Chimeric antigen receptor modified cells for the treatment of CLDN6-expressing cancer

Abstract

The present disclosure relates to immune effector cells expressing chimeric antigen receptors (CARs), which show highly specific and sensitive recognition of CLDN6-expressing cancer cells and high potential to treat cancer in humans.

Description

[Technical field]

[0001] Adoptive cell transfer (ACT)-based immunotherapy can be broadly defined as a form of passive immunization with pre-primed T cells that are transferred into non-immune recipients or autologous hosts after ex vivo expansion from low precursor frequencies to clinically relevant cell numbers. The use of genetic engineering approaches to insert antigen-targeting receptors of defined specificity into T cells has greatly expanded the potential capabilities 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) derived from a monoclonal antibody. CARs directly recognize cell surface antigens, independent of MHC-mediated presentation. [Background technology]

[0002] Attempts to treat cancer by using genetically modified T cells that target antigens expressed on tumor cells by expressing CARs have met with only limited success.Despite the remarkable responses in patients with B-cell malignancies, the success of clinical responses after targeting solid tumors using CAR T cells with various specificities is 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 the treatment of cancer using CARs. The present invention addresses this need.

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

[0006] CLDN6 is a druggable extracellular loop, and the cell surface level is high enough to allow recognition by CAR T cells. Moreover, CLDN6 expression correlates with disease progression, as it can be detected more frequently in metastatic lesions and dedifferentiated cells, indicating a role in the oncogenic process. The safety of CLDN6 targeting was demonstrated by a phase I / II clinical trial using anti-CLDN6 monoclonal antibody IMAB027 in patients with advanced ovarian cancer (OVAR, NCT02054351), and no IMAB027-related adverse events were detected.

[0007] Based on the results of in vitro and in vivo experiments, the inventors selected a second generation CAR (CLDN6-CAR-CD8h-BBz) with 4-1BB domain as a lead structure for preclinical and clinical trials. The inventors were able to demonstrate highly specific and sensitive recognition of CLDN6-expressing target cells and high potential for 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 progressive tumors. Furthermore, these results could be reproduced with cryopreserved CAR T cells generated in a GMP facility.

[0009] As previously demonstrated, clinical outcomes of CAR T cell therapy positively correlate with the persistence of infused CAR T cells in vivo (Robbins et al. (2004) J Immunol. 173(12):7125-30; Huang et al. (2005) 28(3):258-67), and the inventors combined their innovative CAR in vivo expansion concept using liposomally formulated mRNA encoding the CAR antigen with CLDN6-CAR therapy (CARVac; WO2016 / 180778).

[0010] The present inventors have (LIP) We could demonstrate in different tumor models that the combination of adoptively transferred CAR T cells together with baseline vaccination can accelerate ongoing antitumor responses and restore the antitumor efficacy of even insufficient CAR T cell doses.

[0011] Finally, in clinical trials evaluating CAR T cells alone and in combination with CARVac, no dose-limiting toxicities (DLTs) or treatment-related serious adverse events (AEs) were observed across three dose levels. Efficacy data revealed participants showing partial responses and stable disease with shrinkage of target lesions. Most encouragingly, some partial responses were achieved in all testicular cancer patients who had relapsed on recent high-dose chemotherapy. [Means for solving the problem]

[0012] The present invention provides a method of treating a solid tumor in a human, comprising: i) a CLDN6 antigen-binding domain; ii) a transmembrane domain; and iii) an intracellular domain containing a 4-1BB costimulatory domain and a CD3-zeta signaling domain; The method includes administering to a human an immune effector cell that expresses a chimeric antigen receptor (CAR) molecule comprising:

[0013] In one embodiment, the solid tumor is a CLDN6-positive solid tumor. In one embodiment, the solid tumor is a progressive solid tumor. In one embodiment, the solid tumor is a recurrent or refractory progressive solid tumor. In one embodiment, the solid tumor is selected from the group consisting of liver cancer, lung cancer, ovarian cancer, gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer, melanoma, head and neck cancer, sarcoma, cholangiocarcinoma, renal cell carcinoma, and bladder cancer. In one embodiment, the solid tumor is testicular cancer (including, for example, their metastatic cancers to the lung and / or liver). In one embodiment, treating a solid tumor in a human includes inducing stable disease, inducing stable disease with shrinkage of target lesions, or inducing a partial response.

[0014] 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 Camelidae 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.

[0015] 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.

[0016] In one embodiment, the CD3-zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 31, or a functional variant thereof.

[0017] In one embodiment, the transmembrane domain is selected from the group consisting of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, 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, C D19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIId, ITGAE, CD103, ITGAL, CDIIa, LFA-1, ITG AM, CDIIb, ITGAX, CDIIc, ITGBI, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAMI(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The transmembrane domain of a protein selected from the group consisting of AM, Ly9 (CD229), CD160 (BY55), PSGLI, 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.

[0018] 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.

[0019] 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.

[0020] 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 a 4-1BB costimulatory domain and a CD3-zeta signaling domain; Includes.

[0021] In one embodiment, the CAR molecule of the invention further comprises a leader sequence.

[0022] 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.

[0023] In one embodiment, the CAR molecule of the present invention is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 41 or a functional variant thereof. Thus, in one embodiment, immune effector cells are transfected, e.g., stably transfected, with such a nucleic acid.

[0024] The nucleic acid encoding the CAR molecule of the present invention can be present in a vector. 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.

[0025] In one embodiment, the immune effector cell is genetically modified to express a CAR molecule. In one embodiment, the immune effector cell is transfected with a nucleic acid or vector as described herein. In one embodiment, the immune effector cell is selected from the group consisting of a T cell, a natural killer (NK) cell, and a cytotoxic T lymphocyte (CTL). In one embodiment, the immune effector cell is a CD8+ T cell. In one embodiment, the immune effector cell is a human cell.

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

[0027] The immune effector cells may be generated ex vivo and administered to a human, or the immune effector cells may be generated in a human.

[0028] In one embodiment, the immune effector cells are autologous or allogeneic to the human.

[0029] In one embodiment, the method further comprises administering an agent that increases the effectiveness of immune effector cells. In one embodiment, the agent is Protein phosphatase inhibitors; Kinase inhibitors; Cytokines; Inhibitors of immune inhibitory molecules; or Agents that decrease the level or activity of TREG cells is selected from one or more of:

[0030] In one embodiment, the method of the invention further comprises contacting immune effector cells, either ex vivo or in a human, with a cognate antigenic molecule that binds to the CLDN6 antigen binding domain. 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 cells are contacted with the cognate antigenic molecule under conditions such that expansion and / or activation of the immune effector cells occurs. In one embodiment, the contacting of the immune effector cells with the cognate antigenic molecule is performed in vivo or ex vivo.

[0031] In one embodiment, the method of the invention comprises administering a cognate antigen molecule or a nucleic acid encoding the same to a human. In one embodiment, the nucleic acid encoding the cognate antigen molecule is expressed in a cell of the human to provide the cognate antigen molecule. In one embodiment, the expression of the cognate antigen molecule is cell surface expression. In one embodiment, the nucleic acid encoding the cognate antigen molecule is transiently expressed in a cell of the human. In one embodiment, the nucleic acid encoding the cognate antigen molecule is RNA. In one embodiment, the immune effector cells and / or the cognate antigen molecule or the nucleic acid encoding the same are 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, there is no or substantially no expression of the nucleic acid encoding the cognate antigen molecule 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 greater than expression in the lung.

[0032] 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 lipoplex.

[0033] In one embodiment, the method of the invention does not include administering a cognate antigen molecule or a nucleic acid encoding same to a human. In one embodiment, the method of the invention does not include administering an anti-cancer therapy to a human other than immune effector cells expressing a CAR molecule.

[0034] In one embodiment, the immune effector cells are about 10 6 ~about 10 9 pieces, about 5×10 6 ~Approx. 5×10 8 Pieces, or about 10 7 ~about 10 8 In one embodiment, a single dose of immune effector cells is administered.

[0035] The present invention relates to i) a CLDN6 antigen-binding domain; ii) a transmembrane domain; and iii) the intracellular domain containing the 4-1BB costimulatory domain and the CD3-zeta signaling domain expressing a chimeric antigen receptor (CAR) molecule comprising about 10 6 ~about 10 9 pieces, about 5×10 6 ~Approx. 5×10 8 Pieces, or about 10 7 ~about 10 8Further provided is a composition or pharmaceutical preparation comprising immune effector cells between the individual. Certain embodiments of the composition or pharmaceutical preparation, in particular the immune effector cells, are as described above for the methods of the invention.

[0036] In one embodiment, the composition or pharmaceutical formulation further comprises an agent that increases the effectiveness of immune effector cells and / or cognate antigenic molecules binding to the CLDN6 antigen binding domain or nucleic acid encoding same. Certain embodiments of the composition or pharmaceutical formulation, in particular the agent that increases the effectiveness of immune effector cells and / or cognate antigenic molecules binding to the CLDN6 antigen binding domain or nucleic acid encoding same, are as described above for the methods of the invention.

[0037] In one embodiment, the composition or pharmaceutical formulation is a kit. In one embodiment, the kit further comprises instructions for use of the kit in the method of the invention.

[0038] In a further aspect, the invention provides agents and compositions such as the immune effector cells or cognate antigenic molecules described herein or nucleic acids encoding same, for use in the methods described herein.

[0039] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. [Brief description of the drawings]

[0040] [Figure 1]Figure 1 shows the generation and characteristics of different CLDN6-CARs. A) 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 Fcγ receptor (FcγR) binding site to prevent activation by innate immune cells expressing FcγR (Hombach A. et al., (2010) Gene Therapy 17, 1206-1213); CD28ΔLck: CD28 transmembrane and cytoplasmic domain with a deletion in the Lck-binding portion of the CD28 endodomain that abrogates IL-2 induction upon CAR binding to prevent unwanted Treg cell expansion at the tumor site (Kofler DM et 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. B) Functionality testing of different CLDN6-CARs in PA1-SC12-A2-eGFP tumor spheroid assay. Lysates of tumor spheroids were 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, showing technically three tumor spheroid deaths. [Diagram 2]FIG. 2 shows dose-dependent CAR-mediated recognition and lysis of CLDN6-expressing target cells. A) CLDN6-CAR-BBz surface expression was analyzed in transduced T cells after staining with fluorochrome-conjugated IMAB206-idiotype-specific antibody. Non-transduced T cells were used as negative control. Cells were gated on single CD4+ or CD8+ lymphocytes. The numbers shown represent the frequency of the parent population (%). B) CLDN6 surface expression on Colo699-N cells transfected with titrated amounts of CLDN6-RNA was analyzed by flow cytometry. C) Specific lysis of RNA-transfected Colo699-N cells by CLDN6-CAR-BBz-transduced T cells was analyzed after 12 h of coculture with an E:T ratio of 20:1 using an xCELLigence instrument. Data are shown as mean ± SD of technical triplicates. [Figure 3-1] Figure 3 shows specific CLDN6-CAR-mediated lysis of CLDN6-expressing tumor cell lines. CLDN6-CAR-BBz transduced human T cells were analyzed by flow cytometry and co-cultured with a panel of CLDN6 positive and negative human tumor cell lines of different origins. A) CAR surface expression was assessed by flow cytometry before co-culture was initiated. The numbers shown represent the frequency (%) of the parental population. B) Using an E:T ratio of 10:1. C) Specific lysis was analyzed using the xCELLigence system after 12 h of co-culture by the formula: % lysis = (CI eGFP-CI effector) / CI eGFP*100; CI: cell index. Data are shown as the mean ± SD of technical triplicates. D) CLDN6 surface expression on tumor cell lines was analyzed by flow cytometry after staining with a CLDN6 specific antibody. The percentage of the parental population is shown. E) Relative CLDN6 mRNA expression levels in the tumor cell lines used in (A-D), as assessed by qRT-PCR, were calculated after normalization to the housekeeping gene HPRT1. Bars represent the mean ± SD of technical triplicates. [Figure 3-2] Same as above. [Figure 4]Figure 4 shows dose-dependent proliferation mediated by CLDN6-CAR-BBz in response to CLDN6-expressing target cells. CAR-transduced T cells were co-cultured with autologous DCs labeled with CFSE and transfected with a volume-set amount of CLDN6-RNA lipoplexes [RNA(LIP)]. Proliferation was analyzed based on CFSE after 5 days of co-culture and staining with fluorochrome-conjugated antibodies against CD4, CD8, and CAR. A) CAR surface expression was assessed by flow cytometry before co-culture was initiated. The numbers shown represent the frequency (%) of the parent population. B) CLDN6 expression on the surface of transfected DCs was assessed by flow cytometry using a fluorochrome-conjugated CLDN6-specific antibody. C) Specific proliferation was analyzed by flow cytometry based on the dilution of the CFSE proliferation dye. Bars indicate the percentage of proliferating CAR-expressing CD8+ and CD4+ T cells. [Figure 5-1]Figure 5 shows the antitumor activity of CLDN6-CAR-BBz transduced T cells in an advanced ovarian cancer (OV90) xenograft tumor model. 5x106 OV90-SC12 tumor cells were engrafted subcutaneously 25 days before adoptive cell transfer (ACT) of CLDN6-CAR-BBz or eGFP transduced human T cells administered a single dose of 1x107 iv (n=10 / group). At this time point of T cell treatment, mice already showed progressive tumors with an average of 170mm3. Tumor volumes were measured three times a week using calipers and calculated using the maximum length and width of the tumor by the formula: V=1 / 2 (length x width2). Animals were sacrificed when the tumor volume exceeded 1500mm3 or when the tumor ulcerated. A) Schematic of the mouse experiments performed. B) 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 (%). C) Mean tumor volume in CLDN6-CAR-BBz and eGFP-T cell treated animals up to 44 days after ACT (indicated by dotted line). Data shown as mean ± SEM of all mice / group. D) 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 indicate frequency of parental population. [Figure 5-2] Same as above. [Figure 6]Figure 6 shows repeated suppression of tumor spheroids by cultured CLDN6-CAR T cells at days 7 and 10. CLDN6-CAR transduced T cells generated within 7 and 10 days, respectively, were evaluated for their potential to repeatedly kill CLDN6- and eGFP-expressing tumor spheroids as assessed by live cell imaging. A) CAR surface expression of cultured CLDN6-CAR T cells at days 7 and 10 was analyzed in transduced T cells after staining with fluorochrome-conjugated IMAB206-idiotype-specific antibody. Non-transduced T cells were used as negative control. Cells were gated on single CD4+ or CD8+ lymphocytes. The numbers shown represent the frequency of the parent population (%). B) Functional testing of cultured CLDN6-CAR T cells at days 7 and 10 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 ​​represent the integrated intensity of green objects from technical triplicates as mean ± SD. [Figure 7-1]Figure 7 shows the antitumor efficiency of GMP manufactured CLDN6-CAR T cells. 5x106 OV90-SC12 tumor cells were subcutaneously engrafted 35 days before iv adoptive cell transfer of either a single dose of 1x107 CLDN6-CAR-BBz or non-transduced human T cells (n=12 / group). The T cell products used were manufactured in a GMP facility with either standard (in vitro culture for 10 days) or short manufacturing procedure (already harvested 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 directly injected into mice already bearing progressive tumors of an average of 160 mm3. Tumor volumes were measured 3 times a week using calipers and calculated using the maximum length and width of the tumor by the formula: V=1 / 2 (length x width2). Animals were sacrificed when tumor volume exceeded 1500 mm3 or tumor ulceration. A) Schematic of mouse experiments performed. B) CAR surface expression 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 (%). C) Tumor volume in CLDN6-CAR-BBz and control T cell treated animals up to day 57. Data shown as mean ± SEM of all mice / group. D) T cell persistence and CAR surface expression were analyzed in peripheral blood 2 weeks after ACT using flow cytometry. Representative dot plots are shown. Numbers indicate frequency of parental population. [Figure 7-2] Same as above. [Figure 8]FIG. 8 shows that in vivo expansion with RNA(LIP) results in enhanced persistence of CLDN6-CAR-BBz T cells. 2.5 Gy irradiated (XRAD320) C57BL / 6BrdCrHsd-Tyrc mice (n=2-3 / group) were iv implanted with 5x106 CLDN6-CAR-BBz-Luc-GFP transduced C57Bl / 6-Thy1.1+ T cells. Eight days after ACT, mice received mRNA lipoplex vaccination (RNA(LIP); 20μg, iv) encoding hCLDN6 or Oval(ctrl RNA) followed by ip administration of nucleoside modified formulation RNA encoding mouse albumin (1μg / mRNA / mouse). Vaccination was repeated on days 15, 22, 50 and 85. Serial bioluminescence imaging was performed to monitor expansion and persistence from day 1 (baseline) to day 92 after ACT. A) Schematic of the mouse experiments performed. B) Examples of bioluminescence imaging of mice in lateral decubitus position at the indicated time points after treatment with ACT and antigen RNA (LIP). Off-color images show light intensity (black, lowest intensity; white to dark grey, highest intensity) superimposed on a greyscale reference image. C) Quantification of bioluminescence during or after expansion rounds with CLDN6-RNA (LIP) / ctrl-RNA (LIP) in the presence of the indicated nucleoside-modified formulations cytokine RNA (LIP) (mean ± s.e.m.). 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: CD3 zeta. [Figure 9]FIG. 9 shows the improved antitumor activity of in vivo expanded CLDN6-CAR-BBz T cells. 5×105 CT26 tumor cells were subcutaneously engrafted into Balb / c mice (n=10 / group) 20 days prior to 4 Gy total body irradiation and 26 days prior to iv ACT of a single dose of 1×106 CLDN6-CAR-BBz or ctrl-CAR-BBz transduced Balb / c-Thy1.1+ T cells. At the time of T cell treatment, mice showed established tumors averaging approximately 80 mm3. Tumor volumes were measured three times a week using calipers and calculated using the maximum length and width of the tumor by the formula: V=1 / 2 (length×width2). A) Schematic of the mouse experiment performed. B) Average tumor volumes are shown in CLDN6-CAR-BBz and ctrl-CAR-BBz-T cell treated animals by day 40. Data are presented as mean ± SEM of all mice / group. ACT is shown as a dotted line and RNA(LIP) treatment is shown as a grey line. [Figure 10-1]Figure 10 shows the recovery of antitumor efficiency of low dose in vivo expanded CLDN6-CAR T cells. 5x106 OV90-SC12 tumor cells were subcutaneously engrafted 30 days before iv ACT of either a single dose of low (1x105) or high (1x106) dose of CLDN6-CAR-BBz or 1x107 non-transduced human T cells (n=9 / group). The T cell products used were manufactured in a GMP facility by a short transduction process (already harvested 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 directly injected into tumor-bearing mice. Additionally, mice receiving low doses of CAR T cells were additionally vaccinated with 20μg 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 by the formula: V=1 / 2 (length x width2). Animals were sacrificed when tumor volumes exceeded 1500 mm3 or when tumors ulcerated. A) Schematic of mouse experiments performed. B) CAR surface expression of transduced human CD4+ and CD8+ T cells was analyzed by flow cytometry on the day of adoptive transfer. The numbers shown represent the frequency of the parental population (%). C) Tumor growth curves of animals treated with different doses of CLDN6-CAR-BBz+ / -RNA (LIP) or control T cells. Data are shown as mean ± SEM of all mice / group. ACT is shown as dotted line and RNA (LIP) treatment as grey line. D) 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. The numbers indicate the frequency of the parental population. [Figure 10-2] Same as above. [Figure 11] Figure 11 shows dose escalation. Abbreviations: CAR-T, chimeric antigen receptor T cells; CLDN6, claudin 6; CARVac, CLDN6 RNA-LPX vaccine promoting CAR T cells; DL, dose level; DLT, dose-limiting toxicity. [Figure 12]Figure 12 shows the treatment schedule. Abbreviations: ACT: adoptive cell transfer (CAR-T cell infusion), CAR-T, chimeric antigen receptor T cells; CARVac, CLDN6 RNA-LPX vaccine promoting CAR T cells; CLDN6, claudin 6; DLT, dose-limiting toxicity; Cy = cyclophosphamide (500 mg / m2 / d); Flu = fludarabine (30 mg / m2 / d). [Figure 13A] Figure 13 shows CAR-T engraftment. Figure 13 shows CAR-T cell frequency at each dose level showing an increasing trend of improved engraftment by adding CARVac or increasing dose. Figure 13A shows part 1 of dose level 1 (107 CAR T cells, no CLDN6 RNA-LPX). Figure 13B shows part 2 of dose level 1 (107 CAR T cells, with CLDN6 RNA-LPX). Figure 13C shows part 1 of dose level 2 (108 CAR T cells, no CLDN6 RNA-LPX). Patients were numbered independently for each dose level. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 14] Figure 14 shows clinical activity. Patients were numbered independently for each dose level. Data cutoff = November 18, 2021, ACT = adoptive cell transfer; CR = complete response; DCR = disease control rate; EoT = end of study (due to PD); ORR = overall response rate; PD = progressive disease; PR = partial response; SD = stable disease, SD+ = SD with shrinkage of target lesions; wks = weeks; * = no lymphodepletion. [Figure 15] Figure 15 shows clinical activity in selected patients. Figure 15 shows CT scans of three different patients before and after treatment at the times indicated. Patients were numbered independently for each dose level. The top three figures show one patient who received adoptive cell transfer at a dose of 107 cells (dose level 1) in combination with administration of CARVac / CLDN6 RNA-LPX. The bottom four figures show two patients who received adoptive cell transfer at high dose level 2 (108 cells) as monotherapy. [Figure 16] FIG. 16 shows the concept of CLDN6 CAR-T therapy by CLDN6 RNA-LPX-mediated in vivo expansion of CLDN6 CAR-T cells. (A) Autologous T cells are engineered to express CLDN6-CAR and reinfused into the (lymphocyte-depleted) patient. (B) mRNA encoding full-length CLDN6 is complexed with cationic liposomes to form lipoplexes (CLDN6 RNA-LPX). (C) RNA-LPX selectively targets APCs in secondary lymphoid organs. The RNA sequence contains optimized 5' caps and naturally occurring sequence elements in the 3'- and 5'-UTRs, significantly increasing the intracellular half-life and translation efficiency of the molecule. Engagement of CAR-T cells with the CAR target leads to CAR-T cell activation and expansion. (D) RNA-LPX encoding the CAR target can be administered repeatedly to extend the lifespan of CAR-T cells. APC = antigen presenting cell; CAR = chimeric antigen receptor; CARVac = RNA vaccine to expand CAR-T cells; CD = cluster of differentiation; CLDN6 = claudin 6; mRNA = messenger ribonucleic acid; RNA = ribonucleic acid; RNA-LPX = RNA formulated in liposomes; UTR = untranslated region. [Figure 17] Figure 17 shows a schematic diagram of the CLDN6-CAR transgene CG_CAR_001.2 encoding retroviral vector CG_CAR_001.2 RVV. CMV hybrid = chimeric 5' promoter consisting of human cytomegalovirus immediate early enhancer element and murine leukemia virus (MLV) core promoter; R, U5 = derived from MLV (wild type); psi = psi-region of MLV, containing native splice donor and containing B2 mutant of PBS; psi+ = psi+region of MLV (wild type), start codon deleted of gag; EFS = short version of eukaryotic elongation factor 1-alpha promoter; pre*s = short version of WPRE; Woodchuck hepatitis virus posttranscriptional control element; "*" represents deletion of ATG in the sequence; ΔU3 = enhancer element in the U3 region of 3'LTR was removed (including CAAT-Box) and TATA-Box sequence was mutated. [Figure 18]Figure 18 shows the study design. In the Phase 1 part of the study, patients are enrolled into one of two parts. Part 1 evaluates CLDN6 CAR-T / CLDN6 CAR-T(A) monotherapy. Part 2 evaluates CLDN6 CAR-T / CLDN6 CAR-T(A)+CLDN6 RNA-LPX. The Phase 2 part of the study consists of an expansion cohort. One intrapatient dose escalation run is included for CLDN6 RNA-LPX, after which CLDN6 RNA-LPX is administered at a fixed dose. Parts 1 and 2, DL1 and DL2, with CLDN6 CAR-T have been completed. Parts 1 and 2 are conducted using CLDN6 CAR-T(A) manufactured using an automated process. CAR-T = CAR-T cells manufactured using a manual process; CAR-T(A) = CAR-T cells manufactured using an automated process; CLDN6 = claudin 6; DL = dose level; DLT = dose-limiting toxicity; ECOG = Eastern Cooperative Oncology Group; RECIST = Response Evaluation Criteria in Solid Tumors; RNA-LPX = ribonucleic acid-lipoplex; RP2D = recommended dose for phase 2 clinical trials. [Figure 19] Figure 19 shows the treatment timeline. In part 1 and part 2, autologous T cells are engineered to express CLDN6 CAR and reinfused into the patient on day 1. Patients enrolled in part 2 receive additional CLDN6 RNA-LPX treatments. From DL2, CLDN6 RNA-LPX treatments are administered on days 24, 36, 51, 72, and 93, and every 6 weeks thereafter. 1q3W = once every 3 weeks; CAR-T = chimeric antigen receptor T cells; CLDN6 = claudin 6; DLT = dose-limiting toxicity; M = month; RNA-LPX = RNA-lipoplex, RNA formulated in liposomes. [Figure 20]FIG. 20 shows serum IL-6 levels in patients with and without CRS. IL-6 levels were evaluated for all 16 patients treated within four predefined phase 1 cohorts, DL1 and DL2 of CLDN6 CAR-T cell monotherapy (part 1), and in combination with CLDN6 RNA-LPX (part 2). (A) All patients with CRS (mild to moderate, ASTCT grade 1-2) showed elevated IL-6 levels, and CRS was always manageable with administration of tocilizumab. (B) Patients without signs of CRS showed low to normal levels of IL-6. ASTCT=American Society for Bone Marrow Transplantation; CRS=cytokine release syndrome; DL=dose level; DL1=1×107 CLDN6 CAR-T cells; DL2=1×105 CLDN6 CAR-T cells; IL-6=interleukin 6. [Figure 21] FIG. 21 shows that CLDN6 RNA-LPX improved CAR-T engraftment and persistence at low doses. Two testicular cancer patients were treated with DL1 (upper horizontal dotted line, 107 CLDN6 CAR-T cells). Patient (A) received multiple CLDN6 RNA-LPX (vertical dashed line) treatments, and patient (B) did not receive further therapeutic intervention. In patient A, the high CAR-T engraftment and persistence improvement translated into clinical benefit (partial response), while the rapid decline of CAR-T cells (below LLQQ, lower horizontal dotted line) led to fast disease progression in patient B. Both patients were re-administered CAR-T cells (vertical solid lines indicate the first and second adoptive cell transfers). CAR-T = chimeric antigen receptor engineered T cells; CLDN6 = claudin 6; DL = dose level; LLQQ = lower limit of quantification; RNA-PLX = ribonucleic acid-lipoplex. [Figure 22]Figure 22 shows CLDN6 CAR-T cell engraftment per cohort. CAR-T cell frequency in peripheral blood was evaluated in all treated patients. The CLDN6-CAR transgene contains an RNA stabilizing element (WPRE), and the amount of CLDN6 CAR+ cells in the PBMC input population was measured by quantitative PCR targeting the WPRE. Total CAR-T cell counts were calculated based on white blood cell counts and an assumed total volume of 5 L. All patients showed robust CAR-T cell engraftment; data collection will continue in some patients. CRS was observed more frequently with high dose levels of CAR-T (DL2). The upper dotted line indicates the applied CAR-T dose, and the lower solid line indicates the LLQQ. Part 1 and Part 2 patients are shown as black dotted and solid lines, respectively. The 16 patients included in the predefined cohort are shown in black. Patients with reduced lymphodepletion are shown in gray, and patients without lymphodepletion are not shown. CAR = chimeric antigen receptor; CAR-T = chimeric antigen receptor engineered T cells; CLDN6 = claudin 6; DL = dose level; DL1 = 1 x 107 CLDN6 CAR-T cells; DL2 = 1 x 108 CLDN6 CAR-T cells; LLQQ = lower limit of quantification; Part 1 = CLDN6 CAR-T monotherapy; Part 2 = CLDN6 CAR-T + CLDN6 RNA-LPX; PBMC = peripheral blood mononuclear cells; PCR = polymerase chain reaction; RNA = ribonucleic acid; RNA-LPX = RNA-lipoplex; WPRE = woodchuck hepatitis virus post-transcriptional regulatory element. [Figure 23] Figure 23 shows a process flow chart for the automated process flow chart for automated manufacturing of CLDN6 CAR-T(A)DS utilizing the Prodigy® system. Starting materials are shown in bold. CAR-T = Chimeric Antigen Receptor T cells; CD = Cluster of Differentiation; CLDN6 = Claudin 6; DS = Drug Substance; GMP = Good Manufacturing Practice; IL = Interleukin; MOI = Multiplicity of Infection; NaCl = Sodium Chloride. [Figure 24]Figure 24 shows a process flow chart for the manufacture of CLDN6 CAR-T(A)DP. [1] Samples are taken, but RCR testing begins only after traces of RCR activation in the patient. CAR-T = Chimeric Antigen Receptor T-cells; CLDN6 = Claudin 6; DS = Drug Substance; DP = Drug Product; NaCl = Sodium Chloride; RCR = Replication-competent Retrovirus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The present invention relates to compositions and methods for treating cancers, such as solid tumors. In particular, 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 with specificity for a desired antigen (e.g., a tumor antigen), preferably an antibody based on the T cell receptor activating intracellular domain, which produces a chimeric protein that exhibits a specific cellular immune activity (e.g., a 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. In one embodiment, the present invention includes 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.

