Chimeric antigen receptor-modified cells for treatment of CLDN6-expressing cancers

Abstract

To provide compositions and methods effective for treatment of cancer using CARs.SOLUTION: The present disclosure relates to a chimeric antigen receptor (CAR) showing highly specific and sensitive recognition of CLDN6 expressing target cells as well as a high potential for survival and repetitive stimulation of CAR T cells. The present disclosure provides a chimeric antigen receptor (CAR) molecule comprising: i) a CLDN6 antigen binding domain; ii) a transmembrane domain; and iii) an intracellular domain that comprises a 4-1BB costimulatory domain, and a CD3-ζ signaling domain.SELECTED DRAWING: None

Description

[Background technology]

[0001] Immunotherapy based on adoptive cell transfer (ACT) can be broadly defined as a form of passive immunization with previously primed T cells that are expanded ex vivo from low precursor cell frequencies to clinically relevant cell numbers and then transferred into non-immune recipients or autologous hosts. The use of genetic engineering approaches to insert antigen-targeting receptors of defined specificity into T cells has greatly expanded the potential of ACT. Chimeric antigen receptors (CARs) are a type of antigen-targeting receptor that consists of an intracellular T cell signaling domain fused to an extracellular antigen-binding domain, most commonly a single-chain variable fragment (scFv) from a monoclonal antibody. CARs directly recognize cell surface antigens, independent of MHC-mediated presentation.

[0002] Attempts to treat cancer by using genetically modified T cells to target antigens expressed on tumor cells via expression of CARs have met with very limited success. Despite significant responses in patients with B-cell malignancies, successful clinical responses following targeting of solid tumors using CAR T cells with various specificities have been much more limited.

[0003] CAR T cell therapy for solid tumors faces many challenges, including physical barriers, an immunosuppressive tumor microenvironment, and importantly, the lack of truly specific and safe tumor targets. Summary of the Invention [Problem to be solved by the invention]

[0004] Thus, there is an urgent need in the art for effective compositions and methods for treating cancer using CARs. The present invention addresses this need.

[0005] To implement CAR-based therapy for the treatment of solid cancers, we selected the carcinoembryonic antigen CLDN6 (claudin 6), which has all the characteristics of an ideal target for CAR-based therapy. CLDN6 is a tetraspin membrane protein involved in the formation of primitive tight junctions during organogenesis, and is therefore expressed at significant levels exclusively during fetal development, absent in adult healthy tissues, but highly overexpressed in a variety of cancers of high medical need, including ovarian, endometrial, testicular and lung cancers.

[0006] CLDN6 has a druggable extracellular loop and cell surface levels are high enough to allow recognition by CAR T cells. Furthermore, CLDN6 expression correlates with disease progression, as it can be detected at higher frequencies in metastatic lesions and dedifferentiated cells, suggesting a role in the oncogenic process. The safety of CLDN6 targeting is suggested by a clinical phase I / II study (OVAR, NCT02054351) using the anti-CLDN6 monoclonal antibody IMAB027 in patients with advanced ovarian cancer, in which no IMAB027-related adverse events were detected.

[0007] Based on the results of in vitro and in vivo experiments, we selected a second-generation CAR with 4-1BB domains (CLDN6-CAR-CD8h-BBz) as a lead structure for preclinical and clinical trials. We were able to demonstrate highly specific and sensitive recognition of target cells expressing CLDN6, as well as high probability of survival and repeated stimulation of CAR T cells.

[0008] We further evaluated the in vivo antitumor potential of CLDN6-CAR using an ovarian cancer xenograft model and were able to demonstrate that adoptive transfer of CLDN6-CAR-transduced T cells resulted in complete eradication of advanced tumors. Furthermore, we were able to reproduce these results using cryopreserved CAR T cells generated in a GMP facility.

[0009] As it could be demonstrated previously, the clinical outcome of CAR T cell therapy positively correlates with the persistence of the CAR T cells infused in the body (Robbins et al. (2004) J Immunol. 173(12):7125-30; Huang et al. (2005) 28(3):258-67), we combined CLDN6-CAR therapy with our innovative CAR in vivo expansion concept (WO 2016 / 180778) using liposomally formulated mRNA encoding the CAR antigen.

[0010] Finally, we demonstrate the efficacy and safety of adoptively transferred CAR T cells and RNA in various tumor models. (LIP) It could be demonstrated that combination with a CAR T cell-based vaccination can accelerate ongoing antitumor responses and also restore the antitumor effect of insufficient CAR T cell doses. [Means for solving the problem]

[0011] The present invention relates to i) a CLDN6 antigen-binding domain; ii) a transmembrane domain; and iii) an intracellular domain containing the 4-1BB costimulatory domain and the CD3ζ signaling domain The present invention provides a chimeric antigen receptor (CAR) molecule comprising:

[0012] In one embodiment, the CLDN6 antigen binding domain comprises an antibody, an antibody fragment, an scFv, an Fv, a Fab, a (Fab')2, a single domain antibody (SDAB), a VH or VL domain, or a camelid VHH domain. In one embodiment, the CLDN6 antigen binding domain comprises an scFv. In one embodiment, the CLDN6 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 35 or a functional variant thereof.

[0013] In one embodiment, the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 30 or a functional variant thereof. In one embodiment, the CAR molecule of the invention does not comprise an additional costimulatory domain.

[0014] In one embodiment, the CD3 zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 31, or a functional variant thereof.

[0015] In one embodiment, the transmembrane domain is selected from the group consisting of the alpha, beta or zeta chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19 , IL2Rβ, IL2Rγ, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAMl(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The present invention includes a transmembrane domain of a protein selected from the group consisting of AM, Ly9 (CD229), CD160 (BY55), PSGLl, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C, or a functional variant thereof.

[0016] In one embodiment, the transmembrane domain comprises a CD8α transmembrane domain. In one embodiment, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 28 or a functional variant thereof.

[0017] In one embodiment, the antigen-binding domain is connected to the transmembrane domain by a hinge domain. In one embodiment, the hinge domain is a CD8α hinge domain. In one embodiment, the hinge domain comprises the amino acid sequence of SEQ ID NO: 27 or a functional variant thereof.

[0018] In one embodiment, the CAR molecule of the invention comprises: i) a CLDN6 antigen-binding domain; ii) the CD8α hinge domain; iii) the CD8α transmembrane domain; and iv) an intracellular domain containing the 4-1BB costimulatory domain and the CD3ζ signaling domain Includes.

[0019] In one embodiment, the CAR molecule of the invention further comprises a leader sequence.

[0020] In one embodiment, the CAR molecule of the present invention comprises the amino acid sequence of SEQ ID NO: 36 or a functional variant thereof.

[0021] The present invention further provides a nucleic acid encoding the CAR molecule of the present invention. In one embodiment, the nucleic acid is DNA or RNA.

[0022] The present invention further provides a vector comprising the nucleic acid of the present invention. In one embodiment, the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, and a retroviral vector. In one embodiment, the vector further comprises a promoter. In one embodiment, the promoter is selected from an EF-1 promoter, a CMV IE gene promoter, an EF-1α promoter, a ubiquitin C promoter, or a phosphoglycerate kinase (PGK) promoter.

[0023] The present invention further comprises: a CAR molecule of the present invention; A nucleic acid of the invention; or Vectors of the Invention The present invention provides an immune effector cell comprising:

[0024] In one embodiment, the immune effector cell is genetically modified to express the CAR. In one embodiment, the immune effector cell is selected from the group consisting of T cells, natural killer (NK) cells, and cytotoxic T lymphocytes (CTLs). In one embodiment, the immune effector cell is a CD8+ T cell. In one embodiment, the immune effector cell is a human cell.

[0025] The present invention further provides a population of immune effector cells comprising a plurality of the immune effector cells of the present invention.

[0026] In one embodiment of the immune effector cells of the invention or population of immune effector cells of the invention, the cells lack or have low expression of a functional TCR or a functional HLA.

[0027] The present invention further provides a method of producing an immune effector cell or a population of immune effector cells comprising introducing a nucleic acid of the invention or a vector of the invention into an immune effector cell under conditions such that the CAR molecule is expressed.

[0028] The present invention further provides a method for stimulating a cell-mediated immune response against a target cell population or tissue expressing CLDN6 in a subject, the method comprising providing an effective amount of an immune effector cell of the present invention or a population of immune effector cells of the present invention to the subject. The immune effector cell or population of immune effector cells can be generated ex vivo and administered to the subject, or the immune effector cell or population of immune effector cells can be generated in the subject.

[0029] The present invention further provides a method for treating a subject having a disease associated with expression of CLDN6, comprising providing an effective amount of the immune effector cells of the present invention or a population of immune effector cells of the present invention to the subject. The immune effector cells or population of immune effector cells can be generated ex vivo and administered to the subject, or the immune effector cells or population of immune effector cells can be generated in the subject. In one embodiment, the disease associated with expression of CLDN6 is selected from the group consisting of proliferative diseases, precancerous conditions, cancer, and non-cancer-related indications associated with expression of CLDN6. In one embodiment, the cancer is selected from the group consisting of ovarian cancer, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, melanoma, head and neck cancer, sarcoma, cholangiocarcinoma, renal cell carcinoma, and bladder cancer.

[0030] The present invention further provides a method for providing anti-tumor immunity in a subject, comprising providing an effective amount of the immune effector cell of the present invention or a population of immune effector cells of the present invention to the subject. The immune effector cell or population of immune effector cells can be generated ex vivo and administered to the subject, or the immune effector cell or population of immune effector cells can be generated in the subject. In one embodiment, the tumor is a tumor that expresses CLDN6.

[0031] In one embodiment of the methods of the invention, the immune effector cell or population of immune effector cells is autologous or allogeneic to the subject.

[0032] In one embodiment of the methods of the invention, the method further comprises administering an agent that enhances the effectiveness of an immune effector cell or population of immune effector cells. In one embodiment, the agent is Protein phosphatase inhibitors; Kinase inhibitors; Cytokines; Inhibitors of immune inhibitory molecules; or Agents that reduce the level or activity of TREG cells is selected from one or more of the following:

[0033] In one embodiment of the method of the present invention, the method further comprises contacting the immune effector cell or population of immune effector cells with a cognate antigenic molecule that binds to the CLDN6 antigen binding domain ex vivo or in a subject. In one embodiment, the cognate antigenic molecule is selected from the group consisting of CLDN6 or a fragment thereof, or a mutant of CLDN6 or a CLDN6 fragment. In one embodiment, the immune effector cell or population of immune effector cells is contacted with the cognate antigenic molecule under conditions that result in proliferation and / or activation of the immune effector cell or population of immune effector cells. In one embodiment, the contacting of the immune effector cell or population of immune effector cells with the cognate antigenic molecule is performed in vivo or ex vivo.

[0034] In one embodiment of the method of the invention, the method comprises administering a cognate antigen molecule or a nucleic acid encoding the same to a subject. In one embodiment, the nucleic acid encoding the cognate antigen molecule is expressed in cells of the subject to provide the cognate antigen molecule. In one embodiment, the expression of the cognate antigen molecule is at the cell surface. In one embodiment, the nucleic acid encoding the cognate antigen molecule is transiently expressed in cells of the subject. In one embodiment, the nucleic acid encoding the cognate antigen molecule is RNA. In one embodiment, the immune effector cell or population of immune effector cells and / or the cognate antigen molecule or the nucleic acid encoding the same is administered systemically. In one embodiment, after systemic administration of the nucleic acid encoding the cognate antigen molecule, expression of the nucleic acid encoding the cognate antigen molecule occurs in the spleen. In one embodiment, after systemic administration of the nucleic acid encoding the cognate antigen molecule, expression of the nucleic acid encoding the cognate antigen molecule occurs in antigen presenting cells, preferably professional antigen presenting cells. In one embodiment, the antigen presenting cells are selected from the group consisting of dendritic cells, macrophages and B cells. In one embodiment, after systemic administration of the nucleic acid encoding the cognate antigen molecule, no or essentially no expression of the nucleic acid encoding the cognate antigen molecule occurs in the lung and / or liver. In one embodiment, following systemic administration of nucleic acid encoding a cognate antigenic molecule, expression of the nucleic acid encoding the cognate antigenic molecule in the spleen is at least 5-fold higher than expression in the lung.

[0035] In one embodiment, the nucleic acid encoding the cognate antigen molecule is formulated in a delivery vehicle. In one embodiment, the delivery vehicle comprises a particle. In one embodiment, the delivery vehicle comprises at least one lipid. In one embodiment, the at least one lipid comprises at least one cationic lipid. In one embodiment, the lipid complexes with and / or encapsulates the nucleic acid encoding the cognate antigen molecule. In one embodiment, the lipid is included in a vesicle that encapsulates the nucleic acid encoding the cognate antigen molecule. In one embodiment, the nucleic acid encoding the cognate antigen molecule is formulated in a liposome.

[0036] In one embodiment of the immune effector cell of the invention, the population of immune effector cells of the invention, or the method of the invention, the immune effector cell or population of immune effector cells is a CAR-expressing immune effector cell or a population of immune effector cells expressing a CAR.

[0037] The present invention further provides a CAR molecule of the present invention, a nucleic acid of the present invention, a vector of the present invention, an immune effector cell of the present invention, or a population of immune effector cells of the present invention for use as a medicament.

[0038] The present invention further provides a CAR molecule of the present invention, a nucleic acid of the present invention, a vector of the present invention, an immune effector cell of the present invention, or a population of immune effector cells of the present invention for use in treating a disease expressing CLDN6.

[0039] The present invention further provides a kit comprising the CAR molecule of the present invention, the nucleic acid of the present invention, the vector of the present invention, the immune effector cell of the present invention, or a population of immune effector cells of the present invention. In one embodiment, the kit further comprises a cognate antigen molecule that binds to the CLDN6 antigen binding domain or a nucleic acid encoding the same. In one embodiment, the kit further comprises instructions for using the kit in the method of the present invention.

[0040] In a further aspect, the invention provides agents and compositions, such as the immune effector cells described herein, for use in the methods described herein.

[0041] Other features and advantages of the invention will become apparent from the following detailed description, and from the claims. [Brief description of the drawings]

