Car-inkt cell technology for effectively killing intrahepatic cholangiocarcinoma

Abstract

Provided are a chimeric antigen receptor containing MSLN, an iNKT cell transducing the chimeric antigen receptor, and the use thereof in treatment of liver cancer, especially intrahepatic cholangiocarcinoma. Utilization of the characteristic of iNKT cells capable of homing and residing in the liver and selection of a suitable MSLN antibody sequence achieve a high tumor-killing efficiency and a high CAR-iNKT cell proliferation speed. The anti-MSLN CAR-iNKT cells can effectively infiltrate into tumor positions in the liver, thereby greatly improving the curative effects, reducing recurrence and relieving toxic and side effects.

Description

A CAR-iNKT cell technology that effectively kills cholangiocarcinoma CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the priority of the Chinese patent application filed with the Chinese Patent Office on January 20, 2023, with application number 202310087774.1 and invention name “A CAR-iNKT cell technology that effectively kills cholangiocarcinoma”, the entire contents of which are incorporated herein by reference. Technical Field The present application belongs to the field of cancer immunotherapy. Specifically, the present application provides a chimeric antigen receptor comprising MSLN, iNKT cells transduced with the chimeric antigen receptor, and use thereof in the preparation of liver cancer, especially cholangiocarcinoma. Background Art According to WHO statistics, there were 900,000 new cases of liver cancer worldwide in 2020[1], of which 410,000 were in China[2], accounting for 45% of the global liver cancer cases. There were 830,000 deaths from liver cancer worldwide in 2020[1], of which 390,000 were in China[2], accounting for 46% of the global liver cancer deaths. Biliary duct cancer accounts for about 10% of all liver cancers[3]. Cholangiocarcinoma is a type of tumor that originates from the epithelial layer of the bile ducts inside or outside the liver. Cholangiocarcinoma is difficult to detect in its early stages, and the prognosis after diagnosis is poor, with a five-year survival rate of only 10%[4]. Only about one-third of patients with diagnosed cholangiocarcinoma are suitable for surgical treatment[5]. Treatments for cholangiocarcinoma include surgical resection of the tumor, liver transplantation, radiotherapy, chemotherapy, local therapy, molecular targeted therapy, and immunotherapy[6]. Existing treatments cannot meet clinical requirements, and cholangiocarcinoma urgently needs safer and more effective treatments. Chimeric antigen receptor technology has achieved significant results in the treatment of hematological tumors. Kymriah[7] and Yescarta[8] were approved by the FDA in 2017 for the treatment of B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma. China also approved the first chimeric antigen receptor cell product for the treatment of hematological tumors in June 2021[9]. Mesothelin (MSLN) is a type of glycosylphosphatidylinositol protein expressed on the cell membrane surface. Under normal physiological conditions, it is expressed in the male pleura, peritoneum, pericardium and tunica vaginalis, and is lowly expressed in the epithelial layer of the ovary, fallopian tube and testis.

[0010] The human MSLN gene is located on chromosome 16 and encodes a precursor protein with a molecular weight of approximately 71 kDa and composed of 628 amino acids

[0014] . The MSLN precursor protein is spliced ​​at Arg295 to form the N-terminal soluble protein megakaryocyte enhancer factor (MPF) and the glycosylphosphatidylinositol-anchored MSLN that binds to the cell membrane surface

[0020] . Compared with the normal state where it is only limitedly expressed in mesothelial cells, MSLN is overexpressed in many tumors, including malignant mesothelioma.

[0011] , ovarian cancer

[0012] , triple-negative breast cancer

[0013] , prostate cancer

[0014] , lung cancer

[0015] , Gastric cancer

[0016] , Endometrial Cancer

[0017] , cervical cancer

[0018] , bile duct cancer

[0019] Etc., are ideal targets for targeted therapy using chimeric antigen receptor technology. Invariant natural killer T cells (iNKT) are a unique subset of thymus-derived T cells that are CD1d-restricted and express surface receptors that are characteristic of both T cells and natural killer cells (NK) lineages. They share common biological characteristics with T cells and NK cells and play an important role in bridging innate immunity and adoptive immunity. In human iNKT cells, Vα24-Jα18 forms a TCRα chain and then forms a TCR with the Vβ11TCRβ chain

