CAR-iNKT cell technology effectively kills cholangiocarcinoma

Abstract

The present application provides a chimeric antigen receptor containing MSLN, iNKT cells transduced with the chimeric antigen receptor, and their use in generating liver cancer, particularly cholangiocarcinoma. In the present application, by utilizing the property that iNKT cells can home and settle in the liver, a high tumor killing efficiency and a fast CAR-iNKT cell proliferation rate are achieved by selecting an appropriate MSLN antibody sequence, and the anti-MSLN CAR-iNKT cells can effectively infiltrate to the site of liver tumors, thereby extremely improving the therapeutic effect, reducing recurrence, and reducing toxicity and side effects.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims priority to a Chinese patent application, application number 202310087774.1, filed with the China Patent Office on January 20, 2023, entitled "CAR-iNKT cell technology for effectively killing cholangiocarcinoma," the entire contents of which are incorporated herein by reference.

[0002] [Technical field] The present application belongs to the field of cancer immunotherapy. Specifically, the present application provides a chimeric antigen receptor comprising MSLN, an iNKT cell transduced with the chimeric antigen receptor, and its use in generating liver cancer, particularly cholangiocarcinoma. [Background technology]

[0003] According to WHO statistics, in 2020, there will be 900,000 new cases of liver cancer worldwide. [1] China reports 410,000 new cases of liver cancer [2] This accounts for 45% of the total number of liver cancer cases worldwide, and in 2020, there were 830,000 deaths from liver cancer worldwide. [1] 390,000 deaths from liver cancer in China [2] It accounts for 46% of all deaths from liver cancer worldwide. Cholangiocarcinoma accounts for approximately 10% of all liver cancer cases. [3] .

[0004] Cholangiocarcinoma is a type of tumor that occurs in the epithelial layer of the bile duct inside or outside the liver. Cholangiocarcinoma is difficult to detect early and has a poor prognosis after diagnosis, with a 5-year survival rate of only 10%. [4] After diagnosis, only about one-third of patients with cholangiocarcinoma are suitable for surgical treatment. [5] Treatment options for cholangiocarcinoma include surgical tumor resection, liver transplantation, radiation therapy, chemotherapy, localized treatment, targeted therapy, and immunotherapy. [6] Current therapeutic approaches cannot meet clinical needs, and safer and more effective therapeutic approaches are urgently needed for cholangiocarcinoma.

[0005] Chimeric antigen receptor technology has shown remarkable therapeutic effects in the treatment of hematological malignancies. [7] and Yescarta [8] In 2017, it was approved by the FDA for the treatment of B-cell acute lymphoblastic leukemia and diffuse large B-lymphoma. In June 2021, China also approved its first chimeric antigen receptor cell product for the treatment of hematological malignancies. [9] .

[0006] Mesothelin (MSLN) is a glycosylphosphatidylinositol protein expressed on the surface of cell membranes. Under normal physiological conditions, it is expressed in the pleura, peritoneum, pericardium, and vaginal tunica in men, and at low levels in the epithelial layers of the ovaries, fallopian tubes, and testes.

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

[14] The MSLN precursor protein is spliced ​​at Arg295 to give the N-terminal soluble protein megakaryocyte potentiating factor (MPF) and the glycosylphosphatidylinositol-anchored MSLN bound to the cell membrane surface.

[20] .

[0007] In normal cases, expression is restricted to mesothelial cells, whereas in malignant mesothelioma

[11] , ovarian cancer

[12] , Sanyin breast cancer

[13] , prostate cancer

[14] ,lung cancer

[15] ,stomach cancer

[16] , endometrial cancer

[17] , cervical cancer

[18] , cholangiocarcinoma

[19] MSLN is overexpressed in many types of tumors, including those described above, and is a suitable target for targeted therapy using chimeric antigen receptor technology.