[0042] The CAR of the present invention combines the CLDN6 antigen binding domain, preferably a domain of a specific antibody, and an intracellular domain comprising a 4-1BB costimulatory domain and a domain of the CD3-zeta 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-zeta 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 the transmembrane domain of the same or different surface membrane protein and to minimize interaction with other members of the receptor complex. Preferably, the transmembrane domain is derived from CD8α.

[0043] 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 contains a desired CAR, for example, a CAR containing anti-CLDN6, CD8α hinge and transmembrane domains, and 4-1BB and CD3 zeta signaling domains. The CAR T cells of the present invention are preferably capable of replicating in vivo, resulting in long-term persistence that can result in sustained tumor control.

[0044] In one embodiment, the present invention generally relates to administering genetically modified T cells expressing a desired CAR for the treatment of patients with or at risk of having cancer.Preferably, autologous cells are used in the treatment.In one embodiment, autologous PBMCs are collected from the patient in need of 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.

[0045] In one embodiment, the present invention generally relates to the treatment of human patients with or at risk of developing CLDN6-expressing cancers, such as CLDN6-expressing solid tumor cancers. The present invention includes the use of T cells expressing anti-CLDN6-CARs that contain both CD3-zeta and 4-1BB costimulatory domains. The CAR T cells of the present invention can undergo robust T cell expansion in vivo, particularly when contacted with their cognate antigen, and can be maintained at high levels for long periods of time. In some instances, the CAR T cells of the present invention infused into a patient can eliminate cancer cells in vivo in the patient.

[0046] definition The present invention will be described in detail below, but it is understood that the present invention is not limited to the specific methods, protocols and reagents described herein, which may be modified. It is also understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the scope of the present invention, but are limited only by the scope of the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0047] The elements of the present invention are described below. Although these elements are listed by specific embodiments, it should be understood that they can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be construed as limiting the present invention to only the embodiments expressly described. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any order and combination of all elements described herein should be considered to be disclosed by the description herein, unless the context indicates otherwise.

[0048] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terms are used.

[0049] Throughout this specification and the claims, unless the context requires otherwise, it will be understood that the term "comprise", and variations such as "comprises" and "comprising", include the inclusion of a stated member, integer, or step, or group of members, integers or steps, but do not exclude any other members, integers or steps, or group of members, integers or steps, and that in some embodiments such other members, integers or steps, or group of members, integers or steps may be excluded, i.e., the subject matter is in the inclusion of a stated member, integer or step, or group of members, integers or steps.

[0050] The articles "a" and "an" are 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.

[0051] As used herein, "about" when referring to a measurable value, such as an amount, period, etc., is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the particular value.

[0052] As used herein, the term "antibody" refers to an immunoglobulin molecule that binds, preferably specifically binds, to an antigen. An antibody may be an intact immunoglobulin from natural or recombinant sources, or may be an immunoreactive portion or a fragment of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. The antibodies of the present invention may exist in a variety of forms, including, for example, polyclonal, monoclonal, Fv, Fab and F(ab)2, as well as single chain 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).

[0053] Antibodies expressed by B cells are sometimes called 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 bodily 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 main immunoglobulin produced in the primary immune response in most subjects. It is the immunoglobulin most effective 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 serve 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.

[0054] The term "antibody fragment" refers to a portion of an intact antibody, typically containing the antigen-determining variable regions of the intact antibody.

[0055] 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.

[0056] As used herein, "antibody heavy chain" refers to the large chain of two types of polypeptide chains present in antibody molecules in their naturally occurring configurations.

[0057] As used herein, "antibody light chain" refers to the smallest of the two types of polypeptide chains present in antibody molecules in their naturally occurring configurations; kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0058] As used herein, the term "antigen" or "Ag" is defined as a molecule that induces an immune response. This immune response may include either 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, antigens may be naturally occurring or recombinant antigens.

[0059] 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.

[0060] Claudins are integral transmembrane proteins located within epithelial and endothelial tight junctions. Claudins are predicted to have four transmembrane segments, with two extracellular loops and N- and C-termini located in the cytoplasm. The first extracellular loop, called EC1 or ECL1, consists of an average of 53 amino acids, and the second extracellular loop, called 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, as well as in the maintenance of the cytoskeleton and cell signaling.

[0061] Claudin 6 (CLDN6) is an oncofetal gene expressed in mouse and human stem cells and embryoid bodies committed to an 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). Tumor-associated antigens can be classified as differentiation antigens due to their expression during early stages of epidermal morphogenesis, which is important for epidermal differentiation and barrier formation. Further 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). Moreover, our data also revealed low or very low expression of CLDN6 in human placenta, bladder, endometrium, prostate and peripheral nerve, and frequent overexpression of CLDN6 in different cancers. CLDN6 has been demonstrated to be overexpressed in tumors including pediatric brain tumors, gastric adenocarcinomas, 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, indicating that CLDN6 is 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 by inhibiting cell proliferation and inducing apoptosis in breast cancer cell lines.

[0062] 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, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly lung cancer including squamous cell 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 polymorphic adenoma, sarcoma, particularly synovial sarcoma and carcinosarcoma, bile duct cancer, bladder cancer, particularly transitional cell carcinoma and papillary carcinoma, renal CLDN6 is a particularly preferred target for preventing and / or treating pancreatic cancer, particularly renal cell carcinoma including renal clear cell carcinoma and papillary cell carcinoma, colon cancer, ileal cancer, particularly small intestinal cancer including small intestinal adenocarcinoma and ileal adenocarcinoma, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, particularly testicular seminoma, testicular teratoma and embryonic testicular cancer, uterine cancer, germ cell tumor, such as teratocarcinoma or embryonic carcinoma, particularly testicular germ cell tumor, 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, and preferably bronchiolar carcinoma, such as bronchiolar carcinoma or bronchiolar adenocarcinoma.

[0063] The term "CLDN6" preferably relates to human CLDN6, and specifically to 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 mutant 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.

[0064] The term "expressed on the cell surface" or "associated with the cell surface" means that a molecule such as CLDN6 is associated with and located on the cell membrane of a cell, with at least a portion of the molecule facing the extracellular space of said cell and capable of being bound 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 binding can be direct or indirect. For example, the binding can be by interaction with one or more transmembrane domains, one or more lipid anchors, or any other protein, lipid, sugar, or other structure that can be found on the outer leaflet of the cell's plasma membrane. For example, a molecule that binds to the surface of a cell can be a transmembrane protein with an extracellular portion, or a protein that binds to the surface of a cell by interaction with another protein that is a transmembrane protein.

[0065] "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 for binding by proteins and other molecules.

[0066] "Cell-mediated immunity," "cell-mediated immunity," "cell-mediated immune response," or similar terms are meant to include a cellular response to cells characterized by expression of an antigen, specifically, 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 controlling the immune response and act as killer cells (cytotoxic T cells, cytolytic T cells, CD8 + These cells (also called T cells or CTLs) kill diseased cells, such as cancer cells, and prevent the production of more diseased cells.

[0067] 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., it is recognized by an antibody or CAR. For example, an epitope is a discrete, three-dimensional portion of an antigen that is recognized by the immune system. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural features, as well as specific charge characteristics. Structural and non-structural epitopes are distinct, and binding to the latter is lost in the presence of denaturing solvents, while binding to the former is not. Preferably, an epitope can induce an immune response against an antigen or a cell expressing the antigen. Preferably, the term relates to an immunogenic portion of an antigen. Epitopes of a protein, such as a tumor antigen, preferably comprise continuous or discontinuous portions of said protein and are preferably between 5 and 100 amino acids in length, preferably between 5 and 50 amino acids in length, more preferably between 8 and 30 amino acids in length, most preferably between 10 and 25 amino acids in length, for example epitopes may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0068] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of said antigen (e.g., proteolysis into peptides), and the binding (e.g., by binding) of one or more of these fragments to an MHC molecule for presentation by a cell, preferably an antigen-presenting cell, to a specific T cell.

[0069] Antigen-presenting cells (APCs) are cells that present antigens on their surface in the context of major histocompatibility complexes (MHC). T cells can recognize this complex using their T cell receptors (TCRs). Antigen-presenting cells process antigens and present them to T cells. According to the present invention, the term "antigen-presenting cells" includes professional and non-professional antigen-presenting cells.

[0070] Professional antigen-presenting cells are highly efficient in internalizing antigens by phagocytosis or by receptor-mediated endocytosis, and then presenting fragments of the antigen, binding to class II MHC molecules on their membranes. T cells recognize and interact with antigen-class II MHC molecule complexes on the membrane of antigen-presenting cells. Further costimulatory signals are then produced by the antigen-presenting cells, leading to T cell activation. The expression of costimulatory molecules defines the characteristics of professional antigen-presenting cells. The main types of professional antigen-presenting cells are dendritic cells, which show a wide range of antigen presentation, and macrophages, B cells, and certain activated epithelial cells, which are probably the most important antigen-presenting cells.

[0071] The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Macrophages activated by inflammation, immune cytokines or microbial products non-specifically engulf and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack, resulting in the degradation of the pathogen. Peptides derived from degraded proteins are presented on the macrophage cell surface where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, resulting in T cell and B cell activation and further stimulation of the immune response. Macrophages belong to a class of antigen-presenting cells. In one embodiment, the macrophages are splenic macrophages.

[0072] The term "dendritic cells" (DC) refers to another subtype of phagocytes belonging to the class of antigen-presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitors. These progenitors are first transformed into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation capacity. Immature dendritic cells regularly sample the surrounding environment for pathogens, such as viruses and bacteria. When they come into contact with presentable antigens, they are activated into mature dendritic cells and begin to migrate to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, break down their proteins into small pieces, and upon maturation, present these fragments on their cell surface using MHC molecules. At the same time, they upregulate cell surface receptors that act as co-receptors in T cell activation, such as CD80, CD86, and CD40, which greatly enhance their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that allows dendritic cells to migrate to the spleen via bloodstream or to lymph nodes via lymphatic system. Here, they act as antigen-presenting cells and activate B cells by presenting antigen to helper T cells and killer T cells, as well as non-antigen-specific co-stimulatory signals. Thus, dendritic cells can actively induce T cell or B cell-related immune responses. In one embodiment, dendritic cells are splenic dendritic cells.

[0073] In accordance with 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 recognizes, i.e., binds (e.g., by binding of an antigen binding domain to an antigen expressed on the surface of a target cell) to a target structure (e.g., an antigen) on a target cell, such as a cancer cell, and may confer specificity to an immune effector cell, such as a T cell, that expresses said CAR on its cell surface. Preferably, recognition of the target structure by a CAR results in activation of an immune effector cell that expresses said CAR. A CAR may comprise one or more protein units, including one or more domains described herein. The term "CAR" does not include T cell receptors.

[0074] Adoptive cell transfer therapy with CAR-engineered T cells expressing chimeric antigen receptors allows for anti-cancer treatment, as CAR-engineered T cells can be engineered to target virtually any tumor antigen. For example, a patient's T cells can be genetically engineered (modified) to express a CAR that specifically targets an antigen on the patient's tumor cells, and then infused back into the patient.

[0075] As used herein, the term "anti-tumor" relates to a biological effect that can be evidenced 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 tumors in the first place, an increase in life expectancy, or an alleviation of various physiological symptoms associated with a cancer condition.

[0076] As used herein, the term "autologous" is meant to refer to any material from the same individual that is subsequently reintroduced into the individual.

[0077] "Allogeneic" refers to graft tissue derived from a different animal of the same species.

[0078] "Xenogeneic" refers to a transplant tissue derived from an animal of a different species.

[0079] The term "syngeneic" is used to describe individuals or tissues that have the same genotype, ie, identical twins or the same inbred animal strain, or tissues derived therefrom.

[0080] As used herein, the term "cancer" is defined as a disease characterized by rapid and uncontrolled proliferation of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. In one embodiment, the cancer comprises a solid tumor. In one embodiment, the cancer comprises testicular cancer. In one embodiment, the cancer comprises cancer metastasis; for example, testicular cancer metastasis, such as metastasis to the lung and / or liver.

[0081] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds that contain 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 comprise a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids connected to each other by peptide bonds. As used herein, the term generally refers to both short chains, also referred to in the art as peptides, oligopeptides and oligomers, for example, and longer chains, generally referred to in the art as proteins, of which there are many types. "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.

[0082] As used herein, the term "polynucleotide" 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 and 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, but not limited to, recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes, using conventional cloning techniques and PCR™, etc., and by synthetic means. As used herein, the term "polynucleotide" is broadly interpreted and includes DNA and RNA, including modified DNA and RNA.

[0083] In some embodiments, the peptides or polypeptides described herein, such as CAR molecules or cognate antigen molecules, are encoded by coding sequences that are codon-optimized and / or have an increased G / C content compared to the wild-type coding sequence. This also includes embodiments in which one or more sequence regions of the coding sequence are codon-optimized and / or have an increased G / C content compared to the corresponding sequence region of the wild-type coding sequence. In one embodiment, the codon optimization and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0084] The term "codon-optimized" refers to the modification of codons in the coding region of a nucleic acid molecule, preferably reflecting the typical codon usage of a host organism, without changing the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present invention, the coding region is preferably codon-optimized for optimal expression in the subject treated with the polynucleotide described herein. Codon optimization is based on the discovery that translation efficiency is also determined by the different frequencies of occurrence of tRNA in a cell. Thus, the sequence of a polynucleotide can be modified so that the codons of frequently occurring tRNAs are available and inserted in place of "rare codons".

[0085] In some embodiments of the present invention, the guanosine / cytosine (G / C) content of the coding region of the polynucleotide described herein is increased compared to the G / C content of the corresponding coding sequence of the wild-type polynucleotide, and the amino acid sequence encoded by the polynucleotide is preferably not modified compared to the amino acid sequence encoded by the wild-type polynucleotide. This modification of the polynucleotide sequence is based on the fact that the sequence of any polynucleotide region to be translated is important for the efficient translation of that polynucleotide. A sequence with an increased G (guanosine) / C (cytosine) content is more stable than a sequence with an increased A (adenosine) / U (uracil) content. With regard to the fact that several codons code for one and the same amino acid (the so-called degeneracy of the genetic code), the most favorable codon for stability can be determined (the so-called alternative codon usage). Depending on the amino acid encoded by the polynucleotide, there are various possibilities for modification of the polynucleotide sequence compared to its wild-type sequence. Specifically, codons containing A and / or U nucleotides can be modified by replacing these codons with other codons that encode the same amino acids but that do not contain A and / or U or that contain a low content of A and / or U nucleotides.