[0021] . iNKT cells differentiate into at least three effector subsets in the thymus, which are similar to subsets of CD4+ T helper cells and can also be similar to subsets of innate lymphoid cells (ILCs) [22-24]. Functional iNKT cell subsets are distinguished based on the expression of different cell surface markers and characteristic transcription factors. NKT1 cells are similar to Th1 cells and ILC1s in that they both highly express the transcription factor T-bet and secrete IFN-γ after activation. NKT1 cells also exhibit greater cytotoxic function than other iNKT cell subsets. NKT1 cells differ from Th1 cells or ILC1s in that in addition to producing IFN-γ through TCR activation, they can also produce factors such as IL-4. Cytokines secreted by NKT2 cells include IL-4 and IL-13, similar to Th2 cells. NKT17 cells are similar to Th17 cells in cytokine secretion [25-27]. Different iNKT cell subsets are enriched in different tissues. NKT1 cells are highly enriched in the liver, while NKT17 cells are mainly located in the lymph nodes, skin, and lungs, with a small number of cells in the spleen

[0028] . NKT2 cells are located in multiple sites, including the lungs and spleen, but they are particularly abundant in the mesenteric lymph nodes

[0028] . In the peripheral lymph nodes, iNKT cells can be rapidly activated and may play a key role in fighting pathogens