[0042] [Figure 1] (FIG. 1A) Generation and characterization of various CLDN6-CARs. Four different CLDN6-targeting CARs were designed and generated based on the variable domains of the heavy (VH) and light (VL) chains of the CLDN6-specific antibody IMAB206-C46S. IgG1ΔFc: human IgG1 hinge-CH2-CH3 Fc domain with a mutated IgG FcγR binding site to prevent activation by innate immune cells expressing Fcγ receptors (FcγR) (Hombach A.et al.,(2010) Gene Therapy 17,1206-1213); CD28ΔLck: CD28 transmembrane and cytoplasmic domain with a deletion into the Lck-binding portion of the CD28 endodomain that abolishes IL-2 induction upon CAR engagement to prevent unwanted Treg cell proliferation at the tumor site (Kofler DMet al.,(2011) Molecular Therapy 19(4),760-767); 4-1BB: 4-1BB costimulatory endodomain; CD3ζ: CD3ζ signaling domain; CD3ζ*: CD3ζ with mutation Q14→K; CD8α: human CD8α hinge domain. (Figure 1B) Generation and characterization of various CLDN6-CARs. Figure 1 shows functional testing of various CLDN6-CARs in PA1-SC12-A2-eGFP tumor spheroid assay. Lysis of tumor spheroids was analyzed based on eGFP expression after 24 hours of co-culture (E:T=10:1) using an IncuCyte® live cell imaging system. Images of wells were scanned with a 4x objective at the start of co-culture (0 hours) and 24 hours later, and represent tumor spheroid killing of technical triplicates. [Diagram 2](Figure 2A) Dose-dependent CAR-mediated recognition and lysis of target cells expressing CLDN6. CLDN6-CAR-BBz surface expression was analyzed on transduced T cells after staining with fluorochrome-conjugated IMAB206 idiotype-specific antibody. Non-transduced T cells served as negative control. Cells were gated on single CD4+ or CD8+ lymphocytes. Numbers shown represent frequency (%) of parent population. (Figure 2B) Dose-dependent CAR-mediated recognition and lysis of target cells expressing CLDN6. CLDN6 surface expression on Colo699-N cells transfected with titrated amounts of CLDN6-RNA was analyzed by flow cytometry. (Figure 2C) Dose-dependent CAR-mediated recognition and lysis of target cells expressing CLDN6. Specific lysis of RNA-transfected Colo699-N cells by CLDN6-CAR-BBz-transduced T cells was analyzed after 12 h of coculture at an E:T ratio of 20:1 using an xCELLigence instrument. Data are presented as the mean ± SD of technical triplicates. [Figure 3A] Figure 3 (A-E) shows specific CLDN6-CAR-mediated lysis of tumor cell lines expressing CLDN6. CLDN6-CAR-BBz transduced human T cells were analyzed by flow cytometry and co-cultured with a panel of CLDN6-positive and CLDN6-negative human tumor cell lines of different origins. (Figure 3A) Before initiating the co-culture, CAR surface expression was assessed by flow cytometry. The numbers shown represent the frequency (%) of the parental population. [Figure 3B] An E:T ratio of 10:1 is used. [Figure 3C] Specific lysis was analyzed using the xCELLIgence system after 12 h of co-culture according to the formula % lysis = (CI eGFP-CI effector) / CI eGFP*100; CI: cell index. Data represent the mean ± SD of technical triplicates. [Figure 3D] CLDN6 surface expression on tumor cell lines was analyzed by flow cytometry after staining with a CLDN6-specific antibody. The percentages of the parental population are shown. [Figure 3E]Relative CLDN6 mRNA expression levels in the tumor cell lines used in (A-D), assessed by qRT-PCR, were calculated after normalization to the housekeeping gene HPRT1. Bars represent the mean ± SD of technical triplicates. [Figure 4] Figure 4 (A-C) shows the dose-dependent proliferation mediated by CLDN6-CAR-BBz in response to target cells expressing CLDN6. CAR-transduced T cells were labeled with CFSE and cocultured with autologous DCs transfected with titrated amounts of CLDN6-RNA lipoplexes (RNA(LIP)). Proliferation was analyzed based on CFSE after 5 days of coculture and staining with fluorochrome-conjugated antibodies against CD4, CD8 and CAR. (Figure 4A) CAR surface expression was assessed by flow cytometry before starting the coculture. The numbers shown represent the frequency (%) of the parental population. (Figure 4B) CLDN6 expression on the surface of transfected DCs was assessed by flow cytometry using a fluorochrome-conjugated CLDN6-specific antibody. (Figure 4C) Specific proliferation was analyzed by flow cytometry based on the dilution of the CFSE proliferation dye. The bars indicate the percentage of proliferating CAR-expressing CD8+ and CD4+ T cells. [Diagram 5]Figure 5 (A-D) shows the antitumor activity of CLDN6-CAR-BBz transduced T cells in an advanced ovarian cancer (OV90) xenograft tumor model. 5 × 106 OV90-SC12 tumor cells were implanted subcutaneously (n = 10 mice / group) 25 days prior to adoptive cell transfer (ACT) of a single dose of 1 × 107 iv administered CLDN6-CAR-BBz or eGFP transduced human T cells. At this time point of T cell treatment, mice already showed advanced tumors of 170 mm3 on average. Tumor volumes were measured three times a week using calipers and calculated according to the formula V = 1 / 2 (length × width 2) using the maximum length and width of the tumor. Animals were sacrificed when the tumor volume exceeded 1500 mm3 or when the tumor ulcerated. (Figure 5A) A schematic diagram of the mouse experiments performed is shown. (Figure 5B) Transgene expression (GFP or CAR) of transduced human CD4+ and CD8+ T cells was analyzed by flow cytometry on the day of ACT. Numbers shown represent frequency (%) of parental population. (Figure 5C) Mean tumor volume in animals treated with CLDN6-CAR-BBz and eGFP-T cells up to day 44 after ACT (indicated by dotted line). Data are presented as mean ± SEM of all mice / group. (Figure 5D) T cell persistence and CAR surface expression were analyzed in peripheral blood 3 weeks after ACT using flow cytometry. Representative dot plots are shown. Numbers indicated frequency of parental population. [Figure 6]Figure 6 (A and B) shows repeated elimination of tumor spheroids by CLDN6-CAR T cells cultured for 7 and 10 days. CLDN6-CAR transduced T cells generated within 7 and 10 days, respectively, were evaluated for their ability to repeatedly kill tumor spheroids expressing CLDN6 and eGFP, as assessed by live cell imaging. (Figure 6A) CAR surface expression of CLDN6-CAR T cells cultured for 7 and 10 days was analyzed on transduced T cells after staining with fluorochrome-conjugated IMAB206 idiotype-specific antibody. Non-transduced T cells served as negative control. Cells were gated on single CD4+ or CD8+ lymphocytes. Numbers shown represent frequency (%) of parental population. (Figure 6B) Functional testing of CLDN6-CAR T cells cultured for 7 and 10 days was performed using the PA1-SC12-A2-eGFP tumor spheroid assay. Lysis of tumor spheroids was analyzed based on eGFP expression using an IncuCyte® live cell imaging system over a total period of 10 days. After 5 days, new tumor spheroids were added. Values ​​are expressed as the mean ± SD of the green object integrated intensity of technical triplicates. [Figure 7]Figure 7 (A-D) shows the antitumor effect of GMP manufactured CLDN6-CAR T cells. 5x106 OV90-SC12 tumor cells were implanted subcutaneously (n=12 mice / group) 35 days before adoptive transfer of a single dose of 1x107 CLDN6-CAR-BBz or non-transduced human T cells iv. The T cell products used were produced in a GMP facility either in the standard way (in vitro culture for 10 days) or by a shortened manufacturing procedure (harvested already after 7 days). Non-transduced T cells treated in the same way as the transduced T cells were used as negative control. The indicated T cell products were thawed, washed with PBS and injected directly into mice that already showed advanced tumors of 160 mm3 on average. Tumor volumes were measured three times a week using calipers and calculated according to the formula V=1 / 2 (length x width2) using the maximum length and width of the tumor. Animals were sacrificed when the tumor volume exceeded 1500 mm3 or when the tumor ulcerated. (Figure 7A) Schematic diagram of the mouse experiment performed is shown. (Figure 7B) CAR surface expression on transduced human CD4+ and CD8+ T cells was analyzed by flow cytometry on the day of ACT. The numbers shown represent the frequency (%) of the parental population. (Figure 7C) Tumor volumes in CLDN6-CAR-BBz and control T cell treated animals up to day 57 are shown. Data are presented as mean ± SEM of all mice / group. (Figure 7D) T cell persistence and CAR surface expression were analyzed in peripheral blood 2 weeks after ACT using flow cytometry. Representative dot plots are shown. The numbers indicated the frequency of the parental population. [Figure 8]Figure 8 (A-C) shows that RNA(LIP)-mediated in vivo expansion results in enhanced persistence of CLDN6-CAR-BBz T cells. 2.5 Gy irradiated (XRAD320) C57BL / 6BrdCrHsd-Tyrc mice (n=2-3 mice / group) were iv transferred with 5x106 CLDN6-CAR-BBz-Luc-GFP transduced C57Bl / 6-Thy1.1+ T cells. Eight days after ACT, mice were vaccinated with mRNA lipoplex (RNA(LIP); 20 μg, iv) encoding hCLDN6 or OvaI (control RNA), followed by ip administration of nucleoside-modified formulated RNA encoding mouse albumin (1 μg / mRNA / mouse). Vaccinations were repeated on days 15, 22, 50 and 85. Serial bioluminescence imaging was performed to monitor proliferation and persistence from day 1 (baseline) to day 92 after ACT. (Figure 8A) Schematic diagram of the performed mouse experiment is shown. (Figure 8B) Examples of bioluminescence imaging of mice in lateral position at the indicated time points after ACT and after treatment with antigen RNA (LIP). Off-color images represent light intensity (black, weakest; white to dark gray, strongest) superimposed on a grayscale reference image. (Figure 8C) Quantification of bioluminescence during and after expansion rounds with CLDN6-RNA (LIP) / control RNA (LIP) in the presence of the indicated nucleoside-modified formulated cytokine RNAs is shown (mean + / - sem). Grey vertical lines indicate the time points of RNA (LIP) vaccination. ACT: adoptive T cell transfer, TBI: total body irradiation, BLI: bioluminescence imaging, Luc: activated firefly luciferase, BBz: 4-1BB; z: CD3ζ. [Figure 9]Figure 9 (A and B) shows the improvement of antitumor activity of in vivo expanded CLDN6-CAR-BBz T cells. 5x105 CT26 tumor cells were implanted subcutaneously in Balb / c mice (n=10 mice / group) 20 days prior to 4 Gy total body irradiation and 26 days prior to ACT of a single dose of 1x106 CLDN6-CAR-BBz or control CAR-BBz transduced Balb / c-Thy1.1+ T cells iv. At the time of T cell treatment, mice showed established tumors of about 80 mm3 on average. Tumor volumes were measured three times a week using calipers and calculated according to the formula V=1 / 2 (length x width2) using the maximum length and width of the tumor. (Figure 9A) A schematic diagram of the mouse experiment performed is shown. (Figure 9B) The average tumor volumes in CLDN6-CAR-BBz and control CAR-BBz-T cell treated animals by day 40 are shown. Data are presented as mean ± SEM of all mice / group. ACT is shown as a dotted line and RNA(LIP) treatment as a grey line. [Figure 10A] Figure 10 (A-D) shows the recovery of antitumor efficacy of low doses of in vivo expanded CLDN6-CAR T cells. 5x106 OV90-SC12 tumor cells were implanted subcutaneously (n=9 mice / group) 30 days prior to ACT of a single dose of either low (1x105) or high (1x106) doses of CLDN6-CAR-BBz or 1x107 non-transduced human T cells iv. The T cell products used were manufactured following an abbreviated transduction process (already harvested after 7 days) in a GMP facility. Non-transduced T cells treated in the same way as the transduced T cells were used as negative control. The indicated T cell products were thawed, washed with PBS and directly injected into tumor-bearing mice. Additionally, mice receiving low doses of CAR T cells were additionally vaccinated with 20 μg of RNA (LIP) encoding either CLDN6 or a control antigen as indicated. Tumor volumes were measured three times a week using calipers and calculated using the maximum length and width of the tumor according to the formula V = 1 / 2 (length x width 2). Animals were sacrificed when the tumor volume exceeded 1500 mm3 or when the tumor ulcerated. (Figure 10A) A schematic diagram of the mouse experiments performed is shown. [Figure 10B]CAR surface expression on transduced human CD4+ and CD8+ T cells was analyzed by flow cytometry on the day of adoptive transfer. Numbers shown represent frequencies (%) of the parental population. [Figure 10C] Tumor growth curves of animals treated with different doses of CLDN6-CAR-BBz+ / -RNA(LIP) or control T cells. Data are presented as mean ± SEM of all mice / group. ACT is shown as dotted line and RNA(LIP) treatment as grey line. [Figure 10D] T cell persistence and CAR surface expression were analyzed in peripheral blood 2.5 weeks after ACT using flow cytometry. Representative dot plots are shown. Numbers indicate frequencies of parental populations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] The present invention relates to compositions and methods for treating cancer, including but not limited to solid tumors. The present invention relates to a strategy of adoptive cell transfer of cells, such as T cells, transduced to express a CAR. A CAR is a molecule that combines specificity for a desired antigen (e.g., tumor antigen), preferably antibody-based, with a T cell receptor activating intracellular domain to generate a chimeric protein that exhibits specific cellular immune activity (e.g., specific anti-tumor cellular immune activity). Preferably, the cell can be genetically modified to stably express a CAR on its surface, conferring a novel antigen specificity that is MHC-independent. The present invention generally relates to T cells genetically modified to stably express a desired CAR. T cells expressing a CAR are referred to herein as CAR T cells or CAR-modified T cells.

[0044] The CAR of the present invention combines a CLDN6 antigen binding domain, preferably a domain of a specific antibody, with an intracellular domain comprising a 4-1BB costimulatory domain and a domain of the CD3 ζ chain into a single chimeric protein. In one embodiment, the CAR of the present invention comprises an extracellular domain comprising a CLDN6 antigen binding domain, a transmembrane domain, and an intracellular domain comprising a 4-1BB costimulatory domain and a domain of the CD3 ζ chain. In one embodiment, the transmembrane domain is not naturally associated with one of the domains in the CAR. In one embodiment, the transmembrane domain is naturally associated with one of the domains in the CAR. In one embodiment, the transmembrane domain is modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. Preferably, the transmembrane domain is derived from CD8α.

[0045] The present invention further provides CAR T cells and their use for adoptive therapy.In one embodiment, the CAR T cells of the present invention can be generated by introducing a retrovirus, such as a lentiviral vector, into cells, which comprises a desired CAR, for example, a CAR comprising anti-CLDN6, CD8α hinge and transmembrane domain, and 4-1BB and CD3ζ signaling domain.The CAR T cells of the present invention can preferably replicate in vivo, resulting in long-term persistence, which can lead to sustained tumor control.

[0046] In one embodiment, the present invention relates to administering genetically modified T cells expressing desired CARs to treat patients with cancer or at risk of having cancer.Preferably, autologous cells are used for treatment.In one embodiment, autologous PBMCs are collected from the patient who needs treatment, and T cells are activated and expanded using the methods described herein and known in the art, and then infused back into the patient.

[0047] In one embodiment, the present invention generally relates to the treatment of patients with cancers expressing CLDN6 or at risk of developing cancers expressing CLDN6. The present invention includes the use of T cells expressing anti-CLDN6-CARs that contain both CD3ζ and 4-1BB costimulatory domains. The CAR T cells of the present invention can undergo strong in vivo T cell proliferation, especially when contacted with their cognate antigen, and can persist at high levels for extended periods of time. In some cases, the CAR T cells of the present invention infused into a patient can eliminate cancer cells in vivo in the patient.

[0048] definition The present invention will be described in detail below, but it should be understood that the present invention is not limited to the specific methodology, protocol and reagent described herein, which may vary.It should also be understood that the terms used herein are only intended to describe specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the scope of the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0049] In the following, the elements of the present invention are described. Although these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be interpreted as limiting the present invention to only the embodiments specifically described. The description should be understood to support and encompass embodiments combining the specifically described embodiments with any number of the disclosed elements and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application, unless otherwise indicated by the context.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein can be used to carry out the testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terms are used.

[0051] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise" and variations such as "comprising" are understood to imply the inclusion of a stated member, integer or step, or group of members, integers or steps, but not the exclusion of any other members, integers or steps, or group of members, integers or steps, although in some embodiments such other members, integers or steps, or group of members, integers or steps may be excluded, i.e., the subject matter resides in the inclusion of a stated member, integer or step, or group of members, integers or steps.

[0052] The article "a" or "an" is used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0053] As used herein, "about" when referring to a measurable value such as an amount, duration, and the like, is meant to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value.

[0054] The term "antibody" as used herein refers to an immunoglobulin molecule that binds, preferably specifically binds, to an antigen. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, and can be an immunoreactive portion or fragment of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. Antibodies in the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0055] Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin with no known antibody function, but can function as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

[0056] The term "antibody fragment" refers to a portion of an intact antibody, typically including the antigen-determining variable region of the intact antibody.

[0057] Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0058] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in antibody molecules in their naturally occurring conformations.

[0059] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in antibody molecules in their naturally occurring conformations; kappa light chain and lambda light chain refer to the two major antibody light chain isotypes.

[0060] The term "antigen" or "Ag" as used herein is defined as a molecule that elicits an immune response. This immune response may include antibody production, or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule may serve as an antigen, including virtually any protein or peptide. Furthermore, the antigen may be a naturally occurring antigen or a recombinant antigen.

[0061] In the context of the present invention, the term "tumor antigen" refers to an antigen that is common to a particular hyperproliferative disorder, such as cancer.

[0062] Claudins are integral membrane proteins located within epithelial and endothelial tight junctions. Claudins are predicted to have four transmembrane segments and two extracellular loops, with N- and C-termini located in the cytoplasm. The first extracellular loop, designated EC1 or ECL1, consists of an average of 53 amino acids, and the second extracellular loop, designated EC2 or ECL2, consists of approximately 24 amino acids. The claudin (CLDN) family of transmembrane proteins plays an important role in the maintenance of epithelial and endothelial tight junctions and may also play a role in cytoskeleton maintenance and cell signaling.

[0063] Claudin 6 (CLDN6) is an oncofetal gene expressed in mouse and human stem cells and embryoid bodies involved in epithelial cell fate (Turksen, K. et al. (2001) Dev Dyn 222, 292-300; Anderson WJ. et al. (2008) Dev Dyn 237, 504-12; Turksen K. et al. (2002) Development, 129, 1775-84; Assou S. et al. (2007) Stem Cells 25, 961-73). As a tumor-associated antigen, CLDN6 can be classified as a differentiation antigen since it is expressed in early stages of epidermal morphogenesis, which is important for epidermal differentiation and barrier formation. Moreover, expression was observed in the epithelial tissues of tongue, skin, stomach and breast or in normal epithelial tissues of newborns (Abuazza G. et al. (2006), Am J Physiol Renal Physiol 291, 1132-1141; Troy TC et al. (2007), Molecular Biotechnology 36, 166-74; Zhao L. et al. (2008), Am J Physiol Regul Integr Comp Physiol 294, 1856-1862). In addition, our data also reveal low or very low expression of CLDN6 in human placenta, bladder, endometrium, prostate and peripheral nerve, as well as frequent overexpression of CLDN6 in various cancers. CLDN6 has been demonstrated to be overexpressed in tumors including pediatric brain tumors, gastric adenocarcinoma and germ cell tumors, as well as visceral cancers such as ovarian cancer. It has also been demonstrated that overexpression of CLDN6 in gastric cancer cells results in increased invasiveness, migration and proliferation, suggesting that CLDN6 may be a marker of poor prognosis and may play a potential role in maintaining the malignant phenotype.Furthermore, CLDN6 has been shown to function as a tumor suppressor through inhibition of cell proliferation and induction of apoptosis in breast cancer cell lines.

[0064] CLDN6 has been found to be expressed in, for example, ovarian cancer, lung cancer, stomach cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, melanoma, head and neck cancer, sarcoma, bile duct cancer, renal cell carcinoma, and bladder cancer. CLDN6 is expressed in ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung carcinoma and adenocarcinoma, stomach cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, particularly basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, particularly malignant pleomorphic adenoma, sarcoma, particularly synovial sarcoma and carcinosarcoma, bile duct cancer, cancer of the bladder, particularly transitional cell carcinoma and papillary carcinoma, kidney cancer, particularly nephroclear cell carcinoma. CLDN6 is a particularly preferred target for the prevention and / or treatment of renal cell carcinoma, including renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestine cancer, including cancer of the ileum, particularly small intestine adenocarcinoma and adenocarcinoma of the ileum, embryonal carcinoma of the testis, placental choriocarcinoma, cervical cancer, testicular cancer, particularly germ cell tumors, such as testicular seminoma, testicular teratoma and embryonal carcinoma of the testis, uterine cancer, teratocarcinoma or embryonal carcinoma, particularly germ cell tumors of the testis, and metastatic forms thereof. In one embodiment, the cancer disease associated with CLDN6 expression is selected from the group consisting of ovarian cancer, lung cancer, metastatic ovarian cancer and metastatic lung cancer. Preferably, the ovarian cancer is carcinoma or adenocarcinoma. Preferably, the lung cancer is carcinoma or adenocarcinoma, preferably bronchiolar carcinoma, such as bronchiolar carcinoma or bronchiolar adenocarcinoma.

[0065] The term "CLDN6" preferably relates to human CLDN6, in particular a protein comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing, or a variant of said amino acid sequence. The first extracellular loop of CLDN6 preferably comprises amino acids 28 to 80, more preferably amino acids 28 to 76 of the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2. The second extracellular loop of CLDN6 preferably comprises amino acids 138 to 160, preferably amino acids 141 to 159, more preferably amino acids 145 to 157 of the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2. The first and second extracellular loops preferably form the extracellular portion of CLDN6.

[0066] The term "expressed on the cell surface" or "associated with the cell surface" means that a molecule such as CLDN6 is located in association with the plasma membrane of a cell, with at least a portion of the molecule facing the extracellular space of said cell and accessible from the outside of said cell, for example, by an antibody located on the outside of said cell. In this context, a portion is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association can be direct or indirect. For example, the association can be by one or more transmembrane domains, one or more lipid anchors, or by interaction with any other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of a cell. For example, a molecule associated with the surface of a cell can be a transmembrane protein with an extracellular portion, or a protein that associates with the surface of a cell by interacting with another protein that is a transmembrane protein.

[0067] "Cell surface" or "surface of a cell" is used according to its ordinary meaning in the art and thus includes the outside of a cell that is accessible to binding by proteins and other molecules.