[0008] Invariant natural killer T cells (iNKT) are a unique subset of thymus-derived T cells that are CD1d-restricted and express surface receptors characteristic of both T cell and natural killer cell (NK) lineages, sharing common biological features with T cells and NK cells and playing an important role in bridging innate and adaptive immunity. In human iNKT cells, Vα24-Jα18 forms the TCRα chain and then Vβ11 TCRβ chain to form the TCR.

[21] .

[0009] In the thymus, iNKT cells differentiate into at least three effector subsets that resemble subsets of CD4+ T helper cells and may also resemble subsets of innate lymphoid cells (ILCs). [22-24] Functional iNKT cell subsets are distinguished by the expression of distinct cell surface markers and characteristic transcription factors. NKT1 cells are similar to Th1 cells and ILC1s, as both highly express the transcription factor T-bet and secrete IFN-γ after activation. NKT1 cells also exhibit greater cytotoxicity than other iNKT cell subsets. NKT1 cells differ from Th1 cells and ILC1s in that they produce IFN-γ upon TCR activation, as well as factors such as IL-4. Cytokines secreted by NKT2 cells include IL-4 and IL-13, similar to Th2 cells. NKT17 cells, on the other hand, are similar to Th17 cells in cytokine secretion. [25-27] .

[0010] Different iNKT cell subsets are enriched in different tissues: NKT1 cells are highly enriched in the liver, whereas NKT17 cells are located primarily in lymph nodes, skin and lungs, with a small number of cells in the spleen.

[28] NKT2 cells are present in multiple sites, including the lung and spleen, but are particularly abundant in mesenteric lymph nodes.

[28] In surrounding lymph nodes, iNKT cells are rapidly activated and can play a key role in fighting pathogens.

[29] .

[0011] Research into NKT cell immunotherapy began late, the content of NKT cells in the body is low, and the selection of specific antibodies is limited. Currently, only research has been done on the design of antibodies against GD2, CD19, CSPG4, and BCMA, and there are few types of specific antibodies to refer to, making both selection and design difficult.

[0012] In Australian Patent Publication No. 2020264343 and Chinese Patent Publication No. 112574953, which are similar to this patent, T cells are genetically modified to express a chimeric receptor for the MSLN antigen, where the CAR contains an MSLN antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3ζ signaling domain, and exhibits a killing effect on cancer cells bearing the MSLN antigen. However, ordinary T cells cannot effectively infiltrate solid tumors, and the tumor microenvironment of solid tumors is oxygen-deficient and relatively acidic, which is very unfavorable to the proliferation and long-term survival of CAR-T cells, and therefore the therapeutic effect of CAR-T cells is seriously affected. Summary of the Invention [Problem to be solved by the invention]

[0013] In response to the above problems, the applicant has utilized the liver homing characteristics and non-specific killing function of iNKT cells to endow iNKT cells with chimeric antigen receptors that can bind to the MSLN antigen through genetic modification, and developed a method for treating cholangiocarcinomas bearing the MSLN antigen using anti-MSLN-CAR-iNKT cells. In addition, by comparing CAR molecules composed of different scFvs, the researchers selected a CAR molecule that binds to the MSLN antigen on the surface of cancer cells and is advantageous in killing cancer cells.

[0014] By introducing an MSLN-specific scFv into a chimeric antigen receptor (CAR) molecule and allowing it to specifically bind to the MSLN antigen, which is specifically expressed in cholangiocarcinoma cells, and by introducing the costimulatory molecule CD28 intracellular signal domain and CD3ζ intracellular signal domain into the CAR molecule, iNKT cells can be stimulated to proliferate, secrete IFN-γ, granulase, etc., and kill cancer cells. By comparing the killing effects of CAR molecules composed of different scFvs against cancer cells expressing the MSLN antigen, a CAR molecule with a greater anti-cancer effect was selected.