[0086] In various embodiments, the G / C content of the coding region of the polynucleotides described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or more compared to the G / C content of the coding region of the wild-type polynucleotide.

[0087] In this disclosure, the term "RNA" refers to a nucleic acid molecule that includes ribonucleotide residues. In a preferred embodiment, the RNA contains all or most 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 within the RNA nucleotides or to the end(s) of the RNA. It is also contemplated herein that the nucleotides within the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In this disclosure, these modified RNAs are considered to be analogs of naturally occurring RNA.

[0088] In certain embodiments of the present disclosure, the RNA is messenger RNA (mRNA), which is related to RNA transcripts that code for peptides or proteins. As established in the art, mRNA generally contains 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 contains deoxyribonucleotides.

[0089] 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 of any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, specifically a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0090] 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-methyl-pseudouridine.

[0091] In some embodiments, the RNA includes a modified nucleoside in place of at least one (eg, all) uridines.

[0092] The term "uracil" as used herein describes one of the nucleobases that can occur in RNA nucleic acids. The structure of uracil is: [ka] It is.

[0093] The term "uridine" as used herein describes one of the nucleosides that can occur in RNA. The structure of uridine is: [ka] It is.

[0094] UTP (uridine 5'-triphosphate) has the following structure: [ka] has.

[0095] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure: [ka] has.

[0096] "Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine in which uracil is attached to the pentose ring through a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0097] Another exemplary modified nucleoside is N1-methyl-pseudouridine (m1Ψ), which has the structure: [ka] has.

[0098] N1-methyl-pseudo-UTP has the following structure: [ka] has.

[0099] Another exemplary modified nucleoside is 5-methyl-uridine (m5U), which has the structure: [ka] has.

[0100] In some embodiments, one or more uridines in the RNA described herein are replaced by modified nucleosides.In some embodiments, the modified nucleosides are modified uridines.In some embodiments, the modified uridines that replace uridines are pseudouridines (Ψ), N1-methyl-pseudouridines (m1Ψ) or 5-methyl-uridines (m5U).

[0101] In some embodiments, the modified nucleoside that replaces one or more, e.g., all, uridines in the RNA is 3-methyl-uridine (m3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0102] In some embodiments, the RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is independently selected from pseudouridine (Ψ), N1-methyl-pseudouridine (m1Ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (Ψ). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1Ψ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m5U). In some embodiments, the RNA may comprise more than one type of modified nucleoside, and the modified nucleoside is independently selected from pseudouridine (Ψ), N1-methyl-pseudouridine (m1Ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides include pseudouridine (Ψ) and N1-methyl-pseudouridine (m1Ψ). In some embodiments, the modified nucleosides include pseudouridine (Ψ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides include N1-methyl-pseudouridine (m1Ψ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides include pseudouridine (Ψ), N1-methyl-pseudouridine (m1Ψ), and 5-methyl-uridine (m5U). In one embodiment, the RNA includes other modified nucleosides or further modified nucleosides, such as modified cytidine. For example, in one embodiment, 5-methylcytidine in the RNA partially or completely replaces cytidine, preferably completely. In one embodiment, the RNA comprises 5-methylcytidine and one or more selected from pseudouridine (Ψ), N1-methyl-pseudouridine (m1Ψ), and 5-methyl-uridine (m5U). In one embodiment, the RNA comprises 5-methylcytidine and N1-methyl-pseudouridine (m1Ψ).In some embodiments, the RNA contains a 5-methylcytidine in place of every cytidine and an N1-methyl-pseudouridine (m1Ψ) in place of every uridine.

[0103] In some embodiments, the RNA described herein comprises a 5'-cap. In one embodiment, the RNA of the present disclosure does not have an uncapped 5'-triphosphate. In one embodiment, the RNA may be modified with a 5'-cap analog. The term "5'-cap" refers to a structure found at the 5'-end of an mRNA molecule, and generally consists of a guanosine nucleotide connected to the mRNA via a 5' to 5' triphosphate linkage. In one embodiment, the guanosine is methylated at position 7. Providing an RNA with a 5'-cap or 5'-cap analog can be achieved by in vitro transcription, where the 5'-cap can be expressed onto the RNA strand co-transcriptionally or attached to the RNA post-transcriptionally using a capping enzyme.

[0104] In some embodiments, the component cap of the RNA is m2 7,3’-O Gppp(m1 2’-O )ApG(m2 7,3’-O G(5')ppp(5')m 2’-O ApG) has the following structure: [ka] has.

[0105] The following is an exemplary Cap1 RNA and m2 7,3’-O G(5')ppp(5')m 2’-O ApG: [ka] Includes.

[0106] The following is another exemplary Cap1 RNA (not a cap analog): [ka] It is.

[0107] In some embodiments, the RNA has the structure: [ka] A cap analog having an anti-reverse cap [ARCA Cap(m2 7,3’-O G(5')ppp(5')G)] is used and modified with the "Cap0" structure.

[0108] The following are RNA and m2 7,3’-O G(5')ppp(5')G: [ka] An exemplary Cap0 RNA includes:

[0109] In some embodiments, the “Cap0” structure has the structure: [ka] Cap analogue beta-S-ARCA (m2 7,2’-O G(5')ppSp(5')G).

[0110] Below is the Beta-S-ARCA(m2 7,2’-O G(5')ppSp(5')G) and RNA: [ka] An exemplary Cap0 RNA includes:

[0111] Particularly preferred Caps are 5'-cap m2 7,2’-O In some embodiments, the RNA described herein comprises a 5'-capped m2 7,2’-OG(5')ppSp(5')G. The "D1" diastereomer of beta-S-ARCA or "beta-S-ARCA(D1)" is the diastereomer of beta-S-ARCA that elutes first on an HPLC column and therefore exhibits a shorter retention time compared to the D2 diastereomer of beta-S-ARCA [beta-S-ARCA(D2)] (see WO 2011 / 015347, incorporated herein by reference). A particularly preferred cap is beta-S-ARCA(D1)(m2 7,2’-O GppSpG).

[0112] In some embodiments, the RNA described in 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. An untranslated region (UTR) can be present at the 5' (upstream) of an open reading frame (5'-UTR) and / or at the 3' (downstream) of an open reading frame (3'-UTR). A 5'-UTR, if present, is located at the 5' end, upstream of the start codon of a protein coding region. A 5'-UTR is downstream of a 5'-cap (if present), e.g., directly adjacent to the 5'-cap. A 3'-UTR, if present, is located at the 3' end, downstream of a stop codon of a protein coding region, although the term "3'-UTR" preferably does not include a poly(A) tail. Thus, a 3'-UTR is upstream of a poly(A) sequence (if present), e.g., directly adjacent to a poly(A) sequence.

[0113] A particularly preferred 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 44. A particularly preferred 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 45.

[0114] In some embodiments, the RNA comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO:44, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:44.

[0115] In some embodiments, the RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO:45, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:45.

[0116] In some embodiments, the RNA described herein comprises a 3'-poly(A) sequence.

[0117] As used herein, the term "poly-A-tail" or "poly-A sequence" refers to an uninterrupted or interrupted sequence of adenylated residues typically located at the 3' end of an RNA molecule. Poly-A tails or poly-A sequences are known to those skilled in the art and can continue into the 3'-UTR in the RNA described herein. Uninterrupted poly-A tails are characterized by continuous adenylated residues. In nature, uninterrupted poly-A tails are typical. The RNA disclosed herein can have a poly-A tail that is attached to the free 3' end of the RNA after transcription by a template-independent RNA polymerase, or a poly-A tail that is encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0118] PolyA tails of approximately 120 A nucleotides have been shown to have beneficial effects on the levels of RNA in transfected eukaryotic cells as well as on the levels of protein translated from open reading frames present upstream (5') of the polyA tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0119] The polyA tail can be of any length. In some embodiments, the polyA tail comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, specifically comprising about 120 A nucleotides. In this context, "consisting essentially of" means that most of the nucleotides in the polyA tail are A nucleotides, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the number of nucleotides in the polyA tail, while allowing for the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridine), G nucleotides (guanylated), or C nucleotides (cytidylated). In this context, "consisting of" means that all nucleotides in the poly A tail, i.e., 100% of the number of nucleotides in the poly A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylation.

[0120] In some embodiments, the polyA tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template that contains repeated dT nucleotides (deoxythymidylic acid) in the strand complementary to the coding strand. The DNA sequence encoding the polyA tail (coding strand) is called a poly(A) cassette.

[0121] In some embodiments, the poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides, but is interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides long. Such cassettes are disclosed in WO2016 / 005324A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in WO2016 / 005324A1 can be used in the present invention. A poly(A) cassette consisting essentially of dA nucleotides, but interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of, for example, 5-50 nucleotides, shows, at the DNA level, a constant growth of plasmid DNA in E. coli, and, at the RNA level, is still associated with beneficial properties in terms of supporting RNA stability and translation efficiency. As a result, in some embodiments, the poly-A tail contained in the RNA molecules described herein consists essentially of A nucleotides, but is interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length.

[0122] In some embodiments, the polyA tail is not flanked at its 3' end by nucleotides other than A nucleotides, i.e., the polyA tail is not masked or is followed at its 3' end by nucleotides other than A. In some embodiments, the polyA tail comprises the sequence of SEQ ID NO:46.

[0123] In some embodiments, the RNA comprises a polyA tail. In some embodiments, the polyA tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the polyA tail may consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the polyA tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the polyA tail may comprise a polyA tail as set forth in SEQ ID NO: 46. In some embodiments, the polyA tail comprises at least 100 nucleotides. In some embodiments, the polyA tail comprises about 150 nucleotides. In some embodiments, the polyA tail comprises about 120 nucleotides.

[0124] In some embodiments, the RNA comprises a polyA tail comprising the nucleotide sequence of SEQ ID NO:46, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:46.

[0125] In the context of the present disclosure, the term "transcription" refers to the process in which the genetic code of a DNA sequence is transcribed into RNA. The RNA can then be translated into peptides or proteins. The term "transcription" includes "in vitro transcription", which refers to the process in which RNA, specifically mRNA, is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is applied for the generation of the transcript. These cloning vectors are generally designed as transcription vectors and are encompassed by the term "vector" according to the present invention. According to the present invention, the RNA used in the present invention is preferably in vitro transcribed RNA (IVT-RNA), which 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. Particular examples of RNA polymerases are T7, T3, and SP6 RNA polymerase. Preferably, the in vitro transcription according to the present invention is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription can be obtained by cloning a nucleic acid, specifically a cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0126] The term "translation" refers to the process within a cell's ribosomes by which a chain of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.

[0127] "Encoding" refers to the inherent property of a particular sequence of nucleotides 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 that have 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 encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the 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 said to encode the protein or other product of that gene or cDNA.

[0128] As used herein, "endogenous" refers to any substance that originates or is produced within an organism, cell, tissue or system.

[0129] As used herein, the term "exogenous" refers to any substance that originates or is produced outside an organism, cell, tissue or system.

[0130] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence.

[0131] "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 the recombinant polynucleotide.

[0132] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, 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 times 100, divided by the number of positions compared. For example, if 6 out of 10 positions in two sequences are matched or homologous, 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 amino acid or nucleotide residues that are at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and preferably at least 95%, at least 98%, or at least 99% identical.

[0133] "Fragment" refers to a portion of an amino acid sequence (peptide or protein), i.e. a sequence that represents an amino acid sequence that is truncated at the N-terminus and / or C-terminus. A fragment that is truncated at the C-terminus (N-terminal fragment) can be obtained, for example, by translation of a shortened open reading frame that lacks the 3'-end of the open reading frame. A fragment that is truncated at the N-terminus (C-terminal fragment) can be obtained, for example, by translation of a shortened open reading frame that lacks the 5'-end of the open reading frame, as long as the shortened open reading frame contains an initiation codon that contributes to initiating the 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.

[0134] By "variant" or "variant protein" or "variant polypeptide" herein is meant a protein that differs from a parent protein by at least one amino acid modification. The parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide, or may be a modified version of a wild-type polypeptide. Preferably, the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, for example, 1 to about 20 amino acid modifications compared to the parent, and preferably 1 to about 10, or 1 to about 5 amino acid modifications. As used herein, the term "variant" may include sequences that are modified versions and / or fragments compared to a parent or reference sequence.

[0135] By "parent polypeptide," "parent protein," "precursor polypeptide," or "precursor protein," as used herein, is meant an unmodified polypeptide that is subsequently modified to produce a variant. A parent polypeptide can be a wild-type polypeptide, or a mutant or engineered version of a wild-type polypeptide.

[0136] 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.

[0137] 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 mutations, splice variants, post-translational modification variants, structural, isoforms, allelic variants, species variants, and species homologs, particularly those that are naturally occurring.

[0138] Amino acid insertion variants include the insertion of a single or two 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 into a specific site in the amino acid sequence, but random insertion is also possible with appropriate screening of the resulting products. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 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 the sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion can be at any position in the protein. Amino acid deletion variants that include deletions at the N-terminus and / or C-terminus of the 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. It is preferred that there are modifications in positions in the amino acid sequence that are not conserved between homologous proteins or peptides, and / or that amino acids are substituted with others that have similar properties. Preferably, the amino acid changes in the peptides and protein variants are conservative amino acid changes, i.e., substitutions of similar charged or uncharged amino acids. Conservative amino acid changes include the substitution of one of a family of amino acids that are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In one embodiment, conservative amino acid substitutions are made in the following groups: Glycine, Alanine; valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, Glutamine; Serine, Threonine; Lysine, arginine; and Phenylalanine, Tyrosine Includes substitutions in .

[0139] In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) or a nucleotide sequence is preferably a "functional fragment" or "functional variant". With respect to a nucleotide sequence, the term "functional fragment" or "functional variant" refers to the fact that the nucleotide sequence encodes an amino acid sequence (peptide or protein) that is or comprises a "functional fragment" or "functional variant" of a parent amino acid sequence.

[0140] The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. Such functions may include the binding and / or immunogenic activity exhibited by the amino acid sequence from which the fragment or variant is derived, for example, in the case of an antigen or antigen sequence, the activity of binding to a receptor, for example, a CAR, and / or activating an immune effector cell expressing said receptor, or in the case of an antigen-binding domain, the activity of binding to an antigen. The term "functional fragment" or "functional variant" as used herein specifically refers to a variant molecule or sequence that includes an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of a parent molecule or sequence, and that is still able to fulfill one or more of the functions of the parent molecule or sequence, for example, the binding and / or activation functions. In one embodiment, the alterations in the amino acid sequence of the parent molecule or sequence do not significantly affect or change the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant is reduced but still significantly present, for example the function, such as binding and / or activation, of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence, however, in other embodiments the function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0141] 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 first amino acid sequence. Preferably, an amino acid sequence that is 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 can be a variant of the particular sequence or a fragment thereof.

[0142] 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 will be 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 over 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 to 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 to the full length of the reference amino acid sequence. Alignment for determining sequence similarity, preferably sequence identity, can be performed by using tools known in the art, preferably best sequence alignment, e.g., Align using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0143] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences.

[0144] The term "% identical", "% identity", or similar terms are specifically intended to refer to the percentage of nucleotides or amino acids that are identical in optimal alignment between the sequences being compared. Said percentage is entirely statistical, and the differences between the two sequences can be, but are not necessarily, randomly distributed over the entire length of the sequences being compared. Comparison of two sequences is usually performed by comparing the sequences after optimal alignment over a segment or "window of comparison" to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually, or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, using the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, using the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or using computer programs that use said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithms available at the National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for the BLASTN algorithm of the NCBI website include (i) prediction threshold 10; (ii) word size 28; (iii) maximum match of query range 0; (iv) match / mismatch score 1, -2; (v) linear gap cost; and (vi) low complexity region filter used. In some embodiments, the algorithm parameters used for the BLASTP algorithm of the NCBI website include (i) prediction threshold 10; (ii) word size 3; (iii) maximum match of query range 0; (iv) BLOSUM62 matrix; (v) gap cost Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.

[0145] Percentage identity is achieved by determining the number of corresponding identical positions in the compared sequences, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence) and multiplying the result by 100.

[0146] In some embodiments, the degree of identity is given to a region that is 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 full length of the reference sequence.For example, if the reference nucleic acid sequence is 200 nucleotides, the degree of identity is given to at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, consecutive nucleotides.In some embodiments, the degree of identity is given to the full length of the reference sequence.

[0147] A nucleic acid sequence or amino acid sequence that has a particular degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, can have at least one functional characteristic of the given sequence, e.g., in some cases, is functionally equivalent to the given sequence.

[0148] As used herein, "instructional material" or "instructions" includes articles, records, diagrams, or any other medium of expression that can be used to communicate the utility of the compositions and methods of the invention. The instructions of the kits of the invention may, for example, be affixed to a container containing the compositions of the invention or shipped together with a container containing the composition. Alternatively, the instructions may be shipped separately from the container with the intention that the instructions and the composition be used conjointly by the recipient.

[0149] "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 can exist in a substantially pure form, or can exist in a non-native environment, such as, for example, a host cell.

[0150] The term "recombinant", in the context of this disclosure, means "produced by genetic engineering." In one embodiment, a "recombinant subject", in the context of this disclosure, is not naturally occurring.

[0151] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature.For example, a peptide or nucleic acid that exists in an organism (including viruses), can be isolated from a natural source, and is not intentionally modified by humans in a laboratory is naturally occurring.The term "naturally occurring" means "naturally occurring", and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0152] In the context of the present invention, the following abbreviations are used for commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine and "U" refers to uridine.

[0153] " Lentivirus " as used herein refers to a genus of the Retroviridae family.Lentivirus is unique among retroviruses in that it can infect non-dividing cells; they can deliver a significant amount of genetic information to the DNA of host cells, and therefore they are one of the most effective methods of gene delivery vectors.HIV, SIV, and FIV are all examples of lentivirus.Lentivirus-derived vectors provide a means to achieve a significant level of gene transfer in vivo.

[0154] The term "operably linked" refers to a functional link between a control sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. 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 the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where two protein coding regions must be connected, are in the same reading frame.

[0155] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to refer to an abnormal level of expression of a tumor antigen in cells derived from a disease area, such as a solid tumor, within a particular tissue or organ of a patient, compared to the level of expression in normal cells derived from that tissue or organ.

[0156] The term "promoter" as used herein is defined as a DNA sequence that is recognized by or introduced into the synthetic machinery of a cell and is required to initiate the specific transcription of a polynucleotide sequence.

[0157] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence that is necessary for the 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 control elements that are necessary for the expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0158] 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.

[0159] 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 substantially only when an inducer corresponding to the promoter is present in the cell.

[0160] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoded 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.

[0161] As used herein, the term "specific binding" refers to a molecule, such as an antibody or CAR, that recognizes a specific 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 can also bind to that antigen from one or more other species. However, such cross-species reactivity does not, in itself, change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen can also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in itself, change the classification of the antibody as specific. In some examples, the term "specific binding" or "specifically binding" is used in reference to the interaction of an antibody, protein, or peptide with a second chemical species, where the interaction may depend on the presence of a specific structure (e.g., an antigenic determinant or epitope) of the chemical species; for example, an antibody is meant to recognize and bind to a specific protein structure rather than to proteins in general. If an antibody is specific for epitope "A," then the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody in a reaction containing labeled "A" and the antibody.

[0162] As used herein, the terms "transfect" or "transformation" or "transduction" 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.

[0163] As used herein, the phrase "under transcriptional control" or "operably linked" means that a promoter is in the correct location and orientation in relation to a polynucleotide that controls the initiation of transcription and expression of the polynucleotide by RNA polymerase.

[0164] A "vector" is a composition of matter that contains an isolated nucleic acid, which can be used for delivery of the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be considered to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0165] explanation The present invention provides compositions and methods for treating cancer. The cancer can be a solid tumor, primary or metastatic tumor. In one embodiment, the cancer is a CLDN6-expressing cancer. In one embodiment, the cancer is ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly lung cancer including lung squamous cell 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 cancer, bladder cancer, particularly bladder ... The cancer disease is selected from the group consisting of epithelial and papillary carcinoma, renal cancer, particularly renal cell carcinoma including renal clear cell carcinoma and papillary renal cell carcinoma, colon cancer, ileal cancer, particularly small intestinal cancer including small intestinal adenocarcinoma and ileal adenocarcinoma, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, particularly testicular seminoma, testicular teratoma and embryonic testicular cancer, uterine cancer, germ cell tumors such as teratocarcinoma or embryonic carcinoma, particularly testicular germ cell tumors, 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, liver 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.

[0166] The chimeric antigen receptor (CAR) described herein comprises an extracellular domain and an intracellular domain. The extracellular domain comprises a target-specific binding element, otherwise referred to as an antigen-binding portion or domain. The intracellular domain, or 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. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, which are necessary for lymphocytes to respond efficiently to antigens.

[0167] 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 of the antigen-binding domain. As used herein, the term "spacer domain" generally refers to any oligo- or polypeptide that functions to link a transmembrane domain to either an extracellular or cytoplasmic domain in 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 contain one or more amino acid substitutions. In some embodiments, the spacer domain is derived from the hinge region sequence of CD8α.

[0168] A cell (e.g., a T cell) is engineered to express a CAR as described herein, and the CAR-engineered cell exhibits anti-tumor properties. When expressed in a cell, the CAR of the present invention can redirect antigen recognition based on the antigen-binding specificity of the CAR. In one embodiment, CLDN6 is expressed in a cell of a cancer type as disclosed herein. When the CAR-engineered cell binds to its cognate antigen, it affects the tumor cell, causing the tumor cell to fail to proliferate, to die, or otherwise reduce or eliminate the tumor burden in the patient.

[0169] In one embodiment, the CLDN6 antigen binding portion is fused to an intracellular domain comprising a combination of the 4-1BB (CD137) signaling domain and the CD3-zeta 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).

[0170] antigen binding part The CAR of the present invention generally comprises a target-specific binding element, which is a part of the extracellular domain of the CAR, otherwise called antigen-binding portion or antigen-binding domain. The antigen-binding domain recognizes a ligand that acts as a cell surface marker on target cells associated with a particular disease state. In particular, the CAR of the present invention targets the tumor antigen CLDN6 on tumor cells.