[0068] "Cell-mediated immunity", "cell-mediated immunity", "cell-mediated immune response", or similar terms, are intended to include a cellular response to cells characterized by expression of an antigen, particularly by presentation of the antigen by class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes that act as either "helpers" or "killers". Helper T cells (CD4 + T cells (also called CD8 T cells) play a central role by regulating the immune response and act as killer cells (cytotoxic T cells, cytolytic T cells, + CTLs, also called T cells or CTLs, kill diseased cells, such as cancer cells, and prevent the production of further diseased cells.

[0069] The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e., a part or fragment of a molecule that is recognized, i.e., bound, by the immune system, e.g., recognized by an antibody or CAR. For example, an epitope is a distinct three-dimensional site on an antigen that is recognized by the immune system. Epitopes usually consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former, but not the latter, is lost in the presence of denaturing solvents. Preferably, an epitope is capable of eliciting an immune response against the antigen or a cell expressing the antigen. Preferably, the term relates to an immunogenic portion of an antigen. An epitope of a protein, such as a tumor antigen, preferably comprises a continuous or discontinuous portion of said protein and is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0070] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of said antigen (e.g., degradation of a protein into peptides), and the association (e.g., by binding) of one or more of these fragments with an MHC molecule for presentation to a specific T cell by a cell, preferably an antigen-presenting cell.

[0071] An antigen-presenting cell (APC) is a cell that displays an antigen on its surface in association with a major histocompatibility complex (MHC). A T cell can recognize this complex using its T cell receptor (TCR). The antigen-presenting cell processes and presents the antigen to the T cell. According to the present invention, the term "antigen-presenting cell" includes professional and non-professional antigen-presenting cells.

[0072] Professional antigen-presenting cells are highly efficient at internalizing antigens, either by phagocytosis or receptor-mediated endocytosis, and then displaying fragments of the antigen bound to class II MHC molecules on their membrane. T cells recognize and interact with the antigen-class II MHC molecule complex on the membrane of the antigen-presenting cell. Further costimulatory signals are then generated by the antigen-presenting cell, leading to T cell activation. The expression of costimulatory molecules is a defining feature of professional antigen-presenting cells. The main types of professional antigen-presenting cells are dendritic cells, macrophages, B cells, and certain activated epithelial cells, which have the most extensive antigen presentation and are probably the most important antigen-presenting cells.

[0073] The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Activated by inflammation, immune cytokines or microbial products, macrophages non-specifically engulf foreign pathogens within the macrophage and kill them by hydrolytic and oxidative attack that results in the degradation of the pathogen. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells and can directly interact with antibodies on the B cell surface, resulting in activation of T and B cells and further stimulation of the immune response. Macrophages belong to a class of antigen-presenting cells. In one embodiment, the macrophages are splenic macrophages.

[0074] The term "dendritic cells" (DC) refers to another subtype of phagocytes that belong to the class of antigen-presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitors. These progenitors initially transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation capacity. Immature dendritic cells are constantly sampling the surrounding environment for pathogens such as viruses and bacteria. When they come into contact with presentable antigens, they are activated to become mature dendritic cells and begin to migrate to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, break down their proteins into small fragments, and upon maturation, present those fragments on the cell surface using MHC molecules. At the same time, they upregulate cell surface receptors that function as co-receptors for T cell activation, such as CD80, CD86 and CD40, greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that directs dendritic cells to migrate through the bloodstream to the spleen or through the lymphatic system to the lymph nodes. Here, they function as antigen-presenting cells, and activate helper T cells and killer T cells and B cells by presenting antigens together with non-antigen-specific costimulatory signals. Thus, dendritic cells can actively induce T cell or B cell-related immune responses. In one embodiment, the dendritic cells are splenic dendritic cells.

[0075] According to the present invention, the term "CAR" (or "chimeric antigen receptor") refers to an artificial receptor comprising a single molecule or a complex of molecules that can recognize, i.e., bind, a target structure (e.g., an antigen) on a target cell, such as a cancer cell (e.g., by binding of an antigen-binding domain to an antigen expressed on the surface of the target cell), and confer specificity to an immune effector cell, such as a T cell, expressing said CAR on its cell surface. Preferably, recognition of the target structure by a CAR results in activation of an immune effector cell expressing said CAR. A CAR can comprise one or more protein units comprising one or more domains described herein. The term "CAR" does not include T cell receptors.

[0076] Adoptive cell transfer therapy using CAR-engineered T cells expressing chimeric antigen receptors is a promising anti-cancer treatment, since CAR-modified T cells can be engineered to target virtually any tumor antigen. For example, a patient's T cells can be genetically engineered (genetically modified) to express a CAR that specifically targets an antigen on the patient's tumor cells, and then infused back into the patient.

[0077] The term "anti-tumor" as used herein refers to a biological effect that may be manifested by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, prevention of the development of the original tumor, an increase in life expectancy, or an amelioration of various physiological symptoms associated with a cancerous condition.

[0078] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is later reintroduced into the individual.

[0079] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0080] "Xenogeneic" refers to a graft derived from an animal of a different species.

[0081] The term "syngeneic" is used to describe individuals or tissues having the same genotype, i.e., derived from identical twins or animals of the same inbred line, or tissues thereof.

[0082] The term "cancer" as used herein is defined as a disease characterized by rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0083] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and longer chains, commonly referred to in the art as proteins, of which there are many varieties. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0084] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide, which can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including, without limitation, recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR™, etc., and synthetic means. As used herein, the term "polynucleotide" should be broadly interpreted and includes DNA and RNA, including modified DNA and RNA.

[0085] In the present disclosure, the term "RNA" refers to a nucleic acid molecule that comprises ribonucleotide residues. In a preferred embodiment, the RNA comprises all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the ends (either or both) of the RNA. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In the present disclosure, these modified RNAs are considered analogs of naturally occurring RNA.

[0086] In certain embodiments of the present disclosure, the RNA is messenger RNA (mRNA), which is related to an RNA transcript that codes for a peptide or protein. As established in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide coding region and a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid that comprises deoxyribonucleotides.

[0087] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling the transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, in particular a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0088] In one embodiment, the RNA may have modified ribonucleotides. Examples of modified ribonucleotides include, but are not limited to, 5-methylcytidine, pseudouridine and / or 1-methylpseudouridine.

[0089] In some embodiments, the RNA according to the present disclosure includes a 5' cap. In one embodiment, the RNA of the present disclosure does not have an uncapped 5'-triphosphate. In one embodiment, the RNA can be modified by a 5' cap analog. The term "5' cap" refers to the structure found at the 5' end of an mRNA molecule, and generally consists of a guanosine nucleotide connected to the mRNA by a 5'-5' triphosphate bond. In one embodiment, the guanosine is methylated at position 7. Providing the RNA with a 5' cap or a 5' cap analog can be achieved by in vitro transcription, where the 5' cap is co-transcriptionally expressed on the RNA strand, or can be attached to the RNA post-transcriptionally using a capping enzyme.

[0090] In some embodiments, the RNA according to the present disclosure comprises a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) may be located 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR). The 5'-UTR, if present, is located at the 5' end, upstream of the start codon of the protein coding region. The 5'-UTR is downstream of the 5' cap (if present), e.g., directly adjacent to the 5' cap. The 3'-UTR, if present, is located at the 3' end, downstream of the stop codon of the protein coding region, although the term "3'-UTR" preferably does not include a poly(A) tail. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence.

[0091] In some embodiments, the RNA according to the present disclosure comprises a 3'-poly(A) sequence. The term "poly(A) sequence" refers to a sequence of adenyl (A) residues typically located at the 3' end of an RNA molecule. According to the present disclosure, in one embodiment, the poly(A) sequence comprises at least about 20, at least about 40, at least about 80, or at least about 100, and up to about 500, up to about 400, up to about 300, up to about 200, or up to about 150 A nucleotides, in particular about 120 A nucleotides.

[0092] "Encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either having a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0093] As used herein, "endogenous" refers to any substance that is produced from or within an organism, cell, tissue or system.

[0094] As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside an organism, cell, tissue or system.

[0095] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.

[0096] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0097] "Homology" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If both positions of two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. Generally, the comparison is performed when the two sequences are aligned to give maximum homology. A homologous sequence, according to the present disclosure, exhibits at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98, or at least 99% identity of amino acid or nucleotide residues.

[0098] A "fragment" in relation to an amino acid sequence (peptide or protein) refers to a portion of the amino acid sequence, i.e. a sequence that represents an amino acid sequence truncated at the N-terminus and / or C-terminus. A fragment truncated at the C-terminus (N-terminal fragment) is obtained, for example, by translation of a truncated open reading frame lacking the 3' end of the open reading frame. A fragment truncated at the N-terminus (C-terminal fragment) is obtained, for example, by translation of a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon that serves to initiate translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50 or at least 100 consecutive amino acids from the amino acid sequence.

[0099] By "mutant" or "mutant protein" or "mutant polypeptide" herein is meant a protein that differs from a parent protein by at least one amino acid modification. The parent polypeptide can be a naturally occurring or wild-type (WT) polypeptide, or can be a modified version of a wild-type polypeptide. Preferably, the mutant polypeptide has at least one amino acid modification compared to the parent polypeptide, such as 1 to about 20 amino acid modifications compared to the parent, preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0100] As used herein, "parent polypeptide," "parent protein," "precursor polypeptide," or "precursor protein" refers to an unmodified polypeptide that is subsequently modified to produce a variant. A parent polypeptide can be a wild-type polypeptide, or a variant or engineered version of a wild-type polypeptide.

[0101] By "wild-type" or "WT" or "native" herein is meant an amino acid sequence found in nature, including allelic variations. A wild-type protein or polypeptide has an amino acid sequence that has not been intentionally modified.

[0102] For the purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. The term "variant" includes all variants, splice variants, post-translational modification variants, conformational variants, isoform variants, allelic variants, species variants and species homologs, particularly those that occur naturally.

[0103] Amino acid insertion variants include the insertion of one or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at a particular site in the amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from a sequence, e.g., the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may be at any position in the protein. Amino acid deletion variants that include deletions at the N-terminus and / or C-terminus of a protein are also called N-terminus and / or C-terminus truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications at positions of the amino acid sequence that are not conserved between homologous proteins or peptides and / or replacement of amino acids with other amino acids with similar properties are preferred. Preferably, the amino acid changes in peptides and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of one of a family of amino acids whose side chains are related. Naturally occurring amino acids are generally divided into four families of acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: Glycine, Alanine; valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, Glutamine; Serine, Threonine; Lysine, arginine; and Phenylalanine, tyrosine.

[0104] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given for the full length of the reference amino acid sequence. Alignment for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using optimal sequence alignment, for example using Align, using standard settings, preferably EMBOSS::Needle, matrix:Blosum62, gap open 10.0, gap extension 0.5.

[0105] "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between these sequences.

[0106] The term "percent identity" is intended to indicate the percentage of amino acid residues that are identical between the two sequences being compared, obtained after optimal alignment, and this percentage is purely statistical, with the differences between the two sequences being randomly distributed over their entire length. Sequence comparison between two amino acid sequences is conventionally carried out by comparing these sequences after optimal alignment, said comparison being carried out segment by segment or "comparison window" to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison can be created manually or by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0107] The percent identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions being compared, and multiplying the result by 100 to obtain the percent identity between the two sequences.

[0108] The term "functional variant" as used herein refers to a variant molecule or sequence that includes an amino acid sequence modified by one or more amino acids compared to the amino acid sequence of a parent molecule or sequence and can still perform one or more functions of the parent molecule or sequence, such as binding to or contributing to binding to a target molecule. In one embodiment, the functional variant competes with the parent molecule or sequence for binding to a target molecule, either alone or in combination with other elements. In other words, the modification in the amino acid sequence of the parent molecule or sequence does not significantly affect or change the binding properties of the molecule or sequence. In different embodiments, the binding of the functional variant may be reduced but still significant, for example, the binding of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, the binding of the functional variant may be enhanced compared to the parent molecule or sequence.

[0109] An amino acid sequence (peptide, protein or polypeptide) "derived from" a specified amino acid sequence (peptide, protein or polypeptide) refers to the origin of the initial amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof.

[0110] As used herein, "instructional material" or "instructions" includes publications, records, drawings, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the present invention. The instructional material of the kits of the present invention can be, for example, affixed to a container containing the composition of the present invention or shipped together with a container containing the composition. Alternatively, the instructional material can be shipped separately from the container, with the intention that the instruction material and the composition are used in conjunction by the recipient.

[0111] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment, such as, for example, a host cell.

[0112] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine and "U" refers to uridine.

[0113] "Lentivirus" as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver large amounts of genetic information to the DNA of host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

[0114] The term "operably linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to link two protein coding regions, are in the same reading frame.

[0115] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to indicate that the expression level of a tumor antigen in cells derived from a disease area, such as a solid tumor, in a particular tissue or organ of a patient is abnormal compared to the expression level in normal cells derived from that tissue or organ.

[0116] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery required to initiate the specific transcription of a polynucleotide sequence.

[0117] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0118] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a gene product to be produced in a cell under most or all physiological conditions of the cell.

[0119] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell only when an inducer corresponding to the promoter is present in the cell.

[0120] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes a gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0121] The term "specifically binds" as used herein refers to a molecule, such as an antibody or CAR, that recognizes a particular antigen but does not substantially recognize or bind other molecules in a sample or subject. For example, an antibody that specifically binds to an antigen from one species may also bind that antigen from one or more other species. However, such species cross-reactivity does not in itself change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity does not in itself change the classification of the antibody as specific. In some cases, the terms "specifically bind" or "specifically bind" can be used with respect to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) of the chemical species; for example, antibodies recognize and bind to a particular protein structure rather than proteins in general. If an antibody is specific for epitope "A", then in a reaction involving labeled "A" and that antibody, the presence of a molecule containing epitope A (or free unlabeled A) reduces the amount of labeled A that binds to the antibody.

[0122] The terms "transfected" or "transformed" or "transduced" as used herein refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0123] As used herein, the phrases "under transcriptional control" or "operably linked" mean that the promoter is in the correct location and orientation with respect to the polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0124] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0125] explanation The present invention provides compositions and methods for treating cancer, among other diseases.Cancer can be solid tumor, primary or metastatic tumor.In one embodiment, cancer is a cancer that expresses CLDN6. In one embodiment, the cancer is selected from the group consisting of ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung carcinoma and adenocarcinoma, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, particularly basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, particularly malignant pleomorphic adenoma, sarcoma, particularly synovial sarcoma and carcinosarcoma, bile duct cancer, bladder cancer, particularly transitional cell carcinoma and papillary carcinoma, kidney cancer, particularly renal cell carcinoma, including renal clear cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestine cancer, including cancer of the ileum, particularly small intestinal adenocarcinoma and adenocarcinoma of the ileum, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, particularly testicular seminoma, testicular teratoma and testicular embryonal carcinoma, uterine cancer, germ cell tumors such as teratocarcinoma or embryonal carcinoma, particularly germ cell tumors of the testis, and metastatic forms thereof. In one embodiment, the cancer disease associated with CLDN6 expression is selected from the group consisting of ovarian cancer, lung cancer, metastatic ovarian cancer and metastatic lung cancer.Preferably, the ovarian cancer is carcinoma or adenocarcinoma.Preferably, the lung cancer is carcinoma or adenocarcinoma, preferably bronchiolar carcinoma, such as bronchiolar carcinoma or bronchiolar adenocarcinoma.

[0126] The present invention provides a chimeric antigen receptor (CAR) comprising an extracellular domain and an intracellular domain. The extracellular domain comprises a target-specific binding element, also referred to as an antigen-binding portion or domain. The intracellular domain, or alternatively, the cytoplasmic domain, comprises a 4-1BB costimulatory signaling region and a CD3 zeta chain portion. The 4-1BB costimulatory signaling region refers to the portion of the CAR that comprises the intracellular domain of the costimulatory molecule 4-1BB. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for the efficient response of lymphocytes to antigens.

[0127] A spacer domain or region may be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR. In embodiments, the spacer domain provides flexibility for the antigen-binding domain. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that serves to link a transmembrane domain to either the extracellular or cytoplasmic domain of a polypeptide chain. A spacer domain may contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. In embodiments, the spacer domain has about 10-300 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids, about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, including any integer between any of the endpoints of the recited ranges. In some embodiments, the spacer domain is derived from a hinge region of an immunoglobulin-like molecule. In embodiments, the spacer domain comprises all or a portion of a hinge region from human IgG1, human IgG2, human IgG3, or human IgG4, and may include one or more amino acid substitutions. In some embodiments, the spacer domain is derived from the hinge region sequence of CD8α.

[0128] In one embodiment, the present invention provides cells (e.g., T cells) engineered to express CARs that exhibit anti-tumor properties. When expressed in cells, the CARs of the present invention can redirect antigen recognition based on the antigen-binding specificity of the CAR. In one embodiment, CLDN6 is expressed on cells of the cancer types disclosed herein. When the CAR-engineered cells bind to their cognate antigen, they affect tumor cells, such that the tumor cells cannot grow, are prompted to die, or are otherwise affected to reduce or eliminate the tumor burden in the patient.

[0129] According to the present invention, the CLDN6 antigen-binding portion is fused to an intracellular domain comprising a combination of the 4-1BB (CD137) signaling domain and the CD3ζ signaling domain via a hinge domain derived from CD8α. The inclusion of the 4-1BB (CD137) signaling domain significantly increases the antitumor activity and in vivo persistence of CAR T cells compared to otherwise identical CAR T cells that have not been engineered to express 4-1BB (CD137) (Milone MC et al., (2009) Molecular Therapy 17(8), 1453-1464).

[0130] antigen binding part The CAR of the present invention comprises a target-specific binding element, also referred to as an antigen-binding portion or antigen-binding domain, which is generally a part of the extracellular domain of the CAR. The antigen-binding domain recognizes a ligand that serves as a cell surface marker on target cells associated with a particular disease state. Specifically, the CAR of the present invention targets the tumor antigen CLDN6 on tumor cells.