[0029] . The research on NKT cell immunotherapy started relatively late, and its content in the body is low, so the selection of its specific antibodies is relatively limited. Currently, there are only related studies on the design of antibodies for GD2, CD19, CSPG4 and BCMA. There are few types of specific antibodies available for reference, and the selection and design are relatively difficult. In AU2020264343A1 and CN112574953A, which are similar to this patent, T cells are genetically modified to express antibodies against MSLN. The CAR contains the MSLN antigen binding domain, the transmembrane domain, the co-stimulatory signal transduction domain and the CD3δ signal transduction domain, and exhibits a killing effect on cancer cells carrying the MSLN antigen. However, since ordinary T cells cannot effectively infiltrate into solid tumors, the tumor microenvironment of solid tumors is hypoxic and acidic, which is very unfavorable for the expansion and long-term survival of CAR-T cells, thus seriously affecting the efficacy of CAR-T cells. Summary of the invention In response to the above problems, the applicant took advantage of the characteristics of iNKT cells homing to the liver and their non-specific killing function, and genetically modified them to carry chimeric antigen receptors that can bind to MSLN antigens, so that anti-MSLN-CAR-iNKT cells can effectively and specifically kill cholangiocarcinoma cells carrying MSLN antigens; and compared CAR molecules composed of different scFvs to select CAR molecules that are more conducive to binding to MSLN antigens on the surface of cancer cells and killing cancer cells. Specifically: A specific scFv targeting MSLN is introduced into the chimeric antigen receptor (CAR) molecule, so that it can specifically bind to the MSLN antigen specifically expressed by cholangiocarcinoma cells. At the same time, the intracellular signaling domain of the co-stimulatory molecules CD28 and CD3δ are introduced into the CAR molecule, thereby stimulating iNKT cells to proliferate and secrete IFN-γ, granzymes, etc. to kill cancer cells. The killing effects of CAR molecules composed of different scFvs on cancer cells expressing MSLN antigen are compared, so as to select CAR molecules with better anti-cancer effects. Through creative work, the inventors continuously carried out amino acid sequence design, sequence arrangement, combination and screening, and conducted random screening tests and targeted function verification on nearly dozens of CAR molecular sequences (such as constructing viral vectors, and further infecting iNKT cells to obtain modified iNKT cells, and detecting the in vitro killing activity of the modified iNKT cells, etc.), and then compared the results of multiple random combinations, adjusted the sequences, and finally screened out the sequences with the best effects, and obtained the anti-human MSLN single-chain antibody chimeric antigen receptor sequence and its functional variants that target the human MSLN protein with high titer of the present application. On the one hand, the present application provides a chimeric antigen receptor, characterized in that it comprises a MSLN single-chain antibody, a spacer domain or a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and a signal region connected in sequence. Optionally, the chimeric antigen receptor comprises a CD8α signal peptide, a MSLN single-chain antibody, a CD8α hinge region, a CD28 transmembrane region, a CD28 co-stimulatory region, and a CD3δ signal region, which are sequentially connected. Optionally, the amino acid sequence of the MSLN single-chain antibody is selected from SEQ ID NO.8, 10, 12. Optionally, the MSLN single-chain antibody nucleotide sequence is selected from SEQ ID NO.9, 11, and 13. Optionally, the amino acid sequence of the MSLN single-chain antibody is SEQ ID NO.8. Optionally, the CD8α signal peptide amino acid sequence is SEQ ID NO.2, the CD8α hinge region amino acid sequence is SEQ ID NO.14, the CD28 transmembrane region amino acid sequence is SEQ ID NO.4, the CD28 co-stimulatory region amino acid sequence is SEQ ID NO.6, and the CD3δ signal region amino acid sequence is SEQ ID NO.16. Optionally, the CD8α signal peptide nucleotide sequence is SEQ ID NO.3, the CD8α hinge region nucleotide sequence is SEQ ID NO.15, the CD28 transmembrane region nucleotide sequence is SEQ ID NO.5, the CD28 co-stimulatory region nucleotide sequence is SEQ ID NO.7, and the CD3δ signal region nucleotide sequence is SEQ ID NO.17. Optionally, the chimeric antigen receptor amino acid sequence is selected from SEQ ID NO.18, 20, 22. Optionally, the chimeric antigen receptor nucleotide sequence is selected from SEQ ID NO.19, 21, 23. On the other hand, the present application provides a vector carrying the above-mentioned chimeric antigen receptor, optionally a pLV300 vector. Optionally, the nucleotide sequence of the pLV300 vector is SEQ ID NO.24. On the other hand, the present application provides an immune cell that transduces the above-mentioned chimeric antigen receptor. Optionally, the immune cells are T cells, NK cells or iNKT cells. Optionally, the