise not fatal and can be adequately managed with intravenous immunoglobulin (IVIG) replacement therapy.

[0006] In stark contrast, expression of CAR-T target antigens on solid tissues has been associated with lethal toxicity. For example, anti-HER2 CAR-T cells administered for the treatment of metastatic colon cancer attacked low HER2-expressing lung epithelium, resulting in a fatal event [Morgan, RA et al. (2010) Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol. Ther. 18(4):843-851]. Similar toxicity was observed in renal cancer therapy, where CAR-T cells against CAIX caused liver damage by attacking CIAX-expressing biliary epithelium [Lamers CH, et al. (2006) Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. Journal of Clinical Oncology. 24(13):e20-2 (Non-Patent Document 4)]. Overall, the success of CAR-T therapy for solid tumors has been limited, and in cases where measurable responses have been observed, dose escalation as a means to improve efficacy has not been possible, in part due to toxicity associated with off-target binding [Wagner, J. (2020) CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality? Molecular Therapy. 28(11):2320-23394 (Non-Patent Document 5)].

[0007] Additional levels of failure to obtain significant responses in solid tumors, even for the most active and specific scFVs, stem from the tumor microenvironment: (1) CAR immune cell infiltration is limited due to extracellular matrix deposition and high interstitial pressure, and (2) expansion and effector function of intratumoral CAR immune cells are blocked due to hypoxia and nutrient deprivation. 3) Immunosuppressive signals produced by other tumor-resident cells, including regulatory T cells (T-reg: TGF-β), cancer-associated fibroblasts (CAF: TGF-β), and myeloid-derived suppressor cells (MDSC: IL-10 and TGF-β), prevent CAR-T cell expansion and effector function. 4) Upregulation of immune checkpoint ligands, such as PDL1, directly inhibits CAR-T cell activity. To develop successful CAR-T products for the treatment of solid tumors, these challenges must be addressed through complex engineering of next-generation CAR-T cells.

[0008] Table 1. Determinants of successful CAR-T therapy in solid tumors TIFF2024517713000001.tif67158

[0009] These challenges, summarized in Table 1, are addressed in the present invention by engineering anti-MUC1 CAR immune cells that target highly specific post-translational versions of tumor-exclusive MUC1 epitopes and by introducing genetic modifications that enable them to combat the immunosuppressive tumor microenvironment associated with MUC1-positive solid tumors.

[0010] MUC1 is a large mucin-type glycoprotein produced by epithelial cells lining several organs, including the lungs, cervix, stomach, and intestine. In normal tissues, MUC1 is localized only to the apical side of the epithelial lining and extends into the lumen, where the glycans on MUC1 form a mucosal barrier that traps water and protects these tissues from injury and pathogens. MUC1 is highly enriched in tumors, as well as other solid tumor targets, but unlike other solid tumor targets, MUC1 is structurally different when produced by tumor cells (abbreviated herein as tMUC1). The structural difference in MUC1 occurs due to differential glycosylation of MUC1 within the VNTR region of the extracellular subunit (see Figure 1). The O-glycans added to tMUC1 are truncated, missing entirely, or prematurely modified with sialic acid. As a result, tMUC1 produced by tumor cells is different at the molecular level, and scFVs produced against underglycosylated tMUC1 should not recognize fully glycosylated MUC1 produced by normal cells. Based on these differences between tMUC1 and MUC1, it is hypothesized that CAR immune cells produced against tMUC1 will be highly specific in recognizing tumors [Naito et al. (2017) Generation of Novel Anti-MUC1 Monoclonal Antibodies with Designed Carbohydrate Specificities Using MUC1 Glycopeptide Library. ACS Omega. 2(11): 7493-7505 (Non-Patent Document 6)].

[0011] Based on the primary selection of 14 anti-MUC1 ScFv candidates from the literature, we developed a CAR scaffold against tMUC1 by including four ScFvs that commonly target the same polypeptide segment of MUC1. Expression of various CARs in immune cells all resulted in positive and specific anti-tMUC1 anti-tumor response activity. However, the CARs showed different degrees of in vivo anti-tumor response.

[0012] Since CAR immune cell activity in solid tumors is often inhibited by the tumor immune suppressive environment, the inventors expressed various CARs of the invention in immune cells engineered to simultaneously address several problems related to solid tumors, such as: 1) Increasing the potency of CAR immune cells through cytokine-induced heterologous expression; 2) limiting CAR-T cell exhaustion by inactivating immune checkpoint genes that inhibit the activation of CAR immune cells; 3) prevention of immunosuppression from the tumor microenvironment by using alternative means to alleviate the immunosuppressive effects of TGF-β; 4) enhancing tumor invasion by expressing hyaluronan (HA), a glycosaminoglycan that is an essential structural and signaling component of the extracellular matrix; and / or 5) Alleviation of hypoxic and nutrient-deficient environments, particularly by overexpressing metabolic enzymes such as glucose-6-phosphate isomerase (e.g., GPI1), lactate dehydrogenase (e.g., LDHA), and phosphoenolpyruvate carboxykinase 1 (e.g., PCK1), to enhance the proliferation capacity of inflammatory Th17 cells in hypoxic and nutrient-deficient environments. During these experiments, gene targeting integration and retroviral approaches were used and combined to ultimately optimize the genetic modification of anti-MUC1 CAR immune cells and provide therapeutic cells with improved efficacy. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] June et al., CAR T cell immunotherapy for human cancer (2018) Science 359:1361-1365 [Non-Patent Document 2] Marofi, F., et al. (2021) CAR T cells in solid tumors: challenges and opportunities. Stem Cell Res Ther. 18(4):843-851 [Non-Patent Document 3] Morgan, RA et al. (2010) Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol. Ther. 18(4):843-851 [Non-Patent Document 4] Lamers CH, et al. (2006) Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. Journal of Clinical Oncology. 24(13):e20-2 [Non-Patent Document 5] Wagner, J. (2020) CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality? Molecular Therapy. 28(11):2320-23394 [Non-Patent Document 6] Naito et al. (2017) Generation of Novel Anti-MUC1 Monoclonal Antibodies with Designed Carbohydrate Specificities Using MUC1 Glycopeptide Library. ACS Omega. 2(11): 7493-7505 Summary of the Invention

[0014] To achieve the present invention, four different CARs against tMUC1 were synthesized: CLS MUC1-A, CLS MUC1-B, CLS MUC1-C, and CLS MUC1-D. The scFVs used in these CAR constructs recognized different epitopes of the tMUC1 polypeptide segment of SEQ ID NO:1, which were screened to be breast cancer cell specific (HCC70 and T47D cell lines). As a negative control, MUC1 / tMUC1 negative HEK293FT cells were used. As shown in the experimental part of this specification, the four different CARs showed high cytotoxic activity in vitro against breast cancer cell lines T47D and HCC70, but were not cytotoxic against normal primary cells from lung, kidney, or cervix. These data demonstrated that the four CARs produced were highly specific to tMUC1. At the same time, we could observe that the four CARs conferred different levels of activity when expressed in primary T cells, and that one of them (CAR MUC1-A) unexpectedly surpassed the others in terms of in vivo antitumor activity.

[0015] The above-mentioned anti-MUC1 CARs of the present invention, all of which were primarily believed to be suitable for eliciting a specific immune response against solid tumors, were tested in genetically modified immune cells, specifically considering their applicability in the allogeneic setting.

[0016] The results demonstrate that the anti-MUC1 CAR can be successfully combined with various genetic signatures, beyond just the four CARs detailed herein, to generate sophisticated gene-edited CAR immune cells with significantly improved anti-tumor efficacy.

[0017] Thus, the present invention encompasses the generation of novel anti-MUCI CARs and engineered immune cells equipped with anti-MUCI CARs that exhibit various genetic signatures that enhance the activity of the CARs for the in vivo elimination of solid tumors.

[0018] The anti-MUC1 chimeric antigen receptor (CAR) of the present invention preferably comprises the MUC1 polypeptide region It is directed against an antigen capable of identifying hypoglycosylated epitopes of TIFF2024517713000002.tif4128.

[0019] One exemplary and preferred anti-MUC1 CAR of the invention comprises: Transmembrane domain The cytoplasmic domain, which contains the CD3 zeta signaling domain, and the costimulatory domain A ligand-binding domain comprising an ScFv having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to one selected from MUC1-A (SEQ ID NO:17), MUC1-B (SEQ ID NO:27), MUC1-C (SEQ ID NO:37), or MUC1-D (SEQ ID NO:37).

[0020] The structure of such CARs used in the present invention generally belongs to the second, third or fourth generation as described in the art by combining an anti-tMUCI ligand binding domain together with a hinge and transmembrane domain from CD8 alpha, a costimulatory domain from 4-1BB and a signaling domain from CD3 zeta [Subklewe, M., et al. (2019) Chimeric Antigen Receptor T Cells: A Race to Revolutionize Cancer Therapy. Transfusion medicine and hemotherapy. 46(1), 15-24].

[0021] The present invention also relates to polypeptide and polynucleotide sequences, and vectors used to introduce and express anti-MUC1 CARs on the surface of immune cells.

[0022] The modified immune cells expressing anti-MUC1-CAR according to the present invention are generally NK cells or T cells originating from human donors, or their precursor cells, suitable for use in allogeneic settings.Such cells can be genetically edited to be less immunoreactive to the patient's host cells and at the same time more aggressive to the patient's tumor cells.In this respect, some or all of the genetic modifications illustrated in Figures 5, 6, 7, and / or 15 can be advantageously combined.

[0023] In some embodiments, expression of TCR alpha and / or TCR beta can be suppressed or inactivated in the modified cell.

[0024] In some embodiments, the modified immune cells can be mutated to confer resistance to at least one immunosuppressant drug, such as an anti-CD52 antibody.

[0025] In some embodiments, the modified immune cells can be mutated to confer resistance to at least one chemotherapeutic agent, particularly a purine analog agent.

[0026] In some embodiments, the modified immune cells can be mutated in the patient, specifically in genes encoding MHCI components such as HLA or B2m, to improve their persistence or their longevity.

[0027] In some embodiments, the engineered immune cells can be mutated to improve their MUC1 CAR-dependent immune activation, specifically to reduce or suppress expression of immune checkpoint proteins and / or their receptors, e.g., PD-1 / PDL1, CTLA4, and / or TIM3.

[0028] In some embodiments, the modified immune cells can be mutated in genes involved in the TGF beta pathway, such as those encoding TGF beta and / or the TGF beta receptor (TGFβRII).

[0029] In some embodiments, the engineered immune cells can express a decoy of a TGF beta receptor, e.g., a dominant negative TGF beta receptor (dnTGFβRII) having at least 80% polypeptide sequence identity to SEQ ID NO:59.

[0030] In some embodiments, the engineered immune cells are transfected with an exogenous polynucleotide that allows for simultaneous expression of a CAR and a decoy of a TGF beta receptor, e.g., comprising a first polynucleotide sequence encoding the anti-MUC1 specific CAR, a second polynucleotide encoding a 2A self-cleaving peptide, and a third polynucleotide encoding the dominant negative TGF beta receptor (dnTGFβRII).

[0031] In some embodiments, the modified immune cells comprise an anti-MUC1 chimeric antigen receptor (CAR); NK cell inhibitors, such as HLAG, HLAE, or ULBP1; a CRS inhibitor, such as mutant IL6Ra, sGP130, or IL18-BP; or · cytochrome P450, CYP2D6-1, CYP2D6-2, CYP2C9, CYP3A4, CYP2C19, or CYP1A2, which confers hypersensitivity of said immune cells to drugs such as cyclophosphamide and / or isophosphamide; · dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin or methylguanine transferase (MGMT), mTORmut or Lckmut, conferring drug resistance; · a chemokine or cytokine, such as IL-12, IL-15, or IL-18; · Hyaluronidases, such as HYAL1, HYAL2, and HYAL3 (SPAM1); Chemokine receptors, such as CCR2, CXCR2, or CXCR4; Tumor-associated macrophage (TAM) secretion inhibitors, such as CCR2 / CCL2 neutralizing agents, to enhance the therapeutic activity of immune cells; and / or Metabolic enzymes, such as glucose phosphate isomerase 1 (GPI1), lactate dehydrogenase (LDHA), and / or phosphoenolpyruvate carboxykinase 1 (PCK1) The mouse is transfected with an exogenous polynucleotide sequence that allows for the simultaneous expression of another polypeptide that exhibits identity to one selected from the group consisting of:

[0032] The present invention also provides a method for producing the above-described population of modified therapeutic immune cells useful for the treatment of solid tumors. Such a method typically comprises the steps of: a) providing immune cells originating from a patient, or preferably a donor; b) expressing an anti-MUC1 chimeric antigen receptor (CAR) in the cell; c) introducing at least one genetic modification into the genome of the cell, the modification comprising: · Reduction or inactivation of TCR expression; · Reduction or inactivation of B2M; ·Reduced interaction between TGFβ and TGFβRII; Reducing the interaction between PD1 and PDL1; and / or Enhancement of expression of IL-12, IL-15, or IL-18 a process selected from those which result in d) Expanding the cells to form a therapeutically effective immune cell population.

[0033] In some embodiments, genetic modifications, particularly to knock out gene expression, are performed by using sequence-specific gene editing reagents, such as rare-cutting endonucleases / nickases or base editors.

[0034] The combination of gene editing modifications described above has been found to be particularly suitable for overcoming the biochemical barrier created by tumors that prevents access of immune cells, specifically T cells, to hot tumor cells.

[0035] The resulting population of cells generally contains at least 25%, preferably at least 50%, more preferably at least 75% modified cells having at least two, preferably at least three, preferably at least four, even more preferably at least five of said genetic modifications.

[0036] In some embodiments, a therapeutic composition can comprise a population of modified immune cells characterized by one, some, or all of the following (phenotypic) properties: Exogenous expression of a CAR targeting the tMUC1 epitope, and a reduction in B2M expression of at least 30%; preferably 50%, more preferably 75%; and / or a reduction in PD1 expression of at least 30%; preferably 50%, more preferably 75%; and / or Optionally, a reduction in TCR expression by at least 50%; preferably 75%; Optionally, an increase in expression of IL-12, IL-15, or IL-18 of at least 50%; preferably 75, more preferably 100%; Optionally, a reduction in expression of TGFβ or TGFβRII by at least 30%; preferably by 50%, more preferably by 75%; Optionally, exogenous expression of a decoy of TGFβR2; Optionally, secretion of HYAL1, HYAL2, and HYAL3 (SPAM1) by introduction of an exogenous coding sequence; and Optionally, expression of GPI1, PCK1, and / or LDHA by introduction of exogenous coding sequences.

[0037] From a genotypic point of view, such therapeutic populations of modified immune cells are characterized by one, several or all of the following (genotypic) properties: at least 50% of the immune cells present an exogenous polynucleotide sequence encoding a CAR that targets the tMUC1 epitope; and at least 50% and preferably at least 75% of the immune cells display a B2M inactive allele; and / or At least 30%, preferably at least 50%, more preferably 75% of the immune cells present a mutated PD1 allele; Optionally, at least 50% of the T cells, preferably at least 75%, display a TCR inactive allele; Optionally, at least 30%, preferably at least 50%, more preferably 75% of the immune cells display an exogenously introduced sequence encoding IL-12, IL-15, or IL-18; Optionally, at least 20%, preferably at least 50%, more preferably 75% of the immune cells present a sequence encoding a decoy of TGFβR2 exogenously inserted into their genome; Optionally, at least 20%, preferably at least 50%, more preferably 75% of the immune cells present a sequence encoding HYAL1, HYAL2 and / or SPAM1 exogenously inserted into their genome; Optionally, at least 20%, preferably at least 50%, more preferably 75% of the immune cells present a sequence encoding GPI1 and / or PCK1 exogenously inserted into their genome; Optionally, at least 30%, preferably at least 50%, more preferably 75% of the immune cells display a mutated allele encoding TGFβ or TGFβRII.

[0038] The modified immune cells of the present invention are particularly suitable for treating conditions characterized by tMUC1-expressing cells, in particular solid tumors, such as typically esophageal cancer, breast cancer, in particular triple-negative breast cancer, gastric cancer, bile duct cancer, pancreatic cancer, colon cancer, lung cancer, thymic cancer, mesothelioma, ovarian cancer, and / or endometrial cancer.

[0039] Thus, the present invention encompasses methods for producing modified cells, the resulting therapeutic cells, cell populations comprising such cells, and therapeutic compositions comprising same, as well as methods of treatment that allow for addressing pathology induced by tMUC1-expressing cells.

[0040] A holistic method of treatment typically includes the following steps: Modifying patient-derived or donor-derived immune cells to express a functional anti-MUC1 CAR. administering the CAR-positive modified immune cells to a patient to eliminate cells expressing the tMUC1 epitope.

[0041] In the allogeneic setting, but not only, such methods of treatment may advantageously combine the administration of (1) a lymphocyte depleting agent and (2) a population of allogeneic engineered immune cells expressing a chimeric antigen receptor (CAR) specifically directed against the tMUC1 epitope.

[0042] In such a setting, it may be advantageous for anti-MUC1 CAR-positive engineered immune cells to have their CD52 gene expression, or any other polypeptide targeted by the lymphodepleting agent, inactivated in order to confer resistance to lymphodepleting therapy.

[0043] The present invention also provides for utilizing dual CAR with any combination of two anti-tMUC1 scFVs, which can target a larger number of cancers and eliminate antigen escape due to glycoform changes under the selective pressure of CAR-T therapy.It also encompasses the use of dual CAR T cells that target two independent antigens of MUC-1 to increase the rate of target cell recognition and the efficiency of CAR-T cell therapy.In a preferred approach, anti-MUC1 CAR is co-expressed with anti-mesothelin CAR in modified T cells. [Brief description of the drawings]

[0044] [Figure 1] A. Schematic of MUC1 expression on the surface of normal and tumor cells. B. Schematic comparison of the glycosylation status of MUC1 on healthy and tumor cells. Cancer-associated MUC1 is underglycosylated along the tandem polypeptide repeat (SEQ ID NO:20). [Diagram 2] Specificity of selected MUC1 scFVs measured on cancer cells and primary cells from kidney, lung, and cervical tissues, which express high levels of normal MUC1 in contrast to breast cancer cell lines (T47D and HCC70), which are not subsequently stained by the candidate scFVs. [Diagram 3] Figure 1 shows the percentage of specific lysis of breast cancer cells T47D and HCC70 obtained by expression of the anti-MUC1-CAR of the invention in primary T cells in vitro (at ratios of 1:1, 2,5:1, and 5:1). The figure shows the high activity response of the novel anti-MUC1 CAR-T, and the superiority of the CLS MUC1-A CAR. [Figure 4]Analysis of the solid tumor microenvironment and various factors that inhibit CAR-T function in solid tumors. [Diagram 5] Schematic diagram of an anti-MUC1-CAR modified immune cell of the present invention with its main characteristic genetic features. A. Representation of a genetic construct for co-expression of an anti-MUC1 CAR and a decoy for TGF beta (dnTGRBR2B). This genetic construct can be included in a lentiviral vector or on a donor template for site-specific gene insertion by homologous recombination or NHEJ using nuclease or nickase gene editing reagents. B. Representation of a genetic insertion at the β2m endogenous locus resulting in inactivation of β2m and expression of HLA-E providing resistance to the patient's NK cells. C. Representation of a genetic insertion at the PDCD1 (PD1) endogenous locus resulting in inactivation of PD1 and expression of IL-12 enhancing the activity of immune cells. D. Representation of a modified immune cell surface showing heterologous expression of an anti-MUC1 CAR, HLA-E as an NK inhibitor, secretion of IL-12, and dnTGFβRII as a decoy for TGFβ. On the other hand, the expression of TCR, PD1, and β2m is suppressed or inactivated. [Figure 6] Detailed mechanism of ΔB2M-HLA-E gene characteristics shown in Figure 5B above. [Figure 7] Detailed mechanism of the ΔPD1-IL12 gene signature shown in Figure 5C above. By inserting an exogenous sequence encoding IL-12 under the transcriptional control of the endogenous PD1 promoter, IL-12 secretion is synchronized with tumor antigen recognition by CAR. [Figure 8] FACS analysis of cells modified according to the present invention (detailed in the experimental section) showing that at least 30% of the cells in the population of modified cells exhibit at least three of the major genetic signatures. At least 30% of the cells in the population were determined to be CAR positive, [PD1] negative, [TCR] negative, [β2m] negative, and [HLAE] positive. [Figure 9]Low score of candidate off-sites observed in modified cells of the present invention (rLV CAR-DNTGFBR2 transduction + triple TALEN® transfection (TRAC / B2M / PD-1) + AAV-mediated KI of HLA-E) when OCA (oligo capture assay) analysis was performed. [Figure 10] FIG. 13 shows the experimental results as detailed in Example 2, showing the robust in vivo intratumoral expansion of UCARTMUC1 achieved with the ΔPD1-IL12 gene signature against HCC70 cancer cells (same experimental setup as illustrated in FIG. 12). [Figure 11] Graph showing in vivo tumor volume in mice treated with each of the four anti-MUC1 CAR-Ts of the invention up to day 28 after inoculation. [Figure 12] Experiments showing that the genetic signature detailed in Figure 5 extends the survival of mice when treated with anti-MUC1 CAR (see Example 2). [Figure 13] Graph showing subcutaneous tumor volume reduction obtained by treatment with CLS MUC1-A CAR-T cells in a mouse study. [Figure 14] Analysis of HCC70 tumors from mice treated with anti-MUC1 CAR T cells with and without the genetic signature. The graph shows the stronger intratumoral UCARTMUC1 expansion achieved with cells bearing the genetic signature of the invention. [Figure 15] Rationale for an anti-MUC1 CAR / anti-mesothelin CAR dual approach aimed at overcoming solid tumor heterogeneity. [Figure 16] Rationale for the anti-MUC1 CAR-T signature of the present invention for optimized immune scenarios. [Figure 17]Schematic diagram of an optimized anti-MUC1-CAR modified immune cell of the invention with its main characteristic genetic features. Anti-MUC1 CAR constructs can be introduced randomly (with lentiviral vectors) or at specific sites (by homologous recombination or NHEJ with gene editing reagents); HLA-E is integrated into the β2m endogenous locus, resulting in host immune escape; IL-12 is integrated into the PDCD1 (PD1) endogenous locus, resulting in inactivation of PD1 and tumor-specific and localized expression of IL-12, and TGFBR2 is inactivated, resulting in blockade of the TGFβ pathway. [Figure 18A] A. Experimental in vivo setup for evaluation of MUC1-A and MUC1-C modified CAR-T cells. B. Tumor volumes obtained after treatment with the indicated doses of MUC1-A and MUC1-C modified CAR-T cells. C. Survival curves. D. Percentage of hCD45+ cells detected in tumors 54 days after treatment. These results reveal the superiority and dose-response sensitivity of MUC1-A over other anti-tMUC1 CARs. [Figure 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 18D] See legend to Figure 18A. [Figure 19] IHC results performed on tumor microarrays with MUC1-A, MUC1-C, and MUC1-D scFV proteins revealed that CAR MUC1-A showed greater affinity to patient tumor cell samples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Detailed Description of the Invention Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of gene therapy, biochemistry, genetics, and molecular biology.