[0131] In one embodiment, the CLDN6 binding domain in the CAR of the present invention specifically binds to CLDN6. In one embodiment, the CLDN6 to which the CLDN6 binding domain in the CAR of the present invention binds is expressed in a cancer cell. In one embodiment, CLDN6 is expressed on the surface of a cancer cell. In one embodiment, the CLDN6 binding domain binds to the extracellular domain of CLDN6 or an epitope within the extracellular domain. In one embodiment, the CLDN6 binding domain binds to a natural epitope of CLDN6 present on the surface of a living cell. In one embodiment, the CLDN6 binding domain binds to the first extracellular loop of CLDN6, preferably amino acid positions 28-76 of CLDN6, or the second extracellular loop of CLDN6, preferably amino acid positions 141-159 of CLDN6. In a particular embodiment, the CLDN6 binding domain binds to an epitope on CLDN6 that is not present on CLDN9. Preferably, the CLDN6 binding domain binds to an epitope on CLDN6 that is not present on CLDN4 and / or CLDN3. Most preferably, the CLDN6 binding domain binds to an epitope on CLDN6 that is not present on CLDN proteins other than CLDN6. The CLDN6 binding domain preferably binds to CLDN6 but not to CLDN9, and preferably not to CLDN4 and / or CLDN3. Preferably, the CLDN6 binding domain is specific for CLDN6. Preferably, the CLDN6 binding domain binds to CLDN6 expressed on the cell surface.

[0132] In one embodiment of the present invention, the CLDN6 antigen-binding domain comprises a variable region (VH) of a heavy chain of an immunoglobulin having specificity for CLDN6 and a variable region (VL) of a light chain of an immunoglobulin having specificity for CLDN6. In one embodiment, the heavy chain variable region (VH) and the corresponding light chain variable region (VL) are connected by a peptide linker, preferably a peptide linker comprising the amino acid sequence (GGGGS)3.

[0133] In one embodiment, the binding domain against CLDN6 comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7 and 9, or a functional variant thereof.

[0134] In one embodiment, the binding domain against CLDN6 comprises a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 23 and 24, or a functional variant thereof.

[0135] In one embodiment, the binding domain for CLDN6 comprises (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: x or a functional variant thereof; and (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: x+1 or a functional variant thereof where x is selected from 3, 5, 7 and 9.

[0136] In one embodiment, the binding domain for CLDN6 comprises (i) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7, and 9, or a functional variant thereof; and (ii) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 23 and 24, or a functional variant thereof; Includes.

[0137] In one embodiment, the binding domain for CLDN6 comprises (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 5 or a functional variant thereof; and (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 4 or a functional variant thereof Includes.

[0138] In one embodiment, the binding domain for CLDN6 comprises (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 5 or a functional variant thereof; and (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 23 or a functional variant thereof Includes.

[0139] In one embodiment, the binding domain for CLDN6 comprises (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 5 or a functional variant thereof; and (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 24 or a functional variant thereof Includes.

[0140] In certain preferred embodiments, the binding domain against CLDN6 comprises a combination of heavy chain variable region (VH) and light chain variable region (VL) selected from the following possibilities (i) to (xi): (i) VH comprises an amino acid sequence represented by SEQ ID NO: 3 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant, and VL comprises an amino acid sequence represented by SEQ ID NO: 4 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant; (ii) VH comprises an amino acid sequence represented by SEQ ID NO: 5 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant, and VL comprises an amino acid sequence represented by SEQ ID NO: 6 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant; (iii) VH comprises an amino acid sequence represented by SEQ ID NO: 7 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant, and VL comprises an amino acid sequence represented by SEQ ID NO: 8 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant; (iv) VH comprises an amino acid sequence represented by SEQ ID NO: 9 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant, and VL comprises an amino acid sequence represented by SEQ ID NO: 10 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant; (v) VH comprises an amino acid sequence represented by SEQ ID NO:5 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant, and VL comprises an amino acid sequence represented by SEQ ID NO:4 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant; (vi) VH comprises an amino acid sequence represented by SEQ ID NO: 5 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant; and VL comprises an amino acid sequence represented by SEQ ID NO: 23 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant; (vii) the VH comprises an amino acid sequence represented by SEQ ID NO:5 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant, and the VL comprises an amino acid sequence represented by SEQ ID NO:24 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant.

[0141] In particularly preferred embodiments, the binding domain against CLDN6 comprises the following combination of heavy chain variable regions (VH) and light chain variable regions (VL): The VH comprises an amino acid sequence represented by SEQ ID NO:5 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant, and the VL comprises an amino acid sequence represented by SEQ ID NO:24 or a functional variant thereof, or a fragment of said amino acid sequence or said functional variant.

[0142] The term "fragment" refers in particular to one or more of the complementarity determining regions (CDRs) of the heavy chain variable region (VH) and / or the light chain variable region (VL), preferably at least the CDR3 variable region. In one embodiment, said one or more of the complementarity determining regions (CDRs) are selected from the set of complementarity determining regions CDR1, CDR2 and CDR3. In a particularly preferred embodiment, the term "fragment" refers to the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and / or the light chain variable region (VL).

[0143] In one embodiment, a binding domain for CLDN6 comprising one or more CDRs, a set of CDRs or a combination of a set of CDRs described herein comprises said CDRs together with their intervening framework regions.Preferably, this portion also comprises at least about 50% of either or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region.The construction of binding domains made by recombinant DNA techniques may result in the introduction of residues at the N- or C-terminal end of the variable region that are encoded by linkers that are introduced to facilitate cloning or other engineering steps, including the introduction of linkers to join the variable region to additional protein sequences, including sequences described herein.

[0144] In one embodiment, a binding domain comprising one or more CDRs, a set of CDRs or a combination of a set of CDRs described herein comprises said CDRs within a human antibody framework.

[0145] In one embodiment, the binding domain against CLDN6 comprises a heavy chain variable region (VH) comprising at least one, preferably two, and more preferably all three of the CDR sequences of the heavy chain variable region (VH) described herein, e.g., the heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7 and 9, or a functional variant thereof.

[0146] In one embodiment, the binding domain against CLDN6 comprises a light chain variable region (VL) comprising at least one, preferably two, and more preferably all three, of the CDR sequences of the light chain variable region (VL) described herein, e.g., the light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 23 and 24, or a functional variant thereof.

[0147] In one embodiment, the binding domain for CLDN6 comprises (i) a heavy chain variable region (VH) comprising at least one, preferably two, and more preferably all three CDR sequences of the heavy chain variable region (VH) of a combination of heavy chain variable region (VH) and light chain variable region (VL) described herein, e.g., a combination in which the VH comprises the amino acid sequence represented by SEQ ID NO: 5 or a functional variant thereof and the VL comprises the amino acid sequence represented by SEQ ID NO: 6 or a functional variant thereof; and (ii) a light chain variable region (VL) comprising at least one, preferably two, and more preferably all three CDR sequences of the light chain variable region (VL) of a combination of a heavy chain variable region (VH) and a light chain variable region (VL) described herein; Includes.

[0148] In one embodiment, the binding domain for CLDN6 comprises (i) a heavy chain variable region (VH) comprising at least one, preferably two, and more preferably all three CDR sequences of the heavy chain variable region (VH) of SEQ ID NO: x or a functional variant thereof; and (ii) A light chain variable region (VL) comprising at least one, preferably two, and more preferably all three CDR sequences of a light chain variable region (VL) of SEQ ID NO: x+1 or a functional variant thereof. where x is selected from 3, 5, 7 and 9.

[0149] In one embodiment, the binding domain for CLDN6 comprises (i) a heavy chain variable region (VH) comprising at least one, preferably two, and more preferably all three CDR sequences of SEQ ID NO: 5 or a functional variant thereof; and (ii) A light chain variable region (VL) comprising at least one, preferably two, and more preferably all three CDR sequences of the light chain variable region (VL) of SEQ ID NO: 6 or a functional variant thereof. Includes.

[0150] In one embodiment, the binding domain for CLDN6 comprises (i) a heavy chain variable region (VH) comprising at least one, preferably two, and more preferably all three CDR sequences of SEQ ID NO: 5 or a functional variant thereof; and (ii) A light chain variable region (VL) comprising at least one, preferably two, and more preferably all three CDR sequences of the light chain variable region (VL) of SEQ ID NO: 4 or a functional variant thereof. Includes.

[0151] The term "at least one, preferably two, more preferably all three of the CDR sequences" preferably relates to at least a CDR3 sequence, optionally in combination with a CDR1 sequence and / or a CDR2 sequence.

[0152] CDR1 of a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 3, 5, 7, and 9 preferably comprises amino acids 26 to 33 of the sequences shown in SEQ ID NOs: 3, 5, 7, and 9, respectively. CDR2 of a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 3, 5, 7, and 9 preferably comprises amino acids 51 to 58 of the sequences shown in SEQ ID NOs: 3, 5, 7, and 9, respectively. CDR3 of a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 3, 5, 7, and 9 preferably comprises amino acids 97 to 106 of the sequences shown in SEQ ID NOs: 3, 5, 7, and 9, respectively.

[0153] CDR1 of a light chain variable region (VL) selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 23 and 24 preferably comprises amino acids 27 to 31 of the sequences shown in SEQ ID NOs: 4, 6, 8, 10, 23 and 24, respectively. CDR2 of a light chain variable region (VL) selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 23 and 24 preferably comprises amino acids 49 to 51 of the sequences shown in SEQ ID NOs: 4, 6, 8, 10, 23 and 24, respectively. CDR3 of a light chain variable region (VL) selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 23 and 24 preferably comprises amino acids 88 to 97 of the sequences shown in SEQ ID NOs: 4, 6, 8, 10, 23 and 24, respectively.

[0154] In one embodiment, the binding domain for CLDN6 comprises a heavy chain variable region (VH) comprising the CDR3 sequence Xaa1 Gly Xaa2 Val Xaa3, where Xaa1 is any amino acid, preferably an aromatic amino acid, more preferably Phe or Tyr, most preferably Tyr, Xaa2 is any amino acid, preferably an aromatic amino acid, more preferably Phe or Tyr, most preferably Tyr, and Xaa3 is any amino acid, preferably Leu or Phe, more preferably Leu. In one embodiment, the binding domain for CLDN6 comprises a heavy chain variable region (VH) comprising the CDR3 sequence Asp Xaa1 Gly Xaa2 Val Xaa3 or Xaa1 Gly Xaa2 Val Xaa3 Asp, where Xaa1, Xaa2 and Xaa3 are as defined above. In one embodiment, the binding domain for CLDN6 comprises a heavy chain variable region (VH) comprising the CDR3 sequence Asp Xaa1 Gly Xaa2 Val Xaa3 Asp, where Xaa1, Xaa2 and Xaa3 are as defined above. In one embodiment, the binding domain for CLDN6 comprises a heavy chain variable region (VH) comprising the CDR3 sequence Ala Arg Asp Xaa1 Gly Xaa2 Val Xaa3 Asp Tyr, where Xaa1, Xaa2 and Xaa3 are as defined above. In one embodiment, the binding domain for CLDN6 according to the preceding embodiment comprises a heavy chain variable region (VH) comprising a CDR1 sequence according to SEQ ID NO: 16 or a functional variant thereof and / or a CDR2 sequence according to SEQ ID NO: 17 or a functional variant thereof.

[0155] In one embodiment, the binding domain for CLDN6 comprises a light chain variable region (VL) comprising the CDR3 sequence Arg Xaa1 Xaa2 Xaa3 Pro, where Xaa1 is any amino acid, preferably Ser or Asn, most preferably Ser, Xaa2 is any amino acid, preferably Tyr, Ser, Ile, Asn or Thr, more preferably Tyr, Ser or Asn, most preferably Asn, and Xaa3 is any amino acid, preferably Ser or Tyr, more preferably Tyr. In one embodiment, the binding domain for CLDN6 comprises a light chain variable region (VL) comprising the CDR3 sequence Gln Arg Xaa1 Xaa2 Xaa3 Pro Pro, where Xaa1, Xaa2 and Xaa3 are as defined above. In one embodiment, the binding domain against CLDN6 comprises a light chain variable region (VL) comprising the CDR3 sequence Gln Gln Arg Xaa1 Xaa2 Xaa3 Pro Pro Trp Thr, where Xaa1, Xaa2 and Xaa3 are as defined above. In one embodiment, the binding domain against CLDN6 according to the above embodiment comprises a light chain variable region (VL) comprising a CDR1 sequence according to SEQ ID NO: 21 or a functional variant thereof and / or a CDR2 sequence according to SEQ ID NO: 22 or a functional variant thereof.

[0156] In one embodiment, the binding domain for CLDN6 comprises: (i) a heavy chain variable region (VH) comprising a CDR3 sequence selected from the group consisting of Xaa1 Gly Xaa2 Val Xaa3, Asp Xaa1 Gly Xaa2 Val Xaa3, Xaa1 Gly Xaa2 Val Xaa3 Asp, Asp Xaa1 Gly Xaa2 Val Xaa3 Asp, and Ala Arg Asp Xaa1 Gly Xaa2 Val Xaa3 Asp Tyr, wherein Xaa1 is any amino acid, preferably an aromatic amino acid, more preferably Phe or Tyr, most preferably Tyr, Xaa2 is any amino acid, preferably an aromatic amino acid, more preferably Phe or Tyr, most preferably Tyr, and Xaa3 is any amino acid, preferably Leu or Phe, more preferably Leu, and (ii) a light chain variable region (VL) comprising a CDR3 sequence selected from the group consisting of Arg Xaa1 Xaa2 Xaa3 Pro, Gln Arg Xaa1 Xaa2 Xaa3 Pro Pro, Gln Gln Arg Xaa1 Xaa2 Xaa3 Pro Pro Trp Thr, where Xaa1 is any amino acid, preferably Ser or Asn, most preferably Ser, Xaa2 is any amino acid, preferably Tyr, Ser, Ile, Asn or Thr, more preferably Tyr, Ser or Asn, most preferably Asn, and Xaa3 is any amino acid, preferably Ser or Tyr, more preferably Tyr.

[0157] In one embodiment, the binding domain against CLDN6 according to the aforementioned embodiment comprises (i) a heavy chain variable region (VH) comprising a CDR1 sequence according to SEQ ID NO: 16 or a functional variant thereof and / or a CDR2 sequence according to SEQ ID NO: 17 or a functional variant thereof, and / or (ii) a light chain variable region (VL) comprising a CDR1 sequence according to SEQ ID NO: 21 or a functional variant thereof and / or a CDR2 sequence according to SEQ ID NO: 22 or a functional variant thereof.

[0158] In one embodiment, the binding domain for CLDN6 competes with the binding domain for CLDN6 described above for CLDN6 binding and / or has the specificity for CLDN6 of the binding domain for CLDN6 described above. In these and other embodiments, the binding domain for CLDN6 may be highly homologous to the binding domain for CLDN6 described above. It is contemplated that the preferred binding domain for CLDN6 has CDR regions that are identical or highly homologous to the CDR regions of the binding domain for CLDN6 described above. By "highly homologous", it is contemplated that 1 to 5, preferably 1 to 4, for example 1 to 3 or 1 or 2 substitutions may be made in each CDR region.

[0159] The term "compete" refers to the competition between two binding molecules for binding to a target antigen. If two binding molecules do not block each other from binding to a target antigen, such binding molecules are non-competitive, which is an indication that said binding molecules do not bind to the same part, i.e., epitope, of the target antigen. Methods for testing the competition of binding molecules, such as antibodies, for binding to a target antigen are well known to those skilled in the art. One example of such a method is the so-called cross-competition assay, which can be performed, for example, as an ELISA or by flow cytometry. For example, an ELISA-based assay can be performed by coating the wells of an ELISA plate with one of the antibodies, adding the competing antibody and the His-tagged antigen / target, and detecting whether the added antibody inhibits the binding of the His-tagged antigen to the coated antibody by, for example, adding a biotinylated anti-His antibody, followed by adding streptavidin-poly-HRP, further developing the reaction with ABTS, and measuring the absorbance at 405 nm. For example, a flow cytometry assay may be performed by incubating cells expressing the antigen / target with an excess of unlabeled antibody, incubating the cells with a suboptimal concentration of biotin-labeled antibody, followed by incubation with fluorescently labeled streptavidin and analysis by flow cytometry.

[0160] Two binding molecules have the "same specificity" if they bind to the same antigen and the same epitope. Whether a molecule to be tested recognizes the same epitope as a particular binding molecule, i.e., whether the binding molecules bind to the same epitope, can be tested by various methods known to those skilled in the art, for example, based on the competition of binding molecules such as antibodies to the same epitope. Competition between binding molecules can be detected by cross-blocking assays. For example, a competitive ELISA assay can be used as a cross-blocking assay. For example, a target antigen can be coated on the wells of a microtiter plate, and an antigen-binding antibody and a candidate competitive test antibody can be added. The amount of antigen-binding antibody bound to the antigen in the wells indirectly correlates with the binding ability of the candidate competitive test antibody that competes for binding to the same epitope. Specifically, the greater the affinity of the candidate competitive test antibody for the same epitope, the less the amount of antigen-binding antibody that binds to the antigen-coated wells. The amount of antigen-binding antibody bound to the wells can be measured by labeling the antibody with a detectable or measurable labeling substance.

[0161] Preferably, the antigen-binding portion of the CAR of the present invention is an anti-CLDN6 scFV, which preferably comprises the sequence shown in SEQ ID NO: 35 or a functional variant thereof.

[0162] Transmembrane domain The CAR of the present invention is designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain is not naturally associated with one of the domains in the CAR. In one embodiment, the transmembrane domain is naturally associated with one of the domains in the CAR. In one embodiment, the transmembrane domain is modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein to minimize interaction with other members of the receptor complex. The transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane region particularly used in the present invention can be derived from (i.e., includes at least one transmembrane region of) the alpha, beta or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain may be synthetic, in which case it comprises predominantly hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine is found at each end of a synthetic transmembrane domain.

[0163] Preferably, the transmembrane domain in the CAR of the present invention is a CD8α transmembrane domain. In one embodiment, the CD8α transmembrane domain comprises the amino acid sequence of SEQ ID NO: 28 or a functional variant thereof.

[0164] In some cases, the CAR of the present invention comprises a CD8α hinge domain that forms a bond between the transmembrane domain and the extracellular domain. In one embodiment, the CD8α hinge domain comprises the amino acid sequence of SEQ ID NO: 27 or a functional variant thereof.

[0165] Cytoplasmic domain The cytoplasmic domain or alternatively the intracellular signaling domain of the CAR of the present invention is responsible for the activation of at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Usually, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits an effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0166] It is known that the signal generated only through TCR is insufficient for the complete activation of T cells, and secondary or costimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through TCR (primary cytoplasmic signaling sequences) and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0167] The CAR of the present invention comprises a primary cytoplasmic signaling sequence derived from CD3ζ. Furthermore, the cytoplasmic domain of the CAR of the present invention is designed to comprise a CD3ζ signaling domain in combination with a costimulatory signaling region derived from 4-1BB.