[0171] In one embodiment, the CLDN6 binding domain of the CAR of the present invention specifically binds to CLDN6. In one embodiment, the CLDN6 to which the CLDN6 binding domain of 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 of CLDN6. 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 acids 28-76 of CLDN6, or the second extracellular loop of CLDN6, preferably amino acids 141-159 of CLDN6. In a particular embodiment, the CLDN6 binding domain binds to an epitope of CLDN6 that is not present in CLDN9. Preferably, the CLDN6 binding domain binds to an epitope of CLDN6 that is not present in CLDN4 and / or CLDN3. Most preferably, the CLDN6 binding domain binds to an epitope of CLDN6 that is not present in 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 to CLDN6. Preferably, the CLDN6 binding domain binds to CLDN6 expressed on the cell surface.

[0172] 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 via a peptide linker, preferably a peptide linker comprising the amino acid sequence (GGGGS)3.

[0173] In one embodiment, the binding domain of 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.

[0174] In one embodiment, the binding domain of 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.

[0175] In one embodiment, the binding domain of CLDN6 is (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 wherein x is selected from 3, 5, 7 and 9.

[0176] In one embodiment, the binding domain of CLDN6 is (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.

[0177] In one embodiment, the binding domain of CLDN6 is (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.

[0178] In one embodiment, the binding domain of CLDN6 is (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.

[0179] In one embodiment, the binding domain of CLDN6 is (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.

[0180] In certain preferred embodiments, the binding domain of CLDN6 comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following possibilities (i) to (xi): (i) the VH comprises an amino acid sequence represented by SEQ ID NO: 3 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant; and the VL comprises an amino acid sequence represented by SEQ ID NO: 4 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant; (ii) the VH comprises an amino acid sequence represented by SEQ ID NO:5 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the VL comprises an amino acid sequence represented by SEQ ID NO:6 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant; (iii) the VH comprises an amino acid sequence represented by SEQ ID NO: 7 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the VL comprises an amino acid sequence represented by SEQ ID NO: 8 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant; (iv) VH comprises an amino acid sequence represented by SEQ ID NO: 9 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and VL comprises an amino acid sequence represented by SEQ ID NO: 10 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant; (v) the VH comprises an amino acid sequence represented by SEQ ID NO:5 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant, and the VL comprises an amino acid sequence represented by SEQ ID NO:4 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant; (vi) VH comprises an amino acid sequence represented by SEQ ID NO: 5 or a functional variant thereof, or a fragment of the amino acid sequence or functional variant; and VL comprises an amino acid sequence represented by SEQ ID NO: 23 or a functional variant thereof, or a fragment of the amino acid sequence or 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 the amino acid sequence or 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 the amino acid sequence or functional variant.

[0181] In certain preferred embodiments, the binding domain of 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 the amino acid sequence or 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 the amino acid sequence or functional variant.

[0182] The term "fragment" specifically refers to one or more complementarity determining regions (CDRs), preferably at least the CDR3 variable region of the heavy chain variable region (VH) and / or the light chain variable region (VL). In one embodiment, said one or more 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).

[0183] In one embodiment, a binding domain of CLDN6 comprising one or more CDRs, a set of CDRs, or a combination of sets of CDRs as described herein comprises said CDRs together with their intervening framework regions. Preferably, the portion also comprises at least about 50% of either or both of the first and fourth framework regions, the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region.

[0184] The construction of binding domains made by recombinant DNA techniques may result in the introduction of N- or C-terminal residues into the variable region that are encoded by linkers introduced to facilitate cloning or other engineering steps, including the introduction of linkers that connect the variable region to additional protein sequences, including those described herein.

[0185] 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 in a human antibody framework.

[0186] In one embodiment, the binding domain of CLDN6 comprises 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) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7 and 9, or a functional variant thereof, as described herein.

[0187] In one embodiment, the binding domain of CLDN6 comprises 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) 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, as described herein.

[0188] In one embodiment, the binding domain of CLDN6 is (i) 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) 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 of the CDR sequences of the light chain variable region (VL) of a combination of heavy chain variable region (VH) and light chain variable region (VL) described herein; Includes.

[0189] In one embodiment, the binding domain of CLDN6 is (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 the light chain variable region (VL) of SEQ ID NO: x+1, or a functional variant thereof. wherein x is selected from 3, 5, 7 and 9.

[0190] In one embodiment, the binding domain of CLDN6 is (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: 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.

[0191] In one embodiment, the binding domain of CLDN6 is (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: 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.

[0192] The term "at least one, preferably two, more preferably all three of the CDR sequences" preferably relates to at least the CDR3 sequence, optionally in combination with the CDR1 and / or CDR2 sequences.

[0193] 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.

[0194] 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.

[0195] In one embodiment, the binding domain of 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 of 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 of 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 of 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 of CLDN6 according to the preceding embodiment comprises a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO: 16 or a functional variant thereof and / or the CDR2 sequence set forth in SEQ ID NO: 17 or a functional variant thereof.

[0196] In one embodiment, the binding domain of 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 of 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 of 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 of CLDN6 according to the preceding embodiment comprises a light chain variable region (VL) comprising the CDR1 sequence set forth in SEQ ID NO: 21 or a functional variant thereof and / or the CDR2 sequence of Ser Thr Ser or a functional variant thereof.

[0197] In one embodiment, the binding domain of CLDN6 is (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, wherein 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. Includes.

[0198] In one embodiment, the binding domain of CLDN6 described in the above embodiment comprises (i) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO: 16 or a functional variant thereof and / or the CDR2 sequence set forth in SEQ ID NO: 17 or a functional variant thereof, and / or (ii) a light chain variable region (VL) comprising the CDR1 sequence set forth in SEQ ID NO: 21 or a functional variant thereof and / or the CDR2 sequence of Ser Thr Ser or a functional variant thereof.

[0199] In one embodiment, the binding domain of CLDN6 competes with said binding domain of CLDN6 for CLDN6 binding and / or has the specificity for CLDN6 of said binding domain of CLDN6. In these and other embodiments, the binding domain of CLDN6 may be highly homologous to said binding domain of CLDN6. It is contemplated that preferred binding domains of CLDN6 have CDR regions that are identical or highly homologous to the CDR regions of said binding domain of CLDN6. 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.

[0200] 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 not competing, which indicates that the binding molecules do not bind to the same part of the target antigen, i.e., epitope. 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. An example of such a method is the so-called cross-competition assay, which can be carried out, for example, as an ELISA or by flow cytometry. For example, an ELISA-based assay can be carried out by coating the wells of an ELISA plate with one antibody; adding a competing antibody and a His-tag antigen / target, and detecting whether the added antibody inhibits the binding of the His-tag antigen to the coated antibody, for example, by adding a biotinylated anti-His antibody, then streptavidin-poly-HRP, and further developing the reaction with ABTS, and measuring the absorbance at 405 nm. For example, a flow cytometry assay can 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.

[0201] 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., a binding molecule that binds to the same epitope, can be tested by different 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 a well of a microtiter plate, and an antigen-binding antibody and a candidate competitive test antibody can be added. The amount of antigen-binding antibody that binds to the antigen in the well indirectly correlates with the binding ability of the candidate competitive test antibody that competes with them for binding to the same epitope. In particular, the greater the affinity of the candidate competitive test antibody to the same epitope, the less the amount of antigen-binding antibody that binds to the well coated with the antigen. The amount of antigen-binding antibody bound to the well can be measured by labeling the antibody with a detectable or measurable labeling substance.

[0202] Preferably, part of the antigen-binding portion of the CAR of the present invention is an anti-CLDN6 scFv, which preferably comprises the sequence set forth in SEQ ID NO: 35 or a functional variant thereof.

[0203] Transmembrane domain The CAR of the present invention is designed to include a transmembrane domain that is 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 and to minimize interaction with other members of the receptor complex. The transmembrane domain can be naturally derived or from a synthetic source. If the source is natural, the domain can be from any membrane-bound or transmembrane protein. A transmembrane region of particular use in the present invention may be derived from (i.e., comprising at least the transmembrane region(s) thereof) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, 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 mainly hydrophobic residues such as leucine and valine. Preferably, a triad of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain.

[0204] 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.

[0205] In some examples, the CAR of the present invention comprises a CD8α hinge domain that forms a link 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.

[0206] Cytoplasmic domain The cytoplasmic domain or otherwise intracellular signaling domain of the CAR of the present invention is responsible for activating 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. The term intracellular signaling domain is therefore meant to include any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0207] It is known that the signal generated by TCR alone is insufficient for the complete activation of T cells, and secondary or costimulatory signals are also required. Therefore, T cell activation is said to be mediated by two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-dependent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0208] The CAR of the present invention comprises a primary cytoplasmic signaling sequence derived from CD3-zeta. Furthermore, the cytoplasmic domain of the CAR of the present invention is designed to comprise a CD3-zeta signaling domain in combination with a costimulatory signaling region derived from 4-1BB.

[0209] The term "4-1BB" refers to a membrane receptor protein, also called CD137, which is expressed on the surface of activated T cells as a type of accessory molecule and is a member of the tumor necrosis factor receptor (TNFR) superfamily. 4-1BB has a molecular weight of 55 kDa and is found as a homodimer.

[0210] The T cell surface glycoprotein CD3-zeta chain is also known as the T cell receptor T3 zeta chain, and CD247 is a protein encoded by the CD247 gene in humans. Together with the T cell receptor alpha / beta and gamma / delta heterodimers and CD3-gamma, -delta, and -epsilon, the T cell receptor zeta (ζ) forms the T cell receptor-CD3 complex. The zeta chain plays an important role in coupling antigen recognition to several intracellular signaling pathways. Low expression of antigens leads to impaired immune responses.

[0211] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present invention can be linked to each other in a random or specific order. A short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, may form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[0212] Thus, the cytoplasmic domain in the CAR of the invention is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In one embodiment, the cytoplasmic domain in the CAR of the invention is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, 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 CD3-zeta comprises the amino acid sequence of SEQ ID NO: 31 or a functional variant thereof.

[0213] 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 the sequence set forth in SEQ ID NO: 25 or a functional variant thereof.

[0214] In one embodiment, a CAR of the invention comprises the following elements in the following order: NH2-CLDN6 antigen binding domain-transmembrane domain-4-1BB costimulatory domain-CD3-zeta signaling domain-COOH.

[0215] 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-CD3-zeta signaling domain-COOH.

[0216] In one embodiment, the CAR of the present invention comprises the amino acid sequence set forth in SEQ ID NO: 36, or a functional variant thereof.

[0217] vector The present invention encompasses nucleic acid constructs, such as DNA constructs, that comprise a sequence encoding a CAR of the present invention. In one embodiment, the nucleic acid construct encoding a CAR of the present invention comprises the nucleotide sequence of SEQ ID NO: 41, or a functional variant thereof.

[0218] In one embodiment, (i) the nucleic acid construct encoding the CAR comprises the nucleotide sequence of SEQ ID NO:41, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:41; and / or (ii) the CAR comprises the amino acid sequence of SEQ ID NO:36, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:36.

[0219] 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, because they allow long-term, stable integration of transgenes and their proliferation in daughter cells.Lentivirus vectors have the added advantage of vector derived from oncoretrovirus, such as mouse leukemia virus, that they can transduce non-proliferating cells, such as hepatocytes.They also have the added advantage of low immunogenicity.

[0220] In brief summary, expression of natural or synthetic nucleic acid encoding CAR is typically achieved by operably linking the nucleic acid encoding the 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.Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for controlling the expression of the desired nucleic acid sequence.

[0221] The nucleic acid of the present invention can be cloned into several types 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. Vectors of particular interest 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 biology and molecular biology manuals. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, a suitable vector contains 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).

[0222] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of a subject, 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.

[0223] Additional promoter elements, such as enhancers, control the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although many promoters have recently been shown to contain functional elements downstream of the start site as well. The frequency of spacing between promoter elements is flexible, and promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased up to 50 bp apart before activity begins to decrease. It is believed that promoters allow individual elements to function 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 factor-1α (EF-1α). However, other constitutive promoter sequences may 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 considered as part of the present invention. The use of inducible promoters provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0224] To assess the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into the cell can also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells that may be transfected or infected by the viral vector. In other embodiments, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate control sequences to allow expression in the host cell. Useful selectable markers include antibiotic resistance genes, such as, for example, neo, etc.

[0225] Reporter genes are used to identify cells that may be transfected and to evaluate the functionality of control sequences. In general, reporter genes are genes that code for polypeptides that are not present in or expressed by recipient organisms or tissues, and whose expression is evidenced by some easily detectable property, such as enzymatic activity. The expression of reporter genes is evaluated at a suitable time after DNA is introduced into recipient cells. Suitable reporter genes may include genes that code for luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and can be prepared and commercially available using known techniques.

[0226] Methods for introducing and expressing genes into cells are known in the art. In the context of 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.

[0227] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0228] Biological methods for introducing a polynucleotide of interest into host cells include the use of DNA vectors and RNA vectors.Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc.

[0229] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0230] cell The cells used in connection with the CAR system of the present invention and into which the nucleic acid (DNA or RNA) encoding the CAR system of the present invention can be introduced include any cell with lytic ability, particularly lymphoid cells, and preferably T cells, particularly cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. When activated, each of these cytotoxic lymphocytes drives the destruction of target cells. For example, cytotoxic T cells drive the destruction of target cells by either or both of the following means: First, when T cells are activated, they release cytotoxins such as perforin, granzymes, and granulysin. Perforin and granulysin create holes in the target cell, and granzymes enter the cell and drive a caspase cascade in the cytoplasm that induces apoptosis (programmed cell death) of the cell. Second, apoptosis can be induced through Fas-Fas ligand interaction between T cells and target cells. The cytotoxic lymphocytes are preferably autologous cells, although xenogeneic or allogeneic cells can be used.

[0231] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include helper T 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 an antigen to which the T cell is targeted and preferably exerts an effector function of the T cell. A T cell is considered specific for an antigen if the cell kills a target cell expressing the antigen. T cell specificity can 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 (e.g., interferon-γ) can be measured.

[0232] 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 T cell receptor (CD3). As provided herein, NK cells may be differentiated from stem or progenitor cells.

[0233] The term "effector function" in the context of the present invention includes any function mediated by a component of the immune system, which results in, for example, the killing of diseased cells, such as tumor cells, or the inhibition of tumor growth and / or tumorigenesis, including the inhibition of tumor proliferation and metastasis. Preferably, the effector function in the context of the present invention is a T cell-mediated effector function. Such a function may be mediated by helper T cells (CD4 + T cells), cytokine release and / or CD8 + It involves activation of lymphocytes (CTLs) and / or B cells, and in the case of CTLs, removal of cells, i.e., cells characterized by expression of the antigen, e.g., by 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.

[0234] The term "immune effector cells" or "immune response cells" in the context of the present invention relates to cells that exert an effector function during an immune response. "Immune effector cells" are 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, "immune effector cells" are T cells, preferably CD4 + T cells and / or CD8 + According to the present invention, the term "immune effector cells" also includes cells that can be matured into immune cells (e.g., T cells, specifically helper T cells or cytolytic T cells) upon appropriate stimulation. Immune effector cells are characterized by the expression of 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.

[0235] Preferably, "immune effector cells" recognize antigens with some degree of specificity, particularly when present on the surface of diseased cells such as cancer cells. Preferably, said recognition causes the cells to recognize antigens to which they are responsive and 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., the cell characterized by expression of the antigen, for example, by apoptosis or perforin-mediated cytolysis. According to the present invention, CTL responsiveness may include sustained calcium influx, cell differentiation, 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 artificial reporters that accurately indicate CTL responsiveness. Such CTLs that recognize and are responsive or reactive to an antigen are also referred to herein as "antigen-responsive CTLs".

[0236] "Lymphoid cells" are cells that may be capable of producing an immune response, such as a cellular immune response, or precursors of such cells after suitable modification, e.g., after transfer of a CAR, and include lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. Lymphoid cells may be immune effector cells as described herein. Preferred lymphoid cells are T cells, which may be modified to express a CAR on the cell surface. In one embodiment, lymphoid cells lack endogenous expression of a T cell receptor.

[0237] The terms "T cells" and "T lymphocytes" are used interchangeably herein and include helper T cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), including cytolytic T cells.

[0238] 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 lymphocyte types, such as B cells and natural killer cells, by the presence of a special receptor on their cell surface 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 are found, each with a different function.

[0239] Helper T cells help 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 control and aid in an active immune response.

[0240] 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.

[0241] Most T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor is composed of two separate peptide chains, independently produced from the T cell receptor alpha and beta (TCRα and TCRβ) genes, called the α- and β-TCR chains. γδ T cells (gamma delta T cells) represent a small subset of T cells that carry different T cell receptors (TCRs) on their surface. However, in γδ T cells, the TCR is made up of one γ chain and one δ chain. This group of T cells is much less common than αβ T cells (2% of all T cells).

[0242] All T cells arise from hematopoietic stem cells in the bone marrow. Hematopoietic stem cell-derived hematopoietic precursors populate the thymus and proliferate by cell division to generate 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 they progress through development, they become double positive thymocytes (CD4+CD8+) and finally mature into single positive (CD4+CD8- or CD4-CD8+) thymocytes, which are then released from the thymus into peripheral tissues.

[0243] 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 a mammal, such as a patient, using a commercially available cell separation system. Alternatively, T cells can be from related or unrelated humans, non-human animals, cell lines, or cultures. Samples containing T cells can be, for example, peripheral blood mononuclear cells (PBMCs).

[0244] 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.

[0245] The term "CAR targeted to an antigen" refers to a CAR that, when present on an immune effector cell, such as a T cell, recognizes an antigen, such as on the surface of an antigen-presenting cell or a diseased cell, such as a cancer cell, and the immune effector cell is stimulated, primed and / or expanded, or exerts an effector function of the immune effector cell as described above.

[0246] 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 an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, several T cell lines available in the art can be used. In certain embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using several techniques known to those skilled in the art, such as Ficoll™ separation. In a preferred embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains 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 may be washed to remove the plasma fraction, and the cells placed 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, but not all, divalent cations. Alternatively, and surprisingly, an initial activation step in the absence of calcium results in expanded 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 by using a semi-automated "flow-through" centrifuge (e.g., Cobe2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) following the manufacturer's instructions. After washing, the cells are washed with, for example, Ca 2+ Free, 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, unwanted components of the apheresis sample may be removed and the cells are resuspended directly in culture medium.

[0247] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, e.g., by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. Specific subpopulations of T cells, e.g., CD3 + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO + T cells may 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 period of time sufficient for positive selection of the desired T cells. In one embodiment, the period ranges from 30 minutes to 36 hours or more. One skilled in the art will recognize that multiple rounds of selection may also be used in the context of the present invention.

[0248] Enrichment of a T cell population by negative selection can be achieved by a combination of antibodies against surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, negative selection can 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 TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, TCR8, TCR9, TCR10, TCR11, TCR12, TCR13, TCR14, TCR15, TCR26, TCR27, TCR38, TCR4, TCR5, TCR6, TCR7, TCR8, TCR11, TCR12, TCR14, TCR15, TCR16, TCR17, TCR28, TCR19, TCR29, TCR28, TCR29, TCR30, TCR40, TCR5, TCR6, TCR10, TCR11, TCR29, TCR12, TCR14, TCR29, TCR28, TCR15, TCR29, TCR29, TCR30, TCR40, TCR10

[0249] Various methods of introducing CAR constructs into T cells can be used, 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 insertion mutation. Transposon-based systems can integrate transgenes more efficiently than plasmids that do not contain integration elements. Virus-based systems include the use of gamma-retrovirus vectors and lentivirus vectors. Gamma-retroviruses are relatively easy to produce, efficiently and persistently transduce T cells, and have previously been shown to be safe in terms of integration in primary human T cells. Lentivirus vectors also efficiently and persistently transduce T cells, but are more expensive to manufacture. They may also be safer than retrovirus-based systems.

[0250] 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 the CAR.

[0251] CAR T cells may be produced in vivo and therefore almost instantaneously using nanoparticles that target T cells. For example, poly(β-amino ester)-based nanoparticles can be coupled to anti-CD3e f(ab) fragments to bind to CD3 on T cells. Upon binding to T cells, these nanoparticles are internalized. Their contents, for example, plasmid DNA encoding antitumor antigen CAR, can be directed to the T cell nucleus by inclusion of a peptide containing a microtubule-associated sequence (MTAS) and a nuclear localization signal (NLS). Inclusion of a transposon with inverted repeats (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.

[0252] Another possibility is to use CRISPR / Cas9 technology to deliberately place a CAR coding sequence at a specific locus. For example, an existing T cell receptor (TCR) may be knocked out, knocking in the CAR and placing it under the dynamic control of an endogenous promoter that otherwise regulates TCR expression; see, e.g., Eyquem et al. (2017) Nature 543:113-117.

[0253] In one embodiment of all aspects of the invention, T cells genetically engineered to express a CAR are stably or transiently transfected with a nucleic acid encoding the CAR, and thus the nucleic acid encoding the CAR may or may not be integrated into the genome of the T cell.

[0254] In one embodiment of all aspects of the invention, the T cells or T cell precursors are from a subject to be treated. In one embodiment of all aspects of the invention, the T cells or T cell precursors are from a subject different from the subject to be treated.

[0255] 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).

[0256] 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.

[0257] T cell activation and expansion Before or after genetic modification of the T cells to express the desired CAR, the T cells can generally be activated and expanded using methods known in the art.

[0258] In general, the T cells of the present invention are expanded by contact with a surface that is attached with an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. Specifically, the T cell population can be stimulated as described herein, for example, by contact with an anti-CD3 antibody, or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. For costimulation of accessory molecules on the surface of the T cells, a ligand that binds to the accessory molecule is used. For example, the T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under appropriate conditions to stimulate the proliferation of the T cells.

[0259] 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 coupled to a surface. When coupled to a surface, the agents can be coupled 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 coupled to a surface and the other agent in solution. In one embodiment, the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution.