[0046] In carrying out or testing the present invention, any methods and materials similar or equivalent to those described herein can be used, but suitable methods and materials are described herein.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

[0047] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art, and such techniques are fully explained in the literature. For example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (MJ Gait ed., 1984); Mullis et al. U.S. Patent No. 4,683,195; Nucleic Acid Hybridization (BD Harries & SJ Higgins eds. 1984); Transcription And Translation (BD Hames & SJ Higgins eds. 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);Methods In ENZYMOLOGY series (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JH Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (DM Weir and CC Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

[0048] The present invention relates to a general method of treating solid tumors by adoptive immune cells directed against the underglycosylated MUC1 tumor antigen, specifically specific epitope regions spanning the polypeptide sequence SEQ ID NO:1 of this polypeptide, and more specifically by using engineered allogeneic CAR immune cells, which have proven particularly efficient in targeting such antigens.

[0049] Described herein are methods for generating engineered immune cells directed against the human MUC1 protein (also referred to as MUCIN-1 and P15941 in the Uniprot database), which is underglycosylated on the surface of malignant cells, and more specifically against the polypeptide region represented by SEQ ID NO: 1. As shown in the experimental section herein, efficient CAR T cells were generated by directing a CAR against this antigen region comprising or consisting of SEQ ID NO: 1, specifically by using one of the binding domains comprising scFv comprising SEQ ID NO: 17 (MUC1-A), SEQ ID NO: 27 (MUC1-B), SEQ ID NO: 37 (MUC1-C), or SEQ ID NO: 47 (MUC1-D).

[0050] The resulting modified immune cells of the invention, generally NK cells or T cells bearing the anti-MUC1 CARs of the invention, demonstrate greater activation, potency, killing activity, cytokine release, and in vivo persistence than their counterparts equipped with other previous anti-MUC1 CARs.

[0051] Thus, the present invention relates to immune cells equipped with a CAR that targets a specific epitope contained in the sequence SEQ ID NO:1 of MUC1, in particular immune cells engineered to treat solid tumors such as triple-negative breast cancer tumors.

[0052] Designing an anti-MUC1-CAR for expression in immune cells : " Chimeric Antigen Receptor"Carbohydrate Antibody (CAR)" refers to a recombinant receptor that includes a targeting moiety that is linked to one or more signaling domains in a single fusion molecule. Generally, the binding moiety of a CAR consists of the antigen-binding domain of a single-chain antibody (scFv), which includes a light chain variable fragment and a heavy chain variable fragment of a monoclonal antibody linked by a flexible linker. These ScFvs are generally characterized by complementarity determining regions (CDRs), which are short invariant polypeptide sequences that are important for the specificity of the interaction with the epitope region. Binding moieties based on receptor or ligand domains have also been successfully used. The signaling domains of CARs are generally derived from the cytoplasmic region of CD3 zeta or Fc receptor gamma chain, which are generally combined with signaling domains from costimulatory molecules, including CD28, OX-40 (CD134), ICOS, and 4-1BB (CD137), to enhance cell survival and increase proliferation. CAR is generally expressed in effector immune cells to redirect their immune activity to antigens expressed on the surface of tumor cells from various malignant tumors, including lymphomas and solid tumors.A component of CAR is any functional subunit of CAR that is encoded by the exogenous polynucleotide sequence introduced into cell.For example, this component can be useful for interacting with target antigen, stabilizing or localizing CAR in cell.

[0053] In general, such CARs are: · an extracellular ligand-binding domain, including VH and VL, derived from a monoclonal anti-MUC1 antibody; · a transmembrane domain; and a signaling domain, preferably a cytoplasmic domain including a CD3 zeta signaling domain, and a costimulatory domain Includes.

[0054] In a preferred aspect, the anti-MUC1 chimeric antigen receptor (CAR) of the invention has an extracellular ligand-binding domain comprising an ScFv having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to MUC1-A ScFv (SEQ ID NO:17), MUC1-B (SEQ ID NO:27), MUC1-C (SEQ ID NO:37), and / or MUC1-D (SEQ ID NO:47), respectively.

[0055] Said ScFv is characterized by a variable light (VL) chain and a variable heavy (VH) chain comprising specific CDRs, and therefore the extracellular ligand binding domain of the anti-MUC1 CAR of the invention can be, as a first example, a variable light (VL) having at least 90% identity to SEQ ID NO:11 (CDR-VL1-A), SEQ ID NO:12 (CDR-VL2-A), and SEQ ID NO:13 (CDR-VL3-A), and a variable heavy (VH) chain comprising CDRs having at least 90% identity to SEQ ID NO:14 (CDR-VH1-A), SEQ ID NO:15 (CDR-VH2-A), and SEQ ID NO:16 (CDR-VH3-A), respectively; may include The extracellular ligand binding domain preferably comprises a VH chain and a VL chain having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO:9 (MUC1-A full VH) and SEQ ID NO:10 (MUC1-A full VL), respectively.

[0056] In a second example, the ScFv comprises a variable light (VL) chain comprising CDRs having at least 90% identity with SEQ ID NO:21 (CDR-VL1-B), SEQ ID NO:22 (CDR-VL2-B), and SEQ ID NO:23 (CDR-VL3-B), respectively; a variable heavy (VH) chain comprising CDRs having at least 90% identity to SEQ ID NO:24 (CDR-VH1-B), SEQ ID NO:25 (CDR-VH2-B), and SEQ ID NO:26 (CDR-VH3-B), respectively; may include The extracellular ligand-binding domain comprises a VH chain and a VL chain having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO:19 (MUC1-B full VH) and SEQ ID NO:20 (MUC1-B full VL), respectively.

[0057] In a third example, the ScFv comprises: a variable light (VL) chain comprising CDRs having at least 90% identity with SEQ ID NO:31 (CDR-VL1-C), SEQ ID NO:32 (CDR-VL2-C), and SEQ ID NO:33 (CDR-VL3-C), respectively; a variable heavy (VH) chain comprising CDRs having at least 90% identity to SEQ ID NO:34 (CDR-VH1-C), SEQ ID NO:35 (CDR-VH2-C), and SEQ ID NO:36 (CDR-VH3-C), respectively; may include The extracellular ligand-binding domain comprises a VH chain and a VL chain having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO:29 (MUC1-C full VH) and SEQ ID NO:30 (MUC1-C full VL), respectively.

[0058] In a fourth example, the ScFv comprises: a variable light (VL) chain comprising CDRs having at least 90% identity with SEQ ID NO: 41 (CDR-VL1-D), SEQ ID NO: 42 (CDR-VL2-D), and SEQ ID NO: 43 (CDR-VL3-D), respectively; and A variable heavy (VH) chain comprising CDRs having at least 90% identity to SEQ ID NO:44 (CDR-VH1-D), SEQ ID NO:45 (CDR-VH2-D), and SEQ ID NO:46 (CDR-VH3-D), respectively. may include The extracellular ligand-binding domain comprises a VH chain and a VL chain having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to SEQ ID NO:39 (MUC1-D full VH) and SEQ ID NO:40 (MUC1-D full VL), respectively.

[0059] Generally, residues in the framework regions of the binding domain can be substituted to improve antigen binding or to humanize these regions. These framework substitutions are identified by methods well known in the art, for example, by modeling the interactions between CDRs and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify unusual framework residues at specific positions (see, for example, Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., (1988) Nature, 332:323, which are incorporated herein by reference in their entirety).

[0060] In some embodiments, the VH and VL chains in the above anti-MUC1 CAR constructs can be replaced by their humanized versions. For example, as a preferred embodiment of a MUC1-A CAR, the VH can be one selected from SEQ ID NOs:228-234 (humanized variants of MUC1 full VH represented by SEQ ID NO:9), and the VL can be selected from SEQ ID NOs:235-239 (humanized variants of MUC1 full VH represented by SEQ ID NO:10), respectively, provided in Table 3 herein.

[0061] Various aspects of the present invention are provided through the features provided in the claims, in view of the ordinary practice and knowledge of those skilled in the art. The detailed sequences of the CARs of the present invention are detailed in Table 2, Table 3, Table 4, Table 5, Table 6, and Table 7, where each row or column should be considered as an independent aspect of the present invention.

[0062] Table 2: Amino acid sequences of various domains other than scFv that constitute the anti-MUC1 CAR of the present invention TIFF2024517713000003.tif98162

[0063] Table 3: Polypeptide sequences contained in CLS MUC1-A CAR TIFF2024517713000004.tif216161

[0064] Table 3 (continued) Humanized Polypeptide Sequences Optionally Included in MUC1-A CAR TIFF2024517713000005.tif217161

[0065] Table 4: Polypeptide sequences contained in CLS MUC1-B CAR TIFF2024517713000006.tif216163

[0066] Table 5: Polypeptide sequences contained in CLS MUC1-C CAR TIFF2024517713000007.tif219161

[0067] Table 6: Polypeptide sequences contained in CLS MUC1-D CAR TIFF2024517713000008.tif229153

[0068] Table 7. Polypeptide structures of MUC1-A, MUC1-B, MUC1-C, and MUC1-D CARs TIFF2024517713000009.tif114166

[0069] The signal transduction domain or intracellular signal transduction domain of the CAR of the present invention is responsible for intracellular signal transduction after the extracellular ligand binding domain binds to the target, resulting in the activation of immune cells and immune responses.In other words, the signal transduction domain is responsible for the activation of at least one of the normal effector functions of the immune cells that the CAR expresses.For example, the effector function of T cells can be cytolytic activity or helper activity, including the secretion of cytokines.Therefore, the term "signal transduction domain" refers to a part of a protein that transmits effector signal function signals to allow cells to perform special functions.

[0070] Preferred examples of signaling domains used in CARs can be cytoplasmic sequences of T cell receptors and co-receptors that act in concert to initiate signaling after binding to antigen receptors, as well as any derivatives or variants of these sequences, and any synthetic sequences with the same functional capabilities. Signaling domains include two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation, and those that act antigen-independently to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences can include signaling motifs known as immunoreceptor tyrosine-based activation motifs, or (of) ITAMs. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that serve as binding sites for tyrosine kinases of the syk / zap70 class. Non-existent examples of ITAMs used in the present invention can include those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signaling domain of the CAR may comprise a CD3 zeta signaling domain, which has an amino acid sequence having at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97%, or 99% sequence identity to an amino acid sequence selected from the group consisting of: (SEQ ID NO:7).

[0071] In certain embodiments, the signaling domain of the CAR of the present invention comprises a costimulatory signal molecule. Costimulatory molecules are cell surface molecules that are not antigen receptors or their ligands that are required for efficient immune responses. "Costimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates T cell responses, such as, but not limited to, proliferation activation and differentiation, in addition to the primary signal provided by, for example, the binding of the TCR / CD3 complex to a peptide-loaded MHC molecule. Costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intracellular adhesion molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, agonists that bind to Toll ligand receptor or Examples of co-stimulatory ligands include antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0072] In a preferred embodiment, the signaling domain of the CAR of the invention comprises a portion of a costimulatory signal molecule selected from the group consisting of fragments of 4-1BB (GenBank: AAA53133.) and CD28 (NP_006130.1). Specifically, the signaling domain of the CAR of the invention comprises an amino acid sequence that comprises at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity to 4-1BB or CD28. Thus, the anti-MUC1 chimeric antigen receptor of the invention preferably comprises a CD3 zeta signaling domain having at least 80% identity to SEQ ID NO:7, and generally comprises a costimulatory domain having at least 80% identity to SEQ ID NO:6 (4-1BB).

[0073] The CAR of the present invention is generally expressed on the membrane surface of a cell. Thus, such a CAR further comprises a transmembrane domain. The distinguishing feature of a suitable transmembrane domain includes the ability to be expressed on the surface of a cell, preferably an immune cell in the present invention, specifically a lymphocyte cell or a natural killer (NK) cell, and to interact with each other to direct the cellular response of the immune cell to a predetermined target cell. The transmembrane domain can be derived from natural or synthetic sources. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. As non-existent examples, the transmembrane polypeptide can be a subunit of the T cell receptor, such as α, β, γ or ζ, a polypeptide that constitutes the CD3 complex, the IL2 receptor p55 (α chain), p75 (β chain), or γ chain, a subunit chain of the Fc receptor, specifically the Fcγ receptor III, or a CD protein. Alternatively, the transmembrane domain can be synthetic and mainly comprise hydrophobic residues such as leucine and valine. In a preferred embodiment, the transmembrane domain is derived from the human CD8 alpha chain (e.g., NP_001139345.1). The transmembrane domain may further comprise a hinge region between the extracellular ligand-binding domain and the transmembrane domain. The term "hinge region" as used herein broadly refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular ligand-binding domain. Specifically, the hinge region is used to provide additional flexibility and accessibility to the extracellular ligand-binding domain. The hinge region may comprise up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. The hinge region may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or may be derived from all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be an entirely synthetic hinge sequence.In preferred embodiments, the hinge domain comprises a portion of the human CD8 alpha chain, the FcγRIIIα receptor, or IgG1, respectively, or a hinge polypeptide exhibiting preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity to these polypeptides.

[0074] Thus, the chimeric antigen receptor (CAR) of the present invention comprises a hinge between the extracellular ligand binding domain and the transmembrane domain, said hinge being generally selected from the CD8α hinge, the IgG1 hinge, and the FcγRIIIα hinge, or a polypeptide having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with these polypeptides, in particular with SEQ ID NO:4 (CD8α).

[0075] The CARs of the invention generally further comprise a transmembrane domain (TM), preferably selected from CD8α and 4-1BB, more preferably from CD8α-TM or a polypeptide exhibiting at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity to SEQ ID NO:5 (CD8α TM).

[0076] In further embodiments, the anti-MUC1 CARs of the invention contain a safety switch that is convenient for sorting, purifying, and / or depleting the modified immune cells. Although the methods of the invention are designed to be performed ex vivo, depletion can be performed in vivo to control immune cell proliferation in the patient, and potentially halt the therapeutic effect by using antibodies approved by regulatory agencies for therapeutic use in humans. Examples of mAb specific epitopes (and their corresponding mAbs) that can be incorporated into the extracellular binding domain of the CARs of the invention are listed in Table 8.

[0077] Table 8. 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. TIFF2024517713000010.tif111146

[0078] Thus, a specific CAR of the invention can comprise a safety switch that comprises at least one exogenous mAb epitope listed in Table 8, preferably a safety switch that comprises the epitope CPYSNPSLC (SEQ ID NO:49) to which rituximab specifically binds. More preferably, such a CAR comprises a safety switch designated "R2" that has at least 90% identity to SEQ ID NO:3.

[0079] The structures of preferred polypeptide structures of the anti-MUC1 CARs of the invention are illustrated in Table 7.

[0080] The CAR of the present invention may also comprise a signal peptide that aids in its expression on the surface of the modified cell. Chimeric antigen receptors (CARs) generally form single-chain polypeptides, but may also be produced in a multi-chain format, for example as described in WO2014039523.

[0081] Thus, an anti-MUC1 CAR of the present invention may exhibit a transmembrane domain that shares 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:5 from CD8α.

[0082] The anti-MUC1 CAR of the present invention may further comprise a hinge between the extracellular ligand binding domain and the transmembrane domain, which is preferably selected from a CD8α hinge, an IgG1 hinge, and an FcγRIIIα hinge. In a preferred embodiment, the hinge shares 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:4 (CD8α), respectively.

[0083] In a preferred embodiment, the anti-MUC1CAR has a polypeptide structure comprising a CD8α hinge having at least 80% identity to the amino acid sequence shown in SEQ ID NO:4, together with a CD8α transmembrane domain having at least 80% identity to the amino acid sequence shown in SEQ ID NO:5.

[0084] In a preferred embodiment, the anti-MUC1CAR of the invention comprises a safety switch, which is a polypeptide sequence that is specifically recognized by an antibody suitable for use in therapy, such as an epitope selected from Table 8. For example, a safety switch containing the epitope CPYSNPSLC (SEQ ID NO:49) can be specifically bound by rituximab (Rituxan, Hoffmann-La Roche), an approved anti-CD20 chimeric monoclonal antibody commonly used in cancer therapy to deplete B cells.

[0085] In a preferred embodiment, the anti-MUC1CAR of the present invention comprises a costimulatory domain from 4-1BB or CD28, preferably 4-1BB, or any functionally similar domain having at least 80% identity with SEQ ID NO:6.

[0086] In a preferred embodiment, the anti-MUC1CAR of the present invention comprises a CD3 zeta signaling domain having at least 80% identity to SEQ ID NO: 7. It may also contain a signal peptide so that it is better addressed at the cell surface.

[0087] As illustrated in the Examples and Tables 2-8, preferred CARs of the invention are anti-MUC1 CARs having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% overall amino acid sequence identity with SEQ ID NO:18 (CLS MUC1-A), SEQ ID NO:28 (CLS MUC1-B), SEQ ID NO:38 (CLS MUC1-C), and SEQ ID NO:48 (CLS MUC1-D), respectively. One preferred anti-MUC1-CAR is CLS MUC1-A, represented by SEQ ID NO:18.

[0088] More generally, the CARs of the invention are produced by assembling various polynucleotide sequences encoding consecutive fragments of the CAR polypeptide in a vector and transfecting and expressing them in immune cells, as described in the art and as outlined, for example, in [Boyiadzis, MM, et al. (2018) Chimeric antigen receptor (CAR) T therapies for the treatment of hematologic malignancies: clinical perspective and significance. J. Immunotherapy Cancer 6, 137].

[0089] The present invention relates to the polynucleotides and vectors, as well as any intermediate products intervening in any step of the immune cell manufacturing process referred to herein.

[0090] "Vector" refers to a nucleic acid molecule capable of transporting another nucleic acid that is linked to it. In the present invention, "vector" includes, but is not limited to, a viral vector, a plasmid, an RNA vector, or a linear or circular DNA or RNA molecule that may consist of chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acid. Preferred vectors are those capable of self-replicating (episomal vectors) and / or capable of expressing a nucleic acid that is linked to it (expression vectors). Many suitable vectors are known to those of skill in the art and are commercially available. Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses (AAV)), coronaviruses, negative-stranded RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses such as adenoviruses, herpesviruses (e.g., herpes simplex viruses types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox viruses). Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include avian leukosis sarcoma viruses, mammalian types C, B, and D viruses, the HTLV-BLV complex, lentiviruses, and spumaviruses (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0091] Specifically, the present invention provides vectors in the form of non-integrating lentiviral vectors (IDLV) or AAV vectors comprising the exogenous polynucleotide sequences described herein encoding, inter alia, CAR, IL-12, dnTGFβR, HLA-E or any other useful transgene, for use as donor polynucleotide templates to perform gene targeted integration.

[0092] Such lentiviral vectors can include a polynucleotide sequence encoding a CAR of the present invention operably linked to a promoter (such as the spleen focus forming virus promoter (SFFV)). "Operably linked" means that the described components are juxtaposed in a relationship allowing them to function in their intended manner. When a gene (such as a polynucleotide sequence encoding a CAR) is "operably linked" to a promoter and its transcription is under the control of the promoter, this transcription results in a product encoded by the gene.

[0093] The lentiviral vector of the present invention typically comprises control elements such as 5' and 3' long terminal repeats (LTR), but may also comprise other structural and functional genetic elements originally derived from lentivirus. Such structural and functional genetic elements are well known in the art. The lentiviral vector may, for example, comprise the genes gag, pol, and env. However, preferably, the lentiviral vector of the present invention does not comprise the genes gag, pol, and env. As further control elements, the lentiviral vector may comprise one or more (such as two or more) packaging signals (such as the packaging signal ψ), a primer binding site, a transactivation response region (TAR), and a rev response region (RRE).

[0094] The 5'-terminal repeat (LTR) and 3'-terminal repeat (LTR), typically located on either side of the lentiviral genome, have promoter / enhancer activity and are essential for the correct expression of full-length lentiviral vector transcripts. LTRs usually contain the repeat sequence U3RU5 present at both the 5'- and 3'-ends of the double-stranded DNA molecule, which is a combination of a 5'R-U5 segment and a 3'U3-R segment of single-stranded RNA, where the repeat R occurs at both ends of the RNA, but U5 (unique sequence 5) occurs only at the 5'-end of the RNA, and U3 (unique sequence 3) occurs only at the 3'-end of the RNA. The safety of lentiviral vectors can be improved by removing the U3 sequence, which can result in a "self-activating" vector that is completely devoid of the viral promoter and enhancer sequences originally present in the LTR. Thus, the vector can infect and integrate into the host genome once, but is not inherited further, increasing the safety of using the vector as a gene delivery vector.