[0168] The term "4-1BB" refers to a membrane receptor protein, also called CD137, which is a member of the tumor necrosis factor receptor (TNFR) superfamily that is expressed on the surface of activated T cells as a type of accessory molecule. 4-1BB has a molecular weight of 55 kDa and is found as a homodimer.

[0169] The T cell surface glycoprotein CD3ζ chain, also known as the T cell receptor T3ζ chain or CD247, is a protein encoded by the CD247 gene in humans. T cell receptor zeta (ζ) forms the T cell receptor-CD3 complex together with T cell receptor α / β and γ / δ heterodimers and CD3γ, δ and ε. The ζ chain plays a key role in linking antigen recognition to several intracellular signaling pathways. Low expression of antigens results in a reduced immune response.

[0170] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present invention can be linked together in a random or specific order. Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, can form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[0171] Thus, the cytoplasmic domain in the CAR of the present invention is designed to include the signaling domain of CD3ζ and the signaling domain of 4-1BB. In one embodiment, the cytoplasmic domain in the CAR of the present invention is designed to include the signaling domain of 4-1BB and the signaling domain of CD3ζ, wherein the signaling domain of 4-1BB comprises the amino acid sequence of SEQ ID NO: 30 or a functional variant thereof, and the signaling domain of CD3ζ comprises the amino acid sequence of SEQ ID NO: 31 or a functional variant thereof.

[0172] In one embodiment, the CAR of the present invention comprises a signal peptide that directs the nascent protein to the endoplasmic reticulum. In one embodiment, the signal peptide precedes the antigen binding domain. In one embodiment, the signal peptide is derived from an immunoglobulin, such as IgG. In one embodiment, the signal peptide comprises a sequence according to SEQ ID NO: 25 or a functional variant thereof.

[0173] In one embodiment, the CAR of the invention comprises the following elements in the following order: NH2-CLDN6 antigen binding domain-transmembrane domain-4-1BB costimulatory domain-CD3ζ signaling domain-COOH.

[0174] In one embodiment, a CAR of the invention comprises the following elements in the following order: NH2-CLDN6 antigen binding domain-CD8α hinge-CD8α transmembrane domain-4-1BB costimulatory domain-CD3ζ signaling domain-COOH.

[0175] In one embodiment, the CAR of the present invention comprises the amino acid sequence of SEQ ID NO: 36 or a functional variant thereof.

[0176] vector The present invention encompasses nucleic acid constructs, such as DNA constructs, that comprise a sequence encoding a CAR of the present invention.

[0177] The present invention also provides a vector in which the DNA of the present invention is inserted.Vector derived from retrovirus such as lentivirus is a suitable tool for achieving long-term gene transfer, since it allows long-term stable integration of transgene and its proliferation in daughter cells.Lentivirus vector has an additional advantage over vector derived from oncoretrovirus such as mouse leukemia virus in that it can transduce non-proliferating cells such as hepatocytes.They also have the additional advantage of low immunogenicity.

[0178] Briefly, expression of natural or synthetic nucleic acid encoding CAR is typically achieved by operably linking the nucleic acid encoding CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes. A typical cloning vector includes transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0179] The nucleic acid of the present invention can be cloned into many kinds of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids or transposons. Particularly interesting vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. Furthermore, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0180] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Using techniques known in the art, a selected gene can be inserted into a vector and packaged into retroviral particles. Recombinant viruses can then be isolated and delivered to target cells either in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0181] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is maintained when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to be able to function either cooperatively or independently to activate transcription. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation growth factor 1 alpha (EF-1α). However, other constitutive promoter sequences may also be used, including, but not limited to, Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters, and inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence to which the promoter is operably linked when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0182] To evaluate the expression of the CAR polypeptide or a part thereof, the expression vector introduced into the cell can also contain a selection marker gene or a reporter gene or both to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected via a viral vector. In other embodiments, the selection marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selection marker and the reporter gene can be flanked by appropriate regulatory sequences that allow expression in the host cell. Useful selection markers include, for example, antibiotic resistance genes such as neo.

[0183] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. In general, reporter genes are genes that code for polypeptides that are not present or expressed in recipient organisms or tissues and whose expression is manifested by some easily detectable property, such as enzymatic activity. The expression of the reporter gene is assayed at a suitable time after DNA is introduced into recipient cells. Suitable reporter genes may include genes that code for luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.

[0184] Methods for introducing and expressing genes into cells are known in the art. In relation to expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art. For example, the expression vectors can be transferred into host cells by physical, chemical or biological means.

[0185] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. A preferred method for introducing a polynucleotide into a host cell is calcium phosphate transfection.

[0186] Biological methods for introducing polynucleotide of interest into host cells include the use of DNA and RNA vectors.Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells.Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus type I, adenovirus and adeno-associated virus, etc.

[0187] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0188] cell The cells used in connection with the CAR system of the present invention and into which a nucleic acid (DNA or RNA) encoding the CAR system of the present invention can be introduced include any cell with lytic potential, particularly lymphoid cells, preferably T cells, particularly cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. Upon activation, each of these cytotoxic lymphocytes causes the destruction of a target cell. For example, cytotoxic T cells cause the destruction of a target cell by either or both of the following means: First, upon activation, T cells release cytotoxins such as perforin, granzymes, and granulysin. Perforin and granulysin create pores in the target cell, and granzymes enter the cell and trigger a cytoplasmic caspase cascade that induces apoptosis (programmed cell death) of the cell. Second, apoptosis can be induced via Fas-Fas ligand interaction between the T cell and the target cell. The cytotoxic lymphocytes are preferably autologous cells, but xenogeneic or allogeneic cells can be used.

[0189] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTL, CD8+ T cells), including cytolytic T cells. The term "antigen-specific T cell" or similar terms refers to a T cell that recognizes the antigen targeted by the T cell and preferably exerts the effector function of the T cell. A T cell is considered specific for an antigen if it kills a target cell expressing the antigen. The specificity of a T cell may be assessed using any of a variety of standard techniques, for example in a chromium release assay or proliferation assay. Alternatively, the synthesis of lymphokines (such as interferon gamma) may be measured.

[0190] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by expression of CD56 or CD16 and the absence of the T cell receptor (CD3). As provided herein, NK cells can also be differentiated from stem or progenitor cells.

[0191] The term "effector function" in the context of the present invention includes any function mediated by components of the immune system that results in the killing of diseased cells, e.g. tumor cells, or the inhibition of tumor growth and / or tumorigenesis, including the suppression of tumor dissemination and metastasis. Preferably, effector functions in the context of the present invention are T cell-mediated effector functions. Such functions include those mediated by helper T cells (CD4 + T cells), cytokine release and / or CD8 + This includes activation of lymphocytes (CTLs) and / or B cells, and in the case of CTLs, elimination of cells, i.e., cells characterized by expression of the antigen, e.g., via apoptosis or perforin-mediated cytolysis, production of cytokines such as IFN-γ and TNF-α, and specific cytolytic killing of target cells expressing the antigen.

[0192] The term "immune effector cell" or "immunoreactive cell" in the context of the present invention relates to a cell that exerts an effector function during an immune response. An "immune effector cell" is preferably capable of binding to an antigen, such as an antigen expressed on the surface of a cell, and mediating an immune response. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, an "immune effector cell" is a T cell, preferably a CD4 + and / or CD8 + According to the present invention, the term "immune effector cells" also includes cells that can be matured into immune cells (such as T cells, in particular T helper cells, or cytolytic T cells) upon appropriate stimulation. Immune effector cells are CD34 +They include hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. The differentiation of T cell precursors into cytolytic T cells resembles the clonal selection of the immune system upon exposure to antigen.

[0193] Preferably, "immune effector cells" recognize antigens with some degree of specificity, especially when present on the surface of diseased cells such as cancer cells. Preferably, said recognition enables the cells that recognize the antigen to be responsive or reactive. The cells are helper T cells (CD4 + In the case of CD4+ T cells, such responsiveness or reactivity may be determined by the release of cytokines and / or CD8 + It may include activation of lymphocytes (CTL) and / or B cells. If the cell is a CTL, such responsiveness or reactivity may include removal of the cell, i.e., cells characterized by expression of the antigen, for example, via apoptosis or perforin-mediated cytolysis. According to the present invention, CTL responsiveness may include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of target cells expressing the antigen. CTL responsiveness may also be determined using an artificial reporter that pinpoints CTL responsiveness. Such CTLs that recognize and are responsive or reactive to an antigen are also referred to herein as "antigen-responsive CTLs."

[0194] "Lymphoid cells" are cells that can generate immune responses, such as cellular immune responses, optionally after appropriate modification, such as after CAR transfer, or precursors of such cells, including lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. Lymphoid cells can be immune effector cells as described herein. Preferred lymphoid cells are T cells, which can be modified to express CAR on the cell surface. In one embodiment, lymphoid cells lack endogenous expression of T cell receptors.

[0195] The terms "T cells" and "T lymphocytes" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), including cytolytic T cells.

[0196] T cells belong to a group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other types of lymphocytes, such as B cells and natural killer cells, by the presence of a special receptor on the surface of these cells, called the T cell receptor (TCR). The thymus is the main organ responsible for the maturation of T cells. Several different subsets of T cells have been discovered, each with distinct functions.

[0197] T helper cells assist other white blood cells in immunological processes, including, among other functions, maturation of B cells into plasma cells and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 protein on their surface. Helper T cells become activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in active immune responses.

[0198] Cytotoxic T cells destroy virus-infected and tumor cells and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of almost every cell in the body.

[0199] The majority of T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor is produced from independent T cell receptor alpha and beta (TCRα and TCRβ) genes and is composed of two separate peptide chains called the α-TCR chain and the β-TCR chain. γδ T cells (gamma delta T cells) are a small subset of T cells that have a different T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR is composed of one γ chain and one δ chain. This group of T cells is much rarer than αβ T cells (2% of all T cells).

[0200] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells reside in the thymus and expand by cell division to give rise to a large population of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8 and are therefore classified as double negative (CD4-CD8-) cells. As development progresses, they become double positive thymocytes (CD4+CD8+) and finally mature into single positive (CD4+CD8- or CD4-CD8+) thymocytes that are released from the thymus into peripheral tissues.

[0201] T cells can generally be prepared in vitro or ex vivo using standard procedures.For example, T cells can be isolated from bone marrow, peripheral blood, or bone marrow or peripheral blood fractions of mammals such as patients using commercially available cell separation systems.Alternatively, T cells can be derived from related or unrelated humans, non-human animals, cell lines, or cultures.The sample containing T cells can be, for example, peripheral blood mononuclear cells (PBMCs).

[0202] T cells used in accordance with the present invention may express an endogenous T cell receptor or may lack expression of an endogenous T cell receptor.

[0203] The term "antigen-targeted CAR" refers to a CAR that recognizes an antigen, such as the surface of an antigen-presenting cell or a diseased cell, such as a cancer cell, such that when present on an immune effector cell, such as a T cell, the immune effector cell is stimulated, primed and / or expanded as described above, or exerts an effector function of the immune effector cell.

[0204] Source of T cells Prior to the expansion and genetic modification of the T cells of the present invention, a source of T cells is obtained from a subject. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, various T cell lines available in the art may be used. In certain embodiments of the present invention, T cells can be obtained from a unit of blood drawn from a subject using various techniques known to those skilled in the art, such as Ficoll™ separation. In a preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In one embodiment of the present invention, the cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium, may lack magnesium, or may lack many, if not all, divalent cations. Again, surprisingly, the initial activation step in the absence of calcium leads to an expansion of activation. As one of skill in the art will readily appreciate, the wash step may be accomplished by methods known to those of skill in the art, such as using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells are washed with, for example, Ca 2+ Contains no Mg 2+The cells may be resuspended in a variety of biocompatible buffers, such as free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in media.

[0205] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example by centrifugation through a PERCOLL™ gradient or counter-flow centrifugal elutriation. + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO + Specific subpopulations of T cells, such as T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3x28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a time sufficient for positive selection of the desired T cells. In one embodiment, the time period ranges from 30 minutes to 36 hours or more. One of skill in the art will recognize that multiple rounds of selection can also be used in the context of the present invention.

[0206] Enrichment of a T cell population by negative selection can be achieved using a combination of antibodies against surface markers unique to the negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, negative selection can be used to enrich for CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, typically CD4 + , CD25 + , CD62L hi , G.I.T.R. + , and FoxP3 +It may be desirable to enrich for or positively select for regulatory T cells expressing T-cell IL-1. Alternatively, in certain embodiments, regulatory T cells are depleted by anti-CD25-conjugated beads or other similar selection methods.

[0207] A variety of methods may be used to introduce the CAR construct into T cells, including non-viral-based DNA transfection, transposon-based systems, virus-based systems, and RNA-based systems. Non-viral-based DNA transfection has a low risk of insertional mutagenesis. Transposon-based systems can integrate transgenes more efficiently than plasmids that do not contain integration elements. Viral-based systems include the use of gamma-retroviruses and lentiviral vectors. Gamma-retroviruses are relatively easy to produce, efficiently and persistently transduce T cells, and have been previously proven to be safe in terms of integration in primary human T cells. Lentiviral vectors also efficiently and persistently transduce T cells, but are more expensive to manufacture. They are also potentially safer than retroviral-based systems.

[0208] In one embodiment of the invention, T cells or T cell precursors are transfected, either ex vivo or in vivo, with a nucleic acid encoding a CAR to provide T cells genetically modified to express a CAR.

[0209] CAR T cells can be produced in vivo, and therefore almost instantly, using nanoparticles that target T cells. For example, poly(β-amino ester)-based nanoparticles can be conjugated to anti-CD3e f(ab) fragments to bind to CD3 on T cells. Upon binding to T cells, these nanoparticles are endocytosed. Their contents, for example, plasmid DNA encoding the antitumor antigen CAR, can be directed to the T cell nucleus because it contains a peptide containing a microtubule-associated sequence (MTAS) and a nuclear localization signal (NLS). The inclusion of a transposon with an inverted repeat (IR) flanking the CAR gene expression cassette and a separate plasmid encoding a hyperactive transposase can allow efficient integration of the CAR vector into the chromosome. Such a system that allows in vivo production of CAR T cells after nanoparticle injection is described in Smith et al. (2017) Nat. Nanotechnol. 12:813-820.

[0210] Another possibility is to use CRISPR / Cas9 technology to purposefully place a CAR coding sequence at a specific genetic locus, for example, to knock out an existing T cell receptor (TCR) while knocking in the CAR and placing it under the dynamic regulatory control of an endogenous promoter that would otherwise silence expression of the TCR; see, e.g., Eyquem et al. (2017) Nature 543:113-117.

[0211] In one embodiment of all aspects of the invention, T cells genetically modified to express a CAR are stably or transiently transfected with a nucleic acid encoding the CAR. Thus, the nucleic acid encoding the CAR may or may not be integrated into the genome of the T cell.

[0212] In one embodiment of all aspects of the invention, the T cells or T cell precursors are derived from the subject to be treated. In one embodiment of all aspects of the invention, the T cells or T cell precursors are derived from a subject different from the subject to be treated.

[0213] In one embodiment of all aspects of the invention, the T cells may be autologous, allogeneic or syngeneic to the subject being treated. The T cells may be genetically modified in vitro to express a chimeric antigen receptor (CAR).

[0214] In one embodiment of all aspects of the invention, T cells genetically modified to express a CAR are inactivated due to expression of endogenous T cell receptors and / or endogenous HLA.

[0215] T cell activation and proliferation Whether before or after genetic modification of the T cells to express a desired CAR, the T cells can be activated and expanded using methods generally known in the art.

[0216] In general, the T cells of the present invention are expanded by contact with a surface to which an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of T cells are attached.In particular, the T cell population can be stimulated as described herein, for example, by contact with surface-immobilized anti-CD3 antibody, or its antigen-binding fragment, or anti-CD2 antibody, or by contact with a protein kinase C activator (e.g., bryostatin) combined with a calcium ionophore.For costimulation of auxiliary molecules on the surface of T cells, a ligand that binds to the auxiliary molecules is used.For example, the T cell population can be contacted with anti-CD3 antibody and anti-CD28 antibody under conditions suitable for stimulating the proliferation of T cells.

[0217] In certain embodiments, the primary and costimulatory signals of T cells can be provided by different protocols. For example, the agents providing each signal can be in solution or bound to a surface. When bound to a surface, the agents can be bound to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one agent can be bound to a surface and the other agent can be in solution. In one embodiment, the agent providing the costimulatory signal is bound to the cell surface, and the agent providing the primary activation signal is in solution or bound to a surface. In certain embodiments, both agents can be in solution.

[0218] In one embodiment, the two agents are immobilized on the beads, either on the same bead, i.e., in "cis," or on separate beads, i.e., in "trans." By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof, and both agents are co-immobilized on the same bead. In one embodiment, the ratio of CD3:CD28 antibodies bound to the beads ranges from 100:1 to 1:100.

[0219] A particle to cell ratio of 1:500 to 500:1 may be used to stimulate T cells or other target cells. As one of skill in the art can readily appreciate, the particle to cell ratio may depend on the particle size relative to the target cells. For example, small sized beads may only be able to bind to a small number of cells, whereas larger beads may bind to many cells. In certain embodiments, the cell to particle ratio ranges from 1:100 to 100:1, and in further embodiments, the ratio includes 1:9 to 9:1.

[0220] Suitable conditions for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI medium 1640 or X-vivo 15 (Lonza)) that may contain factors necessary for growth and survival, including serum (e.g., fetal bovine serum or human serum), interleukin 2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α, or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Target cells are maintained under conditions necessary to support growth, for example, at an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

[0221] therapeutic use The invention encompasses cells (e.g., T cells) containing a CAR molecule of the invention that have been transduced with a retrovirus, such as a lentiviral vector (LV) encoding a CAR of the invention. Thus, in some cases, the transduced T cells can elicit a CAR-mediated T cell response.

[0222] The present invention provides the use of CARs to redirect the specificity of primary T cells to the tumor antigen CLDN6. Thus, the present invention also provides a method for stimulating a T cell-mediated immune response against a target cell population or tissue in a mammal, comprising administering to the mammal a T cell expressing a CAR of the present invention, wherein the CAR comprises a binding moiety that specifically interacts with CLDN6 as a predetermined target.