[0260] In one embodiment, the two agents are immobilized on beads, either on the same bead, i.e., "cis," or on separate beads, i.e., "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; 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.

[0261] A particle to cell ratio of 1:500 to 500:1 can be used to stimulate T cells or other target cells. One skilled in the art can readily appreciate that the particle to cell ratio can depend on the particle size compared to the target cells. For example, small size beads can only bind to a few cells, while large size beads can bind to many cells. In certain embodiments, the particle to cell ratio ranges from 1:100 to 100:1, and in further embodiments, the ratio includes 1:9 to 9:1.

[0262] Suitable conditions for T cell culture include an appropriate medium [e.g., Minimum Essential Medium or RPMI medium 1640 or X-vivo15, (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 additive for the growth of cells known to those skilled in the art. Other additives for the growth of cells 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, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% CO2).

[0263] therapeutic application The present invention encompasses cells (e.g., T cells) that contain the CAR molecules of the present invention, transduced by a retrovirus, such as a lentiviral vector (LV) that encodes the CAR of the present invention. Thus, in some instances, the transduced T cells can induce a CAR-mediated T cell response.

[0264] 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 T cells expressing a CAR of the present invention, wherein the CAR comprises a binding moiety that specifically interacts with CLDN6 as a predetermined target.

[0265] In one embodiment, the present invention includes a type 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 a recipient in need thereof. The infused cells can kill tumor cells in the recipient. Unlike antibody therapy, CAR T cells can replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0266] In one embodiment, the CAR T cells of the invention undergo robust in vivo T cell expansion and can 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 any further tumor formation or growth.

[0267] Cancers that can be treated include tumors that are not or are not yet substantially vascularized, as well as vascularized tumors. In one embodiment, the cancer comprises a solid tumor.

[0268] A solid tumor is an abnormal mass of tissue that does not usually contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcomas and carcinomas, are fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal cancer, lymphatic tumors, 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, hepatoma, cholangiocarcin ... Hair carcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors (e.g., gliomas (e.g., brain stem gliomas and mixed gliomas), glioblastomas, (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germinomas, medulloblastomas, Schwannomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendroglioma, meningiomas, neuroblastomas, retinoblastomas, and brain metastases).

[0269] In one embodiment, the cancer that can be treated is a CLDN6-expressing cancer, such as those described herein.

[0270] 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 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.

[0271] Combination therapy 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, specifically an 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, specifically an 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 and the nucleic acid encoding the cognate antigen molecule are administered, providing 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 cell surface expression. 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 an RNA. Preferably, contacting the CAR modified cells of the present invention with the cognate antigen molecule results in expansion and / or activation of the cells.

[0272] The peptide and protein antigen suitable for use according to the present invention typically comprises a peptide or protein that comprises an 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.

[0273] The peptide and protein antigens provided to the subject according to the present invention (by administering either the peptide and protein antigens or the nucleic acid, specifically RNA, encoding the peptide and protein antigens), i.e., the vaccine antigen, preferably results in the stimulation, priming and / or expansion of CAR modified cells and the antigen or nucleic acid in the subject to which the nucleic acid has been administered. The stimulated, primed and / or expanded CAR modified cells are preferably directed against the CLDN6 target antigen, specifically 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, and the stimulated, primed and / or expanded CAR modified cells specifically target the disease-associated antigen 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 comprise a disease-associated antigen, may correspond to or comprise a fragment of a disease-associated antigen, or may correspond to or comprise 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, and 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 refers to a fragment of an antigen that can stimulate, prime and / or expand CAR-modified cells. The vaccine antigen (similar to a 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.Vaccine antigens (similar to disease-associated antigens) are also preferably expressed on the surface of cells, such as antigen-presenting cells, so as to provide relevant epitopes for binding by the CAR. Vaccine antigens according to the present invention can be recombinant antigens.

[0274] 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, for example, in terms of the type of immunological effect. In the context of the present disclosure, the term "immunologically equivalent" is preferably used in reference 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, e.g., CLDN6, if, when exposed to a CAR-modified cell that binds to the reference amino acid sequence or a cell expressing the reference amino acid sequence, said amino acid sequence induces an immune response with the specificity of the reaction with the reference amino acid sequence, specifically the stimulation, priming and / or expansion of the CAR-modified cell. 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 in terms of the stimulation, priming and / or expansion of the CAR-modified cell as the antigen to which the CAR-modified cell is targeted.

[0275] As used herein, "activation" or "stimulation" refers to the state of T cells that are sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers to T cells that are undergoing cell division.

[0276] The term "priming" refers to the process by which a T cell first contacts its particular antigen, triggering differentiation into an effector T cell.

[0277] The term "clonal expansion" or "expansion" refers to the process by which a particular entity increases. 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.

[0278] 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 cytoplasm 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 of the lymphatic system, specifically the secondary lymphoid 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 of the spleen. In one embodiment, the target cell is a dendritic cell of the spleen.

[0279] The "lymphatic system" is part of the circulatory system, including the network of lymphatic vessels that transport lymph, and is an important part of the immune system. The lymphatic system consists of lymphoid organs, which carry the lymphatic vessels and circulate 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.

[0280] RNA may be delivered to the spleen by so-called lipoplex formulations, in which RNA is bound to liposomes containing cationic lipids and optionally additional 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-targeting RNA lipoplex particles are described in WO2013 / 143683, which is incorporated herein by reference. It has been found that RNA lipoplex particles with a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen-presenting cells, particularly 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 the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in antigen-presenting cells, such as professional antigen-presenting cells in the spleen. Thus, 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.

[0281] In the context of the present disclosure, the term "particle" refers to a structure formed by a molecule or a molecular complex. In one embodiment, the term "particle" refers to a micro- or nano-sized structure, e.g., a micro- or nano-sized small structure.

[0282] As used in this disclosure, a "nanoparticle" includes a particle comprising RNA and at least one cationic lipid and having an average diameter suitable for intravenous administration.

[0283] In the context of the present disclosure, the term "RNA lipoplex particles" refers to particles containing lipids, specifically 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 particles are nanoparticles.

[0284] The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected through a needle into an aqueous solution. This action disperses the lipids in the solution and promotes lipid structure formation, e.g., lipid vesicle formation, such as liposome formation. In general, the RNA lipoplex particles described herein are obtained by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such a colloidal liposome dispersion is formed in one embodiment as follows: an ethanol solution containing lipids, such as a cationic lipid like DOTMA and additional lipids, is injected into an aqueous solution under stirring. In one embodiment, the RNA lipoplex particles described herein can be obtained without a step of extrusion formation.

[0285] The term "extrude" or "extrusion" refers to the creation of particles having a fixed cross-sectional shape. Specifically, it refers to the downsizing of particles whereby the particles are forced through a filter with defined pores.

[0286] As used herein, "cationic lipid" refers to a lipid that has a net positive charge. Cationic lipids bind negatively charged RNA through electrostatic interactions to the lipid matrix. 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, but are not limited to, 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(tetradecyloxy)-3-dimethylammonium propane (DOCTAC ... Cationic lipids include 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA).Preferably, DOTMA, DOTAP, DODAC, and DOSPA.In certain embodiments, cationic lipid is DOTMA and / or DOTAP.

[0287] Additional lipids can be incorporated to adjust the overall positive to negative charge ratio and physical stability of RNA lipoplex particles.In certain embodiments, the additional lipid is a neutral lipid.As used herein, "neutral lipid" refers to a lipid with a net charge of zero.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.

[0288] 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.

[0289] 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.

[0290] In one embodiment, the RNA lipoplex particles described herein have an average diameter in the range of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 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.

[0291] The term "average diameter" refers to the average hydrodynamic diameter of the particles, as measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, resulting in the so-called Z 平均 , and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, the "average diameter", "diameter" or "size" of a particle is Z 平均 Used synonymously with this value.

[0292] The term "polydispersity index" is used herein as a measure of the overall size distribution of particles, e.g., nanoparticles. The polydispersity index is calculated based on dynamic light scattering measurements by so-called cumulant analysis.

[0293] The charge of the RNA lipoplex particles of the present disclosure is the sum of the charge present on at least one cationic lipid and the charge present on the RNA. The charge ratio is the ratio of the positive charge present on at least one cationic lipid to the negative charge present on the RNA. The charge ratio of the positive charge present on at least one cationic lipid to the negative charge present on the RNA is calculated by the following formula: charge ratio = [(cationic lipid concentration (mol)) * (total number of positively charged cationic lipids)] / [(RNA concentration (mol)) * (total number of negative charges in RNA)].

[0294] 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 of 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.

[0295] In one embodiment, immune effector cells expressing a CAR molecule described herein are co-administered with RNA encoding an amino acid sequence comprising claudin 6 (CLDN6), a functional variant thereof, or a functional fragment of CLDN6 or a functional variant thereof.

[0296] In one embodiment, the RNA encoding an amino acid sequence comprising claudin 6 (CLDN6), a functional variant thereof, or a functional fragment of CLDN6 or a functional variant thereof, comprises as an active ingredient a single-stranded RNA that can be translated into the respective protein upon entry into the recipient's cells. In addition to the wild-type or codon-optimized sequence encoding the amino acid sequence, the RNA may contain one or more structural elements (5' cap, 5' UTR, 3' UTR, poly(A) tail) that are optimized for maximum effectiveness of the RNA in terms of stability and translation efficiency. In one embodiment, the RNA contains all of these elements. In one embodiment, the RNA encoding beta-S-ARCA(D1)(m2 7,2’-O GppSpG) may be utilized as a specific capping structure at the 5' end of the RNA. As the 5'-UTR sequence, the 5'-UTR sequence of human alpha-globin mRNA, which may have an optimized "Kozak sequence" that increases translation efficiency, may be used. As the 3'-UTR sequence, a combination of two sequence elements (FI elements) from the "amino-terminal enhancer of split" (AES) mRNA (called F) and the mitochondrially encoded 12S ribosomal RNA (called I), placed between the coding sequence and the poly(A)-tail, which ensures higher maximum protein levels and extended persistence of the mRNA, may be used. These were identified by an ex vivo selection process of sequences that confer RNA stability and increase total protein expression (see WO2017 / 060314, incorporated herein by reference). Additionally, a poly(A)-tail measuring 110 nucleotides in length may be used, consisting of a stretch of 30 adenosine residues followed by a 10 nucleotide linker sequence (of random nucleotides) and another 70 adenosine residues. This poly(A)-tail sequence was designed to enhance RNA stability and translation efficiency.

[0297] RNA can be complexed with liposomes to generate serum-stable RNA lipoplexes [RNA(LIP)] for intravenous (iv) administration. RNA(LIP) targets antigen-presenting cells (APCs) in lymphoid organs, resulting in sufficient stimulation of CAR-expressing immune effector cells. RNA lipoplex particles may be prepared using liposomes, which may be obtained by injecting a solution of lipids in ethanol into water or a suitable aqueous phase. In one embodiment, the aqueous phase is at an acidic pH. In one embodiment, the aqueous phase includes acetic acid, for example in an amount of about 5 mM. Liposomes may be used to prepare RNA lipoplex particles by mixing liposomes with RNA. In one embodiment, liposomes and RNA lipoplex particles include at least one cationic lipid and at least one additional lipid. In one embodiment, the at least one cationic lipid includes 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA). In one embodiment, the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the liposome and RNA lipoplex particles comprise 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1. In one embodiment, at physiological pH, the charge ratio of positive to negative charges in the RNA lipoplex particles is from about 1.6:2 to about 1:2, or from about 1.6:2 to about 1.1:2.In certain embodiments, the charge ratio of positive to negative charges in the RNA lipoplex particles at physiological pH is 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.

[0298] In one embodiment, (i) the RNA encoding the amino acid sequence comprising claudin 6 (CLDN6), a functional variant thereof, or a functional fragment of CLDN6 or a functional variant thereof comprises the nucleotide sequence of SEQ ID NO: 42 or 43, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 42 or 43; and / or (ii) the amino acid sequence comprising claudin 6 (CLDN6), a functional variant thereof, or a functional fragment of CLDN6 or a functional variant thereof comprises the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 2. In one embodiment, the RNA encoding the amino acid sequence comprising claudin 6 (CLDN6), a functional variant thereof, or a functional fragment of CLDN6 or a functional variant thereof comprises the nucleotide sequence of SEQ ID NO: 43. In one embodiment, the amino acid sequence comprising claudin 6 (CLDN6), a functional variant thereof, or a functional fragment of CLDN6 or a functional variant thereof is encoded by a coding sequence that is codon-optimized and / or has an increased G / C content compared to a wild-type coding sequence, and the codon optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence. In one embodiment, the RNA encoding the amino acid sequence comprising claudin 6 (CLDN6), a functional variant thereof, or a functional fragment of CLDN6 or a functional variant thereof is 5'-capped m2 7,2’-O Gpp sp(5')G. In one embodiment, the RNA encoding an amino acid sequence comprising claudin 6 (CLDN6), a functional variant thereof, or a functional fragment of CLDN6 or a functional variant thereof comprises a 5'UTR comprising a nucleotide sequence of SEQ ID NO: 44, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 44. In one embodiment, the RNA encoding an amino acid sequence comprising claudin 6 (CLDN6), a functional variant thereof, or a functional fragment of CLDN6 or a functional variant thereof comprises a 3'UTR comprising a nucleotide sequence of SEQ ID NO: 45, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 45. In one embodiment, the RNA encoding an amino acid sequence comprising claudin 6 (CLDN6), a functional variant thereof, or a functional fragment of CLDN6 or a functional variant thereof comprises a polyA sequence. In one embodiment, the polyA sequence comprises at least 100 nucleotides. In one embodiment, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 46. In one embodiment, the RNA is formulated as a liquid, formulated as a solid, or a combination thereof. In one embodiment, the RNA is formulated for injection. In one embodiment, the RNA is formulated for intravenous administration. In one embodiment, the RNA is formulated as lipoplex particles. In one embodiment, the RNA lipoplex particles are obtainable by mixing the RNA with liposomes.

[0299] Below, embodiments of RNA encoding cognate antigens are described, and certain terms used when describing elements thereof have the following meanings: hAg-Kozak: 5'-UTR sequence of human alpha-globin mRNA with an optimized "Kozak sequence" that increases translation efficiency. Antigen: The sequence encoding the cognate antigen. FI elements: 3'-UTRs are a combination of two sequence elements, derived from the "amino-terminal enhancer of split" (AES) mRNA (termed F) and the mitochondrially encoded 12S ribosomal RNA (termed I). These were identified by an ex vivo selection process for sequences that confer RNA stability and increase total protein expression. A30L70: a poly(A)-tail measuring 110 nucleotides in length, consisting of a chain of 30 adenosine residues followed by a 10-nucleotide linker sequence and another 70 adenosine residues, was designed to enhance RNA stability and translation efficiency in dendritic cells.

[0300] In one embodiment, the RNA encoding the cognate antigen has the structure: Beta-S-ARCA(D1)-hAg-Kozak-Antigen-FI-A30L70.

[0301] In one embodiment, hAg-Kozak comprises the nucleotide sequence of SEQ ID NO: 44. In one embodiment, the antigen comprises a nucleotide sequence encoding the amino acid sequence of CLDN6 (e.g., SEQ ID NO: 1). In one embodiment, the antigen comprises the nucleotide sequence of SEQ ID NO: 42. In one embodiment, FI comprises the nucleotide sequence of SEQ ID NO: 45. In one embodiment, A30L70 comprises the nucleotide sequence of SEQ ID NO: 46. In one embodiment, the RNA encoding the cognate antigen comprises the nucleotide sequence of SEQ ID NO: 43.

[0302] 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.

[0303] The term "pharmaceutical composition" refers to a formulation containing a therapeutically effective agent, preferably together with a pharma- ceutically acceptable carrier, diluent and / or excipient. The pharmaceutical composition is useful for treating, preventing or reducing the severity of a disease or disorder by administration of the pharmaceutical composition to a subject. Pharmaceutical compositions are also known in the art as pharmaceutical formulations.

[0304] The pharmaceutical compositions of the present disclosure may include one or more adjuvants or may be administered with one or more adjuvants. The term "adjuvant" refers to a compound that prolongs, enhances, or accelerates immune response. Adjuvants include a heterogeneous group of compounds, such as oil emulsions (e.g., Freund's adjuvant), mineral compounds (e.g., alum), bacterial products (e.g., 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.

[0305] The pharmaceutical compositions described in this disclosure are generally applied in a "pharmaceutically effective amount" and in a "pharmaceutically acceptable preparation."

[0306] The term "pharmaceutical acceptable" refers to the non-toxicity of a material that does not interact with the action of the active ingredients of the pharmaceutical composition.

[0307] The term "pharmacologically effective amount" or "therapeutically effective amount" refers to an amount that achieves a desired response or a desired effect, either alone or together with further administrations. In the case of the treatment of a particular disease, the desired response preferably relates to the inhibition of the cause of the disease. This includes the delay of the progression of the disease, and in particular the halting or reversal of the progression of the disease. The desired response in the treatment of a disease can also be the delay of the onset of said disease or said condition or the prevention of their onset. The effective amount of the compositions described herein depends on the condition to be treated, the severity of the disease, the individual parameters of the patient, including age, physiological condition, 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 dose administered of the compositions described herein can depend on various such parameters. If the response in the patient is insufficient with the initial administration, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) can be used.

[0308] The immune effector cells described herein are generally administered in an amount that results in a desired pharmacological effect, e.g., an amount that results in a clinical effect on a tumor, such as a solid tumor, which may include inducing shrinkage of target lesions, inducing stable disease, inducing stable disease with shrinkage of target lesions, or inducing a partial response. In some embodiments, the immune effector cells described herein are administered in an amount that results in partial, complete, or essentially complete elimination of a solid tumor. In some embodiments, the immune effector cells described herein are administered in an amount that results in, e.g., about 10 to 20% of a tumor, in a single or multiple dose. 6 ~about 10 9 , about 5×10 6 ~Approx. 5×10 8 , or about 10 7 ~about 10 8 CAR T cells are administered to humans in an amount of 10 immune effector cells. Some therapeutic approaches involve repeated administration of CAR T cells for a certain period of time (e.g., less than 4 weeks). This results in a "cumulative dose" within such a period. Thus, about 10 6 ~about 10 9 , about 5×10 6 ~Approx. 5×10 8, or about 10 7 ~about 10 8 The amount of immune effector cells can be, for example, the amount of such cells administered as a single dose or cumulative doses over a period of at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 14 days, at least 21 days, at least 28 days, or more. 6 ~about 10 9 , about 5×10 6 ~Approx. 5×10 8 , or about 10 7 ~about 10 8 The amount of immune effector cells refers to a single dose of such cells. The term "single dose" means that one dose of a therapeutic agent is administered for an extended period of time. The term "extended period" includes at least 14 days, at least 21 days, at least 28 days, at least 3 months, at least 6 months or more. In some embodiments, the immune effector cells described herein are administered in a single dose of about 10 8 ~about 10 10 , about 5×10 8 ~Approx. 5×10 9 , or about 10 9 The immune effector cells are administered to a human in an amount such that a total amount of immune effector cells is achieved in the human.

[0309] In one embodiment, lymphodepletion treatment may be applied prior to administration of immune effector cells, for example by administering cyclophosphamide and fludarabine, which may increase cell persistence and the occurrence and duration of clinical responses.

[0310] The term "partial response (PR)" preferably relates to at least a 30% reduction in the sum of the diameters of the target lesions with reference to the baseline sum diameter. Partial remission is synonymous with partial response.

[0311] The pharmaceutical compositions of the present disclosure may contain salts, buffers, preservatives, and other therapeutic agents as appropriate. In one embodiment, the pharmaceutical compositions of the present disclosure include one or more pharma- ceutically acceptable carriers, diluents and / or excipients.

[0312] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0313] As used herein, the term "excipient" refers to a substance that may be present in the pharmaceutical compositions 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, flavorings, or coloring agents.

[0314] The term "diluent" refers to a diluting and / or thinning agent. 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.

[0315] The term "carrier" refers to an ingredient, 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. The carrier used herein may be one or more suitable solid or liquid fillers, diluents, or encapsulating materials 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 specifically biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxy-propylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure includes isotonic saline.

[0316] Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known to those skilled in the art of medicine and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

[0317] Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0318] 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 by the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to administration in any manner other than by 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 directly injected into a tumor or lymph node.

[0319] As used herein, the term "co-administration" refers to a process by which different compounds or compositions are administered to the same patient. For example, the CAR modified cells and antigen or the nucleic acid encoding it described herein can be administered simultaneously, essentially at the same time, or sequentially. When administration is performed sequentially, the CAR modified cells can be administered before or after the administration of the antigen or the nucleic acid encoding it. When administration is performed simultaneously, the CAR modified cells and antigen or the nucleic acid encoding it do not need to be administered in the same composition. The CAR modified cells and antigen or the nucleic acid encoding it can be administered once or multiple times, and the number of administrations of each component can be the same or different. Furthermore, the CAR modified cells and antigen or the nucleic acid encoding it do not need to be administered to the same site.

[0320] 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 can be caused by factors arising from external sources, such as infectious diseases, or by internal malfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, suffering, social problems, or death to the afflicted individual, or similar problems that are in contact with the individual. In this broad sense, it can include injuries, physical disabilities, disorders, syndromes, infections, single symptoms, deviant behaviors, and atypical variations in structure and function, although for other contexts and purposes, these may be considered as distinct categories. A disease usually affects an individual not only physically, but also psychologically, as many diseases are suffered and living with them can change the patient's outlook on life and the patient's personality.

[0321] In the present context, the term "treatment", "treating" or "therapeutic intervention" relates to the management and care of a subject for the purpose of combating a condition, such as a disease or disorder. The term is intended to include the full range of treatments for a given condition suffered by a subject, such as the administration of a therapeutically effective compound to alleviate symptoms or complications, delay the progression of a disease, disorder or condition, relieve or alleviate symptoms and complications, and / or cure or eliminate a disease, disorder or condition, as well as to prevent a condition, where prevention is understood as the management and care of an individual for the purpose of combating a disease, condition or disorder, and includes the administration of an active compound to prevent the onset of symptoms or complications.