immune cells are iNKT cells. On the other hand, the present application provides the use of the above-mentioned chimeric antigen receptor, vector or immune cell in the preparation of a drug for treating cancer. On the other hand, the present application provides a method for treating cancer, characterized in that the above-mentioned chimeric antigen receptor, vector or immune cell is used. Optionally, the cancer is a cancer in which MSLN is overexpressed. Optionally, the cancer is liver cancer. Optionally, the cancer is cholangiocarcinoma. In another aspect, the present application provides a transduction system comprising the above expression vector. Optionally, the transduction system is a viral transduction system and a non-viral transduction system. Optionally, the transduction system is a lentiviral transduction system. The present application includes a nucleic acid expression vector containing one or more of the above elements for constructing a chimeric antigen receptor protein expressed on the surface of T cells, NK cells or NKT cells. Various commercially available vectors can be selected as needed, or vectors can be constructed according to conventional techniques in the field of molecular biology. In a specific embodiment, the vector used in the present application is a lentiviral plasmid vector pLV300. The plasmid belongs to the fourth generation self-inactivating lentiviral vector system, which has four plasmids, namely, a packaging plasmid encoding protein Gag / Pol, a packaging plasmid encoding Rev protein, an envelope plasmid encoding VSV-G protein, and an empty vector pLV300, which can be used to recombinantly introduce a target nucleic acid sequence, i.e., a nucleic acid sequence encoding a chimeric antigen receptor protein. The expression of the chimeric antigen receptor protein is regulated by the pGK-300 promoter in the vector pLV300. The present application includes viruses comprising the above-mentioned vectors, including but not limited to lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and the like. The viruses of the present application include viruses with infectious power after packaging, and also include viruses to be packaged that contain the necessary components for packaging as infectious viruses. Other viruses known in the art that transfect T cells, NK cells, or NKT cells and their corresponding plasmid vectors may also be used in the present application. In one embodiment of the present application, the virus is a lentivirus comprising the above-mentioned pLV300-anti-GPC3 CAR recombinant vector. The present application includes a transgenic T lymphocyte, NK cell or iNKT cell, which is transduced with the nucleic acid of the present application or the recombinant plasmid containing the nucleic acid or the viral system containing the plasmid. Conventional nucleic acid transduction methods in the art, including non-viral and viral transduction methods, can be used in the present application. Non-viral transduction methods include electroporation and transposon methods. The nucleofector nuclear transfection instrument recently developed by Amaxa can directly introduce exogenous genes into the cell nucleus to obtain efficient transduction of the target gene. In addition, the transduction efficiency of transposon systems such as Sleeping Beauty transposon (Sleeping Beauty system) or PiggyBac transposon is greatly improved compared with ordinary electroporation. It has been reported that the nucleofector transfection instrument is combined with the SB Sleeping Beauty transposon system. This method can have both high transduction efficiency and site-specific integration of the target gene. In one embodiment of the present application, the transduction method of iNKT cells modified with chimeric antigen receptor genes is a lentiviral transduction method. This method has the advantages of high transduction efficiency, stable expression of exogenous genes, and shortening the time for in vitro cultured iNKT lymphocytes to reach clinical-level numbers. The nucleic acid introduced by lentiviral transfection is expressed on the surface of the iNKT cell membrane through transcription and translation. In vitro cytotoxicity experiments on various cultured tumor cells have proved that the transgenic iNKT cells expressing chimeric antigen receptors on the surface of the present application have a highly specific tumor cell killing effect (also known as cytotoxicity). Therefore, the nucleic acid encoding the chimeric antigen receptor protein of the present application, the plasmid containing the nucleic acid, the virus containing the plasmid, and the transgenic iNKT cells, T lymphocytes or NK cells transfected with the above nucleic acid, plasmid or virus can be effectively used for tumor immunotherapy. The nucleic acid of the present application may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The nucleic acid codons encoding the amino acid sequence of the chimeric antigen receptor protein of the present application may be degenerate, that is, multiple degenerate nucleic acid sequences encoding the same amino acid sequence are included