[0223] In one embodiment, the present invention includes a kind of cell therapy, in which T cells are genetically modified to express the CAR of the present invention, and the CAR T cells are infused into recipients in need of it. The infused cells can kill tumor cells in the recipients. Unlike antibody therapy, CAR T cells can replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0224] In one embodiment, the CAR T cells of the invention can undergo robust in vivo T cell expansion and persist for extended periods of time, hi another embodiment, the CAR T cells of the invention evolve into specific memory T cells that can be reactivated to inhibit further tumor formation or growth.

[0225] The cancers that can be treated include tumors that are not vascularized or are not yet substantially vascularized, as well as vascularized tumors.Cancers can include solid tumors.The types of cancers that can be treated with the CAR of the present invention include, but are not limited to, carcinomas, blastomas and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas and melanomas.Also include adult tumors / cancers and pediatric tumors / cancers.

[0226] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Various types of solid tumors are named for the type of cells that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors such as sarcoma and carcinoma include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, These include liver cancer, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors, such as gliomas (such as brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases.

[0227] In one embodiment, the cancer that may be treated is a CLDN6-expressing cancer as described herein.

[0228] In one embodiment of the present invention, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic to the recipient.

[0229] The CAR modified cells of the invention may be administered alone or in combination with other components such as IL-2 or other cytokines or cell populations. In one embodiment, the CAR modified cells of the invention are administered in combination with a cognate antigen molecule or a nucleic acid, particularly RNA, encoding the cognate antigen molecule. The cognate antigen molecule may be CLDN6, recombinant CLDN6, a CLDN6 fragment, or a variant of any of the foregoing. In one embodiment, a nucleic acid, particularly RNA, encoding a cognate antigen molecule (e.g., CLDN6, recombinant CLDN6, a CLDN6 fragment, or a variant of any of the foregoing) is administered. Preferably, the nucleic acid encoding the cognate antigen molecule is expressed in the cells of the subject to which the CAR modified cells of the invention are administered, and the nucleic acid encoding the cognate antigen molecule provides the cognate antigen molecule for binding by the CAR antigen binding domain. In one embodiment, the expression of the cognate antigen molecule of the CAR antigen binding domain is at the cell surface. In one embodiment, the nucleic acid encoding the cognate antigen molecule is transiently expressed in the cells of the subject. In one embodiment, the nucleic acid encoding the cognate antigen molecule is RNA. Preferably, contacting the CAR modified cells of the invention with the cognate antigen molecule results in cell proliferation and / or activation.

[0230] The peptide and protein antigens suitable for use according to the present invention typically comprise a peptide or protein that comprises the epitope to which the CLDN6 antigen-binding domain of the CAR of the present invention binds.The peptide or protein or epitope can be derived from CLDN6.For example, the peptide or protein antigen or the epitope contained within the peptide or protein antigen can be CLDN6 or a fragment or variant of CLDN6.

[0231] The peptide and protein antigens provided to a subject according to the present invention (by administering the peptide and protein antigens or nucleic acids, in particular RNA, encoding said peptide and protein antigens), i.e. vaccine antigens, preferably result in the stimulation, priming and / or expansion of CAR modified cells in the subject to which the antigen or nucleic acid is administered. Said stimulated, primed and / or expanded CAR modified cells are preferably directed against the CLDN6 target antigen, in particular a target antigen expressed by diseased cells, tissues and / or organs, i.e. a disease-associated antigen. Thus, the vaccine antigen may comprise a disease-associated antigen, or a fragment or variant thereof. In one embodiment, such a fragment or variant is immunologically equivalent to the disease-associated antigen. In the context of the present disclosure, the term "antigen fragment" or "antigen variant" refers to an agent that results in the stimulation, priming and / or expansion of CAR modified cells, where the stimulated, primed and / or expanded CAR modified cells target the disease-associated antigen, in particular when expressed on the surface of diseased cells, tissues and / or organs. Thus, the vaccine antigen administered according to the present disclosure may correspond to or include a disease-associated antigen, may correspond to or include a fragment of a disease-associated antigen, or may correspond to or include an antigen that is homologous to a disease-associated antigen or a fragment thereof. When the vaccine antigen administered according to the present disclosure comprises a fragment of a disease-associated antigen or an amino acid sequence that is homologous to a fragment of a disease-associated antigen, said fragment or amino acid sequence may comprise an epitope of the disease-associated antigen or a sequence that is homologous to an epitope of the disease-associated antigen, where the CAR-modified cell binds to said epitope. Thus, according to the present disclosure, the antigen may comprise an immunogenic fragment of a disease-associated antigen or an amino acid sequence that is homologous to an immunogenic fragment of a disease-associated antigen. An "immunogenic fragment of an antigen" according to the present disclosure preferably relates to a fragment of an antigen that can stimulate, prime and / or expand CAR-modified cells. The vaccine antigen (like the disease-associated antigen) preferably provides a relevant epitope for binding by the CLDN6 antigen-binding domain present in the CAR of the CAR-modified cell.It is also preferred that vaccine antigens (as well as disease-associated antigens) are expressed on the surface of cells, such as antigen-presenting cells, to provide relevant epitopes for binding by the CAR. Vaccine antigens according to the present invention may be recombinant antigens.

[0232] The term "immunologically equivalent" means that an immunologically equivalent molecule, such as an immunologically equivalent amino acid sequence, exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effect, e.g., with respect to the type of immunological effect. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effect or properties of an antigen or antigen variant. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if, when exposed to a CAR-modified cell that binds to a reference amino acid sequence, e.g., CLDN6, or a cell expressing the reference amino acid sequence, it induces an immune response with specificity that reacts with the reference amino acid sequence, in particular the stimulation, priming and / or expansion of CAR-modified cells. Thus, a molecule that is immunologically equivalent to an antigen exhibits the same or essentially the same properties and / or exerts the same or essentially the same effect, with respect to the stimulation, priming and / or expansion of CAR-modified cells, as the antigen targeted by the CAR-modified cell.

[0233] As used herein, "activation" or "stimulation" refers to the state of T cells that are stimulated sufficiently to induce detectable cell proliferation. Activation can also be associated with the induction of cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells undergoing cell division.

[0234] The term "priming" refers to the process by which a T cell first contacts its specific antigen and triggers differentiation into an effector T cell.

[0235] The term "clonal expansion" or "expansion" refers to the process of increasing a particular entity. In the context of the present disclosure, the term is preferably used in the context of an immunological response in which lymphocytes are stimulated by an antigen, proliferate, and the particular lymphocytes that recognize said antigen are amplified. Preferably, clonal expansion results in differentiation of lymphocytes.

[0236] According to the present invention, it is particularly preferred that the cognate antigen is administered in the form of RNA encoding the antigen. After administration of the RNA, at least a portion of the RNA is delivered to the target cell. In one embodiment, at least a portion of the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is translated by the target cell to produce the encoded peptide or protein. Some embodiments include targeted delivery of the RNA to a specific tissue. In one embodiment, the delivery includes targeting the lymphatic system, in particular the secondary lymphatic organs, more specifically the spleen. In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen presenting cell, such as a professional antigen presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell of the spleen.

[0237] The "lymphatic system" is a part of the circulatory system and an important part of the immune system that includes the network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphoid organs, the conducting network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs generate lymphocytes from immature precursor cells. The thymus and bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs, including lymph nodes and the spleen, maintain mature naive lymphocytes and initiate adaptive immune responses.

[0238] RNA can be delivered to the spleen by so-called lipoplex formulations, in which RNA is bound to liposomes containing cationic lipids and optionally further lipids or helper lipids to form an injectable nanoparticle formulation. Liposomes can be obtained by injecting a solution of lipids in ethanol into water or a suitable aqueous phase. RNA lipoplex particles can be prepared by mixing liposomes with RNA. Spleen-targeted RNA lipoplex particles are described in WO 2013 / 143683, which is incorporated herein by reference. It has been found that RNA lipoplex particles with a net negative charge can be used to selectively target spleen tissue or spleen cells, such as antigen-presenting cells, especially dendritic cells. Thus, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression in the spleen occurs. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and / or RNA expression occurs in the lung and / or liver. In one embodiment, after administration of RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in antigen-presenting cells, such as professional antigen-presenting cells in the spleen.Therefore, the RNA lipoplex particles of the present disclosure can be used to express RNA in such antigen-presenting cells.In one embodiment, the antigen-presenting cells are dendritic cells and / or macrophages.

[0239] In the context of the present disclosure, the term "RNA lipoplex particle" refers to a particle that includes lipids, particularly cationic lipids, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA result in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes can generally be synthesized using cationic lipids such as DOTMA and additional lipids such as DOPE. In one embodiment, the RNA lipoplex particle is a nanoparticle.

[0240] As used herein, "cationic lipid" refers to a lipid that has a net positive charge. Cationic lipids bind negatively charged RNA to lipid matrices through electrostatic interactions. In general, cationic lipids have a lipophilic moiety, such as a sterol, acyl or diacyl chain, and the head group of the lipid typically carries a positive charge. Examples of cationic lipids include 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 2,3-di(tetradecoxy)propane. Cationic lipids include, but are not limited to, pyr-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamide trifluoroacetate (DOSPA).DOTMA, DOTAP, DODAC, and DOSPA are preferred.In certain embodiments, the cationic lipid is DOTMA and / or DOTAP.

[0241] Additional lipids may be incorporated to adjust the overall ratio of positive and negative charges and the physical stability of the RNA lipoplex particles. In certain embodiments, the additional lipid is a neutral lipid. As used herein, "neutral lipid" refers to a lipid with zero net charge. Examples of neutral lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, and cerebroside. In certain embodiments, the additional lipid is DOPE, cholesterol, and / or DOPC.

[0242] In certain embodiments, the RNA lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0243] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In certain embodiments, the molar ratio can be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.

[0244] The RNA lipoplex particles described herein, in one embodiment, have an average diameter in the range of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In certain embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In one embodiment, the RNA lipoplex particles have an average diameter in the range of about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter ranging from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0245] The charge of the RNA lipoplex particle of the present disclosure is the sum of the charge present in at least one cationic lipid and the charge present in RNA.The charge ratio is the ratio of the positive charge present in at least one cationic lipid and the negative charge present in RNA.The charge ratio of the positive charge present in at least one cationic lipid and the negative charge present in RNA is calculated by the following formula: charge ratio = [(cationic lipid concentration (mol)) * (total number of positive charges in cationic lipid)] / [(RNA concentration (mol)) * (total number of negative charges in RNA)].

[0246] The spleen-targeted RNA lipoplex particles described herein at physiological pH preferably have a net negative charge, such as a charge ratio of positive to negative charges of about 1.9:2 to about 1:2. In certain embodiments, the charge ratio of positive to negative charges in the RNA lipoplex particles at physiological pH is about 1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0.

[0247] The CAR modified cells and additional agents described herein may be administered in a pharmaceutical composition or medicament for therapeutic or prophylactic treatment, and may be administered in the form of any suitable pharmaceutical composition.

[0248] The term "pharmaceutical composition" relates to a formulation containing a therapeutically active agent, preferably together with a pharma- ceutically acceptable carrier, diluent and / or excipient. Said pharmaceutical composition is useful for treating, preventing or reducing the severity of a disease or disorder by administering said pharmaceutical composition to a subject. Pharmaceutical compositions are also known in the art as pharmaceutical formulations.

[0249] The pharmaceutical compositions of the present disclosure may include or be administered with one or more adjuvants. The term "adjuvant" refers to a compound that extends, enhances or accelerates an immune response. Adjuvants include a heterogeneous group of compounds, such as oil emulsions (e.g., Freund's adjuvant), inorganic compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune stimulating complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, and chemokines. Chemokines can be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, LT-a. Further known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils such as Montanide® ISA 51. Other suitable adjuvants for use in the present disclosure include lipopeptides such as Pam3Cys.

[0250] Pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in a "pharmaceutically acceptable formulation."

[0251] The term "pharmaceutical acceptable" refers to the non-toxicity of a material that does not interact with the action of the active ingredients of a pharmaceutical composition.

[0252] The term "pharmacologically effective amount" or "therapeutically effective amount" refers to an amount that alone or together with further doses achieves the desired response or the desired effect. In the case of the treatment of a particular disease, the desired response preferably relates to the inhibition of the course of the disease. This includes slowing down the progression of the disease, in particular interrupting or reversing the progression of the disease. The desired response in the treatment of a disease can also be the delay of the onset or prevention of the onset of said disease or said condition. The effective amount of the compositions described herein depends on the condition being treated, the severity of the disease, the individual parameters of the patient, including age, physiological state, size and weight, the duration of the treatment, the type of concomitant treatment (if any), the specific route of administration and similar factors. Thus, the dosage of the compositions described herein may depend on such various parameters. If the patient's response is inadequate with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0253] The pharmaceutical compositions of the present disclosure may include salts, buffering agents, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure include one or more pharma- ceutically acceptable carriers, diluents, and / or excipients.

[0254] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0255] The term "excipient" as used herein refers to a substance that may be present in the pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0256] The term "diluent" refers to an agent that dilutes and / or thins. Furthermore, the term "diluent" includes any one or more of a fluid, liquid or solid suspension and / or mixed medium. Examples of suitable diluents include ethanol, glycerol and water.

[0257] The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, with which the active ingredient is combined to facilitate, enhance, or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and especially biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure comprises isotonic saline.

[0258] Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985).

[0259] Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0260] In one embodiment, the pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical compositions are formulated for local or systemic administration. Systemic administration may include enteral administration, including absorption via the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to administration in any manner other than via the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical compositions are formulated for systemic administration. In another preferred embodiment, the systemic administration is by intravenous administration. The compositions may be injected directly into a tumor or lymph node.

[0261] The term "co-administration" as used herein refers to the process of administering different compounds or compositions to the same patient. For example, the CAR modified cells and antigens or nucleic acids encoding them described herein can be administered simultaneously, essentially simultaneously, or sequentially. When administration is sequential, the CAR modified cells can be administered before or after the administration of the antigens or nucleic acids encoding them. When administration is simultaneous, the CAR modified cells and antigens or nucleic acids encoding them do not need to be administered in the same composition. The CAR modified cells and antigens or nucleic acids encoding them can be administered one or more times, and the number of administrations of each component can be the same or different. Furthermore, the CAR modified cells and antigens or nucleic acids encoding them do not need to be administered to the same site.

[0262] The term "disease" refers to an abnormal condition that affects an individual's body. A disease is often interpreted as a medical condition associated with certain symptoms and signs. A disease may be caused by an external agent, such as an infection, or it may be caused by an internal malfunction, such as an autoimmune disease. In humans, "disease" is often used more broadly to refer to a condition that causes pain, impairment, distress, social problems, or death in the affected individual, or causes similar problems in people who come into contact with the individual. In this broader sense, disease sometimes includes damage, incapacity, disability, syndrome, infection, isolated symptoms, deviant behavior, and atypical changes in structure and function, although in other contexts and for other purposes, these may be considered distinct categories. Diseases usually affect individuals not only physically but also emotionally, as suffering from and living with many diseases can change one's outlook on life and personality.

[0263] In the present context, the term "treatment", "treat" or "therapeutic intervention" relates to the management and care of a subject with the aim of combating a condition, such as a disease or disorder. This term is intended to include the full range of treatments for a given condition suffered by a subject, such as the administration of therapeutically effective compounds to alleviate symptoms or complications, to slow the progression of a disease, disorder or condition, to relieve or alleviate symptoms and complications, and / or to cure or eliminate a disease, disorder or condition, as well as to prevent a condition, where prevention is to be understood as the management and care of an individual with the aim of combating a disease, condition or disorder, and includes the administration of active compounds to prevent the onset of symptoms or complications.

[0264] The term "therapeutic treatment" relates to any treatment that improves the health status and / or extends (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, halt or delay the onset of the disease in an individual, inhibit or delay the onset of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual who currently has or has previously had the disease.

[0265] The term "prophylactic treatment" or "preventative treatment" relates to any treatment aimed at preventing the occurrence of a disease in an individual. The terms "prophylactic treatment" or "preventative treatment" are used interchangeably herein.

[0266] The terms "individual" and "subject" are used interchangeably herein. They refer to a human or another mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may or may not have a disease or disorder (e.g., cancer), but may be afflicted or susceptible to the disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderly people, children, and newborns. In an embodiment of the present disclosure, an "individual" or "subject" is a "patient."

[0267] The term "patient" refers to an individual or subject for treatment, particularly an affected individual or subject.