[0322] The term "therapeutic treatment" refers to any treatment that improves the health status and / or extends (increases) the life span of an individual. Said treatment can eliminate disease in an individual, halt or slow the onset of disease in an individual, inhibit or slow the onset of 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.

[0323] The term "prophylactic treatment" or "preventive treatment" relates to any treatment intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used interchangeably herein.

[0324] The terms "individual" and "subject" are used interchangeably herein. They refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that suffers from or is susceptible to a disease or disorder (e.g., cancer), but may or may not have the disease or disorder. In many embodiments, an individual is a human. Unless otherwise stated, the terms "individual" and "subject" do not specify a particular age, and thus include adults, elderly, children, and newborns. In an embodiment of the present disclosure, an "individual" or "subject" is a "patient," e.g., a human patient.

[0325] The term "patient" refers to an individual or subject for treatment, particularly a diseased individual or subject.

[0326] Combination strategies for cancer treatment may be desirable due to the resulting synergistic effects, which may be significantly stronger than the impact of monotherapy approaches. In one embodiment, the pharmaceutical composition is administered by 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 by various mechanisms, including inducing apoptosis, blocking components of signaling pathways, or inhibiting the proliferation of tumor cells. In certain embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies may induce cell death by binding complement proteins, which leads to direct cytotoxicity, known as antibody-dependent cellular cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). Non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) 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 mafumab (MAF-100), and cefotaxime (CEA). Enatox (TAG-72), apolizumab (HLA-DR), arcitumomab (CEA), atezolizumab (PD-L1), bavituximab (phosphatidylserine), bectumomab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), bivatuzumab-mertansine (CD44v6), blinatumomab (CD19), brentuximab-vedotin (CD30 TNFRSF8), cantuzumab mertansine (mucin CanAg), cantuzumab ravtansine (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 (RNAKL), 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 αvβ3), 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 (IL1β), dilentuximab [carbonic anhydrase 9 (CA-IX)], grembatumumab vedotin (GPNMB), ibritumomab tiuxetan (CD20), icrucumab (VEGFR-1), igovoma (CA-125), indatuximab ravtansine (SDC1), intetumumab (CD51), inotuzumab ozogamicin sine (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), mitumomab (GD3 ganglioside), mogamulizumab (CCR4), moxetumomab-passudotoxin (CD2 2), nacolomab-tafenatox (C242 antigen), naptumomab-estafenatox (5T4), namatumumab (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), sibrotuzumab (FAP), siltuximab (IL6), tabalumab (BAFF), tacatuzumab-tetraxetan (alpha-fetoprotein), tapritumumab These include: tsb-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 CTAA16.88), zalutumumab (EGFR), and zanolimumab (CD4).

[0327] Citation of the documents and works 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 the information available to applicants and do not constitute any admission as to the accuracy of the contents of these documents.

[0328] The present invention will be described in more detail by referring 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.

[0329] 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. Accordingly, the following examples 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. EXAMPLES

[0330] [Example 1] material and method Techniques and methods used herein are performed as described herein or as known methods and described, for example, 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 performed according to manufacturer's information unless otherwise specified.

[0331] 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, a short intronless version of the human elongation factor 1-alpha promoter (EFS-213 / +31). The vector backbone contains MLV wild-type sequences of the R- and U5 regions, as well as the packaging region (psi and psi+) in the 5'- and 3'-LTR. The enhancer element in the U3 region of the 3'-LTR is removed (including the CAAT-Box) and the TATA-Box sequence is 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 unwanted viral proteins.

[0332] CLDN6-CAR-BBz expresses a signaling peptide from human IgG, VL Position 46 of (V shown in the sequence table) L The heavy chain (V H ) and light chain (V L The scFv fragment comprises a single chain Fv fragment of the CLDN6-specific antibody IMAB206 (Ganymed Pharmaceuticals) with a (G4S)3 linker between the chains. The scFv fragment is fused to the human CD8α hinge and transmembrane domain, followed by human 4-1BB and human CD3 zeta (Q14K) signaling moieties.

[0333] 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 is HLA-A * 0201, which overexpresses luciferase and GFP.

[0334] The ovarian cancer cell line OV-90-SC12 was cultured in 41.5% (v / v) MCDB105 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.

[0335] The culture medium for the human melanoma cell line SK-MEL-37 consisted of 90% DMEM GlutaMAX™ (Gibco) supplemented with 10% (v / v) FCS.

[0336] The culture medium for MDA-MB-231 consisted of 88% (v / v) RPMI1640 GlutaMAX™ (Gibco) supplemented with 10% FCS, 1 mM sodium pyruvate, 1 mM MEM non-essential amino acid solution.

[0337] The human adenocarcinoma cell line 23132-87 and the human melanoma cell line MEL-526 were cultured in 90% (v / v) RPMI1640 GlutaMAX™ (Gibco) supplemented with 10% (v / v) heat-inactivated FCS.

[0338] Culture medium for HEK-293 consisted of 90% (v / v) Eagle's Minimum Essential Medium (EMEM) (ATCC) and 10% (v / v) FCS.

[0339] SKOV-3 was cultured in 90% (v / v) McCoy's 5A medium (ATCC) supplemented with 10% (v / v) FCS.

[0340] The human ovarian cell line NIH-OVCAR-3 was cultured in 80% (v / v) RPMI1640 GlutaMAX™ (Gibco, Cat. No. 61870) supplemented with 20% (v / v) FCS and 0.1% (w / v) insulin (Sigma-Aldrich).

[0341] Human tumor cell lines LCLC-103H, COLO-699-N, JAR and NEC-8 were cultured in 90% (v / v) RPMI1640 GlutaMAX™ (Gibco) supplemented with 10% (v / v) FCS. Cell lines were seeded and / or split every 2 or 3 days.

[0342] 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 RPMI1640 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 hGM-CSF (Essex, Lucerne, Switzerland) and 1000 U / mL hIL-4 (Strathmann Biotech, Hamburg, Germany).

[0343] Transduction of T cells T cells were purified using Dynabeads® Human T-Expander CD3 / CD28 CTS. + / CD28 high+ T cells were enriched from PBMCs by magnetic separation. Cells were enriched using a 3:1 ratio of 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-VIVO15 medium supplemented with 5% (v / v) human serum in the presence of 450U / mL rhIL-7 and 50U / mL rhIL-15 (both from Miltenyi Biotec). After 3 days, CD3 / CD28 beads were removed using a magnet and preactivated T cells were transduced with retroviral vectors in the presence of Protransduzin®-A at a final concentration of 25μg / mL. Cells were expanded up to 7 or 10 days in complete culture medium and used directly to evaluate CAR surface expression, T cell phenotype and effector function or cryopreserved.

[0344] Flow cytometric 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 in 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.).

[0345] 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-for: CTT ATC TCC TTC GCA GTG CAG, CLDN6-rev: AAG GAG GGC GAT GAC ACA GAG, HPRT1-for: TGA CAC TGG CAA AAC AAT GCA, HPRT1-rev: GGT CCT TTT CAC CAG CAA GCT (annealing temperature: 62°C).

[0346] 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 plated at 2–10 per well in E-plate 96PET (ACEA Biosciences Inc.). 4 After 24 hours, various numbers of CAR-transduced T cells were added in a final volume of 200 μL and monitored every 30 minutes for up to 48 hours by the xCELLigence system.

[0347] Percent specific lysis was calculated as follows: (CI L min -CI sample ) / CI L min ×100 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 ]} Minimum dissolution (L min ) was assessed after incubation of target cells with effector T cells expressing a control antigen (e.g., eGFP, control CAR).

[0348] CFSE (Carboxyfluorescein succinimidyl ester) proliferation assay To determine the percentage of T cells proliferating 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 (protected from light) at 37° C. To remove free dye, pure FCS was added to the cells and incubated for another 5 min. The 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 a 96-well round-bottom plate. After 5 days of co-culture, the 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 half-life of CFSE fluorescence in daughter cells after cell division using a BD FACSCanto™ II flow cytometer (Becton Dickinson).

[0349] 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) Na-Pyruvat, 1% (v / v) MEM NEAA, and 2% (v / v) Na-Biacarbonate for 48 h after centrifugation. 4 The cells were generated by culturing 1×10 PA1-SC12-A2-eGFP cells. CAR-T cells were added (1×10 5 Cells / well), eGFP-expressing tumor spheroids 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 for 10 days. Data were analyzed using IncuCyte analysis software and the integrated intensity of all green objects (GCU×μm 2Green objects were detected and quantified (Figure 1A,b) and the mean number of green objects with SD at each time point was plotted using IncuCyte analysis software.

[0350] Generation of in vitro transcribed (IVT) mRNA In vitro transcription of antigen coding mRNA is based on 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 α-globin, two consecutive 3' human β-globin UTRs and a 100 nucleotide poly(A) tail with a linker after 70 nucleotides. Antigen coding mRNA was generated by in vitro transcription as described by Holtkamp S. et al. (2006) Blood 108(13):4009-17. In vitro transcription of all described mRNA constructs was carried out at BioNTech RNA Pharmaceuticals GmbH.

[0351] IVT RNA encoding a liposomally formulated antigen [RNA (LIP) Generation of ] and in vitro transfection of dendritic cells Complexation of antigen-encoding IVT RNA with liposomes has been previously described in Kranz et al (2016) Nature 534(7607):396-401. A 1.3:2 charge ratio of cationic DOTMA to RNA was used. In addition to DOTMA, the lipid fraction contains the helper lipid DOPE at a molar ratio of 2:1 DOTMA per DOPE.

[0352] Animal Experimental Techniques animal 9- to 21-week-old female immunodeficient NOD.Cg-Prkdc mice scid IL2rg tm1WjlC57BL / 6BrdCrHsd-Tyr / SzJ (NSG) mice were used for in vivo studies. They were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and bred in the breeding 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 bred in the breeding facilities of BioNTech AG, Germany. All experiments were performed under specific pathogen-free (SPF) conditions and in accordance with the German Animal Experimentation Regulations.

[0353] Tumor cell engraftment 5×10 6 OV90-SC12 or 5×10 5 Mice were injected subcutaneously into the right hind 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 calculated using the formula V=1 / 2(length×width). 2 Prior to adoptive transfer of human CAR-engineered T cells, tumor bearing was stratified using Daniels' XL Toolbox Add-in in Microsoft Excel to achieve a large tumor volume distribution among the different treatment groups.

[0354] Adoptive transfer (ACT) of human T cells Different amounts of gamma-retrovirally transduced total human T cells (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 in vitro activation and transduction process, or cryopreserved transduced T cells were thawed and directly adoptively transferred into mice after two washes with PBS. The survival of all T cell products used in the experiments was >90%.

[0355] In vivo monitoring of human CAR T cells in blood At the indicated time points, 50 μL of peripheral blood was collected from the retro-orbital vein and collected into a reaction tube containing heparin (Sarstedt). Red blood cells were lysed using BD FACS lysing solution (BD). CAR expression on the surface of transplanted human T cells was analyzed using hCD45-PE-Cy7 (HI30, BD), hCD4-APC-Cy7 (OKT4, BioLegend), hCD8-BV421 (RPA-T8, BD) and Alexa-Fluor647-conjugated idiotype-specific antibodies (Ganymed Pharmaceuticals). Dead cells were differentiated from the analysis using 7-AAD (Beckman Coulter). Flow cytometric measurements were performed on a FACSCanto™ II flow cytometer using FACSDiva™ software (BD Bioscience), and analysis was performed using FlowJo® V10 (Treestar inc.).

[0356] Retroviral genetic engineering and preparation of CAR T cells for 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 at a 1:1 bead-to-T cell ratio (invitrogen) 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, supernatants of MLV-E pseudotyped retroviruses were transferred to RetroNectin (2 μg / cm) according to the manufacturer's instructions. 2 )-coated non-tissue culture-treated well plates (Takara Bio Inc., Otsu, Japan), and three cycles of virus loading and centrifugation (1300 × g, 15 °C, 15 min) were repeated to increase binding. After 24 h of preactivation, 0.5–0.6 × 10 6 Cells / cm 2The cells were spun down (300×g, 37° C., 1 h) into the viral particle-coated wells. After overnight culture, the spin-down transduction was repeated in new viral particle-coated plates. After 72 h of preactivation, Dynabeads™ Mouse T-Activator CD3 / CD28 were removed from the cultures and the cells were expanded in the presence of 5 ng / mL rh IL-7 and 5 ng / mL rh IL-15. After Ficoll cleaning, the 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 CLDN6-CAR-BBz, encoding enhanced firefly luciferase (effLuc; Rabinovich et al.BA, PNAS (2008) PNAS 105(38):14342-6) and eGFP (enhanced green fluorescent protein) reporter genes, separately expressed using a 2A splice element (Szymczak et al. AL, Nature Biotechnology, (2004) Nat Biotechnol. 22(5):589-94), was used for transduction.

[0357] Adoptive T cell transfer and RNA transfer of mouse T cells (LIP) vaccine Gamma-retroviral transduction of CAR congenic Thy1.1 + T cells were derived from total body irradiated (XRAD320) C57BL / 6BrdCrHsd-Tyr c or BALB / c donor mice. Mice were then administered the antigen-encoding RNA at various time points after ACT. (LIP) Mice were vaccinated intravenously (iv) with an F12:RNA ratio of 1.3:2. In vivo expansion of CAR was analyzed by whole-body bioluminescence imaging, and antitumor efficacy was analyzed by tumor monitoring.

[0358] In vivo luciferase imaging (BLI) The expansion 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, an aqueous solution of D-luciferin (80 mg / kg body weight; Perkin Elmer) was injected i.p. at the indicated time points after adoptive transfer of transduced T cells. Five minutes later, emitted photons were quantified (integration time 1 min, binning 8). In vivo bioluminescence in regions of interest (ROIs) was quantified as total flux (photons / sec) using IVIS Living Image4.0 software. The intensity of transmitted light arising from luciferase-expressing cells within the animals was represented as a grayscale image, with black being the weakest intensity and white to dark gray being the strongest bioluminescent signal. Grayscale reference images of mice were obtained under low-light illumination of the LED. Images were overlaid using Living Image4.0 software.

[0359] Statistical analysis and depiction of data All results are expressed as the mean + / - SD of technical replicates or the mean + / - SEM of biological replicates. The number of replicates is stated in the graphic description of each experiment. An unpaired two-tailed Student's t-test was used for area under the curve (AUC) comparison of two groups. All statistical analyses were performed using GraphPad PRISM 6.04. *** P ≤ 0.001; **** P ≤ 0.0001. [Example 2]

[0360] Generation and in vitro characterization of CLDN6-specific CARs For lead structure selection, we generated different CAR scaffolds that all share the same scFv fragment from the CLDN6-specific antibody IMAB206-C46S, but differ in their hinge and costimulatory domains (Figure 1A). Because 4-1BB costimulation has been shown to increase CAR T cell persistence and antitumor efficacy, we included second and third generation CAR scaffolds containing the 4-1BB endodomain. Alternatively or additionally, a modified CD28 domain [Kofler DM et al., (2011) Molecular Therapy 19 (4), 760-767] was incorporated, which is 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.

[0361] Different CLDN6-CARs were extensively characterized in vitro for their ability 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 CARs containing 4-1BB compared to CARs with the CD28 domain could be detected using IncuCyte® real-time imaging (Figure 1B).

[0362] Based on the summary of the results of different functional characterization tests, we selected CLDN6-CAR-CD8h-BBz as a lead structure for preclinical and clinical trials, as we could demonstrate highly specific and sensitive recognition of CLDN6, as well as high potential for survival and repeated stimulation of engineered CAR T cells. Importantly, this CAR scaffold has already been successfully used in several CD19-CAR T cell studies. For stable integration of our CLDN6-CAR into the T cell genome, we selected the gamma-retroviral self-inactivating (SIN) vector pES12.6 for stable integration of therapeutic CLDN6-CAR into the T cell genome (Loew et al., Gene Therapy (2010) 17, 272-280). [Example 3]

[0363] Sensitivity of CLDN6-CAR-BBz To analyze the sensitivity of CAR-mediated recognition in more detail, CLDN6-RNA titration experiments were performed. Therefore, 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- 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 correlated with the number of CLDN6 molecules on the target cell surface, evaluated by flow cytometry (Figure 2C). Even when very low numbers of CLDN6 molecules were expressed on the target cell surface after transfection with 0.01 μg of CLDN6-RNA alone, CAR T cells mediated lysis, but this was not detectable by flow cytometry. [Example 4]

[0364] Safety of CLDN6-CAR-BBz To evaluate the safety of CLDN6-CAR-BBz, a screening assay of cell lines was performed. To that end, 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, E). It could be demonstrated that the recognition and lysis of the target cell lines strictly correlated with the CLDN6 mRNA and protein expression levels. [Example 5]

[0365] CAR T cell expansion A prerequisite for the antitumor effect of CLDN6-CAR-BBz-engineered T cells is their ability to proliferate and persist in patients. To analyze if 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 by CFSE and co-transfected with titrated amounts of RNA lipoplexes encoding either CLDN6 or a control antigen [RNA (LIP) [Figure 4A,B]. Surface expression of CLDN6-CAR on T cells and CLDN6 on target cells was examined by flow cytometry (Figure 4A,B). 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).

[0366] CLDN6-CAR was transfected with CLDN6-RNA (LIP) mediated a dose-dependent proliferation that correlated with the amount of IL-1, whereas iDCs were treated with control RNA. (LIP)Only background proliferation was detectable when CLDN6-CAR-BBz T cells were transfected with . These data confirm that efficient antigen-specific expansion of CLDN6-CAR-BBz T cells is induced following antigen-specific stimulation. [Example 6]

[0367] 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 aggressive xenograft tumor model. To this end, immunodeficient NOD.Cg-Prkdcscid IL2rgtm1Wjl / SzJ (NSG) mice were subcutaneously implanted with human ovarian cancer cells (OV90) expressing endogenous CLDN6. OV90 tumor-bearing NSG mice were then treated with 1 × 10 7 CLDN6-CAR or eGFP transduced T cells (approximately 5 × 10 8 The CLDN6-CAR was expressed in approximately 16–18% of human CD4 T cells. + and CD8 + T cells (Figure 5B). Of note, adoptive transfer of CLDN6-CAR T cells resulted in a 170 mm 3 The treatment resulted in complete regression of large tumors with a mean tumor volume of 1000 μg / kg, whereas no antitumor effect was observed in the control group (Figure 5C). This remarkable antitumor effect was associated with the persistence of CLDN6-CAR-BBz T cells in the peripheral blood of treated mice (Figure 5D). [Example 7]

[0368] In vitro functionality of short-term and long-term cultured CAR T cells Reproducible production of high-quality, clinical-grade CAR T cell products is a prerequisite for clinical trials, and GMP manufacturing processes have been optimized to achieve high transduction efficiency and sufficient numbers and quality of CLDN6-CAR-expressing T cells. The transduction procedure can also be simplified by reducing the number of transductions from two to one, and by shortening the ex vivo culture time from 10 to 7 days. T cells cultured for a shortened period of time have been shown to show improved efficacy compared to T cells expanded and exhausted for a long period of time (PMID:30030295). Thus, shortening the culture time should not only reduce time and costs, but most importantly, result in an enhanced ability of engineered T cells to expand and persist in patients.

[0369] 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 ability of repeated killing, CAR T cells were co-cultured with CLDN6 and eGFP-expressing PA1-SC12-A2-eGFP tumor spheroids, and the killing of tumor spheroids was monitored in real time based on eGFP signal using the IncuCyte® system. After complete eradication of tumor spheroids, new tumor spheroids were added (Figure 6B). It could be demonstrated that short-term cultured CAR T cells have a comparable ability of repeated killing to long-term cultured T cells. [Example 8]

[0370] 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 important to confirm the antitumor potential of thawed CLDN6-CAR-BBz T cells engineered in a GMP facility in the future. Furthermore, as the final GMP manufacturing process may be shortened (harvested in 7 days instead of 10 days), this modification of the final protocol must be evaluated by performing in vivo studies. Most importantly, thawed human CLDN6-CAR-BBz T cells showed remarkable antitumor efficacy comparable to freshly generated CAR T cells, suppressing tumor growth in an ovarian cancer xenograft model with a mean tumor volume of 160 mm. 3 Both CAR T cell products eradicated advanced tumors with CLDN6-CAR-BBz T cells (Figure 7A). Furthermore, CAR T cells harvested on day 7 or day 10 showed similar CAR surface expression (Figure 7B), and their antitumor effects were compared in this experiment (Figure 7C). In relation to the results of the long-term tumor spheroid experiment, 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). [Example 9]

[0371] Increased persistence of CLDN6-CAR-BBz-transduced T cells The clinical success of adoptively transferred tumor-reactive T cell therapy was also positively correlated 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. Since xenograft models are not sufficient to test the long-term persistence of CAR T cells due to the graft-versus-host disease of human T cells in mouse tissues as well as 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, CAR-transduced mouse T cells of CLDN6-CAR-BBz co-expressing luciferase were adoptively transferred into mildly irradiated (2.5 Gy) mice and then transfected with RNA encoding CLDN6 or a control antigen. (LIP) was administered repeatedly and the expansion of the CAR T cell population was subsequently monitored by bioluminescence imaging (Figure 8A).