[0095] In some embodiments, the lentiviral vector is a self-inactivating (SIN) lentiviral vector. In certain embodiments, the lentiviral vector comprises a 3'LTR, wherein the 3'LTR enhancer-promoter sequence (i.e., the U3 sequence) is modified (e.g., deleted).

[0096] In some embodiments, the lentiviral vector comprises the following elements in 5' to 3' order: · 5' long terminal repeat (5'LTR); · promoters (e.g. EF1-alpha promoter); a polynucleotide sequence encoding a chimeric antigen receptor of the invention; and / or 3' long terminal repeat (3'LTR), preferably a 3' self-inactivating LTR The polynucleotide sequence includes one or more of:

[0097] In certain embodiments, the lentiviral vector comprises, in 5' to 3' order, the following elements: · 5' long terminal repeat (5'LTR); · promoters (e.g. EF1-alpha promoter); The CAR of the present invention may include a safety switch such as R2, a polynucleotide sequence encoding the 2A peptide; a polynucleotide sequence encoding one or any further polypeptides, such as dnTGFβR, to be co-expressed with the CAR; and / or 3' long terminal repeat (3'LTR), preferably a 3' self-inactivating LTR The polynucleotide sequence may further comprise at least one of:

[0098] Alternatively, the lentiviral vector may comprise, in 5' to 3' order, the following elements: · 5' long terminal repeat (5'LTR); · promoters (e.g. EF1-alpha promoter); a polynucleotide sequence encoding one or any further polypeptides, such as dnTGFβR, to be co-expressed with the CAR; a polynucleotide sequence encoding the 2A peptide; The CAR of the present invention may include a safety switch such as R2, and / or 3' long terminal repeat (3'LTR), preferably a 3' self-inactivating LTR It may include at least one of.

[0099] Generally, the resulting vector forms a single transcription unit operably linked to the promoter of item (b) and all are transcribed under the control of said promoter.

[0100] AAV vectors, especially 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 convenient for introducing the anti-MUC1 CAR of the invention into the genome by site-specific homologous recombination. Generally, site-specific homologous recombination is induced in immune cells by expression of a rare-cutting endonuclease, such as a TALEN, as already taught for other CARs to treat hematological cancers in EP3276000 and WO2018073391. CAR site-specific integration may have several advantages, including more stable integration, integration that places the transgene under the transcriptional control of the endogenous promoter at the selected locus, and integration that can inactivate the endogenous locus. These latter aspects are discussed in more detail in the following section on genome engineering of therapeutic immune cells.

[0101] As an object of the present invention, an AAV vector is provided that comprises a polynucleotide sequence encoding an anti-MUC1 CAR as described above, and optionally another sequence encoding a cis-regulatory element (e.g., a 2A peptide cleavage site) or an internal ribosome entry site (IRES) that allows for the simultaneous expression of a third sequence encoding a product that improves the therapeutic efficacy of the modified immune cell. As an example herein, overexpression of dnTGFβRII has been shown to reduce SMAD2-3 phosphorylation, which reduces TGFβ-induced cellular exhaustion in the tumor environment.

[0102] " therapeutic propertiesThe term "improvement of cells" encompasses various ways in which such cells can be improved in terms of their use in therapeutic treatments. This means that genetic engineering can confer a therapeutic benefit to the cells (i.e., therapeutic efficacy) or facilitate their use or production. For example, genetic engineering can result in effector cells that have better survival rates, faster growth, shorter cell cycles, improved immune activity, are more functional, are more differentiated, are more specific to target cells, are more sensitive or resistant to drugs, are more sensitive to glucose, oxygen, or amino acid deprivation (i.e., are more adaptable to the tumor microenvironment). Progenitor cells can be more productive, can be tolerated by the recipient patient, and can be more likely to produce cells that differentiate into the desired effector cells. These examples of "therapeutic properties" are listed as non-limiting examples.

[0103] Genomic engineering of anti-MUC1 CAR immune cells for cell therapy By "immune cell" is meant a cell of hematopoietic origin that is functionally involved in the initiation of innate and / or adaptive immune responses, typically a CD3 or CD4 positive cell. The immune cell of the present invention may be a dendritic cell, a killer dendritic cell, a mast cell, a NK cell, a B cell, or a T cell selected from the group consisting of an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte.

[0104] "Primary cells" refer to cells taken directly from a living tissue (e.g., a biopsy) and grown in vitro for a limited time, i.e., to allow a limited number of population doublings, to become a cell line. Primary cells are the opposite of persistent tumorigenic or artificially immortalized cell lines. Non-existent examples of such cell lines are CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; and Molt4 cells.

[0105] Primary immune cells can be obtained from several sources, including, but not limited to, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of inflammation, ascites, pleural effusion, spleen tissue, and from tumors, such as tumor-infiltrating lymphocytes. In some embodiments, the immune cells can be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with an infectious disease. In another embodiment, the cells are part of a mixed population of immune cells exhibiting different phenotypic characteristics, including CD4, CD8, and CD56 positive cells. Primary immune cells are provided from a donor or patient by various methods known in the art, such as leukapheresis as outlined by Schwartz J.et al. (Guidelines on the use of therapeutic apheresis in clinical practice-evidence-based approach from the Writing Committee of the American Society for Apheresis: the sixth special issue (2013) J Clin Apher. 28(3):145-284).

[0106] Stem cell-derived immune cells, specifically immune cells derived from induced pluripotent stem cells (iPS) [Yamanaka, K. et al. (2008). "Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors". Science. 322 (5903): 949-53], are also considered as primary immune cells of the present invention. Lentiviral expression of reprogramming factors has been used to induce pluripotent cells from human peripheral blood cells [Staerk, J. et al. (2010). "Reprogramming of human peripheral blood cells to induced pluripotent stem cells". Cell stem cell. 7 (1): 20-4] [Loh, YH. et al. (2010). "Reprogramming of T cells from human peripheral blood". Cell stem cell. 7 (1): 15-9].

[0107] In a preferred embodiment of the invention, immune cells are derived from human embryonic stem cells by techniques known in the art that do not destroy the human fetus [Chung et al. (2008) Human Embryonic Stem Cell lines generated without embryo destruction, Cell Stem Cell 2(2):113-117].

[0108] "Genetic engineering" means any method aimed at introducing, modifying, and / or removing genetic material from cells. Gene editing " refers to genetic engineering that allows for the addition, removal, or modification of genetic material at specific locations (locuses) within a genome, including punctual mutations. Gene editing generally involves sequence-specific reagents.

[0109] By "sequence specific reagent" is meant any active molecule capable of specifically recognizing a selected polynucleotide sequence, called a "target sequence", generally at least 9 bp, more preferably at least 10 bp, even more preferably at least 12 pb in length at a genomic locus, with a view to modifying the expression of said genomic locus. Said expression can be modified by mutations, deletions or insertions in coding or regulatory polynucleotide sequences, by epigenetic changes such as by methylation or histone modifications, or by interfering with the transcription level by interaction with transcription factors or polymerases.

[0110] Examples of sequence-specific reagents are endonucleases, RNA guides, RNAi, methylases, exonucleases, histone deacetylases, endonucleases, terminal processing enzymes such as exonucleases, deaminases, more specifically cytidine deaminases, such as those that are combined with the CRISPR / cas9 system or the TALE system to perform base editing (i.e., nucleotide substitution), but without necessarily relying on nuclease cleavage, as described, for example, in Hess, GT et al. [Methods and applications of CRISPR-mediated base editing in eukaryotic genomes (2017) Mol Cell. 68(1): 26-43] or Mok et al. [A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing (2020) Nature 583:631-637].

[0111] In a preferred aspect of the invention, the sequence-specific reagent is a sequence-specific nuclease reagent, such as an RNA guide coupled to a guided endonuclease.

[0112] The present invention aims to improve the therapeutic potential of immune cells by gene editing methods, in particular targeted gene integration.

[0113] By "targeted gene integration" is meant any known site-specific method that allows for the insertion, replacement or modification of a genomic coding sequence in a living cell.

[0114] In a preferred aspect of the invention, said targeted gene integration comprises resulting in the insertion or replacement of at least one exogenous nucleotide, preferably a sequence of several nucleotides (i.e. a polynucleotide), more preferably a coding sequence, by homologous recombination at the locus of the targeted gene.

[0115] By "DNA target", "DNA target sequence", "target DNA sequence", "nucleic acid target sequence", "target sequence", or "processing site" is intended a polynucleotide sequence that can be targeted and processed by the sequence-specific nuclease reagents of the present invention. These terms refer to specific DNA locations within a cell, preferably genomic locations, but also to portions of genetic material that can exist independently of the body of genetic material, such as non-existent examples being portions of plasmids, episomes, viruses, transposons, or organelles such as mitochondria. Non-existent examples of RNA-guided target sequences include genomic sequences that can hybridize with a guide RNA that directs an RNA-guided endonuclease to a desired locus.

[0116] "Low-cutting endonucleases" are sequence-specific endonuclease reagents of choice so long as their recognition sequence is in the range of approximately 10-50 contiguous base pairs, preferably 12-30 bp, more preferably 14-20 bp.

[0117] In a preferred aspect of the invention, the endonuclease reagent is a homing endonuclease, e.g. as described in Arnould S., et al. [WO2004067736], a zinc finger nuclease (ZFN), e.g. as described in Urnov F., et al. [Highly efficient endogenous human gene correction using designed zinc-finger nucleases (2005) Nature 435:646-651], a TALE nuclease, e.g. as described in Mussolino et al. [A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity (2011) Nucl. Acids Res. 39(21):9283-9293], or a TALE nuclease, e.g. as described in Boissel et al. [MegaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering (2013) Nucleic Acids Research 42(4):2591-2601].

[0118] In another embodiment, the endonuclease reagent is RNA-guided, inter alia, used in conjunction with an RNA-guided endonuclease such as Cas9 or Cpf1 as taught by Doudna, J., and Chapentier, E., [The new frontier of genome engineering with CRISPR-Cas9 (2014) Science 346 (6213):1077] (incorporated herein by reference).

[0119] In a preferred aspect of the invention, the endonuclease reagent is expressed transiently in the cell, i.e., said reagent is not integrated into the genome or persists long term, as is the case for RNA, more specifically mRNA, proteins, or mixed complexes of proteins and nucleic acids (e.g. ribonucleoproteins).

[0120] The mRNA form of the endonuclease is preferably synthesized with a cap to enhance stability, using techniques well known in the art, such as those described in Kore AL, et al. [Locked nucleic acid (LNA)-modified dinucleotide mRNA cap analogue: synthesis, enzymatic incorporation, and utilization (2009) J Am Chem Soc. 131(18):6364-5].

[0121] In general, the electroporation process used to transduce genes into primary immune cells such as PBMCs is typically carried out in a closed chamber containing parallel plate electrodes, as described in WO2004083379, which is incorporated herein by reference (in particular, page 23, line 25 to page 29, line 11), which generates a substantially uniform pulsed electric field between the parallel plate electrodes throughout the treatment volume, greater than 100 volts / cm and less than 5000 volts / cm. One such electroporation chamber preferably has a volume equal to the quotient of the electrode gap squared (cm2) to the chamber volume (cm2). 3 ) is the shape factor (cm -1 ), which has a view factor of 0.1 cm -1 where the suspension of cells and sequence-specific reagent is in a medium adjusted to have a conductivity in the range of 0.01 to 1.0 millisiemens. Generally, the cell suspension is subjected to one or more pulsed electric fields. In this method, the processing volume of the suspension is scalable and the cell processing time in the chamber is substantially uniform.

[0122] For example, Mussolino et al. As reported in [facilitate targeted genome editing in human cells with high specificity and low cytotoxicity (2014) Nucl. Acids Res. 42(10): 6762-6773], TALE nucleases or TALE base editors have proven to be particularly suitable sequence-specific nuclease reagents for therapeutic applications due to their relatively high specificity, especially when acting in the form of heterodimers, i.e. as a pair of a "right" (also called "5'" or "forward") and a "left" (also called "3'" or "reverse") monomer.

[0123] As mentioned above, the sequence-specific reagent is preferably in the form of a nucleic acid, e.g. coding for a low-frequency-cutting endonuclease, its subunits, in the form of DNA or RNA, but may also be part of a conjugate comprising polynucleotides and polypeptides, such as so-called "ribonucleoproteins". Such conjugates may be formed with the reagent, such as Cas9 or Cpf1 (RNA-guided endonuclease), as described in Zetsche, B. et al. [Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System (2015) Cell 163(3): 759-771] and Gao F. et al. [DNA-guided genome editing using the Natronobacterium gregoryi Argonaute (2016) Nature Biotech], respectively, which comprises an RNA or DNA guide that can be complexed with the respective nuclease.

[0124] "Exogenous sequence" refers to any nucleotide or nucleic acid sequence that is not originally present at the selected locus. This sequence may be homologous to or a copy of a genomic sequence, or may be an exogenous sequence introduced into the cell. In contrast, "endogenous sequence" refers to the genomic sequence of the cell that is originally present at the locus. The exogenous sequence preferably encodes a polypeptide that, when expressed, confers a therapeutic advantage over a sister cell that does not have the exogenous sequence integrated into the locus. An endogenous sequence that has been genetically edited by the insertion of a nucleotide or polynucleotide according to the method of the present invention to express a different polypeptide is broadly referred to as an exogenous coding sequence.

[0125] By using the reagents and techniques described above, and as detailed in the experimental section, the present invention provides: providing immune cells, preferably primary cells, from a donor or a patient; expressing in such cells the anti-MUC1 CAR as described above, typically by introducing the anti-MUC1 CAR coding sequence into the genome of the cells via a viral vector; introducing into such immune cells a sequence-specific reagent, such as a rare-cutting endonuclease or a base editor, to induce an alteration (mutation or coding sequence insertion) in the endogenous gene locus; and / or Introducing an exogenous coding sequence into said cells in order to improve their therapeutic efficacy, in particular their immunological properties. By implementing one or more of these steps, a method for producing therapeutic cells will be developed.

[0126] In some aspects of the invention, immune cells are derived from the patient or a matched donor and express the anti-MUC1 CAR in the cells, in view of performing a so-called "autologous" infusion of engineered CAR-positive immune cells. The immune cells may also be derived from stem cells, such as iPS cells, derived from such a patient or matched donor, or from tumor infiltrating lymphocytes (TILL).

[0127] In some aspects of the invention, methods are directed to providing "off-the-shelf" compositions of immune cells, said immune cells being modified for allogeneic therapeutic treatment.

[0128] "Allogeneic" means that the cells are derived from a donor. They can be harvested directly by apheresis or can be produced or differentiated from stem cells. Used in an allogeneic setting means that the modified cells are infused into a patient with a different haplotype.

[0129] Such immune cells are generally modified to be less alloreactive and / or more persistent with respect to the patient host.More specifically, the method comprises reducing or inactivating the expression of TCR in T cells or in stem cells derived from T cells.This can be achieved by various sequence-specific reagents, for example by gene silencing or gene editing methods (nuclease, base editing, RNAi, etc.).

[0130] Applicant has previously made available robust protocols and gene editing strategies for the production of allogeneic therapeutic grade T cells from PBMCs, in particular by providing highly safe and specific endonuclease reagents in the form of TALE nucleases (TALEN®), which are capable of encoding donor-derived [TCR] negProduction of T cells, so-called "universal T cells," was achieved and successfully injected into patients with mild graft-versus-host disease (GVhD) [Poirot et al. (2015) Multiplex Genome-Edited T-cell Manufacturing Platform for "Off-the-Shelf" Adoptive T-cell Immunotherapies. Cancer. Res. 75 (18): 3853-3864] [Qasim, W. et al. (2017) Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Science Translational 9(374)].

[0131] In a preferred embodiment, the invention provides a method of modifying an immune cell, in which at least one gene encoding TCR alpha or TCR beta is inactivated, preferably by expression of a rare-cutting endonuclease, while an exogenous polynucleotide encoding an anti-MUC1 CAR is introduced into the genome of said cell for stable expression, as described above. The exogenous sequence may be integrated into the locus encoding TCR alpha or TCR beta, more preferably under the transcriptional control of the endogenous promoter of TCR alpha or TCR beta.

[0132] In a further embodiment, the modified immune cells can be further modified to confer resistance to at least one immunosuppressant, for example by inactivating CD52, the target of an anti-CD52 antibody (e.g., alemtuzumab), which has been described, for example, in WO2013176915 for the treatment of hematological cancers.

[0133] While the use of anti-CD52 lymphocyte depleting agents has so far been limited to liquid tumor cancers [Quasim W. et al. (2019) Allogeneic CAR T cell therapies for leukemia Am J Hematol. 94:S50-S54.], one key aspect of the present invention is the use of genetically modified lymphocytes rendered resistant to lymphocyte depletion regimens for the treatment of solid tumors.

[0134] The present invention also provides modified lymphocytes with chimeric antigen receptors directed against solid tumors, particularly against MUC1-positive cells, which are used in the treatment of solid tumor cancers in conjunction with or following a lymphocyte depletion treatment step.

[0135] Such lymphocyte depletion regimens may include anti-CD52 reagents, such as alemtuzumab or purine analogs used in the treatment of hematological cancers.

[0136] In preferred embodiments, modified lymphocytes equipped with an anti-MUC1 CAR described herein are rendered resistant to such lymphodepletion regimens by inhibiting or preventing expression of the molecule targeted by the lymphodepletion reagent, e.g., the antigen CD52 in the case of alemtuzumab.

[0137] In a further embodiment, the modified immune cells can be further modified to confer resistance to chemotherapeutic agents, particularly purine analogue agents, and / or by inactivating DCK, for example as described in WO201575195.

[0138] As mentioned above, it is an important aspect of the present invention to treat solid tumor cancers with genetically modified lymphocytes with chimeric antigen receptors that are resistant to chemotherapy or lymphocyte depletion regimens. Such regimens may include antibodies targeting antigens such as CD52, CD3, CD4, CD8, CD45, or other specific markers present on the surface of immune cells, as well as less specific drugs such as purine analogs (e.g., fludarabine and / or chlorofarabine) and glucocorticoids. One aspect of the present invention is to render modified lymphocytes resistant to such regimens by inactivating or reducing the expression of genes that code for at least one molecular target of these lymphocyte depletion reagents, such as the gene DCK that metabolizes purine analogs, or the gene that codes for the glucocorticoid receptor (GR).

[0139] Thus, the present invention more specifically focuses on CAR-positive cells and renders them less alloreactive and resistant to lymphodepletion regimens by reducing, inactivating or impairing their expression of TCR, CD52, and / or DCK and / or GR for their allogeneic use in the treatment of solid tumors.

[0140] To increase the efficacy of anti-MUC1 CAR-T cells and overcome resistance mechanisms of solid tumors, the inventors combined several genetic modifications, referred to herein as genetic signatures, specifically dedicated for immunotherapy of MUC1-positive tumors. These genetic modifications, illustrated in FIG. 5, are more specifically selected from the following:

[0141] 1) Limiting CAR-T cell exhaustion by genetically inhibiting the activation of immune checkpoint genes such as PD1 Tumor cells are known to upregulate the expression of PDL1, a ligand that interacts with PD-1 on T cells. This interaction effectively blocks T cell-mediated immune responses and prevents tumor clearance. The success of immune checkpoint therapy depends on blocking the interaction between PD1 and PDL-1, which allows the immune system to kill cancer cells. Therefore, the present invention provides for knocking out PD1 in CAR-T cells to prevent PD-1 / PDL-1-mediated immune suppression. This modification, in parallel with other properties such as inducible IL-12 release as suggested below, can alleviate the problem of CAR-T cell exhaustion within the tumor microenvironment.

[0142] 2) Induction of expression of IL-12 (and / or IL-2, IL-15, and / or IL-18) IL-12 is a cytokine produced by various immune cells, including dendritic cells, monocytes, and macrophages (as well as some B cells), that positively regulates Th1 responses and promotes T cell survival and expansion. Delivery of IL-12 by CAR-T cells is particularly important for the efficacy of anti-tMUC1 CAR-T cells in the allogeneic setting.

[0143] Since IL-12 is toxic when delivered at systemic levels, its coding sequence can be integrated under an inducible IL-12 cassette, which allows exclusive IL-12 release upon activation of CAR-T cells in the presence of tumor targets. In this regard, it is advantageous to express IL12 by using an exogenous sequence that includes IL-12a and IL-12b separated by a 2A self-cleaving peptide (IL-12a-P2A-IL-12b). The same can be applied to sequences that code for IL-2, IL-15, or IL-18. Thus, inducible cytokine release provides a safe way to increase CAR-T efficacy as required by cells. In a preferred embodiment, exogenous polynucleotide sequences expressing IL-2, IL-12, IL-15, and / or IL-18 can be delivered by AAV vectors and integrated into the PD1 locus, preferentially under the transcriptional control of the endogenous PD1 promoter, as described in the examples. Because PD-1 expression is dependent on CAR T cell activation, IL-12 expression is restricted to tumor regions, thereby enhancing CAR T cell function.

[0144] As provided in the Examples, PD-1 knockout together with IL-12 release significantly enhanced CAR-T cell function and intratumoral expansion, resulting in antitumor activity against solid tumors that could not be treated by CAR-T cells without such modifications.

[0145] 3) Enhancement of tumor invasion by inducible expression of hyaluronidase enzyme Hyaluronan (HA) is a glycosaminoglycan that is an essential structural and signaling component of the extracellular matrix. Accumulation of HA has been observed in various solid tumors, including prostate, bladder, lung, and breast cancer, and is associated with poor clinical outcomes. HA capsules increase hydrostatic pressure to prevent T cell infiltration and diffusion of small drugs in solid tumors, making treatment more difficult. Small HA fragments (HA-oligo: 1-10 kda) have been shown to induce apoptosis of cancer cells, whereas larger HA fragments enhance tumor progression in many cancer types by promoting proliferation and preventing T cell infiltration, and by enhancing tumor cell migration. We have successfully expressed hyaluronidase enzymes capable of degrading hyaluronan matrix in CAR-T cells to enhance CAR-T infiltration of solid tumors. Secretion of hyaluronidase enzymes by CAR-T cells is also useful to induce killing of bystander tumor cells through local production of HA-oligo. Importantly, oligo-HA-mediated apoptosis is independent of CAR target antigen recognition and therefore has the potential to act on tumor cells that lack or express low levels of antigen on the cell surface.