[0015] The inventors made creative efforts to repeatedly design amino acid sequences and combine and screen sequences, and conducted random screening tests and target function verification for approximately several dozen CAR molecule sequences (e.g., constructing a viral vector, infecting iNKT cells, obtaining modified iNKT cells, and measuring the ex vivo killing activity of the obtained modified iNKT cells, etc.), and then aligned based on the results of multiple random combinations, further adjusting the sequences, and finally selecting the most effective sequence, thereby obtaining the sequence of a highly potent anti-human MSLN single-chain antibody chimeric antigen receptor targeting the human MSLN protein of the present application and its functional variants. [Means for solving the problem]

[0016] In one aspect, the present application provides a chimeric antigen receptor comprising, in sequence, an MSLN single-chain antibody, a spacer domain or hinge region, a transmembrane region, an intracellular costimulatory domain, and a signal region.

[0017] Optionally, the chimeric antigen receptor comprises, in sequence, a CD8α signal peptide, an MSLN single chain antibody, a CD8α hinge region, a CD28 transmembrane region, a CD28 costimulatory domain, and a CD3ζ signal region.

[0018] Optionally, the amino acid sequence of the MSLN single chain antibody is selected from SEQ ID NOs. 8, 10, and 12.

[0019] Optionally, the nucleotide sequence of the MSLN single chain antibody is selected from SEQ ID NOs. 9, 11, and 13.

[0020] Optionally, the amino acid sequence of the MSLN single chain antibody is SEQ ID NO.8.

[0021] Optionally, the amino acid sequence of the CD8α signal peptide is SEQ ID NO.2, the amino acid sequence of the CD8α hinge region is SEQ ID NO.14, the amino acid sequence of the CD28 transmembrane region is SEQ ID NO.4, the amino acid sequence of the CD28 costimulatory domain is SEQ ID NO.6, and the amino acid sequence of the CD3ζ signal region is SEQ ID NO.16.

[0022] Optionally, the nucleotide sequence of the CD8α signal peptide is SEQ ID NO.3, the nucleotide sequence of the CD8α hinge region is SEQ ID NO.15, the nucleotide sequence of the CD28 transmembrane region is SEQ ID NO.5, the nucleotide sequence of the CD28 costimulatory domain is SEQ ID NO.7, and the nucleotide sequence of the CD3ζ signal region is SEQ ID NO.17.

[0023] Optionally, the amino acid sequence of the chimeric antigen receptor is selected from SEQ ID NOs. 18, 20, and 22.

[0024] Optionally, the nucleotide sequence of the chimeric antigen receptor is selected from SEQ ID NOs. 19, 21, and 23.

[0025] In another aspect, the present application provides a vector carrying the chimeric antigen receptor, optionally a pLV300 vector.

[0026] Optionally, the pLV300 vector has the nucleotide sequence of SEQ ID NO.24.

[0027] In another aspect, the present application provides an immune cell transduced with the chimeric antigen receptor.

[0028] Optionally, the immune cell is a T cell, a NK cell, or an iNKT cell.

[0029] Optionally, the immune cell is an iNKT cell.

[0030] In another aspect, the present application provides a use of the chimeric antigen receptor, vector or immune cell described above in the manufacture of a medicament for the treatment of cancer.

[0031] In another aspect, the present application provides a method for treating cancer, comprising using the chimeric antigen receptor, vector, or immune cell described above.

[0032] Optionally, the cancer is a MSLN-overexpressing cancer.

[0033] Optionally, the cancer is liver cancer.

[0034] Optionally, the cancer is cholangiocarcinoma.

[0035] In another aspect, the present application provides a transduction system comprising the above-described expression vector.

[0036] Optionally, the transduction system is a viral transduction system or a non-viral transduction system.

[0037] Optionally, said transduction system is a lentiviral transduction system.