in the scope of the present application. Degenerate nucleic acid codons encoding corresponding amino acids are well known in the art. The present application also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present application. The variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the polypeptide encoded by it. The cancers in which MSLN is overexpressed include, but are not limited to, malignant mesothelioma, ovarian cancer, triple-negative breast cancer, prostate cancer, lung cancer, gastric cancer, endometrial cancer, cervical cancer, bile duct cancer, etc. The overexpression of MSLN refers to the situation where the content of MSLN-related nucleic acid or protein or other indicators are higher than normal levels by various detection methods including but not limited to molecular level and protein level. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of the anti-MSLN CAR structure; Figure 2 is a schematic diagram of the structure of the pLV300 lentiviral vector; FIG3 is a proliferation curve of total iNKT cells; Figure 4 shows the proportion of CD8+CAR+iNKT cell subsets at different times of anti-MSLN CAR-iNKT cell culture; Figure 5 shows the proportion of CD4+CAR+iNKT cell subsets at different times of anti-MSLN CAR-iNKT cell culture; Figure 6 shows the proportion of CD4-CD8-CAR+iNKT cell subsets at different times of anti-MSLN CAR-iNKT cell culture; FIG7 shows the killing effect of anti-MSLN CAR-iNKT on cholangiocarcinoma cell KMCH at different effector-target ratios; Figure 8 shows the IFN-γ content in the cell culture supernatant after co-culture of anti-MSLN CAR-iNKT cells with cholangiocarcinoma cell KMCH for 24 hours; FIG9 shows the IL-2 content in the cell culture supernatant after co-culture of anti-MSLN CAR-iNKT cells with cholangiocarcinoma cells KMCH for 24 hours. DETAILED DESCRIPTION Example 1 Preparation of lentivirus containing chimeric antigen receptor molecules HEK-293T cells were passaged, and after the cells grew to 60%-70% confluence, the expression vector containing the CAR molecule and the packaging plasmid were transfected into the HEK-293T cells using pEI reagent, and fresh culture medium was replaced 4 hours after transfection. The cell culture supernatant was collected 48-72 hours after transfection. The supernatant was ultracentrifuged to concentrate the lentivirus containing the CAR molecule after packaging. The concentrated lentivirus was titered and frozen at -80°C for use. Example 2 Effects of anti-MSLN CAR molecules on total iNKT cell proliferation and CAR-iNKT cell differentiation After isolating iNKT cells from human peripheral blood mononuclear cells using anti-iNKT mircrobeads, 2 × 10 5Cell inoculation, add X-VIVO complete medium (containing 100 IU / ml and 100 ng / ml α-Galcer) to each well and culture for 48 hours, collect cells from each well, centrifuge at 400×g for 5 minutes, discard the supernatant, add fresh X-VIVO complete medium to resuspend and re-inoculate in 24-well culture plates, add different lentivirus containing anti-MSLN1 CAR, anti-MSLN2 CAR, anti-MSLN3 CAR to each well for infection. After 24 hours, collect cells from each well in a centrifuge tube, centrifuge at 400×g for 5 minutes, count, and calculate 5×10 5 cells / ml were added with fresh X-VIVO complete medium for culture, and the medium was changed every 48 hours. On day 7, day 14, and day 21, samples were taken for counting and flow cytometry to verify the expression of chimeric antigen receptor molecules. The results showed that different anti-MSLN CAR molecules had an effect on the proliferation and differentiation of iNKT cells. After anti-MSLN1 CAR and anti-MSLN2 CAR were transferred into iNKT cells, they were more conducive to cell growth than anti-MSLN3 CAR molecules (Figure 3). After anti-MSLN1 CAR and anti-MSLN2 CAR were transferred into iNKT cells, they were more conducive to the differentiation of CAR-iNKT cells into CD8+CAR+iNKT cells than anti-MSLN3 CAR molecules (Figures 4-6). Example 3 Effects of different anti-MSLN CAR molecules on the tumor killing ability of CAR-iNKT cells When the CAR-iNKT cells of different groups were cultured to day 21, 0.33×10 5 CAR-iNKT cells, 1×10 5 CAR-iNKT cells, 3×10 5 CAR-iNKT cells, and 1×10 5 After 6 hours of co-culture with KMCH cells (KMCH is a mesothelin-positive cholangiocarcinoma cell line. Mix and incubate with fluorescent dye CFSE for 10 minutes), the culture supernatant of each group was taken and the fluorescence intensity in the supernatant was detected by microplate reader (the stronger the killing ability of CAR-iNKT cells, the more CFSE released in KMCH cells, the greater the fluorescence intensity), and the killing efficiency of CAR-iNKT cells in each group was calculated (Figure 7). The results showed that all three anti-MSLN CAR-iNKT cells could effectively kill KMCH cells, but anti-MSLN1 CAR-iNKT cells had the strongest killing ability. Example 4 Comparison of IFN-γ and IL-2 secretion after co-incubation of different anti-MSLN CAR-iNKT cells with KMHC cells expressing MSLN After culturing CAR-iNKT cells in each group for 21 days, the ratio of effector cells to target