[0146] The combination of CAR with hyaluronidase activity is similar to antibody-drug conjugates that can kill bystander cancer cells lacking the necessary antigen levels upon exposure to oligo-HA. This strategy would minimize tumor escape due to antigen scarcity or tumor heterogeneity.

[0147] The present invention provides modified immune cells, preferably but not limited to anti-MUC1 CAR-T cells, into which an exogenous sequence encoding a hyaluronidase enzyme is introduced. The hyaluronidase enzyme is preferably HYAL1 (Uniprot Q12794), HYAL2 (Q12891), and / or HYAL3 (SPAM1-Uniprot P38567), or a similar functional hyaluronidase having at least 80% identity thereto. The sequences of HYAL1, HYAL2, and SPAM1 can also be modified with secretion tags, which can be implemented to enhance secretion into the tumor microenvironment.

[0148] In some preferred embodiments, immune cells can be advantageously modified to secrete a hyaluronidase enzyme upon immune cell activation by incorporating a hyaluronidase coding sequence at a locus selected from PD1, CD69, CD25, or GMCSF, or any such locus whose expression is induced upon binding of a CAR to a target antigen.

[0149] The present invention also provides AAV, preferably AAV6, vectors for mediated insertion of HYAL1, HYAL2, or SPAM1 into immune cells, comprising an exogenous sequence encoding a functional hyaluronidase.

[0150] In a further embodiment, the additional glycosidases human β-glucuronidase (GUSB) and β-N-acetylglucosaminidase (ENGASE) can be co-expressed to further shorten the HA chains cleaved by the hyaluronidase enzyme.

[0151] The resulting hyaluronidase-expressing CAR immune cells exhibit better infiltration into solid tumors and can act synergistically with CAR-T-mediated killing by directly inducing tumor cell death through oligo-HA-mediated apoptosis while sparing healthy tissue.

[0152] 4) Alleviation of hypoxic and nutrient-poor environments The tumor microenvironment is not only hypoxic but also lacks nutrients, two characteristics that hinder the expansion and proliferation of CAR-T cells. Recent studies have explained the mechanism by which proinflammatory Th17 cells effectively expand and proliferate in hypoxic and nutrient-deficient environments, leading to inflammation [Wu, L. et al. (2020) Niche-Selective Inhibition of Pathogenic Th17 Cells by Targeting Metabolic Redundancy. Cell 182(3):641-654]. Th17 lymphocytes upregulate glucose phosphate isomerase 1 (GPI1) to overcome hypoxic and low-nutrient environments and gain a proliferation advantage. Knockout of GPI1, a metabolically redundant enzyme, had no effect on homeostatic Th17 cells present in nutrient-sufficient and normoxic conditions, but significantly impaired the proliferation ability of inflammatory Th17 cells in hypoxic and nutrient-deficient environments. During anaerobic glycolysis, lactate is produced. Until recently, lactate was considered a waste product. However, new findings have demonstrated that it can also be metabolized for energy, most likely through gluconeogenesis, with the help of phosphoenolpyruvate carboxykinase 1 (PCK1), which increases glycolytic flux, resulting in lactose consumption and increased intracellular glucose [Yuan, Y. et al. (2020) PCK1 Deficiency Shortens the Replicative Lifespan of Saccharomyces cerevisiae through Upregulation of PFK1. Biomed Res. Int. Article ID 3858465]. Furthermore, overexpression of PCK1 has been shown to promote the formation and maintenance of CD8+ Tm cells, overall increasing the competitiveness of T cells in obtaining nutrients [Ho, PC et al. (2015) Phosphoenolpyruvate Is a Metabolic Checkpoint of Anti-tumor T Cell Responses. Cell. 162(6):1217-1228].

[0153] The present invention provides modified immune cells, preferably but not limited to anti-MUC1 CAR T cells, into which an exogenous sequence has been introduced that encodes the GPI1 (Uniprot Q9BRB3), PCK1 (Uniprot P35558), and / or LDHA (Uniprot P00338) enzymes or any functional enzyme with at least 80% identity thereto. The inventors expressed GPI1 and PCK1 to boost immune cells in the hypoxic and nutrient-deprived environment of solid tumors. The genes encoding the GPI1 and PCK1 enzymes, or any functional similar enzymes, are preferably delivered using AAV vectors, in particular AAV6, for their targeted insertion at the TRAC locus. Alternatively, the exogenous sequence can be delivered under the control of a strong promoter, such as EF1 alpha, by using lentiviral vectors when it is preferred to obtain its constitutive expression. Expression of the two genes can be achieved through delivery of a bicistronic construct with both genes separated by a self-cleaving 2A peptide.

[0154] The above-mentioned modifications to enhance CAR-T efficacy in solid tumors can be performed in combination with standard modifications of allogeneic CAR-T, which more specifically include the following modifications: ·TRAC knockout using gene editing to prevent graft-versus-host disease; B2M knockout using gene editing or silencing techniques to prevent host-versus-graft activity; and / or Overexpression of HLA-E or HLA-G through targeted gene integration, for example by using AAV6 or by rLV delivery, to prevent NK cells from attacking B2M-negative cells.

[0155] Taken together, combined engineering implementing several of the above-mentioned strategies has the revolutionary potential to generate next-generation CAR-T cells capable of treating solid tumors.

[0156] The present invention also provides a method for producing a population of modified therapeutic immune cells for the treatment of solid tumors, comprising the steps of: a) providing immune cells originating from a patient, or preferably a donor; b) expressing an anti-MUC1 chimeric antigen receptor (CAR) in the cell; c) introducing at least one genetic modification into the genome of the cell, the modification comprising: · Reduction or inactivation of TCR expression; · Reduction or inactivation of B2M; ·Reduced interaction between TGFβ and TGFβR2; Reducing the interaction between PD1 and PDL1; and / or Enhancement of expression of IL-12, IL-15, or IL-18 a process selected from those which result in d) Expanding the cells to form a therapeutically effective immune cell population.

[0157] The genetic modification in this method is generally obtained by using sequence-specific gene editing, such as a slow-cutting endonuclease / nickase or a base editor. The method can also include further genetically modifying the cells to enhance secretion of hyalurinases, such as HYAL1, HYAL2, and / or HYAL3 (SPAM1), or to enhance expression of other enzymes, such as GPI1, PCK1, and / or LDHA.

[0158] Table 9. Genetic signature polypeptide sequences TIFF2024517713000011.tif135158TIFF2024517713000012.tif236158TIFF2024517713000013.tif238158TIFF2024517713000014.tif92158

[0159] In further embodiments, the modified immune cells can be further modified to improve persistence or longevity in the patient, specifically by inactivating genes encoding MHC-I components such as HLA or β2m, as described in WO2015136001 or Liu, X. et al. [CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells (2017) Cell Res 27:154-157].

[0160] In a preferred aspect of the invention, the engineered immune cells are mutated to improve their CAR-dependent immune activation, in particular to reduce or suppress the expression of immune checkpoint proteins and / or their receptors, e.g., PD1 or CTLA4, as described in WO2014184744.

[0161] Combining anti-MUC1 CAR expression with TGFbRII signaling pathway disruption in therapeutically engineered immune cells The present invention more specifically combines the expression of an exogenous sequence encoding the aforementioned anti-MUC1 CAR with another exogenous sequence encoding an inhibitor of a TGF beta receptor, in particular an inhibitor of TGFβRII.

[0162] The TGF beta receptor (Uniprot - P37173) has been described as having a major role in the tumor microenvironment [Papageorgis, P. et al. (2015). Role of TGFβ in regulation of the tumor microenvironment and drug delivery (Review). International Journal of Oncology, 46, 933-943].

[0163] Although the exact role of TGF beta receptors in tumor development remains controversial, the inventors have found that co-expressing anti-MUC1 chimeric antigen receptors (CARs) with another exogenous gene sequence encoding an inhibitor of TGFBRII signaling and / or inactivating or reducing TGF beta receptor signaling using sequence-specific reagents results in improved therapeutic efficacy of engineered immune cells. Specifically, the inventors have used two different approaches to inhibit the TGF beta RII signaling pathway, which may be used in combination: Expression of an inactive ligand of TGFβRII, such as a dominant negative TGFβRII (SEQ ID NO:26), as described in Hiramatsu, K., et al. [Expression of dominant negative TGF-β receptors inhibits cartilage formation in conditional transgenic mice (2011) J. Bone. Miner. Metab. 29: 493], or a similar inactive form of TGFβRII having at least 80%, preferably at least 90%, more preferably at least 95% identity to the polypeptide sequence SEQ ID NO:26. and / or Inactivation of the endogenous gene sequence of TGFβRII, specifically by using low-frequency-cutting endonucleases such as TALE nucleases or RNA-guided endonucleases (e.g., Cas9 or Cpf1).

[0164] Anti-TGFβRII IgG1 monoclonal antibodies such as LY3022859, which inhibit receptor-mediated signaling activation [Tolcher, AW et al. (2017) A phase 1 study of anti-TGFβ receptor type-II monoclonal antibody LY3022859 in patients with advanced solid tumors Cancer Chemother Pharmacol.79(4):673-680], can also be used in combination with the CAR of the invention to inhibit TGFbeta receptor signaling.

[0165] The present application discloses herein a set of TALE nucleases that are specifically specific for a set of target sequences within the TGFβRII gene. These TALE nucleases exhibited the highest TGFβRII knockout efficiency while exhibiting minimal off-target cleavage, resulting in a large population of modified live cells sufficient for administration to several patients. These preferred TALE nucleases and their corresponding target sequences are listed in Table 10.

[0166] Table 10: TALE nuclease target sequence for the TGFβRII gene TIFF2024517713000015.tif72158

[0167] RNA guides were also designed to inactivate the TGFβRII gene by using Cas9 nuclease reagents, and the corresponding target sequences are disclosed in Table 11.

[0168] Table 11. CRISPR target sequence for TGFβRII gene TIFF2024517713000016.tif164128TIFF2024517713000017.tif237110TIFF2024517713000018.tif211110

[0169] The present invention therefore encompasses the use of TALE nucleases or RNA-guided endonucleases designed to bind to any of the target sequences SEQ ID NOs:90-204 set forth in Table 10 or Table 11 to inactivate or reduce expression of TGFβRII to produce therapeutic immune cells within the teachings of the present specification.

[0170] The present invention also relates to modified immune cells comprising an exogenous polynucleotide encoding a nuclease as described above to inactivate or reduce the expression of the endogenous TGFβRII gene in the modified immune cells. This strategy is more specifically illustrated in Figures 16 and 17.

[0171] As shown in the experimental section herein, inactivation of TGFBRII by expression in cells of sequence-specific reagents such as low-cutting endonucleases or base editors results in anti-MUC1 CAR T cells with higher anti-tumor activity.

[0172] Preferred specific reagents in this regard are TALE nucleases or TALE base editors for introducing mutations into one target sequence of TGFBRII selected from SEQ ID NO:86, 87, 88, or 89, more preferably SEQ ID NO:89, in particular a TALE nuclease having at least one monomer exhibiting at least 95% identity to one of the polypeptide sequences SEQ ID NO:223 and SEQ ID NO:224.

[0173] In such an embodiment, the present invention provides a method for generating CAR immune cells active against solid tumors, in which at least one allele of each TCR (e.g., TRAC), PD1, B2M, and TGFBRII gene is mutated by using a sequence-specific reagent (nuclease or base editor) to reduce or inactivate their expression. Such a method preferably comprises two electroporation steps in which polynucleotide sequences encoding those sequence-specific reagents are introduced into cells, preferably under mRNA form. These two electroporation steps are preferably spaced apart by at least 48 hours, more preferably at least 72 hours, and even more preferably at least 96 hours. The inventors have obtained particularly good results by introducing specific reagents targeting TCR and B2M during the first electroporation step and specific reagents targeting PD1 and TGFBRII during the second electroporation step. This occurs in a way that reduces cytotoxicity, making it possible to generate a higher yield of quadruple-mutation modified cells.

[0174] However, it may occur that some tumor-resident cells produce TGF-β, which has a strong immunosuppressive effect, and can block both the proliferation and effector function of CAR-T. To prevent this blockage, the present invention provides for the delivery of dominant-negative TGFBR2 (dnTGFBR2). Dominant-negative TGFBR2 is a receptor that lacks intracellular signaling domains, and therefore retains high affinity for TGF-β, but cannot transmit TGF-B signals. Thus, dnTGFBR2 acts as a sponge that sequesters TGF-β from CAR-T, and also improves the immunosuppressive effect of TGF-β1. The preferred delivery of dnTGFBR2 has been successfully achieved through rLV, either as a single entity or as a bicistronic construct expressing CAR and separated by 2A self-cleaving peptide. However, this can also be achieved via targeted integration, for example with AAV vectors, at inducible loci (e.g., PD-1, CD25...) to help reprogram the immunosuppressive solid tumor microenvironment.

[0175] This application therefore reports modified immune cells, in particular CAR immune cells, into which an exogenous sequence encoding an inhibitor of TGF beta receptor, more particularly a sequence encoding a dominant negative TGF beta receptor, is introduced. Such cells are more particularly dedicated to the treatment of solid tumors, in particular MUC1 positive tumors.

[0176] Thus, the present application also claims a vector, in particular a viral vector, such as a lentiviral vector or an AAV vector as described in the art, comprising at least a polynucleotide sequence encoding a dominant negative TGFβRII and, optionally, an anti-MUC1 chimeric antigen receptor. In a preferred embodiment, said vector comprises a first polynucleotide sequence encoding said dominant negative TGFβRII, a second polynucleotide sequence encoding a 2A self-cleaving peptide, and a third polynucleotide sequence encoding said specific anti-MUC1 chimeric antigen receptor.

[0177] Targeted insertion into immune cells can be greatly improved by using AAV vectors, in particular vectors of the AAV6 family, or the chimeric vector AAV2 / 6 already described by Sharma A., et al. [Transduction efficiency of AAV 2 / 6, 2 / 8 and 2 / 9 vectors for delivering genes in human corneal fibroblasts. (2010) Brain Research Bulletin. 81 (2-3): 273-278].

[0178] One aspect of the present invention is therefore the transduction of AAV vectors comprising anti-MUC1 CAR coding sequences in human primary immune cells in conjunction with the expression of sequence-specific endonuclease reagents, such as TALE endonucleases, to increase gene integration at the aforementioned loci.

[0179] In a preferred aspect of the invention, the sequence-specific endonuclease reagent may be introduced into the cell by gene transfer, more preferably by electroporation of mRNA encoding said sequence-specific endonuclease reagent.

[0180] The insertion of an exogenous nucleic acid sequence can result in the introduction of genetic material, correcting or replacing an endogenous sequence, more preferably "in frame" with the endogenous gene sequence at that locus.

[0181] In another aspect of the invention, 10 5 ~10 7 , preferably 10 6 ~10 7 , more preferably about 5.10 6 The viral genome is transduced.

[0182] In another aspect of the invention, cells can be treated with a proteasome inhibitor, such as bortezomib, or an HDAC inhibitor, to further favor homologous recombination.

[0183] As one object of the present invention, the AAV vector used in the method may comprise a promoterless exogenous coding sequence, said coding sequence being any of the sequences mentioned herein.

[0184] The present invention also provides an efficient method for obtaining primary immune cells, which can be edited at various gene loci, more specifically involved in host-graft interaction and recognition. Other loci can also be edited with a view to improving the activity, survival or longevity of modified primary cells, especially primary T cells.

[0185] Figure 2 maps the main cellular functions that can be modified by gene editing of the present invention to improve the efficiency of modified immune cells. Any of the gene inactivations listed under each function can be combined with each other to obtain a synergistic effect on the overall therapeutic efficacy of the immune cells.

[0186] The present invention more specifically provides combinations of genetic modifications (genotypes) in immune cells, which are useful in the treatment of solid tumors, in particular ·[Anti-MUC1 CAR] + , ·[Anti-MUC1 CAR] + [dnTGFβRII] + ·[Anti-MUC1 CAR] + [dnTGFβRII] + [TCR] - , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [TGFβRII] - [TCR] - , ·[Anti-MUC1 CAR] + [TGFβRII] - , ·[Anti-MUC1 CAR] + [TGFβRII] - [TCR] - , ·[Anti-MUC1 CAR] + [β2m] - , [Anti-MUC1 CAR] + [dnTGFβRII] + [β2m] - , [Anti-MUC1 CAR] + [TGFβRII] - [β2m] - , [Anti-MUC1 CAR] + [dnTGFβRII] + [β2m] - [TCR] - , [Anti-MUC1 CAR] + [TGFβRII] - [β2m] - [TCR] - , [Anti-MUC1 CAR] + [dnTGFβRII] + [TGFβRII] - [β2m] - [TCR] - , [Anti-MUC1 CAR] + [PD1] - , [Anti-MUC1 CAR] + [TGFβRII] - [PD1] - , [Anti-MUC1 CAR] + [dnTGFβRII] + [PD1] - , [Anti-MUC1 CAR] + [dnTGFβRII] + [TGFβRII] - [PD1] - , [Anti-MUC1 CAR] + [dnTGFβRII] + [β2m] - [PD1] - , [Anti-MUC1 CAR] + [TGFβRII] - [PD1] - [TCR] - , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [TGFβRII] - [PD1] - [TCR] - , ·[Anti-MUC1 CAR] + [PD1] - [β2m] - , ·[Anti-MUC1 CAR] + [TGFβRII] - [PD1] - [β2m] - , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [PD1] - [β2m] - , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [TGFβRII] - [PD1] - [β2m] - , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [β2m] - [PD1] - [TCR] - , ·[Anti-MUC1 CAR] + [TGFβRII] - [PD1] - [TCR] - [β2m] - ,or ·[Anti-MUC1 CAR] + [dnTGFβRII] + [TGFβRII] - [PD1] - [TCR] - [β2m] - This will immediately improve the efficacy of immune cells against anti-MUC1 positive malignant cells such as

[0187] In a further preferred embodiment, the above genotypes are combined with overexpression of IL-12, preferably via the IL-12a-2A-IL-12b construct and insertion of an HLAE as described herein and shown in FIG. 5, to obtain one of the following genotypes: ·[Anti-MUC1 CAR] + [HLAE] + [IL12] + , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [HLAE] + [IL12] + , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [TCR] - [HLAE] + [IL12] + , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [TGFβRII] - [TCR] - [HLAE] + [IL12] + , ·[Anti-MUC1 CAR] + [TGFβRII] - [HLAE] + [IL12] + , ·[Anti-MUC1 CAR] + [TGFβRII] - [TCR] - [HLAE] + [IL12] + , ·[Anti-MUC1 CAR] + [β2m] - [HLAE] + [IL12] + , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [β2m] - [HLAE] + [IL12] + , · [Anti-MUC1 CAR] + [TGFβRII] - [β2m] - [HLAE] + [IL12] + 、 · [Anti-MUC1 CAR] + [dnTGFβRII] + [β2m] - [TCR] - [HLAE] + [IL12] + 、 · [Anti-MUC1 CAR] + [TGFβRII] - [β2m] - [TCR] - [HLAE] + [IL12] + 、 · [Anti-MUC1 CAR] + [dnTGFβRII] + [TGFβRII] - [β2m] - [TCR] - [HLAE] + [IL12] + 、 · [Anti-MUC1 CAR] + [PD1] - [HLAE] + [IL12] + 、 · [Anti-MUC1 CAR] + [TGFβRII] - [PD1] - [HLAE] + [IL12] + 、 · [Anti-MUC1 CAR] + [dnTGFβRII] + [PD1] - [HLAE] + [IL12] + 、 · [Anti-MUC1 CAR] + [dnTGFβRII] + [TGFβRII] - [PD1] - [HLAE] + [IL12]+ , · [Anti-MUC1 CAR] + [dnTGFβRII] + [β2m] - [PD1] - [HLAE] + [IL12] + , · [Anti-MUC1 CAR] + [TGFβRII] - [PD1] - [TCR] - [HLAE] + [IL12] + , · [Anti-MUC1 CAR] + [dnTGFβRII] + [TGFβRII] - [PD1] - [TCR] - [HLAE] + [IL12] + , · [Anti-MUC1 CAR] + [PD1] - [β2m] - [HLAE] + [IL12] + , · [Anti-MUC1 CAR] + [TGFβRII] - [PD1] - [β2m] - [HLAE] + [IL12] + , · [Anti-MUC1 CAR] + [dnTGFβRII] + [PD1] - [β2m] - [HLAE] + [IL12] + , · [Anti-MUC1 CAR] + [dnTGFβRII] + [TGFβRII] - [PD1] - [β2m] - [HLAE] + [IL12] + , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [β2m] - [PD1] - [TCR] - [HLAE] + [IL12] + , ·[Anti-MUC1 CAR] + [TGFβRII] - [PD1] - [TCR] - [β2m] - [HLAE] + [IL12] + ,or ·[Anti-MUC1 CAR] + [dnTGFβRII] + [TGFβRII] - [PD1] - [TCR] - [β2m] - [HLAE] + [IL12] + .