[0268] Combination strategies in cancer treatment may be desirable due to the resulting synergistic effects, which may be significantly more potent than the impact of monotherapy approaches. In one embodiment, the pharmaceutical composition is administered with an immunotherapeutic agent. As used herein, "immunotherapeutic agent" refers to any agent that may be involved in the activation of a specific immune response and / or immune effector function(s). The present disclosure contemplates the use of antibodies as immunotherapeutic agents. Without wishing to be bound by theory, antibodies can achieve therapeutic effects on cancer cells through a variety of mechanisms, including inducing apoptosis, blocking components of signal transduction pathways, or inhibiting tumor cell proliferation. In certain embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies may induce cell death via antibody-dependent cell-mediated cytotoxicity (ADCC) or may bind to complement proteins resulting in direct cytotoxicity known as complement-dependent cytotoxicity (CDC). Non-limiting examples of anti-cancer antibodies and potential antibody targets (in brackets) that may be used in combination with the present disclosure include abagovomab (CA-125), abciximab (CD41), adecatumumab (EpCAM), afutuzumab (CD20), alacizumab pegol (VEGFR2), altumomab pentetate (CEA), amatuximab (MORAb-009), anatumomab mafenatox (TAG-72), apolizumab (HLA-DR), arcitumomab (CEA), atezolizumab (PD-L1), bavituximab (phosphatidylserine), bectumomab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), bivatuzumab mertansine (CD44 v6), blinatumomab (CD19), brentuximab vedotin (CD30TNFRSF8), cantuzumab mertansine (mucin CanAg), cantuzumab mertansine (MUC1), capromab pendetide (prostate cancer cells), carlumab (CNT0888), catumaxomab (EpCAM, CD3), cetuximab (EGFR), sitatuzumab bogatox (EpCAM), cixutumumab (IGF-1 receptor), claudiximab (claudin), clivatuzumab tetraxetan (MUC1), conatumumab (TRAIL-R2), dacetuzumab (CD40), dalotuzumab (insulin -like growth factor I receptor), denosumab (RANKL), detumomab (B lymphoma cells), drozitumab (DR5), ecromeximab (GD3 ganglioside), edrecolomab (EpCAM), elotuzumab (SLAMF7), enavatuzumab (PDL192), ensituximab (NPC-1C), epratuzumab (CD22), ertumaxomab (HER2 / neu, CD3), etaracizumab (integrin ανβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficlatuzumab (SCH900105), figitumumab (IGF-1 receptor), framvotumab (glycoprotein 75), fresolimumab (TGF-β), galiximab (CD80), ganitumab (IGF-I), gemtuzumab ozogamicin (CD33), gevokizumab (ILΙβ), girentuximab (carbonic anhydrase 9 (CA-IX)), glembatumumab vedotin ( GPNMB), ibritumomab tiuxetan (CD20), icrucumab (VEGFR-1), igoboma (CA-125), indatuximab ravtansine (SDC1), intetumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab (CD152), iratumumab (CD30), labetuzumab (CEA), lexatumumab (TRAIL-R2), ribivirumab (Hepatitis B surface antigen), lintuzumab (CD33), lorvotuzumab mertansine (CD56), lucatumumab (CD40), rumiliximab (CD23), mapatumumab (TRAIL-R1), matuzumab (EGFR), mepolizumab (IL5), milatuzumab (CD74), mitsumomab (GD3 ganglionic antigen) Osido), mogamulizumab (CCR4), moxetumomab passudotox (CD22), nacolomab butafenatox (C242 antigen), naptumomab estafenatox (5T4), naptumomab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumab (PDGF-Ra), onartuzumab (human scatter factor receptor kinase), oportuzumab monatox (EpCAM), oregovomab (CA-125), oxelumab (OX-40), panitumumab (EGFR), patritumab (HER3), pemtumomab (MUC1), pertuzumab (HER2 / neu), pintumomab (adenocarcinoma antigen), pritumumab (vimentin), racotumomab (N-glycolylneuraminic acid), radletumab (fibronectin extra domain B), rafivirumab (rabies virus glycoprotein), ramucirumab (VEGFR2), rilotumumab (HGF), rituximab (CD20), lobatumumab (IGF-1 receptor), samaryzumab (CD200), sib Lotuzumab (FAP), siltuximab (IL6), tabalumab (BAFF), tacatuzumab tetraxetan (α-fetoprotein), taplitumomab paptox (CD19), tenatumomab (tenascin-C), teprotumumab (CD221), ticilimumab (CTLA-4), tigatuzumab (TRAIL-R2), TNX-650 (IL13), tositumomab (CD20), trastuzumab (HER2 / neu), TRBS07 (GD2), tremelimumab (CTLA-4), tucotuzumab celmoleukin (EpCAM), ublituximab (MS4A1), urelumab (4-1BB), volociximab (integrin α5β1), votumumab (tumor antigen CTAA 16.88), zalutumumab (EGFR), and zanolimumab (CD4).

[0269] Citation of documents and tests referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements regarding the contents of these documents are based on information available to applicant and do not constitute an admission as to the correctness of the contents of these documents. EXAMPLES

[0270] The present invention will be described in more detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0271] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following examples therefore specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0272] Example 1 material and method The techniques and methods used herein are carried out as described herein or in a manner known per se, for example as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. All methods, including the use of kits and reagents, are carried out according to the manufacturer's information, unless otherwise indicated.

[0273] CAR constructs The gamma-retroviral self-inactivating (SIN) vector pES.12-6 was used to stably overexpress CLDN6-CAR-BBz in human T cells under the control of an internal eukaryotic promoter, the short intronless human elongation factor 1 alpha promoter (EFS-213 / +31). The vector backbone contains MLV wild-type sequences in the R and U5 regions and packaging regions (ψ and ψ+) in the 5'- and 3'-LTRs. The enhancer element in the U3 region of the 3'-LTR was removed (including the CAAT box) and the TATA box sequence was mutated to prevent transcription initiation. A truncated version of the posttranscriptional regulatory element (PRE) of the woodchuck hepatitis virus (WHV) is used to prevent expression of undesired viral proteins.

[0274] CLDN6-CAR-BBz expresses the signaling peptide of human IgG, the heavy chain (V H) and light chain (V L ) with a (G4S)3 linker between them, and V L Position 46 of (V shown in the sequence table) L The scFv fragment is fused to the human CD8α hinge and transmembrane domain, followed by human 4-1BB and human CD3ζ(Q14K) signaling moieties.

[0275] Cell lines and reagents The teratoma cell line PA-1-SC12_A0201_luc_gfp expressing human CLDN6 was cultured in MEM-GlutaMAX medium supplemented with 10% (v / v) FCS (Biochrom), 1 mM sodium pyruvate (Gibco), 1 mM MEM non-essential amino acid solution (Gibco) and 2% (v / v) sodium bicarbonate solution (Gibco). The cell line overexpresses HLA-A*0201, luciferase and GFP.

[0276] The ovarian cancer cell line OV-90-SC12 was cultured in 41.5% (v / v) MCDB 105 medium (Sigma-Aldrich), 41.5% (v / v) Medium 199 (Sigma Aldrich) supplemented with 15% (v / v) FCS and 2% (v / v) of 7.5% sodium bicarbonate solution.

[0277] Culture medium for the human melanoma cell line SK-MEL-37 consists of 90% DMEM GlutaMAX™ (Gibco) supplemented with 10% (v / v) FCS.

[0278] The medium for MDA-MB-231 consisted of 88% (v / v) RPMI 1640 GlutaMAX™ (Gibco) supplemented with 10% FCS, 1 mM sodium pyruvate, 1 mM MEM non-essential amino acid solution.

[0279] The human adenocarcinoma cell line 23132-87 and the human melanoma cell line MEL-526 were cultured in 90% (v / v) RPMI 1640 GlutaMAX™ (Gibco) supplemented with 10% (v / v) heat-inactivated FCS.

[0280] The culture medium for HEK-293 consisted of 90% (v / v) Eagle's Minimum Essential Medium (EMEM) (ATCC) and 10% (v / v) FCS.

[0281] SKOV-3 were cultured in 90% (v / v) McCoy's 5A medium (ATCC) supplemented with 10% (v / v) FCS.

[0282] The human ovarian cell line NIH-OVCAR-3 was cultured in 80% (v / v) RPMI 1640 GlutaMAX™ (Gibco, Cat. No. 61870) supplemented with 20% (v / v) FCS and 0.1% (w / v) insulin (Sigma-Aldrich).

[0283] Human tumor cell lines LCLC-103H, COLO-699-N, JAR and NEC-8 were cultured in 90% (v / v) RPMI 1640 GlutaMAX™ (Gibco) supplemented with 10% (v / v) FCS.

[0284] Cell lines were seeded and / or split every 2 or 3 days.

[0285] Peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) PBMCs were isolated from buffy coats by Ficoll®-Hypaque (1.077 g / mL Amersham Biosciences) density gradient centrifugation. Monocytes were enriched with anti-CD14 microbeads (Miltenyi Biotec). Immature DCs (iDCs) were differentiated by culture in DC medium consisting of RPMI 1640 GlutaMAX™, 100 U / mL penicillin, 100 μg / mL streptomycin, 1 mM sodium pyruvate, non-essential amino acids, and 5% (v / v) heat-inactivated human AB serum (all from Invitrogen, Karlsruhe, Germany), supplemented with 1000 U / mL h GM-CSF (Essex, Lucerne, Switzerland) and 1000 U / mL h IL-4 (Strathmann Biotech, Hamburg, Germany).

[0286] Transduction of T cells CD3 using Dynabeads® Human T-Expander CD3 / CD28 CTS + / CD28 high+ T cells were enriched from PBMCs by magnetic separation of T cells. Cells were enriched 3:1 with beads to CD3 +T cells were incubated at 100x the CD3 / CD28 ratio and separated using a CTS DynaMag magnet. Enriched T cells were cultured in X-VIVO 15 medium supplemented with 5% (v / v) human serum in the presence of 450U / mL rh IL-7 and 50U / mL rh IL-15 (both from Miltenyi Biotec). After 3 days, CD3 / CD28 beads were removed using a magnet and pre-activated T cells were transduced with retroviral vectors in the presence of Protransduzin®-A at a final concentration of 25μg / mL. Cells were expanded in complete medium until day 7 or 10 and either used directly or cryopreserved to assess CAR surface expression, T cell phenotype and effector function.

[0287] Flow cytometry analysis of in vitro cultured cells Cell surface expression of transduced CAR was analyzed using an Alexa-Fluor-647-conjugated idiotype-specific antibody (Ganymed Pharmaceuticals) that recognizes the scFv fragment contained in all CLDN6-CAR constructs. CLDN6 surface expression on target cells was analyzed by staining with Alexa-Fluor647-conjugated CLDN6-specific antibody IMAB027 (Ganymed Pharmaceuticals). Flow cytometry measurements were performed on a FACSCanto™ II flow cytometer using FACSDiva™ software (BD Biosciences), and analysis was performed using FlowJo® V10 (treestar inc.).

[0288] Quantitative real-time PCR (qRT-PCR) Total RNA was isolated from the indicated cell lines using the RNeasy® Mini Kit (QIAGEN). For reverse transcription of RNA to obtain cDNA for qRT-PCR, the PrimeScript™ RT Reagent Kit with gDNA Eraser (TAKARA) was used starting with 1 μg of total RNA. Quantitative real-time PCR was performed using the QuantiTect SYBR® Green PCR Kit (QIAGEN) with the following primers (5'-3'): CLDN6-forward: CTT ATC TCC TTC GCA GTG CAG, CLDN6-reverse: AAG GAG GGC GAT GAC ACA GAG, HPRT 1-forward: TGA CAC TGG CAA AAC AAT GCA, HPRT 1-reverse: GGT CCT TTT CAC CAG CAA GCT (annealing temperature: 62°C).

[0289] xCELLigence Cytotoxicity Assay For the evaluation of CAR-mediated cytotoxicity, the xCELLigence system (OMNI Life Science) was used. Cell index (CI) impedance measurements were performed according to the supplier's instructions. The optimal cell density resulting in an exponential growth curve was determined for each tumor cell line. Target cells were cultured at 2–10 per well. 4 Cells were seeded onto E-Plate 96 PET (ACEA Biosciences Inc.) at a concentration of 100 mM NaCl, 100 mM MgCl, 100 mM T cells, 100 mM NaCl, 100 mM MgCl ...

[0290] Percent specific lysis was calculated as follows: (CI L min -CI sample ) / CI L min ×100

[0291] The standard deviation was calculated as follows: 100×(CI sample / CI L min)×{√[(CI sample STDEV / CI sample ) 2 +(CI L min STDEV / CI L min ) 2 ]}

[0292] Minimum dissolution (L min ) was assessed after incubating target cells with effector T cells expressing a control antigen (e.g. eGFP, control CAR).

[0293] CFSE (Carboxyfluorescein succinimidyl ester) proliferation assay To determine the percentage of proliferating T cells after antigen-specific stimulation, CAR-expressing T cells were labeled with 1.6 μM of the fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) for 10 min at 37° C. (protected from light). To remove free dye, pure FCS was added to the cells and incubated for another 5 min. Cells were washed, resuspended in culture medium, and co-cultured with target cells using different effector-to-target (E:T) ratios in a total volume of 200 μL DC medium in 96-well round-bottom plates. After 5 days of co-culture, cells were stained with fluorescent dye-conjugated antibodies against, for example, CD4, CD8, and CAR. The percentage of proliferating T cells was analyzed by flow cytometry based on the progressive half-life of CFSE fluorescence in daughter cells after cell division using a BD FACSCanto™ II flow cytometer (Becton Dickinson).

[0294] Spheroid Assay (IncuCyte®) Tumor spheroids were cultured at 1 × 10 per well in Corning® Costar® Ultra-Low Attachment 96 round-bottom well plates in MEM-GlutaMAX medium supplemented with 10% (v / v) FCS, 1% (v / v) sodium pyruvate, 1% (v / v) MEM NEAA, and 2% (v / v) sodium bicarbonate for 48 h after centrifugation. 4PA1-SC12-A2-eGFP cells were cultured in vitro and then supplemented with CAR T cells (1 × 10 5 Tumor spheroids expressing eGFP (100 μg / well) were imaged at 4x magnification and 300 ms exposure time to detect green fluorescence in an IncuCyte Zoom Live-content imaging system (Essen Bioscience) at 37 °C and 5% CO2. Images were acquired hourly over a 10-day period. Data were analyzed using IncuCyte analysis software to determine total green object integrated intensity (GCU × μm 2 Green objects were detected and quantified (Figure 1A,b) and the mean and SD of the number of green objects at each time point were plotted using IncuCyte analysis software.

[0295] Preparation of in vitro transcribed (IVT) mRNA In vitro transcription of antigen-encoding mRNA was based on the pST1-T7-GG-hAg-MCS-2hBg-A30LA70 plasmid backbone and derived DNA constructs. These plasmid constructs contain, in addition to the full-length ORF, 5' human alpha globin, two consecutive 3' human beta globin UTRs and a 100-nucleotide poly(A) tail with a linker after 70 nucleotides. Antigen-encoding mRNA was generated by in vitro transcription as described in Holtkamp S.et al.(2006)Blood 108(13):4009-17. In vitro transcription of all described mRNA constructs was performed at BioNTech RNA Pharmaceuticals GmbH.

[0296] IVT RNA encoding a liposomally formulated antigen (RNA (LIP) ) Generation and in vitro transfection of dendritic cells Complexation of IVT RNA encoding antigens with liposomes was previously described by Kranz et al (2016) Nature 534(7607):396-401. A charge ratio of cationic DOTMA to RNA of 1.3 to 2 was used. In addition to DOTMA, the lipid fraction contains the helper lipid DOPE at a molar ratio of 2:1 DOTMA per DOPE.

[0297] Animal Experimental Techniques animal 9-21 week old female immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl C57BL / 6BrdCrHsd-Tyr / SzJ (NSG) mice were used for in vivo studies. Breeding pairs were purchased from the Jackson laboratory (Bar Harbour, ME, USA) and kept in the animal facility of BioNTech AG, Germany. c Mice were purchased from Envigo Labs. Age (8–10 weeks) and sex (male or female)-matched animals were used throughout the experiments. Congenic C57Bl / 6-Thy1.1 mice were kept in the animal facility of BioNTech AG, Germany. All experiments were performed under specific pathogen-free (SPF) conditions and in accordance with German animal experimentation regulations.

[0298] Tumor cell transplantation 5×10 6 OV90-SC12 or 5×10 5 Mice were injected subcutaneously into the right dorsal flank with either 100 μL of CT26 tumor cells (in 100 μL of PBS). Tumor growth monitoring and volume calculations were determined using calipers and inserted into the formula V=1 / 2(length×width2). Prior to adoptive transfer of human CAR-engineered T cells, tumor-bearing mice were stratified using Daniels' XL Toolbox Add-in for Microsoft Excel to achieve gross tumor volume distribution among different treatment groups.

[0299] Adoptive cell transfer (ACT) of human T cells Total human T cells transduced with different amounts of γ-retrovirus (number and frequency of T cells expressing CAR or GFP transgenes are shown in the respective figures) were injected intravenously into the retrobulbar plexus in 200 μL of PBS. Depending on the experimental setup, either transduced T cells were used directly after the in vitro activation and transduction process, or cryopreserved transduced T cells were thawed and adoptively transferred directly into mice after washing twice with PBS. The viability of all T cell products used in the experiments was >90%.

[0300] Monitoring human CAR T cells in blood in vivo At the indicated time points, 50 μL of peripheral blood was withdrawn from the retro-orbital vein and collected in heparin-containing reaction tubes (Sarstedt). Red blood cells were lysed using BD FACS lysis solution (BD). CAR expression on the surface of transferred human T cells was analyzed using hCD45-PE-Cy7 (HI30, BD), hCD4-APC-Cy7 (OKT4, BioLegend), hCD8-BV421 (RPA-T8, BD) and Alexa-Fluor 647-conjugated idiotype-specific antibodies (Ganymed Pharmaceuticals). 7-AAD (Beckmann Coulter) was used to distinguish dead cells from the analysis. Flow cytometry measurements were performed on a FACSCanto™ II flow cytometer using FACSDiva™ software (BD Biosciences) and analysis was performed using FlowJo® V10 (Treestar inc.).

[0301] Preparation of CAR T cells for retroviral genetic manipulation and adoptive T cell transfer Either naive C57Bl / 6-Thy1.1+ or Balb / c-Thy1.1+ splenocytes were isolated and preactivated with Dynabeads™ mouse T activator CD3 / CD28 (Invitrogen) at a bead to T cell ratio of 1:1 in the presence of 5 ng / mL recombinant human (rh)IL-7 and 5 ng / mL rh IL-15 (Miltenyi Biotec). For transduction of mouse cells, MLV-E pseudotyped retroviral supernatants were incubated with RetroNectin (2 μg / cm 2 )-coated non-tissue culture-treated well plates according to the manufacturer's instructions (Takara Bio Inc., Otsu, Japan), and three cycles of virus loading and centrifugation (1,300 × g, 15 °C, 15 min) were repeated to increase binding. After 24 h of preactivation, 0.5–0.6 × 10 6 cells / cm 2 were spun down onto viral particle-coated wells (300xg, 37°C, 1 h). After overnight culture, spin-down transduction was repeated with freshly viral particle-coated plates. After 72 h of preactivation, Dynabeads™ mouse T activator CD3 / CD28 were removed from the cultures and cells were expanded in the presence of 5ng / mL rh IL-7 and 5ng / mL rh IL-15. After Ficoll washing, cells were washed twice with PBS to remove serum proteins and then prepared for adoptive cell transfer (ACT). A pES12.6-based retroviral vector containing the encoded CLDN6-CAR-BBz as well as enhanced firefly luciferase (effLuc; Rabinovich et al. BA, PNAS (2008) PNAS 105(38):14342-6) and eGFP (enhanced green fluorescent protein) reporter genes expressed separately using the 2A splice element (Szymczak et. AL, Nature Biotechnology, (2004) Nat Biotechnol. 22(5):589-94) was used for transduction.