[0372] CLDN6-CAR-BBz T cells can persist in vivo (>3 months) after repeated vaccination with liposomally formulated CAR antigen in vivo. In the control group, CAR T cells disappear over time, whereas antigen-specific restimulated CAR T cells retain all RNA even after treatment has stopped for 3-4 weeks after the third and fourth boosts. (LIP) These data were obtained from BioNTech's RNA (LIP) We show that the technique supports sufficient CLDN6-CAR-BBz T cell activation and proliferation by providing natural costimulation in situ, and also results in increased persistence of in vivo expanded CLDN6-CAR-BBz T cells. [Example 10]

[0373] Improved antitumor activity of in vivo expanded CLDN6-CAR-BBz T cells After CAR-mediated antigen-specific expansion and persistence was demonstrated, the question arose as to whether those in vivo expanded CLDN6-CAR-BBz T cells would also exhibit enhanced antitumor potency compared to their non-expanded counterparts. dim Balb / c mice inoculated with the -expressing colon cancer cell line CT26 received a medium dose of 1 × 10 6 Mouse T cells (3–4 × 10) transduced with either CLDN6-CAR-BBz or control-CAR-BBz 5 After iv administration of CAR T cells, mice were treated with 20 μg of RNA encoding either full-length CLDN6 or a full-length control antigen. (LIP) (Figure 9A). (LIP) No antitumor effect was observed in control-CAR-BBz-treated animals treated with (LIP) When treated with a moderate dose of CLDN6-CAR-BBz T cells in combination with CLDN6-RNA, only slight tumor regression was achieved. However, to achieve in vivo expansion of CLDN6-CAR-BBz T cells, (LIP) Mice that received the combination of a medium dose of CAR T cells along with the vaccine showed clearly enhanced tumor regression (Figure 9B). [Example 11]

[0374] Restoration of antitumor efficacy of low-dose in vivo expanded CAR T cells RNA improves ongoing antitumor responses (LIP) Apart from its ability to base in vivo CAR T cell expansion, this technique has also been thoroughly studied and shown to restore the antitumor potential of insufficient CAR T cell doses. Tumor-bearing mice were treated with CAR T cell doses 10-fold lower than those required for tumor rejection (Figure 10A). Tumor growth was significantly higher than that of control RNA. (LIP) However, after the first transplant, the RNA encoding the CAR antigen was not detected. (LIP)All animals receiving the vaccine showed tumor rejection (Figure 10C). Thus, the frequency of engrafted CAR T cells in the peripheral blood was clearly higher after vaccination (Figure 10D). (LIP) Compensating for insufficient CAR T cell doses after transplantation by further treatment with CAR T-cell therapy could be very useful in at least two different scenarios: 1) poor productivity of GMP manufactured CAR T cell product (e.g., due to low lymphocyte counts in the initial apheresis or other uninfluenced reasons) or 2) avoidance of SAEs due to reduction of the starting CAR T cell dose (especially if the required CAR is known to cause toxicity or in the case of first-in-human dose escalation studies). [Example 12]

[0375] Clinical Trials material and method Construction of chimeric antigen receptors (CARs) CLDN6-CAR was constructed by linking the signal peptide sequence of the immunoglobulin heavy chain variable region (Genebank no. MC18316.1) to the human CLDN6-specific single chain variable fragment (scFv) from the IMAB206-C46S antibody (WO20121560018). The scFv fragment is fused to the human CD8a hinge and transmembrane region (Genebank no. NP_001759.3, aa138-206), then to the human 4-1BB (Genebank no. NP_001552.2, aa214-255) and human CD3ζ (Genebank no. NP_000725, aa52-163, Q65K) signaling moieties. The codon-optimized and synthetic sequence (Eurofins Genomics) was cloned into the gamma-retroviral self-inactivating (SIN) vector pES.12-6 for stable overexpression in human and mouse T cells under the control of a short intronless version of the human elongation factor 1-alpha promoter (EFS).

[0376] CLDN6 RNA-LPX The plasmid template for in vitro transcription of RNA encoding claudin 6 was the pST4-hAg-Kozak-CLDN6-Fl-A30L70 vector. Similar to the vector pST1-T7-GG-hAg-MCS-2hBg-A30LA70 used for preclinical experiments, this vector features 5' and 3' UTRs and a poly(A) tail, specifically the 5' human α-globin, pharmacodynamically optimized for stability and protein translation, and a poly(A) tail measuring 110 nucleotides in length, consisting of 30 adenosine residues, followed by a 10-nucleotide linker sequence and another stretch of 70 adenosines. However, in contrast to pST1-T7-GG-hAg-MCS-2hBg-A30LA70, the vector pST4-hAg-Kozak-CLDN6-Fl-A30L70 contains a 3'-UTR that is a combination of two sequence elements from the AES mRNA (designated F) and the mitochondrial-encoded 125 ribosomal RNA (designated I). The coding sequence cloned into this vector was a codon-optimized sequence encoding the target antigen human claudin 6 (NP_067018.2).

[0377] In vitro transcription and capping with β-S-anti-reverse cap analog (ARCA) were performed as previously described [Holtkamp S. et al., Blood 108, 4009-4017 (2006)].

[0378] autologous T cells Autologous CD3+ T cells were enriched from parent-derived PBMCs and contain various ratios of CD3+CD4+ to CD3+CD8+ cells. CD3+CD4+ and CD3+CD8+ cells were characterized for cell surface expression of T stem cell and differentiation markers CD62L, CD197, and CD45RA before and after processing.

[0379] Manufacturing of CAR-T cell preparations (CLDN6 CAR-T) The starting material for production of CAR-T cell drug substance is autologous patient apheresis collected from a board-certified apheresis center.

[0380] The manufacturing process begins with the isolation of PBMCs from the apheresis material (step 1). The PBMCs were cryopreserved and stored in the vapor phase of liquid nitrogen as an intermediate until further processing. PBMCs not used for the first manufacturing can be stored and used to manufacture further batches at a later time. PBMC processing involves thawing and isolation of the CD3+ cell population using magnetic CD3 / CD28 beads (step 2), followed by CD3+ cell stimulation and culture for 3 days (step 3). Bead removal (step 4) is performed to remove the magnetic beads. The stimulated and bead-removed CD3+ cells were transduced with the supernatant containing the retroviral vector in the presence of the transduction enhancer RetroNectin (step 5). The transduced CD3+ cells were transferred to a G-Rex culture unit and replenished with fresh culture medium. The CD3+ cells were expanded until day 7 (step 6). Finally, the expanded cells are harvested, bead-removed a second time, and pooled to yield the CAR-T cell drug substance (step 7). The CAR-T cell drug substance was immediately manufactured into the CAR-T cell formulation. The CAR-T cell formulation was diluted to 1 × 10 in 100, 100, and 20 mL of 50% sodium chloride solution (0.9%) and 50% Cryostor CS10 as cryoprotectant for frozen transport. 9 pieces, 1×10 8 pcs and 1×10 7 Contains a dose of viable CAR-T cells.

[0381] All processing steps are performed in a class B cleanroom environment. All cell culture steps are performed in an incubator at 37°C and 5% CO2. All remaining processing steps are performed at room temperature. The cell culture medium is X-VIVO15 (gentamicin and phenol red free) supplemented with 5% CTS immune cell serum replacement and 2 mM GlutaMAX. Cytokines promoting cell expansion, i.e., interleukin-7 (IL-7; 450 U / mL) and interleukin-15 (IL-15; 50 U / mL), are added on days 0, 3, and 4 for full volume.

[0382] Study design The study is a Phase I / IIa, FIH, open-label, multicenter, dose-escalation study with expansion cohorts to evaluate the safety and preliminary efficacy of CLDN6 CAR-T + / - CLDN6 RNA-LPX in patients with CLDN6-positive recurrent or refractory advanced solid tumors. All patients will undergo leukopheresis for collection of blood products to manufacture CLDN6 CAR-T. The actual study performed consists of two parts: Part 1 will be CLDN6 CAR-T dose escalation in lymphocyte-depleted patients until the maximum tolerated dose (MTD) and / or recommended phase 2 dose (RP2D) of CLDN6 CAR-T is defined. Part 2 is a vaccine-adjusted dose escalation using a branched design until the MTD and / or RP2D of CLDN6 CAR-T+CLDN6 RNA-LPX are defined. The branching will consist of CLDN6 CAR-T dose level 1 (1×10 7 Part 2 will begin if the first CLDN6 CAR-T is deemed safe. At this point, part 2 will begin at the removed CLDN6 CAR-T dose level. CLDN6 RNA-LPX will be administered at a fixed step-up dose. A cohort testing CLDN6+ / - CLDN6 RNA-LPX without lymphocyte depletion (LD) will also be activated in this part to inform the determination of the preferred phase 2 recommended dose (RP2D).

[0383] If the dose level in part 1 is deemed safe, further treatment with CLDN6 RNA-LPX is possible. The dose level of CLDN6 CAR-T in part 2 at any given time will not exceed that of part 1. This approach allows for fast and safe dose escalation with vaccine adjustments.

[0384] At the same time, patient safety will be protected by generating data for CLDN6 CAR-T before branching, and clear rules for parallel dose escalation will be outlined. Patient enrollment into either the CLDN6 CAR-T or vaccine-modulated dose escalation cohorts will be modified. More patients will be enrolled into selected dose levels for back-up in both part 1 and part 2, so that safety and antitumor activity can be further explored. The study design and status are shown in Figure 1.

[0385] Group Patients with any of the following tumor types: testicular, ovarian, gastric, endometrial, non-squamous small cell lung cancer (NSCLC), advanced or metastatic cancer, who met other inclusion criteria, had appropriate CLDN6 status determined for which no standard treatments that may confer clinical benefit are available or for which the patient is not a candidate for such available treatments. Only patients with a determined ≥2+ ≥50% positive tumor cells expressing CLDN6 protein are eligible to undergo further screening to meet the remaining eligibility criteria for enrollment in this study.

[0386] Important inclusion criteria Patients who meet the following inclusion criteria are eligible for study entry: Each patient enrolled in the study must have a CLDN6-positive tumor regardless of tumor histology, defined as ≥50% tumor cells expressing ≥2+ CLDN6 protein using a semi-quantitative immunohistochemistry (IHC) assay for the specific detection of CLDN6 protein expression in formalin-fixed, paraffin-embedded neoplastic tissue. Patients must have measurable disease per RECIST 1.1 (excluding germ cell tumors). Patients with germ cell cancer without early measurable disease per RECIST 1.1 and evaluable by cancer antigen (CA)-125, alpha-fetoprotein (AFP) or hCG (if applicable) are eligible for the study. Patients must have metastatic or unresectable histologically confirmed solid tumors and have no available standard treatment that could confer clinical benefit to them or are not candidates for such available treatments.

[0387] Important Exclusion Criteria Patients meeting at least one of the following exclusion criteria were ineligible for study entry: Previous CAR-T therapy, except for CLDN6 CAR-T therapy. -Receipt of vaccination with a live virus vaccine within 6 weeks prior to the initiation of lymphodepletion (LD). Receiving concurrent systemic (oral or intravenous [iv]) steroid treatment with >10 mg of prednisolone daily, or its equivalent.

[0388] Current evidence of new or growing brain or spine metastases during screening. Patients with known brain or spinal metastases may be eligible if: have received radiation therapy or another appropriate treatment for brain or spinal metastases, -No neurological symptoms, - have stable brain or spinal disease as determined by computed tomography or magnetic resonance imaging scan within 4 weeks prior to signing the informed consent; - Not receiving acute corticosteroid treatment or steroid tapering. Long-term steroid treatment is acceptable (≤10 mg prednisolone or equivalent daily) provided the dose has been stable for at least 14 days prior to screening. - Not requiring steroid treatment within 7 days prior to the first dose of CLDN6 CART - Urgent fracture or spinal cord compression due to spinal bone metastasis is expected, History of another primary cancer within 2 years prior to enrollment, except for the following: nonmelanoma skin cancer, cervical intraepithelial neoplasia, superficial bladder cancer, prostate cancer with currently undetectable prostate-specific antigen, or other nonmetastatic cancer in complete remission that has not been treated for 2 years or more.

[0389] Lymphodepleting chemotherapy Cyclophosphamide: 500 mg / m2 / d iv on days 5, 4, and 3 according to institutional standards and Fludarabine: 30 mg / m2 / d iv infusion according to institutional standards on days 5, 4, and 3. In case of moderate impairment of renal function, the dose should be reduced by 50% and the patient's renal function should be monitored daily.

[0390] Low-dose lymphodepleting chemotherapy may be used based on Safety Review Committee (SRC) decision: Cyclophosphamide: 250 mg / m2 / d iv infusion on days 5, 4, and 3 according to institutional standards, and Fludarabine: 25 mg / m2 / d iv infusion according to institutional standards on days 5, 4, and 3. In case of moderate impairment of renal function, the dose should be reduced by 50% and the patient's renal function should be monitored daily.

[0391] Investigational Treatments CLDN6 CAR-T are autologous CD4+ and CD8+ T cells engineered with a CAR specific for CLDN6.

[0392] CLDN6 CAR-T will be cryopreserved in injectable freezing medium and administered as a single iv infusion. Each bag contains an aliquot (volume dependent on dose) of CLDN6 CAR-T in freezing medium containing the following injectable grade reagents (% v / v): 50% CryoStor CS10, 50% sodium chloride (0.9%). The total amount of dimethyl sulfoxide is 5%.

[0393] A fixed dose administered by the iv route will be used in this study. The starting dose selected is 10 7 CLDN6 CAR-T, then 10 8 Pieces and 10 9Two additional dose levels of CLDN6 CAR-T. CLDN6 RNA-LPX is a cancer vaccine, a liposome-formulated messenger ribonucleic acid (mRNA) that delivers mRNA encoding CLDN6 systemically to antigen-presenting cells (APCs) such as dendritic cells in secondary lymphoid tissues. CLDN6 RNA-LPX is injected as a bolus dose into a peripheral vein using a peripheral venous catheter. CLDN6 RNA-LPX injection is performed as slowly as possible or over a period of 2 minutes. 5 mL of isotonic sodium chloride solution (0.9%) should be administered before and after CLDN6 RNA-LPX injection (flushing). The starting dose of CLDN6 RNA-LPX cancer vaccine is 25 μg, after which all subsequent doses are 50 μg.

[0394] statistics Statistical hypotheses The primary objectives of Parts 1 and 2 are to evaluate the safety profile and identify the MTD and / or RP2D. Therefore, no statistical assumptions are made for Parts 1 and 2 trials.

[0395] Sample size determination The sample size for parts 1 and 2 will be determined by the 3+3 study design. For parts 1 and 2 with CLDN6 CAR-T, the sample size will be up to 18 DLT-evaluable patients in each part, depending on the number of possible DLTs.

[0396] General Considerations Continuous variables will be summarized by cohort using the following descriptive statistics: number of patients (n), mean, standard deviation, median, minimum and maximum. Categorical variables will be summarized by cohort, showing the absolute and relative frequencies (n and %) of patients in each category.

[0397] Primary Endpoint Adverse events (AEs) will be coded using the latest version of the Medical Dictionary for Regulatory Affairs [MedDRA®] coding system, which captures the system organ class (SOC) and preferred term (PT) for each AE, as well as a severity grade using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0.m.

[0398] TEAEs are defined as AEs with onset date either at or after the first dose of CLDN6 CAR-T (if the AE was absent before the first dose of CLDN6 CAR-T) or worsening after the first dose of CLDN6 CAR-T (if the AE was present before the first dose of CLDN6 CAR-T). AEs with onset 90 days or more after the last dose were considered treatment-emergent only if assessed by the investigator. TEAEs are summarized overall and by cohort.

[0399] The numbers and percentages of patients reporting at least one AE are summarized by PT within SOC for each of the following AE types: Any AE Related AE Grade ≥ 3 AEs Related grade ≥3 AEs Any serious adverse events (SAEs) Related SAEs · SAE causing death AEs leading to dose reduction AEs resulting in sustained interruption of treatment DLT The number and percentage of patients with any AE will be summarized by PT within SOC and by worst NCI CTCAE grade.

[0400] DLTs are presented in terms of the reported term and a list showing MedDRA PT and SOC, their time of onset, duration, and severity including outcome, associations, NCI CTCAE grade, and dose-exposure data.

[0401] Secondary endpoints Objective response rate ORR is defined as the proportion of patients who observed a CR or PR (per RECIST 1.1) as the best overall response. Patients who did not meet the criteria for CR or PR, including those without any post-baseline tumor assessments, were considered non-responders. ORR is summarized by absolute and relative frequencies along with two-sided 95% Clopper-Person confidence intervals by cohort.

[0402] Disease control rate DCR is defined as the proportion of patients who observe CR or PR or SD (SD assessed at least 6 weeks after the first dose per RECIST 1.1) as the best overall response. Patients who do not meet the criteria for CR or PR or SD, including those who do not have any post-baseline tumor assessments, are considered non-responders.

[0403] DCRs will be summarized by absolute and relative frequencies along with two-sided Clopper-Person 95% confidence intervals by cohort.

[0404] Duration of response DOR is defined as the time from first objective response (CR or PR per RECIST 1.1) to first occurrence / recurrence of PD or death from any cause, whichever occurs first. Only patients who observe a CR or PR are analyzed for DOR. DOR is analyzed by cohort using the Kaplan-Meier method.

[0405] Exploratory Endpoints The exploratory endpoints iORR, iDCR and iDOR will be analyzed in the same manner as ORR, DCR and DOR.

[0406] Progression-free survival PFS is defined as the time from first administration of CLDN6 CAR-T to first confirmed PD (per RECIST 1.1, iCPD per RECIST 1.1) or death from any cause, whichever occurs first. PFS will be analyzed by cohort using the Kaplan-Meier method.

[0407] overall survival OS is defined as the time from the first administration of CLDN6 CAR-T to death from any cause. OS will be analyzed by cohort using the Kaplan-Meier method. Patients who are alive or lost to follow-up at the analysis cutoff date will be reviewed on the date of their last data known to be alive.

[0408] result As of November 18, 15 patients with different tumors and extensively pretreated have been treated. A summary of the treatments and patients in whom adverse events (AEs) were observed is shown in the table below. So far, in only one patient, a single dose-dependent toxicity (DLT), mainly prolonged cytopenias, was observed, the patient had recently undergone a stem cell transplant after high-dose chemotherapy. As a consequence, a cohort without lymphodepletion will be opened. Furthermore, only few AEs > grade 2 were observed, considered related to CAR T IMP, most of them also related to lymphodepletion or asymptomatic increase. Interestingly, seven patients experienced cytokine release syndrome (CRS) grade 1-2 with high levels of IL-6, manageable with tocilizumab if necessary, but without signs of neurotoxicity. (Even in one patient with DL1 who developed CRS after administration of CARVac).

[0409] Figure 13 shows the CAR-T cell frequency at each dose level, showing an increasing trend in improved engraftment with the addition of CARVac or increasing the dose. CAR-T cells were approximately 10 9A peak concentration of CAR-T cells is reached, translating into clinical activity as shown in Figure 14. Figure 14 shows the evaluation of clinical efficacy. Ten of the 15 treated patients were evaluable for efficacy assessment at the time of data cutoff. Four patients show partial responses, an additional four patients show stable disease with shrinkage of target lesions marked with a plus, while only one patient shows no signs of clinical activity.

[0410] Most encouragingly, three out of four partial responses were achieved in all patients with recurrent testicular cancer following recent high-dose chemotherapy.

[0411] Figure 15 shows CT scans of these three patients before treatment and at the indicated time points after CAR-T administration. In patient #3, who received DL2 as monotherapy and had a high tumor burden in the liver, tumor shrinkage was remarkably consistent with a reduction in the tumor marker alpha-fetoprotein (AFP) of greater than 99%.

[0412] The lung metastases of patient #4, who also received DL2 as monotherapy, also nearly disappeared, with normalization of AFP levels. In addition, the patient who received dose level 1 boosted with CARVac showed durable responses in many metastases.

[0413] Patient characteristics and outcomes [Table A-1] [Table A-2]

[0414] summary: As monotherapy or in combination with CARVac, CLDN6 CAR-T cells were well tolerated. CRS was seen in 7 of 15 patients but was manageable and therefore a CARVac schedule could be applied.

[0415] As monotherapy or in combination with CARVac, CLDN6 CAR-T cells provided clinical efficacy as demonstrated by partial responses and stable disease with shrinkage of target lesions, with some partial responses seen in all testicular cancer patients who had relapsed following recent high-dose chemotherapy.

[0416] Update results: As of June 15, 22 patients have been treated with CLDN6 CAR-T+ / -CLDN6 RNA-LPX. Among them, 20 patients were assigned to predefined cohorts (including 1 patient treated with a product containing fewer cells than required for DL1 in combination with CLDN6 RNA-LPX and 1 patient with modified lymphodepletion regimen [50% reduction]) and 2 patients were treated within the exploratory cohort without lymphodepletion. Thirteen patients had testicular cancer, 4 had ovarian cancer, with 1 each of fallopian tube, endometrium, gastric cancer, sarcoma, and 1 of unknown primary cancer (most commonly breast cancer). All treated patients were included in the safety evaluation. Two patients experienced dose-limiting toxicities, 1 / 6 (pancytopenia) in DL2 part 1 and 1 / 7 (hemophagocytic lymphohistiocytosis) in DL2 part 2. Other adverse events ≧grade 2 were primarily related to lymphodepletion or asymptomatic elevations of transaminases or lipases. One case of cytokine release syndrome (CRS) was grade 2 or below and resolved with appropriate management. At the time of data cutoff, one CRS event was confirmed as grade 3 (in one patient without lymphodepletion). However, based on reported symptoms, this qualified as a grade 2 event; however, final evaluation has not been completed.

[0417] Tumor response was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 using CT or magnetic resonance imaging. Twenty-one patients were evaluable for efficacy assessment (patients who received products containing fewer cells than required for DL1 were not included in the efficacy analysis). Overall, 7 / 21 patients showed a partial response 6 weeks after infusion (overall response rate [ORR] 35%), while an additional 8 patients had stable disease, 6 of whom showed shrinkage of tumor lesions (disease control rate [DCR] 75%). Six patients had progressive disease. Focusing on testicular cancer, 5 / 9 evaluable patients who had at least 50% lymphodepletion response at 6 weeks were observed to have CAR-T cell persistence and further tumor shrinkage. One testicular cancer patient was assessed by the investigators as a complete response by PET CT after 18 weeks. The remaining cavernous lesions had no metabolic activity, the tumor-specific marker alpha-fetoprotein (AFP) was within the normal range, and the patient's clinical evaluation did not reveal any signs of tumor activity. However, the cavernous lesions remained measurable on CT, the patient did not show any signs of tumor activity, and AFP remained within the normal range.

[0418] The disclosures of each and every patent, patent application, and article cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to certain embodiments, it is apparent that other embodiments and variations of the present invention may be devised by others 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.