[0188] As mentioned above, the present invention is also particularly focused on the use of CAR-positive cells in the treatment of solid tumors, which may be used in the allogeneic setting, in conjunction with or following lymphodepleting regimens, since the cells are rendered resistant to lymphodepleting agents. Such cells preferably exhibit the following genotype: Partial or complete resistance to anti-CD52 antibodies: ·[Anti-MUC1 CAR] + [CD52] - [TCR] - , ·[Anti-MUC1 CAR] + [CD52] - [TCR] - [β2m] - ,, ·[Anti-MUC1 CAR] + [TGFβRII] - [CD52] - [TCR] - , ·[Anti-MUC1 CAR] + [TGFβRII]- [CD52] - [TCR] - [β2m] - , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [CD52] - [TCR] - , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [CD52] - [TCR] - [β2m] - , Partial or complete resistance to purine analogues: ·[Anti-MUC1 CAR] + [DCK] - [TCR] - , ·[Anti-MUC1 CAR] + [DCK] - [TCR] - [β2m] - ,, ·[Anti-MUC1 CAR] + [TGFβRII] - [DCK] - [TCR] - , ·[Anti-MUC1 CAR] + [TGFβRII] - [DCK] - [TCR] - [β2m] - , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [DCK] - [TCR] - , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [DCK] - [TCR] - [β2m] - , Partial or complete resistance to glucocorticoids: ·[Anti-MUC1 CAR] + [GR]- [TCR] - , ·[Anti-MUC1 CAR] + [GR] - [TCR] - [β2m] - ,, ·[Anti-MUC1 CAR] + [TGFβRII] - [GR] - [TCR] - , ·[Anti-MUC1 CAR] + [TGFβRII] - [GR] - [TCR] - [β2m] - , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [GR] - [TCR] - , ·[Anti-MUC1 CAR] + [dnTGFβRII] + [GR] - [TCR] - [β2m] - .

[0189] Further improving therapeutic immune cells by expressing transgenes at inactivated loci The above preferred genotypes may be obtained by targeted gene integration, preferably at the PD1, TCR (TCR alpha and / or TCR beta), or TGFβRII locus, but also at further selected loci as described below.

[0190] "Targeted gene integration" refers to any known site-specific method that allows for the insertion, replacement, or modification of a genomic sequence in a living cell. Targeted gene integration usually involves the mechanism of homologous recombination or NHEJ (non-homologous end joining), which is enhanced by endonuclease sequence-specific reagents, resulting in the insertion or replacement of at least one exogenous nucleotide, preferably a sequence of several nucleotides (i.e., a polynucleotide), more preferably a coding sequence, at a given locus.

[0191] Expression of additional transgenes may improve efficacy of immune cells against solid tumors The methods of the invention may be combined with other methods involving gene conversion, such as viral transduction, and may also be combined with other transgene expression methods that do not necessarily involve integration.

[0192] In one aspect, the method of the invention comprises: a) polynucleotide sequences whose expression is involved in the decrease in glycolysis and calcium signaling in response to low glucose conditions, such as SERCA3, which increases calcium signaling, miR101 and mir26A, which increase glycolysis, and BCAT, which mobilizes glycolytic stores; and / or b) a polynucleotide sequence whose expression upregulates immune checkpoint proteins (e.g., TIM3, CEACAM, LAG3, TIGIT), such as IL27RA, STAT1, STAT3; and / or c) a polynucleotide sequence, the expression of which mediates interaction with HLA-G, such as ILT2 or ILT4; and / or d) polynucleotide sequences whose expression is involved in downregulation of T cell proliferation, such as SEMA7A, SHARPIN, which reduces Treg proliferation, STAT1, which reduces apoptosis, PEA15, which increases IL-2 secretion, and RICTOR, which supports CD8 memory differentiation; and / or e) a polynucleotide sequence, the expression of which is involved in the downregulation of T cell activation, such as mir21; and / or f) polynucleotide sequences whose expression is involved in signal transduction pathways responsive to cytokines, such as JAK2 and AURKA; and / or g) Polynucleotide sequences whose expression is involved in T cell exhaustion, such as DNMT3, miRNA31, MT1A, MT2A, and PTGER2. into an endogenous locus of an immune cell, preferably by expressing within said cell a sequence-specific reagent that specifically targets said selected endogenous locus.

[0193] In a further aspect, the modified immune cells of the present invention can be further modified to obtain co-expression of an anti-MUC1 CAR and another exogenous gene sequence in said cells, said exogenous gene sequence being CRS inhibitors, such as mutant IL6Ra, sGP130, or IL18-BP; · cytochrome P450, CYP2D6-1, CYP2D6-2, CYP2C9, CYP3A4, CYP2C19, or CYP1A2, which confers hypersensitivity of said immune cells to drugs such as cyclophosphamide and / or isophosphamide; ·Dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin, or methylguanine transferase (MGMT), mTORmut, or Lckmut, conferring drug resistance; a chemokine receptor, such as CCR2, CXCR2, or CXCR4; and / or Tumor-associated macrophage (TAM) secretion inhibitors, such as CCR2 / CCL2 neutralizers, to enhance the therapeutic activity of immune cells The nucleic acid sequence is selected from those encoding the following:

[0194] The transgene or exogenous polynucleotide sequence is preferably inserted such that its expression is under the transcriptional control of at least one endogenous promoter present in one of the loci.

[0195] Targeting one such locus by performing gene integration is beneficial to further improve the efficacy of the therapeutic immune cells of the present invention.

[0196] Examples of such exogenous sequences or transgenes that can be expressed or overexpressed at a selected locus are detailed below.

[0197] Further expression of transgenes conferring resistance to drugs or immunoablation agents In one aspect of this method, the exogenous sequence integrated into a genomic locus of the immune cell encodes a molecule that confers resistance to a drug on said immune cell.

[0198] Examples of preferred exogenous sequences are variants of dihydrofolate reductase (DHFR) that confer resistance to folate analogues such as methotrexate, variants of inosine monophosphate dehydrogenase 2 (IMPDH2) that confer resistance to IMPDH inhibitors such as mycophenolic acid (MPA) or its prodrug mycophenolate mofetil (MMF), variants of calcineurin or methylguanine transferase (MGMT) that confer resistance to calcineurin inhibitors such as FK506 and / or CsA, variants of mTOR such as mTORmut that confer resistance to rapamycin, and variants of Lck such as Lckmut that confer resistance to imatinib and Gleevec.

[0199] The term "drug" is used herein to refer to a compound or its derivatives, preferably a standard chemotherapeutic agent that is generally used to interact with cancer cells and thereby reduce the state of proliferation or survival of said cells. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, ifosamide), antimetabolites (e.g., purine nucleoside antimetabolites, such as clofarabine, fludarabine, or 2'-deoxyadenosine, methotrexate (MTX), 5-fluorouracil, or their derivatives), antitumor antibiotics (e.g., mitomycin, adriamycin), plant-derived antitumor agents (e.g., vincristine, vindesine, taxol), cisplatin, carboplatin, etoposide, etc. Such agents may further include, but are not limited to, the anti-cancer agents TRIMETHOTRIXATE™ (TMTX), TEMOZOLOMIDE™, RALTRITREXED™, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzyguanidine (6-BG), bis-chloronitrosourea (BCNU), and CAMPTOTHECIN™, or any therapeutic derivative thereof.

[0200] As used herein, an immune cell is "resistant or tolerant" to a drug when the cell or cell population is modified such that it is capable of proliferation, at least in vitro, in culture medium containing a 50% maximal inhibitory concentration (IC50) of the drug (the IC50 being determined for an unmodified cell or cell population).

[0201] In a particular embodiment, said drug resistance can be conferred to an immune cell by expression of at least one "drug resistance coding sequence", which relates to a nucleic acid sequence that confers "resistance" to an agent, such as one of the chemotherapeutic agents mentioned above. The drug resistance coding sequence of the present invention can code for resistance to antimetabolites, methotrexate, vinblastine, cisplatin, alkylating agents, anthracyclines, cytotoxic antibiotics, anti-immunophilins, analogs or derivatives thereof, and the like (Takebe, N., SC Zhao, et al. (2001) "Generation of dual resistance to 4-hydroperoxycyclophosphamide and methotrexate by retroviral transfer of the human aldehyde dehydrogenase class 1 gene and a mutated dihydrofolate reductase gene". Mol. Ther. 3(1): 88-96), (Zielske, SP, JS Reese, et al. (2003) "In vivo selection of MGMT(P140K) lentivirus-transduced human NOD / SCID repopulating cells without pretransplant irradiation conditioning." J. Clin. Invest. 112(10): 1561-70). (Nivens, MC, T. Felder, et al. (2004) "Engineered resistance to camptothecin and antifolates by retroviral coexpression of tyrosyl DNA phosphodiesterase-I and thymidylate synthase" Cancer Chemother Pharmacol 53(2): 107-15), (Bardenheuer, W., K. Lehmberg, et al. (2005)."Resistance to cytarabine and gemcitabine and in vitro selection of transduced cells after retroviral expression of cytidine deaminase in human hematopoietic progenitor cells". Leukemia 19(12): 2281-8), (Kushman, ME, SL Kabler, et al. (2007) "Expression of human glutathione S-transferase P1 confers resistance to benzo[a]pyrene or benzo[a]pyrene-7,8-dihydrodiol mutagenesis, macromolecular alkylation and formation of stable N2-Gua-BPDE adducts in stably transfected V79MZ cells co-expressing hCYP1A1" Carcinogenesis 28(1): 207-14). .

[0202] The expression of such drug resistance exogenous sequences in immune cells according to the invention allows the use of said immune cells more particularly in treatment schemes of cell therapy combining cell therapy with chemotherapy or in patients who have undergone these drug treatments.

[0203] Several drug resistance coding sequences have been identified that may be used to confer drug resistance in the present invention. One example of a drug resistance coding sequence may be, for example, a mutant or variant of dihydrofolate reductase (DHFR). DHFR is an enzyme involved in regulating the amount of tetrahydrofolate in cells and is essential for DNA synthesis. Folate analogs, such as methotrexate (MTX), inhibit DHFR and are therefore used clinically as anti-cancer drugs. Various mutant forms of DHFR have been described that have increased resistance to inhibition by therapeutic antifolates. In certain embodiments, the drug resistance coding sequence of the present invention may be a nucleic acid sequence that encodes a mutant form of human wild-type DHFR (GenBank: AAH71996.1), which contains at least one mutation that confers resistance to antifolate therapy, such as methotrexate. In certain embodiments, 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 U.S. Patent No. 6,642,043). In certain embodiments, said DHFR mutant form contains two mutant amino acids at positions L22 and F31. The correspondence of amino acid positions described herein is often expressed as the position of the amino acid in the form of the wild-type DHFR polypeptide. In certain embodiments, 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 with an amino acid that prevents the mutant DHFR from binding to antifolates, preferably with an uncharged amino acid residue such as phenylalanine or tyrosine, In another specific embodiment, the phenylalanine residue at position 31 or 34 is preferably replaced with a small hydrophilic amino acid such as alanine, serine, or glycine.

[0204] Another example of drug resistance coding sequence can also be a mutant or variant of ionisine-5'-monophosphate dihydrogenase II (IMPDH2), the rate-limiting enzyme in the de novo synthesis of guanosine nucleotides. The mutant or variant of IMPDH2 is an IMPDH inhibitor resistance gene. The IMPDH inhibitor can be mycophenolic acid (MPA) or its prodrug, mycophenolate mofetil (MMF). The mutant IMPDH2 can contain at least one, preferably two mutations in the MAP binding site of wild-type human IMPDH2 (Genebank: NP_000875.2), thus greatly increasing resistance to IMPDH inhibitors. The mutations in these variants are preferably at positions T333 and / or S351 (Yam, P., M. Jensen, et al. (2006) “Ex vivo selection and expansion of cells based on expression of a mutated inosine monophosphate dehydrogenase 2 after HIV vector transduction: effects on lymphocytes, monocytes, and CD34+ stem cells” Mol. Ther. 14(2): 236-44)(Jonnalagadda, M., et al. (2013) “Engineering human T cells for resistance to methotrexate and mycophenolate mofetil as an in vivo cell selection strategy.” PLoS One 8(6): e65519).

[0205] Another drug-resistant coding sequence is a mutant form of calcineurin. Calcineurin (PP2B - NCBI: ACX34092.1) is a ubiquitously expressed serine / threonine protein phosphatase that is involved in many biological processes and is important for T cell activation. Calcineurin is a heterodimer composed of a catalytic subunit (CnA; three isoforms) and a regulatory subunit (CnB; two isoforms). After binding to the T cell receptor, calcineurin dephosphorylates the transcription factor NFAT, allowing it to translocate to the nucleus and activate key target genes such as IL2. FK506 complexed with FKBP12 or cyclosporine A (CsA) complexed with CyPA prevents NFAT from accessing the active site of calcineurin, preventing its dephosphorylation and thereby inhibiting T cell activation (Brewin et al. (2009) "Generation of EBV-specific cytotoxic T cells that are resistant to calcineurin inhibitors for the treatment of posttransplantation lymphoproliferative disease" Blood 114(23): 4792-803). In a particular embodiment, said mutant form may contain at least one mutated amino acid of the wild-type calcineurin heterodimer at positions V314, Y341, M347, T351, W352, L354, K360, preferably a double mutation at positions T351 and L354, or V314 and Y341.In particular aspects, the valine residue at position 341 may be replaced with a lysine or arginine residue, the tyrosine residue at position 341 may be replaced with a phenylalanine residue; the methionine at position 347 may be replaced with a glutamic acid, arginine, or tryptophan residue; the threonine at position 351 may be replaced with a glutamic acid residue; the tryptophan residue at position 352 may be replaced with a cysteine, glutamic acid, or alanine residue, the serine at position 353 may be replaced with a histidine or asparagine residue, the leucine at position 354 may be replaced with an alanine residue; and the lysine at position 360 may be replaced with an alanine or phenylalanine residue. In another particular embodiment, said mutant form may contain at least one mutated amino acid of wild-type calcineurin heterodimer b at position V120, N123, L124 or K125, preferably a double mutation at positions L124 and K125. In a particular embodiment, the valine at position 120 may be replaced with a serine, aspartic acid, phenylalanine or leucine residue; the asparagine at position 123 may be replaced with a tryptophan, lysine, phenylalanine, arginine, histidine or serine; the leucine at position 124 may be replaced with a threonine residue; the lysine at position 125 may be replaced with an alanine, glutamic acid, tryptophan or two residues such as leucine-arginine or isoleucine-glutamic acid may be added after the lysine at position 125 of the amino acid sequence. The amino acid position correspondences described herein are often expressed as the amino acid position in the form of the wild-type human calcineurin heterodimer b polypeptide (NCBI: ACX34095.1).

[0206] Another drug resistance coding sequence is O(6)-methylguanine methyltransferase (MGMT - UniProtKB: P16455), which encodes human alkylguanine transferase (hAGT). AGT is a DNA repair protein that confers resistance to the cytotoxic effects of alkylating agents such as nitrosoureas and temozolomide (TMZ). 6-benzylguanine (6-BG) is an AGT inhibitor that enhances the toxicity of nitrosoureas and is co-administered with TMZ to enhance the cytotoxic effects of the same agent. Some MGMT mutant forms encoding variants of AGT are highly resistant to inactivation by 6-BG, but retain the ability to repair DNA damage (Maze, R. et al. (1999) "Retroviral-mediated expression of the P140A, but not P140A / G156A, mutant form of O6-methylguanine DNA methyltransferase protects hematopoietic cells against O6-benzylguanine sensitization to chloroethylnitrosourea treatment" J. Pharmacol. Exp. Ther. 290(3): 1467-74). In certain embodiments, AGT mutant forms may contain a mutant amino acid at position P140 of wild-type AGT. In a preferred embodiment, the proline at position 140 is replaced with a lysine residue.

[0207] Another drug resistance coding sequence can be the multidrug resistance protein (MDR1) gene. This gene encodes a membrane glycoprotein known as P-glycoprotein (P-GP), which is involved in the transport of metabolic by-products across the cell membrane. The P-Gp protein exhibits broad specificity for several structurally unrelated chemotherapeutic agents. Thus, expression of a nucleic acid sequence encoding MDR-1 (Genebank NP_000918) can confer drug resistance to cells.

[0208] Alternative drug resistance coding sequences may contribute to the production of cytotoxic antibiotics, such as those derived from the ble or mcrA genes. Ectopic expression of the ble gene or mcrA in immune cells confers a selective advantage upon exposure to the chemotherapeutic agents bleomycin and mitomycin C, respectively (Belcourt, MF (1999) "Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA". PNAS. 96(18):10489-94).

[0209] Another drug resistance coding sequence may be derived from a gene encoding a mutated version of a drug target, such as a mutated variant of mTOR (mTOR mut) that confers resistance to rapamycin as described 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 a specific mutated variant of Lck (Lckmut) that confers resistance to Gleevec as described in Lee KC et al. (2010) "Lck is a key target of imatinib and dasatinib in T-cell activation", Leukemia, 24: 896-900.

[0210] As mentioned above, the genetic modification step of the method can include introducing into cells an exogenous nucleic acid that comprises at least one sequence that codes for a drug resistance coding sequence and a portion of an endogenous gene, so that homologous recombination occurs between the endogenous gene and the exogenous nucleic acid.In certain embodiments, the endogenous gene can be a wild-type "drug resistance" gene, so that after homologous recombination, the wild-type gene is replaced with a mutant form of the gene that confers resistance to the drug.

[0211] Transgene expression enhances in vivo persistence of immune cells In one aspect of this method, the exogenous sequence integrated into the immune cell genomic locus encodes a molecule that enhances immune cell persistence, particularly in vivo persistence in a tumor environment.

[0212] By "enhanced persistence" is meant increasing the survival time of the immune cells in terms of life span, particularly after the modified immune cells have been injected into a patient, for example, enhanced persistence is observed if the average survival time of the modified cells is significantly, at least 10%, preferably 20%, more preferably 30%, and even more preferably 50% longer than unmodified cells.

[0213] This is especially true when the immune cells are allogeneic. It can be achieved by creating local immune protection by introducing coding sequences that allow immunosuppressive polypeptides to be ectopically expressed and / or secreted at or through the cell membrane. Such polypeptides, specifically a panel of immunosuppressive peptides derived from immune checkpoint antagonists, viral envelopes or NKG2D ligands, can enhance the persistence and / or engraftment of allogeneic immune cells in the patient's body.

[0214] In one embodiment, the immunosuppressive polypeptide encoded by the foreign coding sequence is a ligand for cytotoxic T-lymphocyte antigen 4 (CTLA-4, also known as CD152, GenBank Accession No. AF414120.1). The ligand polypeptide is preferably an anti-CTLA-4 immunoglobulin, such as CTLA-4a Ig and CTLA-4b Ig, or a functional variant thereof.

[0215] In one embodiment, the immunosuppressive polypeptide encoded by the exogenous coding sequence is an antagonist of PD1, such as PD-L1 (also known as CD274, programmed cell death 1 ligand; see UniProt for human polypeptide sequence Q9NZQ7), which encodes a 290 amino acid type I transmembrane protein consisting of a 30 amino acid Ig V-like domain, an Ig C-like domain, a hydrophobic transmembrane domain, and a cytoplasmic tail. Such membrane-bound forms of PD-L1 ligands are intended for the purposes of the present invention to be either intact (wild type) forms or truncated forms, e.g., by removal of the intracellular domain or one or more mutations (Wang S et al., 2003, J Exp Med. 2003; 197(9): 1083-1091). It is noted that PD1 is not considered for the purposes of the present invention to be a membrane-bound form of PD-L1 ligand. In another embodiment, the immunosuppressive polypeptide is a secreted form. Such recombinant secreted PD-L1 (or soluble PD-L1) can be made by fusing the extracellular domain of PD-L1 to the Fc portion of immunoglobulin (Haile ST et al., 2014, Cancer Immunol. Res. 2(7): 610-615; Song MY et al., 2015, Gut. 64(2):260-71). This recombinant PD-L1 can neutralize PD-1 and abrogate PD-1-mediated T cell inhibition. PD-L1 ligand can be co-expressed with CTLA4 Ig to further enhance the persistence of both.

[0216] In another embodiment, the exogenous sequence encodes a non-human MHC homologue, in particular a viral MHC homologue, or a chimeric β2m polypeptide, as described in Margalit A. et al. (2003) "Chimeric β2 microglobulin / CD3ζ polypeptides expressed in T cells convert MHC class I peptide ligands into T cell activation receptors: a potential tool for specific targeting of pathogenic CD8+ T cells" Int. Immunol. 15 (11): 1379-1387.

[0217] In one embodiment, the foreign sequence encodes an NKG2D ligand. Some viruses, such as cytomegalovirus, have acquired mechanisms to avoid NK cell-mediated immune surveillance and interfere with the NKG2D pathway by secreting proteins capable of binding NKG2D ligands and preventing their surface expression (Welte, SA et al. (2003) "Selective intracellular retention of virally induced NKG2D ligands by the human cytomegalovirus UL16 glycoprotein". Eur. J. Immunol., 33, 194-203). Tumor cells also have evolved mechanisms to evade NKG2D responses by secreting NKG2D ligands such as ULBP2, MICB, or MICA (Salih HR, Antropius H, Gieseke F, Lutz SZ, Kanz L, et al. (2003) Functional expression and release of ligands for the activating immunoreceptor NKG2D in leukemia. Blood 102: 1389-1396).

[0218] In one embodiment, the exogenous sequence encodes a cytokine receptor, such as the IL-12 receptor. IL-12 is a well-known activator of immune 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).

[0219] In one embodiment, the foreign sequence encodes an antibody directed against the inhibitory peptide or protein. The antibody is preferably secreted in soluble form by immune cells. In this respect, nanobodies from sharks and camels are advantageous because they are structured as single-chain antibodies (Muyldermans S. (2013) "Nanobodies: Natural Single-Domain Antibodies" Annual Review of Biochemistry 82: 775-797). They are also believed to be more easily fused with secretion signal polypeptides and soluble hydrophilic domains.

[0220] Various aspects that enhance the persistence of the expanded cells described above, as described in further detail below, are particularly preferred when the exogenous coding sequence is introduced by disrupting the endogenous gene encoding β2m or another MHC component.

[0221] Transgene expression to enhance the therapeutic activity of immune cells In one aspect of this method, the exogenous sequence integrated into the immune cell genomic locus encodes a molecule that enhances the therapeutic activity of the immune cell.