[0038] The present application includes a nucleic acid expression vector comprising one or more of the above elements for simultaneous use in constructing a chimeric antigen receptor protein expressed on the surface of T cells, NK cells or NKT cells. Various commercially available vectors may be selected as needed, or a vector may be constructed based on conventional techniques in the field of molecular biology. In one specific embodiment, the vector used in the present application is a lentiviral plasmid vector, pLV300 vector. This plasmid belongs to a fourth generation self-inactivating lentiviral vector system, which includes four plasmids, namely, a packaging plasmid encoding Gag / Pol proteins, a packaging plasmid encoding Rev protein, an envelope plasmid encoding VSV-G protein, and an empty pLV300 vector that can be used to recombinably introduce a nucleic acid sequence of interest, i.e., a nucleic acid sequence encoding a chimeric antigen receptor protein. The expression of the chimeric antigen receptor protein is regulated in the pLV300 vector by the pGK-300 promoter.

[0039] The present application includes viruses that contain the above vectors, including, but not limited to, lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, etc. The viruses of the present application include packaged infectious viruses, and also packaged viruses that contain the necessary components to be packaged as infectious viruses. Other viruses and their corresponding plasmid vectors known in the art that transfect T cells, NK cells, or NKT cells can also be used in the present application. In one embodiment of the present application, the virus is a lentivirus that contains the above pLV300-anti-GPC3 CAR recombinant vector.

[0040] The present application includes a transgenic T lymphocyte, NK cell, or iNKT cell into which the nucleic acid of the present application, or the recombinant plasmid of the present application containing the recombinant plasmid containing the nucleic acid, or a viral system containing the plasmid, has been introduced. Conventional nucleic acid introduction methods in the field, including non-viral introduction methods and viral introduction methods, can be used in the present application. Non-viral introduction methods include electroporation and transposon methods. Recently, the nucleofector nuclear transfection device developed by Amaxa can directly introduce a foreign gene into a cell nucleus, thereby achieving highly efficient transduction of a target gene. In addition, the transduction efficiency of transposon systems such as the Sleeping Beauty system and PiggyBac transposon is significantly improved compared to ordinary electroporation, and the combined use of the nucleofector transfection device and the SB transposon system has already been reported, which can realize the integration of a target gene into a specific site in addition to high transduction efficiency. In one embodiment of the present application, the transduction method for realizing chimeric antigen receptor gene-modified iNKT cells is a lentivirus-based transduction method. This method has advantages such as high transduction efficiency, stable expression of foreign genes, and shortening the time required to culture iNKT lymphocytes in vitro to a clinical level. The nucleic acid introduced by lentivirus transfection is expressed on the iNKT cell membrane surface by transcription and translation. As a result of performing in vitro cytotoxicity experiments on various different cultured tumor cells, transgenic iNKT cells expressing the chimeric antigen receptor of the present application on their surface have a highly specific tumor cell killing effect (also called cytotoxicity). For this reason, the nucleic acid of the present application encoding the chimeric antigen receptor protein, a plasmid containing the nucleic acid, a virus containing the plasmid, and transgenic iNKT cells, T lymphocytes, or NK cells transfected with the above nucleic acid, plasmid, or virus can be effectively used in tumor immunotherapy.

[0041] The nucleic acid of the present application may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The nucleic acid codons of the present application that code for the amino acid sequence of the chimeric antigen receptor protein may be degenerate, i.e., multiple degenerate nucleic acid sequences that code for the same amino acid sequence are included within the scope of the present application. Degenerate nucleic acid codons that code for the corresponding amino acids are known in the art. The present application also relates to variants of the above polynucleotides that code for polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as the present application. The variants of the polynucleotides may be naturally occurring or non-naturally occurring isotopic variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, isotopic variants are substitution forms of polynucleotides that may include substitutions, deletions, or insertions of one or more nucleotides, but do not substantially change the function of the polypeptide they encode.

[0042] The MSLN-overexpressing cancers include, but are not limited to, malignant mesothelioma, ovarian cancer, trigeminal breast cancer, prostate cancer, lung cancer, gastric cancer, endometrial cancer, cervical cancer, bile duct cancer, and the like.