[0419] Addendum [Table B-1] [Table B-2]

[0420] 1. Summary of the product and its development program 1.1 Product description and mechanism of action 1.1.1 Product Description CAR T-cell therapy has demonstrated robust antitumor responses, especially in patients with B-cell malignancies (Porter et al. 2011, Grupp et al. 2013, Brentjens et al. 2013, Brentjens et al. 2011). The clinical efficacy of CAR-T cell approaches in solid tumors, however, has been limited so far (Fesnak et al. 2016, Scarfo and Maus 2017, Yu and Hua 2019).

[0421] Two important hurdles for CAR-T cell therapy in solid tumors are: (i) loss of cancer cell-specific targets, and (ii) poor expansion and lack of long-term persistence of CAR-T cells in vivo, the latter likely resulting from the fact that adoptively transferred CAR-T cells against solid tumor targets do not encounter their antigen in the patient's circulation, providing survival and expansion signals.

[0422] To overcome these hurdles, immunotherapies targeting claudin 6 (CLDN6), a four-spanning membrane protein involved in the formation of primitive tight junctions, have been developed. CLDN6 has emerged as an attractive therapeutic target for cancer immunotherapy because it is highly expressed in a variety of cancers with high unmet medical need and absent from healthy adult tissues.

[0423] In a preclinical immunohistological study of 104 testicular cancer samples, CLDN6 protein expression was detected in 97 (93%) patients, making CLDN6 a well-suited target for CAR-T cell therapy in testicular cancer patients.

[0424] Immunotherapy includes two products: · CLDN6 CAR-T (manufactured using a manual process) or CLDN6 CAR-T(A) (manufactured using an automated process). ·CLDN6 liposomal formulation of ribonucleic acid (RNA-LPX)

[0425] CLDN6 CAR-T / CLDN6 CAR-T(A) is composed of autologous patient-derived CD4+ and CD8+ T cells expressing a CAR with specificity for CLDN6 (see section 3.1.3 below for further information on CAR lead structure). The CLDN6 CAR consists of four domains: a single-chain variable fragment derived from the monoclonal CLDN6 antibody, a CD8 hinge region, a 4-1BB costimulatory domain, and an intracellular CD3 zeta domain.

[0426] CLDN6 RNA-LPX is a liposomally formulated intravenous (IV) RNA cancer vaccine with an RNA component encoding the CAR-targeted antigen CLDN6. The RNA vaccine is designed for systemic delivery of the CAR antigen to antigen-presenting cells present in the lymphatic compartment and subsequent stimulation of adoptively transferred CAR-T cells.

[0427] 1.1.2 Mechanism of action An overview of the mechanism of action is shown in Figure 1. Autologous T cells are engineered ex vivo to express CLDN6-CAR (see section 3.1.4 below for more information on retroviral vectors) and reinfused into the patient. CAR redirects T cells to eradicate tumors by specific recognition of a natural surface protein expressed on tumor cells in a manner restricted by non-major histocompatibility complexes. Thus, CAR-T cells can be used for the treatment of all patients whose tumors express the respective target, regardless of their individual human leukocyte antigen genotype. To improve the expansion and long-term persistence of CLDN6 CAR-T cells in vivo, patients receive a CLDN6 RNA-LPX vaccine. After injection, the RNA-LPX is taken up by antigen-presenting cells present in the lymphatic compartment, the RNA is released into the cytoplasm, and the RNA is translated into the encoded protein, CLDN6, which is transiently expressed on the cell surface. By repeated RNA-LPX treatment and exposure to CLDN6, CAR-T cells are stimulated and expanded in vivo in a controlled manner, resulting in CAR-T cell persistence. [ka]

[0428] 1.1.3 CAR structure A CLDN6-specific second generation CAR was developed, which is composed of the extracellular single chain variable fragment (scFv) of IMAB206-C46S linked via a CD8 hinge domain to the intracellular 4-1BB and CD3ζ signaling domains. The different domains of this CLDN6-CAR construct (CG_CAR_001.2) and their respective functions are described in Table 1 below: [Table 1]

[0429] 1.1.4 Retroviral vectors The gamma retroviral SIN vector CG_CAR_001.2RVV is used for stable integration of CG_CAR_001.2 into the T cell genome. A schematic representation of the retroviral vector is shown in FIG. 2. [ka]

[0430] 1.2 Quality Development CLDN6 CAR-T drug substance and drug product were manufactured using a manual process that was improved to a functionally closed and automated platform with the ability to manufacture drug substance and drug product bulk in a single device [CliniMACS Prodigy® instrument] and single process setup that yields CLDN6 CAR-T(A) drug substance and drug product bulk.

[0431] The product manufactured using the manual manufacturing process was named CLDN6 CAR-T, while the product manufactured using the automated manufacturing process is referred to as CLDN6 CAR-T(A).

[0432] 1.3 Preclinical Development 1.3.1 Important findings Key findings from preclinical trials of CLDN6 CAR-T and CLDN6 RNA-LPX are summarized in the Table. [Table 2-1] [Table 2-2]

[0433] 1.3.2 Characterization of CLDN6 CAR-T(A) The manual manufacturing process was improved to a functionally closed and automated platform with the ability to manufacture drug substance and drug product bulk on a single device (CliniMACS Prodigy® instrument), in a single process setup.

[0434] In addition to the comprehensive nonclinical data for the manually manufactured CAR-T product shown in Table 2, a characterization dataset was generated for the CAR-T(A) product as part of the analytical comparability run between the manual and automated manufacturing processes. In addition to characterization, formal risk assessment, evaluation of routine batch analysis, and functional analysis were performed. In this regard, three representative clinical-scale runs were performed using apheresis material from the same healthy donor and using the manual and automated processes to manufacture CAR-T cells.

[0435] Compared with CLDN6 CAR-T cells, CLDN6 CAR-T(A) cells exhibited faster killing kinetics of endogenous CLDN6-expressing tumor cells and enriched more efficiently after tumor challenge. The improved enrichment of CAR-T cells was due to the CD4 + CAR + This coincided with a higher percentage of T cells. Furthermore, CLDN6 CAR-T(A) cells expressed more CD25, CD218a, inducible T cell immunoglobulin domain and mucin domain 3 (TIM3) and inducible T cell costimulator (ICOS), correlating with a more activated phenotype. Together, these results indicate that CLDN6 CAR-T(A) cells may have cells that express more CD25, CD128a, inducible T cell immunoglobulin domain and mucin domain 3 (TIM3) and inducible T cell costimulator (ICOS), correlating with a more activated phenotype. Together, these results indicate that CLDN6 CAR-T(A) cells may have improved ability to persist and eliminate tumor cells in patients, but at the same time, they may also have a higher risk of potential on-target / off-target toxicities.

[0436] 1.4 Clinical Development Clinical development began with a first-in-human, open-label, dose-escalation Phase I / II study with expansion cohorts evaluating safety and preliminary efficacy in patients with CLDN6-positive recurrent or refractory advanced solid tumors (EudraCT number 2019-004323-20).

[0437] Patients will be enrolled in one of three study parts (Figure). The Phase I part of the study (Parts 1 and 2) consists of dose escalation cohorts. Part 1 will evaluate CLDN6 CAR-T / CLDN6 CAR-T(A) monotherapy, and Part 2 will evaluate CLDN6 CAR-T / CLDN6 CAR-T(A)+CLDN6 RNA-LPX. The Phase II part of the study will consist of an expansion cohort of a predefined patient population. [ka]

[0438] The timeline of study treatment is shown in the figure. In parts 1 and 2, a lymphodepletion regimen is applied prior to CLDN6 CAR-T administration and consists of cyclophosphamide (500 mg / m2 / d infused IV on days -5, -4, and -3) and fludarabine (30 mg / m2 / d infused IV on days -5, -4, and -3). CLDN6 CAR-T is administered on day 1. Only in part 2, patients also receive CLDN6 RNA-LPX, administered at a starting dose of 25 μg and increasing to 50 μg from the second dose. Subsequently, doses are administered every 2-3 weeks until 5 doses have been administered. The injection interval is then increased to 6 weeks for the first year after CAR-T administration. Administration may continue until the end of the 22-month study. [ka]

[0439] 2. Effects in humans 2.1 Safety and efficacy of CLDN6 CAR-T+ / -CLDN6 RNA-LPX As of June 15, 2022, 22 patients have been treated with manually manufactured CLDN6 CAR-T + / - CLDN6 RNA-LPX. Among them, 20 patients were assigned to predefined cohorts (including 1 patient treated with a product containing fewer cells than required for DL1 in combination with CLDN6 RNA-LPX and 1 patient with modified lymphodepletion regimen [50% reduction]), and 2 patients were treated within the exploratory cohort without lymphodepletion. Thirteen patients had testicular cancer, 4 had ovarian cancer, with 1 each of fallopian tube, endometrium, gastric cancer, sarcoma, and 1 of unknown primary cancer (most commonly breast cancer). All treated patients were included in the safety evaluation. Two patients experienced dose-limiting toxicities, 1 / 6 (pancytopenia) in DL2 part 1 and 1 / 7 (hemophagocytic lymphohistiocytosis) in DL2 part 2. Other adverse events ≥ grade 2 were primarily related to lymphodepletion and asymptomatic elevations of transaminases or lipases. One instance of cytokine release syndrome (CRS) was grade 2 or below and resolved with appropriate management. At the time of data interruption, one CRS event was confirmed as grade 3 (in one patient without lymphodepletion); however, based on reported symptoms, this would have qualified as a grade 2 event. At the time of completion of this profile, this event was under discussion at the site and final evaluation had not been completed.

[0440] Tumor response was assessed by the investigators according to Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 using CT or magnetic resonance imaging. At the time of data cutoff, 21 patients were evaluable for efficacy assessment (patients who received products containing fewer cells than required for DL1 were not included in the efficacy analysis). Overall, 7 / 21 patients showed a partial response 6 weeks after infusion (overall response rate [ORR] 35%), while an additional 8 patients had stable disease, 6 of whom showed shrinkage of tumor lesions (disease control rate [DCR] 75%). Six patients had progressive disease. Focusing on testicular cancer, 5 / 9 evaluable patients who had at least 50% lymphodepletion response at 6 weeks were observed to have CAR-T cell persistence and further tumor shrinkage. One testicular cancer patient was assessed by the investigators as a complete response by PET CT after 18 weeks. The remaining cavernous lesions had no metabolic activity, the tumor-specific marker alpha-fetoprotein (AFP) was within the normal range, and the patient's clinical evaluation did not reveal any signs of tumor activity. However, the cavernous lesions remained measurable on CT. At the time of data cutoff, the patient did not show any signs of tumor activity, and AFP remained within the normal range.

[0441] 2.2 Clinical Pharmacology 2.2.1 Pharmacodynamics of CLDN6 CAR-T During the first 28 days after infusion (DLT period), different cytokines (e.g., IFNγ, IL-6, IL-10, and tumor necrosis factor [TNFα]) and serum biomarkers (e.g., C-reactive protein [CRP] and soluble IL-2 receptor, sIL-2R) will be evaluated to monitor the pharmacodynamics of CLDN6 CAR-T cells. The analysis focused on IL-6 as a marker of cytokine release syndrome (CRS) (Lee et al. 2019). Analysis of all other cytokines will be performed and included in the next version of IB. CRS development (mild to moderate, American Society of Bone Marrow Transplantation [ASTCT] grades 1–2) was observed in 8 of 16 patients in the predefined cohort, especially in patients receiving CLDN6 CAR-T in DL2, but was manageable by anti-IL-6 treatment (tocilizumab), if necessary. All patients with CRS showed elevated IL-6 levels (Figure 5). [ka]

[0442] 2.2.2 Pharmacodynamics of RNA-LPX vaccines Pharmacodynamic (PD) analyses performed in the Lipo-MERIT clinical trial will include analyses of various cytokines as well as cellular immune responses.

[0443] RNA-LPX activates APCs via TLR signaling upon IV application. With this MoA, activation of an IFNα-dominant pattern of cytokines including IFNγ followed by IP-10, as well as activation of IL-12, IL-6, and TNFα, was also observed. Patients showed a vaccine dose-dependent, transient increase in plasma levels of a distinct spectrum of cytokines, and an increase in body temperature. Preliminary data show RNA-LPX dose-dependent, pulsatile, transient release of cytokines with peak plasma levels approximately 2-6 hours after administration, returning to baseline at the latest 24 hours. In line with the self-limited increase in cytokines, a class-specific AE profile of RNA-LPX was observed with a predominance of grade 1 and 2 flu-like symptoms, mostly manageable by defervescence. Similar effects were observed, with patients developing mild and transient flu-like symptoms that resolved within 24 hours after further treatment with CLDN6 RNA-LPX.

[0444] Within the Lipo-MERIT trial, the immunogenicity of RNA-LPX was demonstrated in several patients studying the immune response to the encoded antigen. + Cytotoxic effector T cells and antigen-specific CD4 + Both T helper (Th) cells and IL-1 induced T cell proliferation and T cell regrowth.

[0445] Preliminary CLDN6 CAR-T cell count data from two patients is shown in Figure 6. The data indicate that CLDN6 RNA-LPX can modify the dynamics of CAR-T cells. Further RNA-LPX treatment appears to support CAR-T engraftment and even prolong their persistence. [ka]

[0446] 2.2.3 Pharmacodynamics of CLDN6 CAR-T In all patients, peripheral blood CAR-T cell frequency was analyzed by qPCR (based on RNA stabilizing the woodchuck hepatitis virus posttranscriptional regulatory element [WPRE] element of the CLDN6-CAR transgene). Dose-dependent, robust engraftment could be observed in all patients (Figure 7). CAR-T cell frequency peaked between days 10 and 17 and then decreased. The addition of CLDN6 RNA-LPX treatment in part 2 induced a sustained prolonged transient expansion at DL1 (patients treated with a mismatched product with CLDN6 CAR-T cell doses below DL1 were included in the analysis and showed comparable kinetics). CAR-T cell numbers were lower in patients with reduced or no lymphodepletion. In patients showing complete disease remission (illustrated as CR), CLDN6 CAR T remained detectable with a plateau longer than 200 days. [ka]

[0447] 2.2.4 Pharmacodynamics of CLDN6 RNA-LPX The lipids used for liposome manufacturing and lipoplex formulation are the naturally occurring phospholipid DOPE and the cationic lipid DOTMA. Synthetic DOPE and DOPE produced by cells in the human body have the same structure and are subject to the same degradation process. Therefore, DOPE is believed to be metabolized like the body's own phospholipids. Cationic DOTMA, as either lipid, is believed to be metabolized at a reduced rate compared to phospholipid DOPE. DOTMA has been safely applied in clinical practice (Wollenberg et al. 1999, Hedman et al. 2003), and the applied dose of DOTMA in the proposed study was very low compared to liposomal products containing similar cationic lipids (Fasol et al. 2012).

[0448] Because synthetic lipids can accumulate in the liver, liver enzymes are monitored in patients treated with RNA-LPX vaccines. No clinically significant changes in liver parameters associated with CLDN6 RNA-LPX were observed.

[0449] 2.3 Safety and efficacy of CLDN6 CAR-T(A) Dose escalation with CLDN6 CAR-T(A) was 1×10 6 CLDN6 CAR-T(A) at DL0, starting June 14, 2022. Two patients were treated at this dose level, one with testicular cancer and one with endometrial cancer. 8 Expansion of CLDN6 CAR T(A) was observed up to 100 patients (all). No DLTs occurred. There were no cases of CRS or ICNAS, and this dose level was considered safe by the safety review committee. Three more patients were already treated at DL1. The DLT period is still ongoing and no conclusions on safety can be made. However, no DLTs have occurred by August 29, 2022. No statements about efficacy can be made at this stage.

[0450] 3.Manufacturing As mentioned above, the CLDN6 CAR-T drug substance and drug product were manufactured using a manual process in the study. This process was improved to a functionally closed and automated platform with the ability to manufacture the drug substance and drug product bulk in a single device (CliniMACS Prodigy® instrument) and in a single process setting that results in the CLDN6 CAR-T(A) drug substance and drug product bulk. Only the final filling of the DP and the medium and transduction solution preparation are performed manually. Both processes include the critical steps of T cell isolation, culture and stimulation, transduction and expansion of T cells with the supernatant (VSN) containing retroviral vectors, harvesting, filling, and cryopreservation of transduced T cells (see comparison in Table 3 below). [Table 3]

[0451] The process flow chart in Figure 8 below describes the automated process and the corresponding IPC / IPM steps. [ka]

[0452] In step 5 (day 7) of the process, the expanded and transduced CD4 + / CD8 + The T cells constitute the drug substance, which is immediately processed into a formulation according to Figure 9, DP process flow chart below. [ka]

[0453] [ka]

Claims

1. 1. A pharmaceutical composition comprising immune effector cells expressing a chimeric antigen receptor (CAR) molecule for use in a method of treating CLDN6-positive testicular cancer in a human, comprising: the method comprising administering the immune effector cells to a human; the CAR molecule i) CLDN6 antigen-binding domain; ii) a transmembrane domain; and iii) an intracellular domain comprising a 4-1BB costimulatory domain, and a CD3-zeta signaling domain; Optionally, the CAR molecule comprises: i) CLDN6 antigen-binding domain; ii) CD8α hinge domain; iii) the CD8α transmembrane domain; and iv) an intracellular domain comprising a 4-1BB costimulatory domain, and a CD3-zeta signaling domain; Further optionally, the immune effector cells are genetically modified to express a CAR molecule.

2. The CLDN6 antigen-binding domain comprises an antibody, an antibody fragment, scFv, Fv, Fab, (Fab')2, a single domain antibody (SDAB), a VH or VL domain, or a Camelidae VHH domain; Optionally, the CLDN6 antigen binding domain comprises an scFv.

3. the CLDN6 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 35 or a functional variant thereof; and / or the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 30 or a functional variant thereof; and / or the CAR molecule does not comprise an additional costimulatory domain; and / or 2. The pharmaceutical composition of claim 1, wherein the CD3-zeta signaling domain comprises the amino acid sequence of SEQ ID NO: 31 or a functional variant thereof.

4. The transmembrane domain may be any of the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, 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, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD 11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226) , SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C, or a functional variant thereof; or the transmembrane domain comprises a CD8α transmembrane domain, or 2. The pharmaceutical composition of claim 1, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 28 or a functional variant thereof.

5. the antigen-binding domain is connected to the transmembrane domain by a hinge domain; Optionally, the hinge domain is a CD8α hinge domain; 2. The pharmaceutical composition of claim 1, further optionally wherein the hinge domain comprises the amino acid sequence of SEQ ID NO: 27 or a functional variant thereof.

6. the CAR molecule further comprises a leader sequence; and / or 2. The pharmaceutical composition of claim 1, wherein the CAR molecule comprises the amino acid sequence of SEQ ID NO: 36 or a functional variant thereof.

7. the immune effector cells are selected from the group consisting of T cells, natural killer (NK) cells, and cytotoxic T lymphocytes (CTLs); and / or the immune effector cells are CD8+ T cells; and / or the immune effector cells are human cells; and / or the immune effector cells lack or have low expression of a functional TCR or a functional HLA; and / or 2. The pharmaceutical composition of claim 1, wherein the immune effector cells are autologous or allogeneic to the human.

8. the method further comprising administering an agent that increases the effectiveness of the immune effector cells; Optionally, the agent is: protein phosphatase inhibitors; kinase inhibitors; cytokines; an inhibitor of an immune inhibitory molecule; or Agents that reduce the level or activity of TREG cells 2. The pharmaceutical composition of claim 1, wherein the compound is selected from one or more of:

9. The method, further comprising contacting the immune effector cell with a cognate antigen molecule that binds to the CLDN6 antigen-binding domain; 2. The pharmaceutical composition of claim 1, wherein optionally the cognate antigen molecule is selected from the group consisting of CLDN6 or a fragment thereof, or a variant of CLDN6 or a CLDN6 fragment.

10. The pharmaceutical composition of claim 9 , wherein the step of contacting the immune effector cells with the cognate antigen molecule is performed in vivo or ex vivo.

11. The method, comprising administering to a human a cognate antigen molecule or a nucleic acid, e.g., RNA, encoding same; Optionally, the nucleic acid encoding the cognate antigen molecule is transiently expressed in a human cell; and optionally, the nucleic acid encoding the cognate antigen molecule is expressed in a cell of a human to provide the cognate antigen molecule; 10. The pharmaceutical composition of claim 9, wherein the expression of the cognate antigen molecule is on the cell surface.

12. Immune effector cells and / or cognate antigen molecules or nucleic acids encoding same are administered systemically, optionally expression of the nucleic acid encoding the cognate antigen molecule in the spleen following systemic administration of the nucleic acid encoding the cognate antigen molecule; and / or systemic administration of said nucleic acid encoding said cognate antigen molecule results in expression of said nucleic acid encoding said cognate antigen molecule in antigen-presenting cells, preferably professional antigen-presenting cells, optionally wherein said antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, and B cells; and / or no or substantially no expression of the nucleic acid encoding the cognate antigen molecule in the lung and / or liver following systemic administration of the nucleic acid encoding the cognate antigen molecule; and / or 2. The pharmaceutical composition of claim 1, wherein after systemic administration of the nucleic acid encoding the cognate antigen molecule, expression of the nucleic acid encoding the cognate antigen molecule in the spleen is at least 5 times greater than expression in the lung.

13. a nucleic acid encoding a cognate antigen molecule is formulated in a delivery vehicle, optionally the delivery vehicle comprises particles, and / or the delivery vehicle comprises at least one lipid, optionally the at least one lipid comprises at least one cationic lipid, and further optionally the lipid complexes with and / or encapsulates the nucleic acid encoding the cognate antigen molecule, and / or 12. The pharmaceutical composition of claim 11, wherein the lipid is contained in a vesicle that encapsulates the nucleic acid encoding the cognate antigen molecule.

14. 12. The pharmaceutical composition of claim 11, wherein the nucleic acid encoding the cognate antigen molecule is formulated in a lipoplex.

15. Immune effector cells are 10 ７ ~10 ８ The pharmaceutical composition of claim 1 , wherein the composition is administered in an amount between 1 and 2 mg / kg.

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