[0222] By "enhanced therapeutic activity" is meant that an immune cell or cell population modified according to the invention is more aggressive against a selected type of target cell compared to an unmodified cell or cell population. Said target cell preferably consists of a given type of cell or cell population characterized by a common surface marker. In this specification, "therapeutic potential" reflects therapeutic activity measured by in vitro experiments. Generally, a sensitive cancer cell line such as Daudi cells is used to assess whether the immune cells are more or less active against said cells by performing measurements of cell lysis or reduced proliferation. This can also be assessed by measuring the levels of immune cell degranulation or chemokine and cytokine production. Experiments can also be performed in mice by injecting tumor cells and observing the resulting tumor growth. The enhanced activity is considered significant if the number of cells generated in these experiments is reduced by the immune cells by more than 10%, preferably more than 20%, more preferably more than 30%, and even more preferably more than 50%.

[0223] In one aspect of the invention, the exogenous sequence encodes a chemokine or cytokine, such as IL-12. Expression of IL-12 is particularly advantageous, as this cytokine has been widely documented in the literature to promote immune cell activation (Colombo MP et al. (2002) "Interleukin-12 in anti-tumor immunity and immunotherapy" Cytokine Growth Factor Rev. 13(2):155-68).

[0224] In a preferred aspect of the invention, the exogenous coding sequence encodes or promotes secreted factors that act on other immune cell populations, such as regulatory T cells, to counteract their inhibitory effects on said immune cells.

[0225] In one aspect of the invention, the exogenous sequence encoding an inhibitor of regulatory T cell activity is a polypeptide inhibitor of forkhead / winged helix transcription factor 3 (FoxP3), more preferably a cell penetrating peptide inhibitor of FoxP3, such as that 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).

[0226] "Inhibitor of regulatory T cell activity" refers to a molecule secreted by a T cell or a precursor of said molecule, which allows the T cell to escape the downregulatory activity of regulatory T cells on the T cell. In general, such an inhibitor of regulatory T cell activity has the effect of reducing FoxP3 transcriptional activity in said cell.

[0227] In one aspect of the present invention, the exogenous sequence encodes a secretion inhibitor of tumor-associated macrophages (TAM), such as a CCR2 / CCL2 neutralizing agent. Tumor-associated macrophages (TAM) are important modifiers of tumor microenvironment. Clinical pathology studies have shown that the accumulation of TAM in tumors correlates with poor clinical outcome. Consistent with this evidence, experimental and animal studies support the notion that TAM can provide a favorable microenvironment for promoting tumor development and progression (Theerawut C. et al. (2014) "Tumor-Associated Macrophages as Major Players in the Tumor Microenvironment" Cancers (Basel) 6(3): 1670-1690). Chemokine ligand 2 (CCL2), also known as monocyte chemotactic protein 1 (MCP1 - NCBI NP_002973.1), is a small cytokine that belongs to the CC chemokine family and is secreted by macrophages to produce chemoattraction in monocytes, lymphocytes, and basophils. CCR2 (CC chemokine receptor type 2 - NCBI NP_001116513.2) is the receptor for CCL2.

[0228] The coding sequence inserted into the locus generally encodes a polypeptide that improves the therapeutic potential of the modified immune cell, but the inserted sequence may also be a nucleic acid capable of directing or blocking the expression of other genes, such as an interfering RNA or a guide RNA. The polypeptide encoded by the inserted sequence may act directly or indirectly, such as a signal transduction factor or transcription factor.

[0229] Engineered immune cells and immune cell populations The present invention also relates to various modified immune cells, which can be obtained in isolated form or as part of a cell population by one of the methods described herein.

[0230] In a preferred aspect of the invention, the modified cells are primary immune cells, such as NK cells or T cells, which are generally part of a cell population that may contain various types of cells, generally a patient or donor derived population, isolated by leukapheresis from PBMCs (peripheral blood mononuclear cells).

[0231] The present invention encompasses immune cells comprising any combination of various exogenous coding sequences and gene inactivations, each of which is described independently above. Particularly preferred among these combinations are those that combine expression of the CAR under the transcriptional control of an endogenous promoter that is active upon immune cell activation, specifically one promoter present at one TCR locus, specifically the TCR alpha promoter.

[0232] Another preferred combination is the insertion of an exogenous sequence encoding a CAR, or one of its components, under the transcriptional control of the hypoxia inducible factor 1 gene promoter (Uniprot: Q16665).

[0233] The present invention also relates to pharmaceutical compositions comprising the modified primary immune cells or immune cell populations described above for the treatment of an infectious disease or cancer, and to methods of treating a patient in need of such treatment, said methods comprising: - providing a population of primary immune cells modified by the method of the invention as described above; Optionally, purifying or sorting the modified primary immune cells; activating the modified primary immune cell population when or after the cells are infused into the patient. Includes.

[0234] The present invention is also directed to modified immune cells resulting from the above method, wherein the cells contain an exogenous polynucleotide or expression vector as referred to herein, in particular for the expression of an anti-MUC1 CAR on the cell surface. Such modified immune cells are preferably T cells or NK cells, derived from primary cells or differentiated from stem cells, such as iPS cells.

[0235] In a particular embodiment, expression of the TCR is reduced or suppressed in said immune cell by inactivation of at least one gene encoding TCR alpha or TCR beta by a rare-cutting endonuclease.

[0236] In certain embodiments, a polynucleotide encoding an anti-MUC1 CAR can be integrated into an endogenous locus under the transcriptional control of an endogenous promoter, preferably into the TCR alpha or TCR beta locus.

[0237] In certain embodiments, the immune cells can be further mutated to confer resistance to at least one immunosuppressant drug, such as an anti-CD52 antibody.

[0238] In certain embodiments, the immune cells can be further mutated to confer resistance to at least one chemotherapeutic agent, particularly a purine analog agent.

[0239] In a preferred embodiment, the modified immune cells of the present invention have been mutated in the patient, specifically in the genes encoding MHCI components such as HLA or B2m, to improve their persistence or their longevity.

[0240] In a preferred embodiment, the modified immune cells of the present invention can be mutated to improve their CAR-dependent immune activation, specifically to reduce or suppress the expression of immune checkpoint proteins and / or their receptors.

[0241] In certain embodiments, the anti-MUC1 CAR can be co-expressed in the cell with another exogenous gene sequence encoding an inhibitor or decoy of the TGF beta receptor, for example, a dominant negative TGF beta receptor (dnTGFβRII) having at least 80% polypeptide sequence identity with SEQ ID NO: 59. This expression can be obtained by introducing into the cell an exogenous polynucleotide comprising a first polynucleotide sequence encoding the anti-MUC1 CAR, a second polynucleotide encoding a 2A self-cleaving peptide, and a third polynucleotide encoding the dominant negative TGF beta receptor.

[0242] Alternatively or supplementally, the modified immune cells have reduced or inactivated expression of at least one TGF beta receptor gene.

[0243] In some further embodiments, the anti-MUC1 chimeric antigen receptor (CAR) is NK cell inhibitors, such as HLAG, HLAE, or ULBP1; a CRS inhibitor, such as mutant IL6Ra, sGP130, or IL18-BP; or · cytochrome P450, CYP2D6-1, CYP2D6-2, CYP2C9, CYP3A4, CYP2C19, or CYP1A2, which confers hypersensitivity of said immune cells to drugs such as cyclophosphamide and / or isophosphamide; · dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin or methylguanine transferase (MGMT), mTORmut or Lckmut, conferring drug resistance; Chemokines or cytokines, such as IL-2, IL-12, and IL-15; · Hyaluronidases, such as HYAL1, HYAL2, and SPAM1; Chemokine receptors, such as CCR2, CXCR2, or CXCR4; Tumor-associated macrophage (TAM) secretion inhibitors, such as CCR2 / CCL2 neutralizing agents, to enhance the therapeutic activity of immune cells; and / or Metabolic enzymes, such as glucose phosphate isomerase 1 (GPI1), lactate dehydrogenase (LDHA), and / or phosphoenolpyruvate carboxykinase 1 (PCK1) The gene can be co-expressed in the cell with another exogenous gene sequence selected from those encoding:

[0244] Dual anti-mesothelin / anti-MUC1 CAR immune cells In a further embodiment, the present invention also features an immune cell or immune cell population that expresses both anti-MUC1 CAR and anti-mesothelin CAR as described above. This particular CAR combination has significant advantages in treating solid tumors that are positive for both mesothelin and MUC1. Targeting these two genetically unrelated antigens reduces the chance of antigen escape. The inventors have found that this combination gives the cell population of the present invention an exceptional ability against solid tumors in vivo. This also allows the scope of anti-tumor activity to be expanded, especially in heterogeneous solid tumors where various types of tumorigenic cells may coexist. For example, dual CAR immune cells expressing anti-mesothelin CAR and anti-MUC1 CAR positive cells can kill either mesothelin positive cells or tMUC1 positive cells, but MUC1 CAR alone cannot.

[0245] Both anti-MUC1 CAR and anti-mesothelin CAR can be co-expressed in immune cells modified by methods such as those described above, and thus can include any of the genetic features mentioned herein, such as reduced expression of TCR, B2M, and PD1. For example, immune cells can be transduced with a polynucleotide construct comprising a sequence encoding either the anti-MUC1 CAR or the anti-mesothelin CAR, or a viral vector comprising a polynucleotide encoding both coding sequences linked by a 2A polypeptide. Alternatively, a therapeutic population of cells can be obtained by mixing immune cells that independently express the anti-MUC1 CAR or the anti-mesothelin CAR.

[0246] Thus, the present invention encompasses therapeutic populations of modified immune cells comprising a mixture of cells expressing either an anti-MUC1 CAR or an anti-mesothelin CAR, or both CARs, for their use in anti-cancer therapy, particularly for the treatment of breast, cervical, endometrial, ovarian, pancreatic, gastric, and lung cancer.

[0247] In a preferred embodiment, a mesothelin-specific chimeric antigen receptor (anti-MESO CAR) is typically ·An extracellular ligand-binding domain containing VH and VL from a monoclonal anti-mesothelin antibody; · a transmembrane domain; and The cytoplasmic domain, which contains the CD3 zeta signaling domain, and the costimulatory domain The compound has a structure comprising:

[0248] In a preferred embodiment, a mesothelin-specific chimeric antigen receptor (anti-MESO CAR) is directed against human mesothelin (MSLN_human, referred to as Q13421 in the Uniprot database), which is displayed on the surface of malignant cells, more specifically against the specific mesothelin polypeptide region represented by SEQ ID NO: 209. An efficient CAR against this antigenic region, specifically one that comprises an extracellular ligand-binding domain comprising a VH chain and a VL chain having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with SEQ ID NO: 210 (Meso1-VH) and SEQ ID NO: 211 (Meso1-VL), respectively, has been developed by the applicant.

[0249] The extracellular ligand-binding domain of the anti-MESO CAR preferably comprises one or several scFv segments from the antibody designated meso1, more specifically, the CDRs therefrom comprising SEQ ID NOs:216, 217, 218, 219, 220, and / or 221.

[0250] In a preferred aspect, the extracellular ligand binding domain of the CAR comprises: a variable heavy VH chain comprising CDRs from antibody meso1 having at least 90% identity to SEQ ID NO:3 (CDRH1-Meso1), SEQ ID NO:4 (CDRH2-meso1), and / or SEQ ID NO:5 (CDRH3-meso1), respectively; and a variable heavy VL chain comprising CDRs from antibody Meso1 having at least 90% identity to SEQ ID NO:6 (CDRL1-meso1), SEQ ID NO:7 (CDRL2-meso1), and / or SEQ ID NO:8 (CDRL3-meso1), respectively; Includes.

[0251] One preferred anti-mesothelin CAR for use in combination with an anti-MUC1 CAR according to the present invention is that designated MESO1 CAR, which has at least 75%, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% identity to SEQ ID NO:222.

[0252] Other anti-MESO CARs, such as MESO2 and P4-R2, or any functional variants thereof, that comprise ScFv sequences at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% identical to the polypeptide sequences referenced in Tables 12 and 13, can also be expressed as part of the present invention.

[0253] Table 12. Amino acid sequences of exemplary binding domains of anti-MESO CARs TIFF2024517713000019.tif228151

[0254] Table 13. Examples of amino acid sequences of P4-R2, Meso1-R2, Meso1, and MESO2-R2 CARs TIFF2024517713000020.tif77156

[0255] T cell activation and proliferation Whether before or after genetic modification, the immune cells of the invention can be activated or expanded, even if they can be activated or expanded independently of an antigen-binding mechanism. In particular, T cells can be activated and expanded using methods such as those described in, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. T cells can be expanded in vitro or in vivo. T cells are generally expanded by contact with an agent that stimulates the CD3 TCR complex and the costimulatory molecules on the surface of the T cells, generating a T cell activation signal. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to generate a T cell activation signal.

[0256] As a non-explanatory example, a T cell population can be stimulated in vitro, for example, by contact with a surface-immobilized anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD2 antibody, or by contact with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. Co-stimulation of an accessory molecule on the surface of T cells is achieved using a ligand that binds to the accessory molecule. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under appropriate conditions to stimulate T cell proliferation. Suitable conditions for T cell culture include an appropriate medium (e.g., minimal essential medium, or RPMI Media1640 or X-vivo 5 (Lonza)), which can contain factors necessary for proliferation and viability, such as serum (e.g., fetal bovine or human fetal serum), interleukin-2 (IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFp, and TNF, or any other additives for cell proliferation known to those skilled in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanates, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Media may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma), or a set of predetermined hormones, and / or cytokines in sufficient amounts for T cell growth and proliferation. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells injected into subjects. Target cells are maintained under the necessary conditions to support growth, such as, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% C02). T cells exposed to various stimulation times may exhibit different characteristics.

[0257] In another particular embodiment, the cells can be expanded in co-culture with tissue or cells. The cells can also be expanded in vivo, for example, in the blood of a subject after administration of the cells to the subject.

[0258] Therapeutic Compositions and Uses The above-mentioned method of the invention allows to produce modified primary immune cells within a limited time frame of about 15-30 days, preferably 15-20 days, most preferably 18-20 days, which therefore retain their maximum immunotherapeutic potential, especially with regard to cytotoxic activity.

[0259] These cells form cell populations, which are preferably derived from a single donor or patient, and which can be expanded in closed culture recipients, thus complying with the most stringent requirements of manufacturing practices, and can be frozen prior to infusion into the patient, providing an "off-the-shelf" or "ready to use" therapeutic composition.

[0260] In the present invention, a large number of cells from the same leukapheresis source can be obtained, which is important in order to obtain a sufficient dose to treat patients. Although differences between cell populations originating from various donors can be observed, the number of immune cells obtained by leukapheresis is generally about 10 8 ~10 10 PBMCs contain several types of cells, including granulocytes, monocytes, and lymphocytes, of which 30-60% are T cells. Primary T cells from a single donor are generally 10 8 ~10 9 The method of the present invention generally comprises about 10 8 More than 10 T cells, more commonly about 10 9 More than 10 T cells, more commonly about 10 10 More than 10 T cells, usually 11 Finally, a population of modified cells is obtained, reaching a super-population of T cells.

[0261] The present invention therefore more particularly relates to therapeutically effective primary immune cell populations, wherein at least 30%, preferably 50%, more preferably 80% of the cells of said population have been modified according to any one of the methods described herein.

[0262] In a preferred aspect of the invention, more than 50% of the immune cells in the population are TCR-negative T cells. In a more preferred aspect of the invention, more than 50% of the immune cells in the population are CAR-positive T cells. Modified immune cells, cell populations, therapeutic compositions, and uses.

[0263] The present invention is particularly focused on populations of cells, which may include mixtures of modified cells comprising one or several properties, obtainable by the methods described herein, for their use as therapeutic compositions.

[0264] Such a population of cells generally comprises at least 25%, preferably at least 50%, more preferably at least 75% of the cells carrying at least one of said genetic modifications.

[0265] Such a population of cells preferably comprises at least 25%, preferably at least 50%, more preferably at least 75% of immune cells having at least two, preferably at least three, preferably at least four, even more preferably at least five of said genetic modifications, also referred to herein as "traits".

[0266] The present invention also encompasses therapeutic compositions comprising a population of modified immune cells characterized by the following (phenotypic) properties: Exogenous expression of a CAR targeting the tMUC1 epitope, and a reduction in B2M expression by at least 30%; preferably at least 50%, more preferably at least 75% of the cells; and / or a reduction in PD1 expression by at least 30%; preferably by 50%, more preferably by at least 75%; and / or Optionally, a reduction in TCR expression by at least 50%; preferably by at least 75%; Optionally, an increase in expression of IL-12, IL-15, or IL-18 of at least 30%; preferably at least 50%, more preferably at least 75%; Optionally, a reduction in TGFβ expression by at least 30%; preferably by 50%, more preferably by at least 75%; Optionally, exogenous expression of a decoy of TGFβR2; Optionally, secretion of HYAL1, HYAL2, and SPAM1 by introduction of an exogenous coding sequence; and Optionally, expression of GPI1, PCK1, and / or LDHA by introduction of exogenous coding sequences.

[0267] The above expression percentages can reflect more than 80%, 90%, 95%, or 100% of the cells in the population that have at least one of these phenotypes. The percentages are based on a comparison with unmodified cells in the same culture conditions, for example, by measuring global protein expression.

[0268] From a genotypic standpoint, the population of cells of the invention can be characterized by the following genetic features: at least 50% of the immune cells present an exogenous polynucleotide sequence encoding a CAR that targets the tMUC1 epitope; and at least 50% and preferably at least 75% of the immune cells display a B2M inactive allele; and / or At least 30%, preferably at least 50%, more preferably 75% of the immune cells present a mutated PD1 allele; Optionally, at least 50% of the T cells, preferably at least 75%, display a TCR inactive allele; Optionally, at least 30%, preferably at least 50%, more preferably at least 75% of the immune cells display an exogenously introduced sequence encoding IL-12, IL-15, or IL-18; Optionally, at least 20%, preferably at least 50%, more preferably at least 75% of the immune cells present a sequence encoding a decoy of TGFβR2 exogenously inserted into their genome; Optionally, at least 20%, preferably at least 50%, more preferably at least 75% of the immune cells present a sequence encoding HYAL1, HYAL2 and / or SPAM1 exogenously inserted into their genome; Optionally, at least 20%, preferably at least 50%, more preferably at least 75% of the immune cells display GPI1, PCK1 and / or LDHA coding sequences exogenously inserted into their genome; Optionally, at least 30%, preferably at least 50%, more preferably at least 75% of the immune cells display a mutated TGFβ allele.

[0269] The above percentages can reflect more than 80%, 90%, 95%, or 100% of the cells in the population that contain at least one of the genetic modifications. The percentages are based on a comparison with non-modified cells, for example, by quantitative PCR performed on the entire cell population or a representative sample thereof.

[0270] In such a population, it may be desirable for at least one TCR alpha allele to be disrupted in more than 90% of cells, preferably more than 95%, for its subsequent allogeneic use in patients. In a preferred embodiment, the TCR alpha gene is disrupted by inserting an exogenous sequence that codes for a genetic characteristic, such as the CAR itself or dnTGFBRII (SEQ ID NO:59) or both. Such a decoy of TGFβR2 can be co-expressed with the CAR during viral vector transduction (rLV or AAV vectors containing both coding sequences).

[0271] In a preferred embodiment, the B2M and / or PD1 alleles are disrupted in said population of cells by insertion of an exogenous sequence encoding an NK inhibitor, such as HLA-E (SEQ ID NO:60) or HLA-G (SEQ ID NO:61), or any functional sequence having at least 80%, 90%, or 95% identity thereto.

[0272] In a preferred embodiment, the B2M and / or PD1 alleles are disrupted in said population of cells by insertion of an exogenous sequence encoding IL-12a (SEQ ID NO:63) and / or IL12b (SEQ ID NO:64), IL-15a (SEQ ID NO:66), or IL-18 (SEQ ID NO:68), or any functional sequence having at least 80%, 90%, or 95% identity thereto.

[0273] In a preferred embodiment, the population of cells comprises an exogenous sequence encoding HYAL1 (SEQ ID NO:69), HYAL2 (SEQ ID NO:70), SPAM1 (SEQ ID NO:71), GPI1 (SEQ ID NO:72), PCK1 (SEQ ID NO:73), or LDHA (SEQ ID NO:74), or any functional sequence having at least 80%, 90%, or 95% identity thereto, preferably inserted into the PD1, CD69, CD25, or GMCSF locus.

[0274] In a preferred embodiment, the population of cells comprises cells that have been further mutated to confer resistance to lymphocyte depletion therapy, for example to inactivate or reduce expression of CD52 and / or dCK gene alleles.

[0275] The population of cells preferably, but not necessarily, comprises an exogenous polynucleotide sequence encoding a CAR that targets the tMUC1 epitope, such as CLS MUC1-A, CLS MUC1-B, CLS MUC1-C, CLS MUC1-D, etc. Such an exogenous polynucleotide sequence encoding a CAR that targets the tMUC1 epitope preferably has at least 80%, 90%, or 95% identity to SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, and SEQ ID NO:208, respectively.

[0276] Such a composition or cell population can therefore be used as a medicament, in particular for cancer treatment, in particular for lymphoma treatment, but also for solid tumors, such as melanoma, neuroblastoma, glioma, or carcinomas, such as lung, breast, rectal, prostate or ovarian tumors, in patients in need thereof.

[0277] The present invention more specifically relates to primary TCR-negative T cell populations of single donor origin, in which at least 20%, preferably 30%, more preferably 50% of the cells in said population are modified with sequence-specific reagents at at least two, preferably three different loci.

[0278] In another aspect, the invention relates to a method of treating a patient in need of treatment, said method comprising: (a) determining specific antigen markers present on the surface of a patient's tumor biopsy; (b) providing a population of modified primary immune cells modified by one of the methods of the invention described above, preferably expressing a recombinant receptor directed against said specific antigen marker; (c) administering the modified primary immune cell population to the patient. Contains at least one of the following:

[0279] Generally, the cell population comprises primarily CD4 and CD8 positive immune cells, such as T cells, which can undergo robust in vivo T cell expansion and survive for extended periods of time in vitro and in vivo.