cells was 3:1, and CAR-iNKT cells (3×10 5 CAR-iNKT cells) were respectively 5 After KMHC cells were co-cultured with 0.5 ml X-VIVO (without IL-2 and α-GalCer) for 24 hours, the cells were collected and centrifuged at 400 x g for 5 minutes to obtain the supernatant. The IFN-γ and IL-2 content in the supernatant was detected by ELISA. The results showed that anti-MSLN1 CAR-iNKT cells had the strongest ability to secrete IFN-γ and IL-2, which was consistent with the strong proliferation ability and tumor killing ability of anti-MSLN1 CAR-iNKT cells (Figures 8-9). Obviously, the above embodiments of the present application are only examples for clearly explaining the present application, and are not intended to limit the implementation methods of the present application. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. However, these obvious changes or modifications derived from the spirit of the present application are still within the scope of protection of the present application. References: 1 https: / / gco.iarc.fr / today / data / factsheets / populations / 900-world-fact-heets.pdf 2 https: / / gco.iarc.fr / today / data / factsheets / populations / 160-china-fact-heets.pdf 3 Sahil Mittal 1,Hashem B El-Serag. Epidemiology of hepatocellular carcinoma:consider the population J Clin Gastroenterol. 2013 Jul; 47 Suppl(0):S2-6.doi:10.1097 / MCG.0b013e3182872f29. 4 Everhart JE,Ruhl CE.Burden of digestive diseases in the United States part III:liver,biliary tract,and pancreas.Gastroenterology 2009 Apr;136(4):1134e44.PubMed PMID:19245868.Epub 2009 / 02 / 28.eng. 5 Shaib YH,Davila JA,Henderson L,McGlynn KA,El-Serag HB.Endoscopic and surgical therapy for intrahepatic cholangiocarcinoma in the United States:a population-based study.J Clin Gastroenterol 2007 NoveDec;41(10):911e7.PubMed PMID:18090160.Epub 2007 / 12 / 20.eng. 6 Khan SA,Davidson BR,Goldin RD,Heaton N,Karani J,Pereira SP,et al.Guidelines for the diagnosis and treatment of cholangiocarcinoma:an update.Gut 2012 Dec;61(12):1657e69.PubMed PMID:22895392.Epub 2012 / 08 / 17.eng. 7 https: / / www.fda.gov / media / 106989 / download 8 https: / / www.fda.gov / media / 108458 / download 9 https: / / www.nmpa.gov.cn / yaowen / ypjgyw / 20210623094232187.html 10 Ordonez NG.Application of mesothelin immunostaining in tumor diagnosis.Am J Surg Pathol.2003;27(11):1418–1428. 11 Ordonez NG.Value of mesothelin immunostaining in the diagnosis of mesothelioma.Mod Pathol.2003;16(3):192–197. 12 Scales SJ,Gupta N,Pacheco G,et al.An antimesothelin-monomethyl auristatin e conjugate with potent antitumor activity in ovarian,pancreatic,and mesothelioma models.Mol Cancer Ther.2014;13(11):2630–2640. 13 Li YR,Xian RR,Ziober A,et al.Mesothelin expression is associated with poor outcomes in breast cancer.Breast Cancer Res Treat.2014;147(3):675–684. 14 Ho M,Bera TK,Willingham MC,et al.Mesothelin expression in human lung cancer.Clin Cancer Res.2007;13(5):1571–1575. 15 Ho M,Bera TK,Willingham MC,et al.Mesothelin expression in human lung cancer.Clin Cancer Res.2007;13(5):1571–1575. 16 Baba K,Ishigami S,Arigami T,et al.Mesothelin expression correlates with prolonged patient survival in gastric cancer.J Surg Oncol.2012;105(2):195–199. 17 Cheng WF,Huang CY,Chang MC,et al.High mesothelin correlates with chemoresistance and poor survival in epithelial ovarian carcinoma.Br J Cancer.2009;100(7):1144–1153. 18 Kawamata F,Kamachi H,Einama T,et al.Intracellular localization of mesothelin predicts patient prognosis of extrahepatic bile duct cancer.Int J Oncol.2012;41(6):2109–2118. 19 Chang K,Pastan I.Molecular cloning of mesothelin,a differentiation antigen present on mesothelium,mesotheliomas,and ovarian cancers.Proc Natl Acad Sci U S A.1996;93(1):136–140. 20 Shimizu A,Hirono S,Tani M,et al.Coexpression of MUC16 and mesothelin is related to the invasion process in pancreatic ductal adenocarcinoma.Cancer Sci.2012;103(4):739–746. 21 Crosby,C.M.and M.Kronenberg,Tissue-specific functions of invariant natural killer T cells.Nat Rev Immunol,2018.18(9):p.559-574. 22 Bennstein,S.B.,Unraveling Natural Killer T-Cells Development.Front Immunol,2017.8:p.1950. 23 Georgiev,H.,et al.,Distinct gene expression patterns correlate with developmental and functional traits of iNKT subsets.Nat Commun,2016.7:p.13116. 24 Lee,Y.J.,et al.,Lineage-Specific Effector Signatures of Invariant NKT Cells Are Shared amongst gammadelta T,Innate Lymphoid,and Th Cells.J Immunol,2016.197(4):p.1460-70. 25 Lee,Y.J.,et al.,Steady-state production of IL-4 modulates immunity in mouse strains and is determined by lineage diversity of iNKT cells.Nat Immunol,2013.14(11):p.1146-54. 26 Verykokakis,M.and B.L.Kee,Transcriptional and epigenetic regulation of innate-like T lymphocyte development.Curr Opin Immunol,2018.51:p.39-45. 27 Engel,I.,et al.,Innate-like functions of natural killer T cell subsets result from highly divergent gene programs.Nat Immunol,2016.17(6):p.728-39. 28 Lee,Y.J.,et al.,Tissue-Specific Distribution of iNKT Cells Impacts Their Cytokine Response.Immunity,2015.43(3):p.566-78. 29 Gaya,M.,et al.,Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells.Cell,2018.172(3):p.517-533 e20.