[0043] The MSLN overexpression refers to a case where the content of MSLN-related nucleic acid or protein, or other indicators detected by various detection methods, including but not limited to the molecular level and protein level, is higher than the normal level. [Brief description of the drawings]

[0044] [Figure 1] FIG. 1 is a structural schematic of the anti-MSLN CAR structure. [Diagram 2] FIG. 1 is a schematic diagram of the structure of the pLV300 lentiviral vector. [Diagram 3] This is a proliferation curve of total iNKT cells. [Figure 4]The percentage of CD8+CAR+iNKT cell subpopulations at different times during anti-MSLN CAR-iNKT cell culture. [Diagram 5] Percentage of CD4+CAR+iNKT cell subpopulations at different times during anti-MSLN CAR-iNKT cell culture. [Figure 6] Percentage of CD4-CD8-CAR+iNKT cell subpopulations at different times during anti-MSLN CAR-iNKT cell culture. [Figure 7] Killing effect of anti-MSLN CAR-iNKT against cholangiocarcinoma KMCH cells at various effector-target ratios. [Figure 8] IFN-γ content in cell culture supernatant 24 hours after co-culture of anti-MSLN CAR-iNKT cells with cholangiocarcinoma KMCH cells. [Figure 9] IL-2 content in cell culture supernatant 24 hours after co-culture of anti-MSLN CAR-iNKT cells and KMCH cholangiocarcinoma cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Example 1: Production of lentiviruses containing chimeric antigen receptor molecules After passage of HEK-293T cells and proliferation to 60%-70% confluence, expression vectors containing CAR molecules were transfected into HEK-293T cells together with packaging plasmids using pEI reagent, and fresh medium was replaced 4 hours after transfection. Cell culture supernatants were collected 48-72 hours after transfection. Lentiviruses containing CAR molecules were concentrated after packaging by ultracentrifugation of the supernatant. The concentrated lentiviruses were titered and frozen at -80°C until use.

[0046] Example 2 Effect of anti-MSLN CAR molecules on total iNKT cell proliferation and CAR-iNKT cell differentiation iNKT cells were isolated from human peripheral blood mononuclear cells using anti-iNKT mircrobeads and then plated in a 24-well plate at 2x10 cells per well. 5 The cells were inoculated and cultured for 48 hours with X-VIVO complete medium (containing 100IU / ml and 100ng / ml a-Galcer) per well, then collected from each well, centrifuged at 400xg for 5 minutes, the supernatant was discarded, and fresh X-VIVO complete medium was added to resuspend the cells, then re-seeded into a 24-well plate, and each well was infected with different lentiviruses containing anti-MSLN1 CAR, anti-MSLN2 CAR, and anti-MSLN3 CAR, respectively. After 24 hours, the cells in each well were collected into a centrifuge tube, centrifuged at 400xg for 5 minutes, counted, and counted at 5×10 5 The cells were cultured in fresh X-VIVO complete medium at 1000 cells / ml, and the medium was replaced every 48 hours. After culturing on days 7, 14, and 21, the cells were sampled and counted and analyzed by flow cytometry to reveal the expression of the chimeric antigen receptor molecule.

[0047] As a result, each anti-MSLN CAR molecule has an effect on the proliferation and differentiation of iNKT cells. When anti-MSLN1 CAR and anti-MSLN2 CAR are transduced into iNKT cells, they are more favorable for cell growth than the anti-MSLN3 CAR molecule (Figure 3). When anti-MSLN1 CAR and anti-MSLN2 CAR are transduced into iNKT cells, they are more favorable for the differentiation of CAR-iNKT cells into CD8+CAR+iNKT cells than the anti-MSLN3 CAR molecule (Figures 4-6).