[0280] Treatments involving the modified primary immune cells of the invention can be ameliorative, curative, or prophylactic. It can be part of an autologous or allogeneic immunotherapeutic treatment.

[0281] In another embodiment, the isolated cells of the invention or cell lines derived from the isolated cells of the invention can be used to treat solid tumors, in particular solid tumors such as, typically, esophageal, breast, gastric, bile duct, pancreatic, colon, lung, thymic, mesothelioma, ovarian, and / or endometrial cancers.

[0282] Adult tumors / cancers and pediatric tumors / cancers are also included.

[0283] Treatment with the modified immune cells of the present invention may be combined with one or more cancer therapies selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiation therapy.

[0284] In a preferred embodiment of the invention, said treatment may be administered to a patient undergoing immunosuppressive treatment. Indeed, the invention preferably relies on cells or cell populations that have been rendered resistant to such immunosuppressive agents by virtue of the inactivation of a gene encoding at least one receptor for such an immunosuppressive agent. In this respect, the immunosuppressive treatment should aid in the selection and expansion in the patient of the T cells of the invention.

[0285] Administration of the cells or cell populations of the present invention can be carried out in any convenient manner, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one aspect, the cell compositions of the present invention are preferably administered by intravenous injection.

[0286] The administration of cells or cell populations was at 10 per kg of body weight. 4 ~10 9 cells, preferably 10 per kg body weight 5 ~10 6 The present invention can therefore comprise the administration of up to 10 cells originating from a single donor or patient sampling, with all integer numbers of cells within these ranges being included. 6 ~10 8 In one embodiment, more than 10, typically more than 50, more typically more than 100, and usually more than 1000 doses containing gene-edited cells may be provided.

[0287] The cells or cell population may be administered in one or more doses. In another embodiment, the effective amount of cells is administered as a single dose. In another embodiment, the effective amount of cells is administered as two or more doses over a period of time. The timing of administration is within the discretion of the attending physician and depends on the clinical condition of the patient. The cells or cell population may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determining the optimal range of effective amounts of a given cell type for a particular disease or condition is within the skill of the art. An effective amount means an amount that provides a therapeutic or prophylactic benefit. The dosage depends on the age, health, and weight of the recipient, the type of concurrent treatment, if any, the frequency of treatment, and the nature of the desired effect.

[0288] In another embodiment, the effective amount of cells or a composition comprising the cells is administered parenterally. The administration can be intravenous. The administration can be by direct tumor injection.

[0289] In certain aspects of the invention, the cells are administered to the patient together with (e.g., before, simultaneously with, or after) any number of relevant treatment modalities, including, but not limited to, antiviral therapy, treatment with agents such as cidofovir, and interleukin-2, cytarabine (also known as ARA-C), or nataliziimab treatment for MS patients, or efaliztimab treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the present invention may be combined with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, or other antibody therapies, cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important in growth factor-induced signal transduction (Henderson, Naya et al. 1991; Liu, Albers et al. 1992; Bierer, Hollander et al. 1993). In further embodiments, the cell composition of the present invention is administered to a patient together with (e.g., before, simultaneously, or after) bone marrow transplantation, chemotherapy such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or T cell depletion therapy using any of antibodies such as OKT3 or CAMPATH. In another embodiment, the cell composition of the present invention is administered following B cell depletion therapy, such as an agent that reacts with CD20, e.g., Rituxan. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following transplantation, the subject receives an infusion of the expanded immune cells of the present invention. In additional embodiments, the expanded cells are administered before or after surgery.

[0290] The present invention also specifically relates to a general method of treating a solid tumor in a patient, said method comprising immunodepleting said patient by a lymphodepleting regimen and injecting genetically modified lymphocytes that have been rendered resistant to the lymphodepleting agent used in the lymphodepleting regimen and that specifically target said solid tumor, such genetically modified lymphocytes being preferably CAR-positive T cells, more preferably equipped with an anti-MUC1 CAR as described herein.

[0291] Lymphocyte depletion regimens preferably include antibodies directed against antigens such as CD52, CD3, CD4, CD8, CD45, or other specific markers present on the surface of immune cells, or drugs such as purine analogues (e.g., fludarabine and / or chlorofarabine) and glucocorticoids.

[0292] In a preferred embodiment of the invention, the method comprises subjecting the patient to a lymphodepletion regimen comprising an antibody directed against CD52 and administering modified CAR T cells comprising an anti-MUC1 CAR with reduced, deficient or inactivated expression of CD52.

[0293] In a preferred embodiment of the invention, the lymphocyte depletion therapy may include an anti-CD52 antibody, such as alemtuzumab, either alone or in combination. The lymphocyte depletion regimen may, for example, include a combination of cyclophosphamide, typically for 1-3 days, fludarabine for 1-5 days, and alemtuzumab for 1-5 days. In general, the lymphocyte depletion regimen may include cyclophosphamide 50-70 mg / kg / day, fludarabine 20-40 mg / m2 / day, and alemtuzumab 0.1-0.5 mg / kg / day, either alone or in combination.

[0294] To that end, the present invention provides a composition for lymphodepletion in patients suffering from solid tumors, comprising an anti-CD52 antibody in combination with a modified population of MUC1-targeted lymphocytes insensitive to said antibody, said population preferably comprising cells expressing an anti-MUC1 CAR and having impaired CD52 expression, wherein an allele of the CD52 gene has preferably been inactivated by a rare-cutting endonuclease, such as a TALE nuclease or an RNA-guided endonuclease as described above.

[0295] The invention is also a method for treating a patient having a condition characterized by MUC1 expressing cells, comprising the steps of: modifying immune cells from the donor to express a functional anti-MUC1 CAR as described above; administering the CAR-positive modified immune cells to a patient to eliminate cells expressing the tMUC1 epitope.

[0296] In a preferred embodiment, the method includes a prior treatment step in which the patient is lymphodepleted. In this regard, the CAR-positive modified immune cells can be mutated to confer resistance to the lymphodepleting treatment. For example, the CAR-positive modified immune cells can be mutated in their CD52 gene to confer resistance to anti-CD52 treatment, such as alemtuzumab.

[0297] When applied in an allogeneic setting, such a protocol can be considered a method for treating patients with a condition characterized by MUC1-expressing cells, which combines the administration of (1) a lymphocyte depleting agent and (2) a population of donor-derived allogeneic modified immune cells expressing a chimeric antigen receptor (CAR) specifically directed against the tMUC1 epitope.

[0298] The present invention also provides a medical kit for use in the treatment of solid tumor cancer, comprising the lymphocyte depletion composition and the modified cell population resistant thereto.

[0299] By "cytolytic activity" or "cytotoxic activity" or "cytotoxicity" is meant the percentage of cytolysis of target cells conferred by immune cells.

[0300] Methods for determining cytotoxicity are described below.

[0301] For adherent target cells: 2.10 4 Specific target antigen (STA)-positive or STA-negative cells were plated in 96-well plates at 0.1 ml per well. The day after plating, STA-positive and STA-negative cells were labeled with CellTrace CFSE and incubated at 4 x 10 for 4 h. 5 The cells were then co-cultured with T cells, harvested, stained with a fixable viability dye (eBioscience), and analyzed using a MACSQuant flow cytometer (Miltenyi).

[0302] For suspension target cells: Label STA-positive and STA-negative cells with CellTrace CFSE and CellTrace Violet, respectively. Approximately 2 x 10 4 2 x 10 ROR1 positive cells 4 STA-negative cells and 4 x 10 in 0.1 ml per well in a 96-well plate 5 After 4 hours of incubation, cells are harvested, stained with a fixable viability dye (eBioscience), and analyzed using a MACSQuant flow cytometer (Miltenyi).

[0303] The percentage of specific lysis can be calculated using the following formula: TIFF2024517713000021.tif25128

[0304] By "increased cytotoxicity" is meant that the % cytolysis of target cells conferred by the modified immune cell is increased by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% or more compared to the % cytolysis of target cells conferred by an unmodified immune cell.

[0305] "Identity" refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence, which may be aligned for comparison. If a position in the compared sequences is occupied by the same base, the molecules are identical at that position. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at the same position common to the nucleic acid sequences. A variety of alignment algorithms and / or programs can be used to calculate the identity between two sequences, including FASTA or BLAST, which are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wisconsin), and can be used, for example, with default settings. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98%, or 99% identity with the specific polypeptides described herein and preferably exhibiting substantially the same function, as well as polynucleotides encoding such polypeptides, are contemplated.

[0306] Unless otherwise specified, the present invention includes polypeptides and polynucleotides that share at least 70%, typically at least 80%, more typically at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 97% with the polypeptides and polynucleotides described herein.

[0307] The term "subject" or "patient", as used herein, generally refers to a mammal, preferably a primate, and more preferably a human.

[0308] The foregoing specification of the invention provides manners and methods for making and using the invention, and is intended to enable any person skilled in the art to make and use the invention, and is provided with particularity in relation to the subject matter of the appended claims, which subject matter forms a part of this specification.

[0309] Where numerical limits or ranges are recited herein, the endpoints are included. Also, every value and subrange within the numerical limit or range is specifically included, as if such values ​​were expressly stated.

[0310] Having broadly described the invention, a further understanding can be obtained by reference to specific embodiments, which are provided herein for illustrative purposes only and are not intended to limit the scope of the invention as claimed. EXAMPLES

[0311] Example 1: In vitro experiments and generation of anti-MUC1 CAR-T Selection of UCARTMUC1 target cell lines for in vitro assays: The target cell line T47D (ATCC® HTB-133 (商標) ) and HCC70 (ATCC® CRL-2315 (商標) ) were purchased from the American Type Culture Collection (ATCC), and the MUC1 negative control cell line 293FT (R70007) was purchased from ThermoFisher Scientific. All cell lines were cultured according to the manufacturers' recommendations.

[0312] Cell surface expression of tumor-associated MUC1 (tMUC1) was measured using flow cytometry. Adherent cells, T47D, HCC70, and 293FT, were detached from cell culture flasks using Accutase Cell Dissociation Reagent (Biolegend: 423201) and then stained with commercial anti-MUC1 antibodies HMFG2 (BD Biosciences: 566590), 16A (Biolegend: 355608), or SM3 (Invitrogen: 53989382). Data were acquired on a BD FACS CANTO II flow cytometer and analyzed with FlowJo.

[0313] The reporter cell lines T47D-NanoLuc-GFP and HCC70-NanoLuc-GFP were generated by transducing wild-type T47D and HCC70 cells with rLV carrying nucleotide sequences encoding NanoLuciferase (NanoLuc) and EGFP separated by the self-cleaving peptide T2A, forming a single bicistronic transcript. MUC1 expression in the reporter cell lines was assessed as described above.

[0314] In vitro specificity of the top four anti-MUC1 scFvs: Anti-MUC1 scFvs (CLS MUC1-A, CLS MUC1-B, CLS MUC1-C, and CLS MUC1-D) as described in Tables 3-6 were produced as recombinant proteins fused to mouse FC by Lake Pharma (201 Industrial Road San Carlos, CA 94070). Primary cells from tissues known to express high levels of normal MUC1: kidney (PCS-400-012), lung (PCS-300-010), and cervix (PCS-480-011) were obtained from ATCC and cultured according to the manufacturer's recommendations. T47D, HCC70, and 293FT cells were used as positive and negative controls (respectively). All cells were detached using Accutase Cell Dissociation solution and stained with recombinant scFV-FC protein followed by PE-conjugated goat anti-mouse FC gamma specific antibody (Jackson Immunoresearch: 115-115-164) or commercial HMFG2 antibody (BD Biosciences: 566590). Data were acquired on a BD FACS CANTO II instrument and analyzed with FlowJo. For comparison, cells were also stained with commercial anti-TROP2 antibodies (Biolegend (NY18): 363804 and Miltenyi (REA916): 130-115-055). A graphical analysis of FlowJo is shown in Figure 2, where it is observed that the commercially available HMFG2 antibody and recombinant scFVs (MUC1-A-scFV, MUC1-B-scFV, MUC1-C and MUC1-D-scFV) stain the tMUC1-expressing breast cancer cell lines T47D and HCC70, but fail to stain primary cells from tissues expressing normal MUC1 (kidney, lung and cervix) or the MUC1-negative control cell line 293FT. These results demonstrate the strong specificity of the four scFV candidates for tMUC1.

[0315] Generation of MUC1 CAR-T cells: On day 0, frozen human peripheral blood mononuclear cells (PBMCs) from AllCells (Alameda, California 94502) were thawed, washed, counted, and resuspended in X-vivo 15 medium (Lonza: 04-418Q) supplemented with 5% AB serum (GeminiBio: 100-318) and 20 ng / mL recombinant human IL-2 (Miltenyi: 130-097-743). The cells were then transferred to an incubator set at 37° C., 5% CO2.

[0316] On day 1, PBMCs were counted, analyzed by flow cytometry to assess % CD3+ cells, centrifuged, and resuspended in X-vivo 15 medium supplemented with 5% AB serum, 20 ng / mL human IL-2, and Dynabeads Human T activator CD3 / CD28 (ThermoFisher #11161D: 25 μl per million CD3+ cells). Cells were then transferred to an incubator set at 37° C., 5% CO2.

[0317] On day 4, T cells and rLV vectors carrying polynucleotide sequences encoding anti-MUC1 CAR were resuspended in X-vivo 15 medium supplemented with 5% AB serum and 20 ng / ml IL-2 and plated onto retronectin-coated plates. Plates were then transferred to an incubator set at 37° C. and 5% CO2.

[0318] On day 5, T cells were passaged into fresh X-vivo 15 medium supplemented with 5% AB serum, 20 ng / ml IL-2, and then transferred to an incubator set at 37° C. and 5% CO2.

[0319] On day 7, T cells were passaged into fresh X-vivo 15 medium supplemented with 5% AB serum, 20 ng / ml IL-2. If CAR-T cells were modified with additional properties (TCR and PD1 knockout, and inducible IL-12 release), cells were passaged at 1e6 cells / mL in X-vivo 15 medium supplemented with 5% AB serum, 20 ng / ml IL-2 in the morning. Six hours later, cells were co-electroporated with mRNAs encoding the right and left arms of TRAC TALEN (SEQ ID NOs: 75 and 76) and PD1 TALEN (SEQ ID NOs: 77 and 78), respectively, to efficiently inactivate TCRα and PD-1 genes and prevent TCRαβ expression on the surface of primary T cells, as previously reported [Poirot et al. (2013) Blood. 122 (21): 1661 and Sachdeva et al. (2019) Nat Commun. 10 (1)]. TALEN® is the registered name for heterodimeric TALE nucleases designed by Cellectis (8, rue de la Croix Jarry, 75013 Paris, France) that use Fok1 as the nuclease catalytic domain as originally described by Voytas et al. in WO2011072246.

[0320] Transfection was performed using AgilePulse technology. Cells were placed in an incubator set at 37°C and 5% CO2 for 15 minutes, following which the cells were pelleted and resuspended in 200 uL of X-vivo 15 medium supplemented with 5% AB serum, 20 ng / ml IL-2. Cells were transduced with 50,000 genomes / cell with AAV6 carrying polynucleotide sequences encoding the IL-12 cytokine and LNGFR reporter gene separated by the self-cleaving peptide T2A.

[0321] On day 8, T cells were transferred to the GRex device for expansion. T cells were expanded in the GRex device between days 8 and 18. When using GRex10 or GRex6 multi-well cell culture plates, 75% of the culture medium was removed on day 13 and replaced with fresh medium containing IL-2, and fresh IL-2 was added on days 11 and 15. During the expansion period, the cell cultures were incubated at 37° C. under 5% CO2.

[0322] On day 18, whole UCART cells were cryopreserved for use in subsequent in vitro and in vivo assays.

[0323] Next generation MUC-1 CAR T cells [dnTGFBR2] pos [TCR] neg [B2M] neg [HLA-E] pos [PD1] neg [IL-12 / LNGFR] pos To modify (Figure 5), the following protocol was used.

[0324] On day 0, frozen human peripheral blood mononuclear cells (PBMCs) from AllCells (Alameda, California 94502) were thawed, washed, counted, and resuspended in X-vivo 15 medium (Lonza: 04-418Q) supplemented with 5% AB serum (GeminiBio: 100-318) and 20 ng / mL recombinant human IL-2 (Miltenyi: 130-097-743). The cells were then transferred to an incubator set at 37° C., 5% CO2.

[0325] On day 1, PBMCs were counted, analyzed by flow cytometry to assess % CD3+ cells, centrifuged, and resuspended in X-vivo 15 medium supplemented with 5% AB serum, 20 ng / mL human IL-2, and Dynabeads Human T activator CD3 CD28 (ThermoFisher #11161D: 25 μl per million CD3+ cells). Cells were then transferred to an incubator set at 37° C., 5% CO2.

[0326] On day 3, T cells and rLV vector carrying a polynucleotide sequence encoding the anti-MUC1 CAR expressed from a bicistronic construct with dnTGFBR2 were resuspended in X-vivo 15 medium supplemented with 5% AB serum and 20 ng / ml IL-2 and plated onto retronectin-coated plates. Plates were then transferred to an incubator set at 37° C. and 5% CO2.

[0327] On day 5, T cells were co-electroporated with mRNAs encoding the right and left arms of the TRAC TALEN (SEQ ID NO:75 and 76) and B2M TALEN (SEQ ID NO:79 and 80), respectively, according to previously reported protocols [Poirot et al. (2013) Blood. 122 (21): 1661 and Sachdeva et al. (2019) Nat Commun. 10 (1)], and 30 min later, transduced with HLA-E AAV6 (SEQ ID NO:85) to efficiently inactivate the TCRα and B2M genes and prevent TCRαβ expression on the surface of primary T cells. TALEN® is the registered name for heterodimeric TALE nucleases designed by Cellectis (8, rue de la Croix Jarry, 75013 Paris, France) that use Fok1 as the nuclease catalytic domain as first described by Voytas et al. in WO2011072246.

[0328] On day 6, the modified T cells were passaged into fresh X-vivo 15 medium supplemented with 5% AB serum and 20 ng / ml IL-2.

[0329] On day 7, T cells were electroporated with mRNA encoding the right and left arms of the PD-1 TALEN (SEQ ID NO:77 and 78), respectively, according to previously reported protocols [Poirot et al. (2013) Blood. 122 (21): 1661 and Sachdeva et al. (2019) Nat Commun. 10 (1)] and 30 min later transduced with IL-12-LNGFR AAV6 (SEQ ID NO:84) as previously reported.

[0330] Transfection was performed using AgilePulse technology. Cells were placed in an incubator set at 37°C and 5% CO2 for 15 minutes, following which the cells were pelleted and resuspended in 200 uL of X-vivo 15 medium supplemented with 5% AB serum, 20 ng / ml IL-2. Cells were transduced at 50,000 genomes / cell on day 5 with AAV6 carrying polynucleotide sequences encoding HLA-E or on day 7 with AAV6 carrying polynucleotide sequences encoding the IL-12 cytokine and LNGFR reporter gene separated by the self-cleaving peptide T2A. After transduction, cells were transferred to an incubator set at 30°C and 5% CO2 and incubated overnight.

[0331] On day 8, T cells were transferred to the GRex device for expansion. T cells were expanded in the GRex device between days 8 and 18. When using GRex10 or GRex6 multi-well cell culture plates, 75% of the culture medium was removed on day 13 and replaced with fresh medium containing IL-2, and fresh IL-2 was added on days 11 and 15. During the expansion period, the cell cultures were incubated at 37° C. under 5% CO2.

[0332] On day 18, whole UCART cells were cryopreserved for use in subsequent in vitro and in vivo assays.

[0333] Next generation MUC-1 CAR T cells [TGFBR2] neg [TCR] neg [B2M] neg [HLA-E] pos [PD1] neg [IL12 / LNGFR] pos To modify (Figure 17), the following protocol was used.

[0334] On day 0, frozen human peripheral blood mononuclear cells (PBMCs) from AllCells (Alameda, California 94502) were thawed, washed, counted, and resuspended in X-vivo 15 medium (Lonza: 04-418Q) supplemented with 5% AB serum (GeminiBio: 100-318) and 20 ng / mL recombinant human IL-2 (Miltenyi: 130-097-743). The cells were then transferred to an incubator set at 37° C., 5% CO2.

[0335] On day 1, PBMCs were counted, analyzed by flow cytometry to assess % CD3+ cells, centrifuged, and resuspended in X-vivo 15 medium supplemented with 5% AB serum, 20 ng / mL human IL-2, and Dynabeads Human T activator CD3 / CD28 (ThermoFisher #11161D: 25 μl per million CD3+ cells). Cells were then transferred to an incubator set at 37° C., 5% CO2.

[0336] On day 2, T cells and rLV vectors carrying polynucleotide sequences encoding anti-MUC1 CAR were resuspended in X-vivo 15 medium supplemented with 5% AB serum and 20 ng / ml IL-2 and plated onto retronectin-coated plates. Plates were then transferred to an incubator set at 37° C. and 5% CO2.

[0337] On day 3, T cells were passaged in fresh X-vivo 15 medium supplemented with 5% AB serum, 20 ng / ml IL-2. If CAR-T cells were modified with additional properties (TCR and B2M knockout, and HLA-E), cells were passaged at 1e6 cells / mL in X-vivo 15 medium supplemented with 5% AB serum, 20 ng / ml IL-2 in the morning. Six hours later, cells were co-electroporated with mRNAs encoding the right and left arms of TRAC TALEN (SEQ ID NOs: 75 and 76) and B2M TALEN (SEQ ID NOs: 79 and 80), respectively, to efficiently inactivate TCRα and B2M genes and prevent TCRαβ expression on the surface of primary T cells, as previously reported [Poirot et al. (2013) Blood. 122 (21): 1661 and Sachdeva et al. (2019) Nat Commun. 10 (1)]. TALEN is the registered name for heterodimeric TALE nucleases designed by Cellectis (8, rue de la Croix Jarry, 75013 Paris, France) that use Fok1 as the nuclease catalytic domain as originally described by Voytas et al. in WO2011072246.