Claims

1. A chimeric antigen receptor, characterized in that It comprises a MSLN single-chain antibody, a spacer domain or hinge region, a transmembrane region, an intracellular co-stimulatory domain, and a signal region which are connected in sequence.

2. The chimeric antigen receptor according to claim 1, comprising a CD8α signal peptide, a MSLN single-chain antibody, a CD8α hinge region, a CD28 transmembrane region, a CD28 co-stimulatory region, and a CD3δ signal region connected in sequence.

3. The chimeric antigen receptor according to claim 1 or 2, wherein the amino acid sequence of the MSLN single-chain antibody is selected from SEQ ID NO. 8, 10, and 12.

4. The chimeric antigen receptor according to claim 3, wherein the MSLN single-chain antibody nucleotide sequence is selected from SEQ ID NO.9, 11, and 13.

5. The chimeric antigen receptor according to claim 3 or 4, wherein the amino acid sequence of the MSLN single-chain antibody is SEQ ID NO.

8.

6. The chimeric antigen receptor according to any one of claims 1 to 5, wherein the CD8α signal peptide amino acid sequence is SEQ ID NO.2, the CD8α hinge region amino acid sequence is SEQ ID NO.14, the CD28 transmembrane region amino acid sequence is SEQ ID NO.4, the CD28 co-stimulatory region amino acid sequence is SEQ ID NO.6, and the CD3δ signal region amino acid sequence is SEQ ID NO.

16.

7. The chimeric antigen receptor according to claim 6, wherein the CD8α signal peptide nucleotide sequence is SEQ ID NO.3, the CD8α hinge region nucleotide sequence is SEQ ID NO.15, the CD28 transmembrane region nucleotide sequence is SEQ ID NO.5, the CD28 co-stimulatory region nucleotide sequence is SEQ ID NO.7, and the CD3δ signal region nucleotide sequence is SEQ ID NO.

17.

8. The chimeric antigen receptor according to any one of claims 1 to 7, wherein the chimeric antigen receptor amino acid sequence is selected from SEQ ID NO. 18, 20, 22.

9. The chimeric antigen receptor according to claim 8, wherein the chimeric antigen receptor nucleotide sequence is selected from SEQ ID NO. 19, 21, and 23.

10. A vector carrying the chimeric antigen receptor according to any one of claims 1 to 9. The vector according to claim 10 , wherein the vector is pLV300, and the nucleotide sequence thereof is SEQ ID NO.

24.

12. An immune cell transduced with the chimeric antigen receptor according to any one of claims 1 to 9. The immune cell according to claim 12 , wherein the immune cell is a T cell, a NK cell or an iNKT cell. The immune cell according to claim 12 , wherein the immune cell is an iNKT cell.

15. Use of the chimeric antigen receptor, vector or immune cell according to any one of claims 1 to 14 in the preparation of a drug for treating cancer.

16. A method for treating cancer, characterized in that: The chimeric antigen receptor, vector or immune cell according to any one of claims 1 to 14 is used.

17. The use according to claim 15 or the method according to claim 17, wherein the cancer is a cancer in which MSLN is overexpressed.

18. The use or method according to claim 17, wherein the cancer is liver cancer.

19. The use or method according to claim 18, wherein the cancer is cholangiocarcinoma.

20. A transduction system comprising the vector according to claim 10 or 11.

21. The transduction system according to claim 20, wherein the transduction system is a viral transduction system and a non-viral transduction system.

22. [Corrected 30.03.2023 according to Rule 26] The transduction system according to claim 21, wherein the transduction system is a lentiviral transduction system.

23. [Corrected 30.03.2023 according to Rule 26] The method according to claim 16, wherein the cancer is a cancer in which MSLN is overexpressed.

24. The method of claim 23, wherein the cancer is liver cancer.

25. The method of claim 24, wherein the cancer is cholangiocarcinoma.

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