[0048] Example 3: Effect of each anti-MSLN CAR molecule on the tumor-killing ability of CAR-iNKT cells When the CAR-iNKT cells in each group were cultured for 21 days, the effector cell to target cell ratios were 1:3, 1:1, and 3:1, respectively, and 0.33 × 10 5 CAR-iNKT cells, 1 x 10 5 CAR-iNKT cells, 3 x 105 CAR-iNKT cells, 1 x 10 5 After co-culturing with KMCH cells (KMCH is a mesothelin-expressing cholangiocarcinoma cell line; homogeneously mixed with the fluorescent dye CFSE and incubated for 10 minutes) for 6 hours, the culture supernatants from each group were collected and the fluorescence intensity in the supernatants was detected using a microplate reader (the stronger the killing ability of CAR-iNKT cells, the more CFSE released from within the KMCH cells and the greater the fluorescence intensity), and the killing efficiency of CAR-iNKT cells in each group was calculated (Figure 7). As a result, it was found that all three types of anti-MSLN CAR-iNKT cells could effectively kill KMCH cells, but that anti-MSLN1 CAR-iNKT cells had the strongest killing ability.

[0049] Example 4 Comparison of IFN-γ and IL-2 secreted by each anti-MSLN CAR-iNKT cell after co-culture with MSLN-expressing KMHC cells After culturing each group of CAR-iNKT cells until day 21, CAR-iNKT cells (3 × 10 per group) were cultured in a 3:1 effector cell to target cell ratio. 5 CAR-iNKT cells) at 1 × 10 5 After co-culture with KMHC cells in 0.5ml X-VIVO (without IL-2 and a-GalCer) for 24 hours, the cells were collected and centrifuged at 400xg for 5 minutes to collect the supernatant, and the contents of IFN-γ and IL-2 in the supernatant were 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 and tumor killing ability of anti-MSLN1 CAR-iNKT cells (Figures 8-9).

[0050] Of course, the above examples of the present application are merely illustrative examples for clarifying the present application, and do not limit the embodiments of the present application. Those skilled in the art can make various other changes and modifications based on the above description. It is not necessary and cannot be possible to cover all the embodiments here. Any obvious changes or modifications from the idea belonging to the present application are still included in the patent scope of the present application.

[0051] References: JPEG2025505477000002.jpg235136

Claims

1. A chimeric antigen receptor comprising, in sequence, an MSLN single-chain antibody, a spacer domain or hinge region, a transmembrane region, an intracellular costimulatory domain, and a signal region.

2. The chimeric antigen receptor of claim 1 , comprising, in sequence, a CD8α signal peptide, an MSLN single-chain antibody, a CD8α hinge region, a CD28 transmembrane region, a CD28 costimulatory domain, and a CD3ζ signal region.

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

5. The chimeric antigen receptor of 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 amino acid sequence of the CD8α signal peptide is SEQ ID NO.2, the amino acid sequence of the CD8α hinge region is SEQ ID NO.14, the amino acid sequence of the CD28 transmembrane region is SEQ ID NO.4, the amino acid sequence of the CD28 costimulatory domain is SEQ ID NO.6, and the amino acid sequence of the CD3ζ signal region is SEQ ID NO.

16.

7. The chimeric antigen receptor of claim 6, wherein the nucleotide sequence of the CD8α signal peptide is SEQ ID NO. 3, the nucleotide sequence of the CD8α hinge region is SEQ ID NO. 15, the nucleotide sequence of the CD28 transmembrane region is SEQ ID NO. 5, the nucleotide sequence of the CD28 costimulatory domain is SEQ ID NO. 7, and the nucleotide sequence of the CD3ζ signal region is SEQ ID NO.

17.

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

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

10. A vector carrying the chimeric antigen receptor according to any one of claims 1 to 9.

11. 11. The vector of 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.

13. The immune cell of claim 12, wherein the immune cell is a T cell, an NK cell, or an iNKT cell.

14. The immune cell of 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 manufacture of a cancer therapeutic agent.

16. A method for treating cancer, comprising using the chimeric antigen receptor, vector, or immune cell according to any one of claims 1 to 14.

17. 17. The use of claim 15 or the method of claim 16, wherein the cancer is an MSLN-overexpressing cancer.

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

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

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

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

22. The transduction system of claim 21 , wherein the transduction system is a lentiviral transduction system.

23. The method of claim 16, wherein the cancer is an MSLN-overexpressing cancer.

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

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

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