[0302] Adoptive T cell transfer and RNA of mouse T cells (LIP) Vaccination γ-retrovirus-transduced CAR congenic Thy1.1 + T cells were cultured in whole-body irradiated (XRAD320) C57BL / 6BrdCrHsd-Tyr c or BALB / c donor mice, respectively. Mice were then intravenously (iv) injected with antigen-encoding RNA at a F12:RNA ratio of 1.3:2 at various time points after ACT. (LIP) The in vivo growth of CAR was analyzed by whole-body bioluminescence imaging, and the antitumor effect was analyzed by tumor monitoring.

[0303] In vivo luciferase imaging (BLI) The proliferation and distribution of CAR-effLuc-GFP transduced T cells were assessed by in vivo bioluminescence imaging using an IVIS Lumina imaging system (Caliper Life Sciences). Briefly, at the indicated time points after adoptive transfer of transduced T cells, an aqueous solution of D-luciferin (80 mg / kg body weight; Perkin Elmer) was injected i.p. After 5 min, emitted photons were quantified (integration time 1 min, binning 8). In vivo bioluminescence in the region of interest (ROI) was quantified as total flux (photons / sec) using IVIS Living Image 4.0 software. The intensity of transmitted light originating from luciferase-expressing cells within the animal is represented as a grayscale image, with black being the weakest and white to dark gray being the strongest bioluminescent signal. Grayscale reference images of mice were acquired under LED low-light illumination. Images were overlaid using Living Image 4.0 software.

[0304] Statistical analysis and data depiction All results are expressed as the mean of technical replicates + / - SD or the mean of biological replicates + / - SEM. The number of replicates is stated in the figure legend for each experiment. An unpaired two-tailed Student's t-test was used for area under the curve (AUC) comparisons of two groups. All statistical analyses were performed using GraphPad PRISM 6.04. ***P ≤ 0.001, ****P ≤ 0.0001.

[0305] Example 2 Generation and in vitro characterization of CLDN6-specific CARs For the selection of lead structures, we generated a variety of CAR scaffolds that all share the same scFv fragment derived from the CLDN6-specific antibody IMAB206-C46S, but differ in hinge and costimulatory domains (Figure 1A). Second and third generation CAR scaffolds containing the 4-1BB endodomain were included, as 4-1BB costimulation has been shown to increase CAR T cell persistence and antitumor efficacy. Alternatively or additionally, we incorporated modified CD28 domains (Kofler DM et al., (2011) Molecular Therapy 19(4), 760-767), known to deliver CD28-mediated costimulation to engineered T cells in the absence of agonistic CD28 ligands such as B7.1 and B7.2. The scFv fragments were fused to the 4-1BB or CD28 costimulatory domains via either a modified IgG Fc (Hombach A. et al., (2010) Gene Therapy 17, 1206-1213) or the CD8α hinge region.

[0306] Various CLDN6-CARs were extensively characterized in vitro for their potential to sensitively and specifically recognize and kill CLDN6-expressing tumor cells. Among other experiments, tumor spheroid assays were performed using CAR-transduced T cells in combination with eGFP-expressing PA1 tumor spheroids. Indeed, accelerated lysis of CLDN6-expressing tumor spheroids by 4-1BB-containing CARs compared to CARs with CD28 domains could be detected using IncuCyte® real-time imaging (Figure 1B).

[0307] Based on the summary of the results of various functional characterization tests, we were able to demonstrate highly specific and sensitive recognition of CLDN6, as well as high probability of survival and repeated stimulation of engineered CAR T cells, so we selected CLDN6-CAR-CD8h-BBz as a lead structure for preclinical and clinical trials. Importantly, this CAR scaffold has already been successfully used in several CD19-CAR T cell tests. To stably integrate our CLDN6-CAR into the T cell genome, we selected the gamma-retroviral self-inactivating (SIN) vector pES12.6 for stably integrating therapeutic CLDN6-CAR into the T cell genome (Loew et al., Gene Therapy (2010) 17, 272-280).

[0308] Example 3 Sensitivity of CLDN6-CAR-BBz To analyze the sensitivity of CAR-mediated recognition in more detail, CLDN6-RNA titration experiments were performed. For this purpose, the CLDN6-negative lung cancer cell line Colo699-N was transfected with titrated amounts of CLDN6-RNA and CLDN6-CAR-mediated target cell lysis was evaluated by xCELLigence cytotoxicity assay. CLDN6-CAR surface expression on transduced T cells (Figure 2A) and CLDN6 protein expression levels on transfected Colo699-N cells (Figure 2B) were evaluated by flow cytometry after staining with CLDN6-CAR-specific and CLDN6-specific antibodies. Specific killing of CLDN6-RNA-transfected target cells by CLDN6-CAR-expressing T cells could be observed depending on the CLDN6-RNA dose used for transfection of Colo699-N cells, which correlates with the number of CLDN6 molecules on the target cell surface, as evaluated by flow cytometry (Figure 2C). When very few CLDN6 molecules were expressed on the target cell surface after transfection with as little as 0.01 μg of CLDN6-RNA, CAR T cells even mediated lysis, which was barely detectable by flow cytometry.

[0309] Example 4 Safety of CLDN6-CAR-BBz To evaluate the safety of CLDN6-CAR-BBz, a cell line screening assay was performed. For this purpose, CLDN6-CAR-BBz-transduced T cells (Figure 3A) were cultured with a panel of CLDN6-positive and CLDN6-negative tumor cell lines of different tissue origins (Figure 3B), and target cell recognition and lysis were analyzed by xCELLigence cytotoxicity assay (Figure 3C). In parallel, CLDN6-mRNA and protein expression levels in the target cell lines were evaluated by qRT-PCR and flow cytometry, respectively (Figure 3D, 3E). It could be demonstrated that the recognition and lysis of the target cell lines strictly correlated with the CLDN6 mRNA and protein expression levels.

[0310] Example 5 CAR T cell expansion An essential prerequisite for the antitumor effect of CLDN6-CAR-BBz engineered T cells is their ability to proliferate and persist in patients. To analyze whether CLDN6-CAR T cells proliferate efficiently in response to ectopically expressed CLDN6 in iDCs, a CFSE-based in vitro co-culture assay was performed. CLDN6-CAR-transduced T cells were labeled with CFSE and co-cultured with titrated amounts of RNA lipoplexes (RNAs) encoding either CLDN6 or a control antigen. (LIP) )-transfected autologous iDCs. Surface expression of CLDN6-CAR on T cells and CLDN6 on target cells was verified by flow cytometry (Figure 4A, 4B). After 5 days of coculture, antigen-specific proliferation of CD4+ and CD8+ T cells expressing CFSE-labeled CAR was analyzed by flow cytometry (Figure 4C).

[0311] CLDN6-CAR was transfected with CLDN6-RNA (LIP) mediated a dose-dependent proliferation that correlated with the amount of (LIP) Only background proliferation could be observed when CLDN6-CAR-BBz T cells were transfected with 100% T cells. These data confirm that efficient antigen-specific proliferation of CLDN6-CAR-BBz T cells is induced after antigen-specific stimulation.

[0312] Example 6 In vivo antitumor efficacy of CLDN6-CAR-BBz After CAR-mediated antigen-specific induction of effector function was demonstrated in vitro, the therapeutic potential of human CLDN6-CAR-BBz T cells was examined in vivo in an advanced xenograft tumor model. To this end, immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice were subcutaneously implanted with human ovarian cancer cells (OV90) that endogenously express CLDN6. OV90 tumor-bearing NSG mice were then treated with 1 × 10 7 CLDN6-CAR or eGFP transduced T cells (approximately 5 × 10 8The CLDN6-CAR was administered to human CD4 T cells / kg of mice (Figure 5A). + and CD8 + It was expressed in approximately 16–18% of T cells (Figure 5B). Of note, adoptive transfer of CLDN6-CAR T cells resulted in a mean tumor volume of 170 mm on the day of adoptive transfer. 3 This resulted in complete regression of large tumors in the control group, whereas no antitumor effect was observed in the control group (Figure 5C). This significant antitumor effect correlated with the persistence of CLDN6-CAR-BBz T cells in the peripheral blood of treated mice (Figure 5D).

[0313] Example 7 In vitro functionality of short-term and long-term cultured CAR T cells As reproducible production of high-quality clinical-grade CAR T cell products is a prerequisite for clinical trials, the GMP manufacturing process was optimized to achieve high transduction efficiency and sufficient number and quality of T cells expressing the CLDN6-CAR. The transduction procedure was also simplified by reducing the number of transductions from 2 to 1 and shortening the ex vivo culture time from 10 to 7 days. It has been shown that T cells cultured for a short period of time show improved efficacy compared to T cells expanded and exhausted for a long period of time (PMID:30030295). Thus, the shortened culture time should not only save time and costs, but most importantly also lead to an improved chance of the engineered T cells expanding and persisting in patients.

[0314] Long-term spheroid experiments were performed to compare the in vitro antitumor efficacy of short-term (7 days) and long-term (10 days) cultured CLDN6-CAR-BBz T cells. Evaluation of CAR surface expression by flow cytometry revealed nearly equivalent CAR expression levels, but the frequency of CAR-positive T cells was slightly lower in T cell samples cultured for 7 days (Figure 6A). To analyze the possibility of repeated killing, CAR T cells were co-cultured with PA1-SC12-A2-eGFP tumor spheroids expressing CLDN6 and eGFP, and the killing of tumor spheroids was monitored in real time based on the eGFP signal using the IncuCyte® system. After complete eradication of the tumor spheroids, new tumor spheroids were added (Figure 6B). It can be demonstrated that short-term cultured CAR T cells have a comparable repeated killing ability to long-term cultured T cells.

[0315] Example 8 In vivo functionality of thawed GMP manufactured CAR T cells As the GMP transduction process intended for clinical use generates a cryopreserved CAR T cell product, it was of great importance to validate the antitumor potential of thawed CLDN6-CAR-BBz T cells engineered in a GMP facility. Furthermore, as the final GMP manufacturing process could be shortened (harvested on day 7 instead of day 10), this change in the final protocol needed to be evaluated by performing in vivo studies. Most importantly, thawed human CLDN6-CAR-BBz T cells showed significant antitumor efficacy comparable to freshly generated CAR T cells, with a mean tumor volume of 160 mm in an ovarian cancer xenograft model. 3 Both CAR T cell products eradicated advanced tumors in the 7-day or 10-day groups (Figure 7A). Furthermore, CAR T cells harvested on day 7 or 10, which showed similar CAR surface expression (Figure 7B), were compared for their antitumor efficacy in this experiment (Figure 7C). Correlating with the results of the long-term tumor spheroid experiments, both CAR T cell products mediated complete tumor rejection with comparable kinetics. The antitumor response of both products correlated with the persistence of CLDN6-CAR-BBz T cells in the peripheral blood 2 weeks after adoptive transfer (Figure 7D).

[0316] Example 9 Increased persistence of CLDN6-CAR-BBz-transduced T cells The clinical success of adoptively transferred tumor-reactive T cell therapy also positively correlates with the persistence of those cells in vivo (Robbins et al. (2004) J Immunol. 173(12):7125-30, Huang et al. (2005) 28(3):258-67). Therefore, we analyzed whether in situ antigen exposure could enhance the persistence of CLDN6-CAR-BBz T cells in vivo. Because xenograft models are not sufficient to examine the long-term persistence of CAR T cells due to graft-versus-host disease of human T cells on mouse tissues and the lack of competing endogenous immune cells, the persistence test of CLDN6-CAR-BBz T cells was performed in a mouse syngeneic mouse model. Therefore, CLDN6-CAR-BBz CAR-transduced mouse T cells co-expressing luciferase were adoptively transferred into mildly stimulated (2.5 Gy) mice, followed by transfection with RNA encoding either CLDN6 or a control antigen. (LIP) was administered repeatedly and the expansion of the CAR T cell population was continuously monitored by bioluminescence imaging (Figure 8A).

[0317] CLDN6-CAR-BBz T cells can persist in vivo (>3 months) after repeated vaccinations with CAR antigen formulated in liposomes. CAR T cells disappear over time in the control group, whereas antigen-specific restimulated CAR T cells remain viable for each RNA even after 3–4 weeks of treatment cessation after the third and fourth boost rounds. (LIP) These data were obtained from BioNTech's RNA (LIP) We demonstrate that the technology supports the activation and proliferation of appropriate CLDN6-CAR-BBz T cells by providing natural costimulation in situ, which also results in increased persistence of in vivo expanded CLDN6-CAR-BBz T cells.

[0318] Example 10 Improved antitumor activity of in vivo expanded CLDN6-CAR-BBz T cells After CAR-mediated antigen-specific proliferation and persistence was demonstrated, the question arose as to whether these in vivo expanded CLDN6-CAR-BBz T cells would also exhibit enhanced antitumor potential compared to their non-expanded counterparts. To this end, we investigated whether CLDN6 dim Balb / c mice inoculated with the colon cancer cell line CT26 expressing 6 CLDN6-CAR-BBz or control CAR-BBz transduced mouse T cells (3–4 × 10 5 After iv administration of CAR T cells, mice were treated with either 20 μg of RNA encoding either full-length CLDN6 or a full-length control antigen. (LIP) The subjects then received a booster vaccination with CLDN6-RNA (Figure 9A). (LIP) No antitumor effects were observed in control CAR-BBz-treated animals treated with non-relevant control RNA (LIP) However, when treated with a moderate dose of CLDN6-CAR-BBz T cells in combination with CLDN6-RNA, only slight tumor regression was achieved. (LIP) Mice receiving the combination of vaccination and intermediate CAR T cell doses showed significantly enhanced tumor regression (Figure 9B).

[0319] Example 11 Restoration of antitumor efficacy of low-dose in vivo expanded CAR T cells RNA (LIP) In addition to the potential for in vivo CAR T cell expansion to improve ongoing antitumor responses, it may also be shown that this technique is well suited to restore the antitumor potential of insufficient CAR T cell doses. Tumor-bearing mice were treated with CAR T cell doses ten times lower than those required for tumor rejection (Figure 10A). Control RNA (LIP) Tumor growth was observed in mice treated with CAR, but after the first implantation, the RNA encoding the CAR antigen (LIP)All animals receiving CAR T cells showed tumor rejection (Figure 10C). Accordingly, the frequency of engrafted CAR T cells in the peripheral blood was significantly higher after vaccination (Figure 10D). Insufficient CAR T cell doses after engraftment could be compensated for by using RNA to achieve in vivo expansion. (LIP) Supplementing with additional treatment with CAR T-cells may be very useful in at least two different scenarios: 1) low yield of GMP manufactured CAR T-cell product (e.g., due to low lymphocyte counts in the first apheresis or other uninfluenced reasons) or 2) avoiding SAEs by reducing the starting CAR T-cell dose (especially if the required CAR is known to cause toxicity or for first-in-human dose-escalation studies).

[0320] The disclosures of any and all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. A chimeric antigen receptor (CAR) molecule comprising: The chimeric antigen receptor (CAR) molecule comprises: i) a CLDN6 antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence having at least 95% identity to SEQ ID NO: 35, and comprising all three CDR sequences of SEQ ID NO: 5 and all three CDR sequences of SEQ ID NO: 24; ii) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 28; iii) a hinge domain connecting the CLDN6 antigen-binding domain to the transmembrane domain, the hinge domain having the amino acid sequence of SEQ ID NO: 27; iv) an intracellular domain comprising a 4-1BB costimulatory domain and a CD3ζ signaling domain, wherein the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 30 and the CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO: 31; Including, The chimeric antigen receptor (CAR) molecule comprises an amino acid sequence having at least 95% identity to SEQ ID NO:

36. Chimeric antigen receptor (CAR) molecules.

2. A nucleic acid encoding the CAR molecule of claim 1.

3. The nucleic acid of claim 2 which is DNA or RNA.

4. A vector comprising the nucleic acid of claim 2 or 3.

5. 5. The vector of claim 4, which is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, and a retroviral vector.

6. The vector of claim 4 or 5, further comprising a promoter.

7. 7. The vector of claim 6, wherein the promoter is selected from the group consisting of an EF-1 promoter, a CMV IE gene promoter, an EF-1α promoter, a ubiquitin C promoter, and a phosphoglycerate kinase (PGK) promoter.

8. The CAR molecule of claim 1; A nucleic acid according to claim 2 or 3; or The vector according to any one of claims 4 to 7. immune effector cells, including

9. The immune effector cell of claim 8, which is genetically modified to express the CAR.

10. The immune effector cell of claim 8 or 9, which is selected from the group consisting of T cells, natural killer (NK) cells, and cytotoxic T lymphocytes (CTLs).

11. The immune effector cell of any one of claims 8 to 10, which is a CD8+ T cell.

12. The immune effector cell of any one of claims 8 to 11, which is a human cell.

13. A population of immune effector cells comprising a plurality of immune effector cells according to any one of claims 8 to 12.

14. The immune effector cell of any one of claims 8 to 12, which lacks or has low expression of a functional TCR or a functional HLA.

15. 14. The population of immune effector cells of claim 13, which lacks or has low expression of a functional TCR or a functional HLA.

16. The immune effector cell of any one of claims 8 to 12 and 14, wherein the immune effector cell is an immune effector cell that expresses a CAR.

17. 16. The population of immune effector cells of claim 13 or 15, wherein the population of immune effector cells is a population of immune effector cells that expresses a CAR.

18. The CAR molecule of claim 1 for use as a pharmaceutical.

19. 4. The nucleic acid of claim 2 or 3 for use as a pharmaceutical.

20. The vector according to any one of claims 4 to 7 for use as a pharmaceutical.

21. An immune effector cell according to any one of claims 8 to 12, 14 and 16 for use as a medicament.

22. 20. A population of immune effector cells according to any one of claims 13, 15 and 17 for use as a medicament.

23. The CAR molecule of claim 1 for use in treating a disease expressing CLDN6.

24. A nucleic acid according to claim 2 or 3 for use in the treatment of a disease in which CLDN6 is expressed.

25. The vector according to any one of claims 4 to 7, for use in treating a disease in which CLDN6 is expressed.

26. An immune effector cell according to any one of claims 8 to 12, 14 and 16 for use in treating a disease expressing CLDN6.

27. A population of immune effector cells according to any one of claims 13, 15 and 17 for use in treating a disease that expresses CLDN6.

28. 19. A kit comprising the CAR molecule of claim 1, the nucleic acid of claim 2 or 3, the vector of any one of claims 4 to 7, the immune effector cell of any one of claims 8 to 12, 14 and 16, or the population of immune effector cells of any one of claims 13, 15 and 17.

29. The kit of claim 28, further comprising a cognate antigen molecule that binds to the CLDN6 antigen-binding domain or a nucleic acid encoding the same.

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