[0338] Transfection was performed using AgilePulse technology. Cells were placed in an incubator set at 37°C and 5% CO2 for 15 minutes, following which the cells were pelleted and resuspended in 200 uL of X-vivo 15 medium supplemented with 5% AB serum, 20 ng / ml IL-2. Cells were transduced with 50,000 genomes / cell with AAV6 carrying a polynucleotide sequence encoding the HLA-E gene separated by the self-cleaving peptide T2A.

[0339] On day 7, T cells were further modified with additional properties (TGFBR2 and PD1 KO, and IL12 expression). T cells were passaged at 1e6 cells / mL in X-vivo 15 medium supplemented with 5% AB serum, 20 ng / ml IL-2. Six hours later, cells were co-electroporated with mRNAs encoding the left and right arms of PD1 TALEN (SEQ ID NOs: 77 and 78) and TGFBR2 TALEN (SEQ ID NOs: 223 and 224), as previously reported, to efficiently inactivate TGFBR2 and PD1 genes and enable specific IL-12 expression upon MUC1 recognition and T cell activation.

[0340] On day 9, the T cells were transferred to a GRex device for expansion. Between days 9 and 18, the T cells were expanded in the GRex device at 37° C. under 5% CO2 with occasional medium changes.

[0341] On day 18, whole UCART cells were cryopreserved for use in subsequent in vitro and in vivo assays.

[0342] To analyze the on-target and potential off-target sites in the genome that arise simultaneously from the co-transfection of several TALENs, we used an oligo-capture assay coupled with next-generation sequencing adapted from a previous assay (Tsai et al. 2015). Only on-targets exhibited high scores, which indicates that any off-targets were not UCART MUC1-A [dnTGFBR2]. pos [TCR] neg [B2M] neg [HLA-E] pos [PD1] neg [IL-12 / LNGFR] pos These results suggest that the combination of TRAC, B2M, and PD1 TALENs used to generate the cells was undetectable (Figure 9).

[0343] Table 14: Polynucleotide and polypeptide sequences used to introduce genetic traits into UCART MUC1 TIFF2024517713000022.tif82156TIFF2024517713000023.tif239156TIFF2024517713000024.tif233156TIFF20245177130 00025.tif236156TIFF2024517713000026.tif238156TIFF2024517713000027.tif238156TIFF2024517713000028.tif39156

[0344] Detection of CAR expression: The different UCART cell products of CLS MUC1-A, CLS MUC1-B, CLS MUC1-C, and CLS MUC1-D were then evaluated in vitro. CAR surface expression was assessed by flow cytometry using either Biotin-SP conjugated goat anti-mouse F(ab)2 fragment-specific antibodies to detect the mouse F(ab)2 fragment of the CAR construct or Biotin-SP conjugated goat anti-human F(ab)2 fragment-specific antibodies to detect the human F(ab)2 fragment of the CAR ScFv, or Alexa Fluor-488 conjugated rituximab, which recognizes the R2 suicide switch portion of all CARs.

[0345] Killing activity assay MUC1 UCART cells with CLS MUC1-A, CLS MUC1-B, CLS MUC1-C, and CLS MUC1-D CARs were generated and CAR expression was tested as described above. On day 0, target cells, either T47D-NanoLuc-GFP or HCC70-NanoLuc-GFP, were plated in flat-bottom 96-well plates at a density of 10,000 target cells per well. Cells were plated in their respective complete medium. On day 1, medium was removed from the target cells and CLS MUC1-A, CLS MUC1-B, CLS MUC1-C, and CLS MUC1-D, or non-transduced (NTD) UCART cells were added to the target cells at a CAR+effector to target (E:T) ratio of 5:1, 2.5:1, or 1:1 in X-Vivo medium supplemented with 5% AB serum. Target cells were co-cultured with CAR or NTD control for 48 hours in an incubator set at 37°C, 5% CO2. NanoLuciferase signal was then developed. After removal of medium, wells were washed once with 100 uL PBS and then incubated with 0.026% Triton X-100 in PBS for 2 minutes with vortexing. A total of 10 uL of lysate was diluted in 40 uL PBS, mixed with 50 uL Nano-Glo substrate and incubated for 3 minutes. Luminescence was read after incubation. Percent specific lysis was calculated using the following formula: lysis%=(1-(target+scFV UCART) / (target+NTD UCART))*100.

[0346] The results are shown in the diagram in Figure 3, which shows the % lysis for each of the four anti-MUC1 CAR-Ts at different ratios of 5:1, 2.5:1, or 1:1. The diagram shows the killing activity of primary T cells equipped with each of the four CAR constructs in a concentration-dependent manner. The killing activity induced by the CLS MUC1-A CAR construct was significantly higher than the other constructs.

[0347] Tumor microarrays for detection of MUC1 expression in breast cancer tumors Proteins expressing CLS MUC1-A, CLS MUC1-C, and CLS MUC1-D scFVs bound to CD8 hinge and mouse IgG1 Fc were produced in CHO cells and purified using protein A. Human paraffin-embedded tissue microarrays (TMAs) containing 84 breast cancer samples were purchased from US Biomax. scFV proteins were used for immunohistochemistry assays. TMAs were dewaxed in successive baths of xylene, 100% ethanol, 96% ethanol, 70% ethanol, and water. Slides were then washed with reaction buffer and stained on a Discovery XT2 instrument for automated staining. Slides were first incubated with reaction buffer for 32 minutes, then incubated with primary antibodies at 3 ug / ml for CAR CLS MUC1-A and CLS MUC1-C, and 9 ug / ml for CAR MUC1-D, at 37C for 60 minutes. Anti-mouse HRP was then applied for 16 minutes and the sections were stained with hematoxylin and bluing reagent. Slides were then washed with soapy water and tap water and finally mounted and analyzed under a microscope. Specific positive staining was graded as follows: grade 1: minimal; grade 2: slight; grade 3: moderate; grade 4: marked; grade 5: strong. Slides were digitized using a Leica hole scanning device AT2 (Figure 19).

[0348] Example 2: In vivo anti-MUC1 CAR activity Rationale for choosing an animal model Due to the human specificity of MUC-1 CAR+ T cells, studies in standard immunocompetent animal models are not applicable due to the rapid targeting and elimination of human T cells by xenogeneic immune responses. The animal model chosen is the highly immunodeficient NSG mouse strain (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ strain from the Jackson laboratory) because it allows the engraftment of both human MUC1+ tumor cells and human CAR T cells.

[0349] Validation of genetic characterization and TCR ablation of UCART MUC1 cells Less than 20% of UCART cells generated according to the protocol described in Example 1 and modified with the CLS MUC1-A CAR remained TCRαβ+, suggesting that the level of TALEN-mediated inactivation of the TCRα gene was highly efficient in the population of cells.

[0350] The remaining TCRαβ+ cells were removed on day 18 of CAR-T cell generation. UCART MUC1 cells were counted, pelleted, and resuspended in PBS containing 0.5% heat-inactivated FBS at a density of 10e8 cells / mL. The cells were then incubated with anti-TCRα / β-biotin antibody (Miltenyi CliniMACS TCRα / β Kit: 200-070-407) at a concentration of 1.875 uL of antibody per 10e7 cells for 30 minutes at 4C. After incubation, the cells were washed with 5 volumes of PBS containing 0.5% FBS, resuspended at 10e8 cells / mL, and incubated with 3.75 uL of anti-biotin beads (Miltenyi CliniMACS TCRα / β Kit: 200-070-407) per 10e7 cells for 30 minutes at 4C. After incubation, cells were washed as before, resuspended at 1.25e8 cells / mL, and passed through a pre-equilibrated LS column. The flow-through containing TCRαβ- cells was collected with five 1 mL washes. Removal of TCRαβ+ cells was analyzed by flow cytometry by staining cells with anti-TCRαβ-PE-Vio770 antibody (Miltenyi: 130-119-617). The efficiency of PD-1 gene inactivation and IL-12 release was tested by direct comparison of cell surface expression of PD-1 and LNGFR reporters on CAR-T cells activated with PMA / ionomycin (40 ng / mL PMA and 2 nM ionomycin) for 24 hours. After activation, cell surface expression of PD-1 and LNGFR was tested by flow cytometry by staining cells with anti-PD-1 or anti-LNGFR antibodies. The editing efficiency of CLS MUC1-A and signature-modified UCART MUC1 was calculated to be 50% PD-1 knockout and 4.7% LNGFR+. Similar values ​​were achieved for NTD control cells. UCART MUC1 cells modified with the signature-free CLS MUC1-A CAR expressed high levels of PD-1 (68%) and no LNGFR.

[0351] Engraftment of HCC70 human tumor cells in NSG mice NSG mice were purchased at 6 weeks of age and shaved one week prior to transplantation. Wild-type HCC70 (ATCC® CRL-2315 (商標) ) cells were cultured according to the supplier's recommendations. On the day of implantation, 80-90% confluent monolayers of HCC70 cells were washed with PBS and then incubated with Accutase Cell Dissociation Regent in an incubator set at 37°C, 5% CO2. Accutase digestion was terminated by adding one volume equivalent of complete growth medium. Cells were pelleted at 300g for 5 minutes, resuspended in 30 mL of PBS, and counted using Vi-cell. Counted cells were pelleted in a 50 mL conical tube at 300g for 5 minutes at 4°C. Pelleted cells were incubated on ice for 5 minutes and then resuspended in a 1:1 mixture of Matrigel (Corning: 356237) and PBS at a density of 100e6 cells / mL or 50e6 cells / mL. On day 0, 100 uL of HCC70 cell suspension was implanted into the mammary fat pad of NSG mice for a total of 10e6 or 5e6 HCC70 cells per mouse. Tumor volumes were measured weekly for 19 days. Good engraftment of HCC70 was observed.

[0352] Tumor implantation, CAR-T treatment, and tumor monitoring As summarized in Figure 12, 100 μL of HCC70-NanoLuc-GFP cells at a density of 100e6 cells / mL were injected into the mammary fat pad of 6-week-old NSG strain mice. On day 0, mice were injected with 10e6 CAR+ UCART CLS MUC1-A and non-transduced matched T cells (NDT) control, respectively. Animals were assigned to each treatment group such that the mean tumor volume per group was most similar. Tumor volumes were measured weekly. Tumors with a tumor volume of 2000 mm were not included. 3 When the tumor volume exceeded 100 mg / kg / day or the tumor ulcerated, the animals were sacrificed.

[0353] Figure 11 and Figure 13 show the results of experiments in which treatment began 7 days after mammary fat transfer or subcutaneous tumor implantation, respectively, and tumor volume was followed over the indicated days. It can be observed that mice treated with T cells equipped with the CLS MUC1-A CAR were tumor-free by about day 28, while controls experienced an exponential increase in the volume of the same tumors.

[0354] Engineered CAR-T cells for in vivo infusion: UCART cells were modified to transduce CLS MUC1-A CAR with or without characteristics and comparisons were performed (CAR-T CLS MUC1-A or UCART CLS MUC1-A+ characteristics). Characteristics consisted of TCR knockout, PD-1 knockout, and IL-12 release. Similarly, non-transduced controls (NTD: CAR negative control T cells) were generated with or without characteristics (NTD and NDT+ characteristics). CAR-T cells were generated as above and infused fresh from generation without prior freezing. Minor modifications to the generation were performed. On day 18 of CAR-T cell generation, cells were removed from G-rex, resuspended at 2e6 cells / mL, and seeded into regular tissue culture flasks. On day 20 of generation, cells were washed once with PBS, counted, and concentrated to 100e6 CAR+ cells / mL. A total of 100 uL of UCART product or control was injected intravenously.

[0355] UCART cells were transduced with CLS MUC1-A or CLS MUC-1 C and further modified for TCR KO, PD1-KO, and IL-12 integration. CAR-T cells were generated as described above. Modified T cells were infused fresh from generation with either 10e6 or 3e6 CAR+ UCARTMUC1-A or UCARTMUC1-C, or 3e6 or 10e6 total non-transduced T cell (NTD) control (from the same donor) or PBS. Tumor volumes were measured weekly after treatment with CLS MUC1-A or CLS MUC1-C modified CAR-T cells. Tumor volumes were measured once a week ...s with tumor volumes of 2000 mm 3Animals were sacrificed when the tumors exceeded 100% or ulcerated (Figure 18A). The results demonstrate that UCART with CLS MUC1-A was able to prevent tumor growth at 3 million injected cells and even at 10 million cells, providing the best survival (Figure 18B and C).

[0356] FACS analysis of tumor samples treated with UCARTMUC1+ / - characteristics Tumors were isolated from mice treated as described above with CLS MUC1-A transduced CAR-T cells with or without characteristics (PD-1 and TCR knockout, and IL-12 release) or NTD cells (with or without characteristics). Tumors were homogenized in Accutase Cell Dissociation Reagent and transferred to an incubator set at 37°C, 5% CO2 for 30 minutes. Dissociated cells were washed with 20 mL ice-cold PBS containing 3% FBS and passed through a 100 um mesh. Tumor isolates were counted on vi-cell and stained for FACS analysis.

[0357] To examine the infiltration of CAR-T cells in tumors and the expression of MUC1 on tumor cells and to determine whether there was a loss of MUC1 expression indicative of antigen escape, cells were stained with the fixable viability dye e780 to gate on viable cells, with anti-human HLA-ABC-VioBlue and anti-mouse MHCII-PE to gate on human cells, and with anti-human CD45 and anti-human EpCAM to gate on epithelial tumor cells, and finally with anti-MUC1 (HMFG2 or 16A) antibodies or their corresponding isotype controls to determine MUC1 expression on tumors isolated from individual mice.

[0358] As depicted in Figure 10 and Figure 14, FACS data was analyzed in FlowJo and values ​​corresponding to the percentage of MUC1 positive cells among viable human epithelial cells from each animal were exported to Excell and plotted as the average for each treatment group. For each tumor isolate, the frequency of CAR-T cells (hCD45+) and tumor cells (hEpCAM+) among all viable human cells (viability e780-, hHLA-ABC+) was plotted both as individual data points and as the average for each treatment group.

[0359] The results showed that CAR-T cells engineered with the characteristics (PD1 / TRAC knockout and IL-12 release) were found at a much higher frequency in HCC70 tumors than CAR-T cells without the characteristics.

[0360] These results suggest that the addition of properties enhances tumor infiltration and / or expansion of intratumoral CAR-T cells. Enhanced tumor infiltration supports the observed anti-tumor response, consistent with the results previously observed in FIG. 12.

[0361] Overall, these results suggest that the addition of properties is significant in achieving complete responses in the treatment of solid tumors.

[0362] Tumors from mice treated with CLS MUC1-A or CLS MUC1-C modified CART cells were isolated from the mice on day 54 (Figure 18A), homogenized in Accutase Cell Dissociation Reagent, and transferred to an incubator set at 37°C and 5% CO2 for 30 minutes. Dissociated cells were washed with 20 mL of ice-cold PBS containing 3% FBS and passed 30 times through a 100 um mesh. Tumor isolates were counted on vi-cell and stained for FACS analysis. To examine CAR-T cell infiltration in the tumor, viable cells were identified using the fixable viability dye e780, and human CAR T cells were identified with human and mouse anti-CD45 antibodies. Both MUC-1A and MUC1-C modified CAR-T cells could be detected in tumors 54 days after challenge (Figure 18D), with a mean value of >40% for 10e6 injected CLS MUC1-A modified CAR-T cells.

[0363] In the hostile tumor microenvironment, the expansion, infiltration and effector function of CAR-T cells may be inhibited by a myriad of intratumoral factors.Thus, even the most active CARs can hardly achieve clinical anti-tumor responses without additional features.Herein, we demonstrate that some of the tumor microenvironment barriers can be successfully eliminated through a combination of features, such as PD1 knockout, which limits CAR-T cell exhaustion, combined with inducible IL-12 release, which enhances T cell efficacy.The features included in the present invention can synergize with the strategy of increasing efficacy in developing successful lines of CAR-T cells against solid tumors.

[0364] The above results also demonstrate that engineering MUC1 CAR T cells is a promising approach to enhance efficacy and improve allogeneic persistence in the setting of solid tumors, particularly for targeting triple-negative breast cancer.

Claims

1. A method for producing a population of modified therapeutic immune cells, comprising the steps of: a) providing immune cells originating from a patient, or preferably a donor; b) expressing an anti-MUC1 chimeric antigen receptor (CAR) in the cell; c) introducing at least one genetic modification into the genome of the cell, the modification comprising: Enhancement of expression of IL-2, IL-12, IL-15, or IL-18 a step selected from those which result in d) expanding the cells to form a therapeutically effective immune cell population.

2. - Reduction or inactivation of TCR expression; - Reduction or inactivation of B2M; - reducing the interaction between TGFβ and TGFβR2; and / or - Reducing the interaction between PD1 and PDL1; The method of claim 1, further comprising introducing a further genetic modification into the genome, which results in:

3. The method according to claim 2, wherein said further genetic modification results in inactivation of expression of at least one TGF beta receptor, preferably TGFβRII.

4. The method of any one of claims 1 to 3, wherein the genetic modification is obtained by using a sequence-specific gene editing reagent, such as a rare-cutting endonuclease / nickase or a base editor.

5. A modified immune cell expressing an anti-MUC1 CAR, obtainable by the method of claim 1.

6. 1. An engineered immune cell expressing an anti-MUC1 CAR, the anti-MUC1 CAR comprising: - an extracellular ligand-binding domain comprising a VH and a VL derived from a monoclonal antibody targeting the tMUC1 epitope; - a transmembrane domain; and - A cytoplasmic domain containing the CD3 zeta signaling domain, and a costimulatory domain At least the extracellular ligand-binding domain is directed against an antigen in the MUC1 polypeptide region HGVTSAPDTRPAPGSTAPPA (SEQ ID NO:1); The CAR is co-expressed with another exogenous sequence encoding a cytokine selected from IL-2, IL-12, IL-15, and IL-18. The modified immune cell.

7. introduced into the genome, - Reduction or inactivation of TCR expression; - Reduction or inactivation of B2M; - reducing the interaction between TGFβ and TGFβR2; and / or - Reducing the interaction between PD1 and PDL1; The modified immune cell of claim 6, comprising at least one further genetic modification that results in:

8. 7. The modified immune cell of claim 6, wherein the anti-MUC1 comprises an extracellular ligand-binding domain ScFv having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to MUC1-A ScFv (SEQ ID NO:17), MUC1-B (SEQ ID NO:27), MUC1-C (SEQ ID NO:37), and / or MUC1-D (SEQ ID NO:47), respectively.

9. the variable light (VL) chain comprises CDRs having at least 90% identity with SEQ ID NO:11 (CDR-VL1-A), SEQ ID NO:12 (CDR-VL2-A), and SEQ ID NO:13 (CDR-VL3-A), respectively; and the variable heavy (VH) chain comprises CDRs having at least 90% identity with SEQ ID NO:14 (CDR-VH1-A), SEQ ID NO:15 (CDR-VH2-A), and SEQ ID NO:16 (CDR-VH3-A), respectively; The modified immune cell of claim 6.

10. 7. The modified immune cell of claim 6, wherein the anti-MUC1 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 to SEQ ID NO:18 (CLS MUC1-A CAR).

11. the variable light (VL) chain comprises CDRs selected from SEQ ID NO:21 (CDR-VL1-B), SEQ ID NO:22 (CDR-VL2-B), and SEQ ID NO:23 (CDR-VL3-B); and the variable heavy (VH) chain comprises CDRs selected from SEQ ID NO:24 (CDR-VH1-B), SEQ ID NO:25 (CDR-VH2-B), and SEQ ID NO:26 (CDR-VH3-B); The modified immune cell of claim 6.

12. 7. The modified immune cell of claim 6, wherein the anti-MUC1 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 to SEQ ID NO:28 (CLS MUC1-B CAR).

13. the variable light (VL) chain comprises CDRs selected from SEQ ID NO:31 (CDR-VL1-C), SEQ ID NO:32 (CDR-VL2-C), and SEQ ID NO:33 (CDR-VL3-C); and the variable heavy (VH) chain comprises CDRs selected from SEQ ID NO:34 (CDR-VH1-C), SEQ ID NO:35 (CDR-VH2-C), and SEQ ID NO:36 (CDR-VH3-C); The modified immune cell of claim 6.

14. 7. The modified immune cell of claim 6, wherein the CAR 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 to SEQ ID NO:38 (CLS MUC1-C CAR).

15. the variable light (VL) chain comprises CDRs selected from SEQ ID NO:41 (CDR-VL1-D), SEQ ID NO:42 (CDR-VL2-D), and SEQ ID NO:43 (CDR-VL3-D); and the variable heavy (VH) chain comprises CDRs selected from SEQ ID NO:44 (CDR-VH1-D), SEQ ID NO:45 (CDR-VH2-D), and SEQ ID NO:46 (CDR-VH3-D); The modified immune cell of claim 6.

16. The modified immune cell of claim 6, wherein the CAR 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 to SEQ ID NO:48 (MUC1-D CAR).

17. The modified immune cell of claim 6 , which is a T cell or a NK cell.

18. A population of cells comprising more than 25%, preferably more than 50%, more preferably more than 75%, even more preferably more than 80% modified immune cells according to any one of claims 5 to 17.

19. A therapeutic composition comprising the modified immune cell of any one of claims 5 to 17.

20. 20. The therapeutic composition of claim 19 for use in the treatment of a solid tumor, preferably a cancer condition selected from esophageal cancer, breast cancer, gastric cancer, bile duct cancer, pancreatic cancer, colon cancer, lung cancer, thymic cancer, mesothelioma, ovarian cancer, and endometrial cancer, more particularly breast cancer.

Images