CAR-iNKT with high proliferation, viability and tumoricidal activity and its use

Abstract

The present application provides a chimeric antigen receptor (CAR) comprising a GPC3 antigen-binding domain, an ICD1, ICD2, or ICD3 intracellular signaling and stimulatory domain having the amino acid sequences of SEQ ID NOs. 29, 31, and 33, respectively, and an IL-15-IL-15a fusion protein having the amino acid sequence of SEQ ID NO. 7, which can effectively improve cell proliferation rate, survival time, and tumoricidal effect after transduction into immune cells, particularly iNKT cells. The present application also provides corresponding expression vectors, transduction systems, pharmaceutical uses, and the individual ICD1, ICD2, and ICD3 intracellular signaling and stimulatory domains and IL-15-IL-15a fusion proteins.

Description

[Technical field]

[0001] The present application belongs to the field of cancer immunotherapy. Specifically, the present application provides a chimeric antigen receptor comprising a GPC3 antigen binding domain, an intracellular signal stimulatory domain and an IL-15-IL-15α fusion protein, and also provides corresponding expression vectors, transduction systems, pharmaceutical applications, etc. [Background technology]

[0002] According to the 2018 World Cancer Report, there were 841,080 new cases of liver cancer worldwide, and 781,631 deaths from liver cancer[1]. Of these, there were 392,868 new cases of liver cancer in China, and 368,960 deaths from liver cancer[2], accounting for nearly half of the new cases and deaths from liver cancer worldwide.

[0003] Existing liver cancer treatments include surgical resection, liver transplantation, local treatment (RFA, TACE, TAE, HIFA, etc.), and systemic treatment (sorafenib, lenvatinib, etc.). Although surgical resection can achieve a cure, the majority of liver cancers are discovered before the optimal time for surgery, and the treatment method of surgical resection is not satisfactory. According to research, the median survival time of liver cancer patients in China is only 23 months, 60 months in Japan, 33 months in North America, 31 months in South Korea, 24 months in Europe, 11 months in Egypt, and only 3 months in other African countries [3]. Safer and more effective treatment methods are urgently needed for the treatment of liver cancer.

[0004] Chimeric antigen receptor technology has achieved remarkable therapeutic effects in the treatment of hematological malignancies, and Kymriah[4] and Yescarta[5] were approved by the FDA in 2017 for the treatment of B-cell acute lymphoblastic leukemia and diffuse large B lymphoma. GPC3 is highly expressed in 70% of hepatocellular carcinoma cells, but not in normal adult tissue cells.[6] GPC3 is also overexpressed in tumors such as hepatoblastoma, squamous cell lung cancer, testicular and ovarian yolk sac tumor, melanoma, and ovarian clear cell carcinoma.[7] This makes it a suitable target for targeted therapy using chimeric antigen receptor technology.

[0005] The GPC3 gene is located on the X chromosome and has 11 exons. The transcript is 2130 bp long, encodes 580 amino acids, and has a molecular weight of approximately 70 kDa. The GPC3 polypeptide contains one Furin restriction enzyme cleavage site, which cleaves the polypeptide into two fragments between Arg358 and Cys359, where the N-terminus is 40 kDa and the C-terminus is 30 kDa. These two subunits may be linked by one or more disulfide bonds. The N-terminal subunit can be further cleaved to give soluble GPC3 in the peripheral circulation. GPC3 can undergo heparin sulfate modification at Cys495 and Cys508. GPC3 Ser560 is anchored to the lipid layer of the cell membrane via phosphatidylinositol [8].

[0006] Under normal physiological conditions, GPC3 was widely expressed in various embryonic cell membranes, but not in normal adult liver tissues. Human GPC3 mutations cause Simpson Golabi Behmel syndrome (SGBS), manifesting as macrosomal neoplasms with multiple organ and bone dysplasia. GPC3 is anchored to the cell membrane and does not have an intracellular domain, but may interact with different growth factors, chemokines, and cytokines, thereby forming a concentration gradient on the cell membrane surface and further facilitating the binding of these ligands to their receptors [8].

[0007] Invariant natural killer T cells (iNKT) are a unique subpopulation of thymus-derived T cells that are CD1d-restricted and express surface receptors characteristic of both T and natural killer cell (NK) lineages, sharing common biological features with T 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 [9].

[0008] iNKT cells differentiate into at least three effector subpopulations in the thymus, some similar to CD4+ T helper cell subpopulations and some similar to innate lymphoid cells (ILCs) [10-12]. Functional iNKT cell subpopulations are distinguished by the expression of various cell surface markers and characteristic transcription factors. NKT1 cells are similar to Th1 cells and ILC1s, because both highly express the transcription factor T-bet and secrete IFN-γ after activation. NKT1 cells also exhibit greater cytotoxicity than other iNKT cell subpopulations. NKT1 cells differ from Th1 cells and ILC1s in that they produce IFN-γ upon TCR activation, as well as other factors such as IL-4. Cytokines secreted by NKT2 cells include IL-4 and IL-13, similar to Th2 cells. On the other hand, NKT17 cells are similar to Th17 cells in cytokine secretion [13-15].

[0009] Different iNKT cell subpopulations are enriched in different tissues: NKT1 cells are highly enriched in the liver, whereas NKT17 cells reside primarily in lymph nodes, skin and lungs, with a small number of cells in the spleen

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

[16] . In the peripheral lymph nodes, iNKT cells are rapidly activated and may play an important role in fighting pathogens

[17] .

[0010] iNKT cells in human blood are classified into DN iNKT (double-negative, DN) cells, CD4+ iNKT cells, and CD8+ iNKT (CD8αα or CD8αβ) [9]. In particular, CD8+ iNKT cells have only been found in humans so far. A study showed that when DN iNKT and CD8+ iNKT cells are activated, their IFN-γ secretion and cytotoxic function are significantly increased [9].

[0011] Current GPC3 chimeric antigen receptors, such as US 10731127B2 and CN 109468279 A, ​​which are close to this patent, use gene modification to make T cells express chimeric receptors for GPC3 antigen, of which CAR contains GPC3 antigen binding domain, transmembrane domain, costimulatory signaling domain and CD3ζ signaling domain, and shows killing effect on liver cancer cells bearing GPC3 antigen. However, considering that normal T cells cannot effectively infiltrate solid tumors, and the tumor microenvironment of solid tumors is hypoxic and more acidic, it is very unfavorable to the expansion and long-term survival of CAR-T cells, which seriously affects the therapeutic effect of CAR-T cells. Summary of the Invention [Problem to be solved by the invention]

[0012] To solve the above problems, the applicant utilizes the homing properties and non-specific killing function of iNKT cells to construct anti-GPC3-CAR-iNKT cells that can specifically kill liver cancer cells that have the GPC3 antigen by genetically modifying them to have a chimeric antigen receptor that can bind to the GPC3 antigen.

[0013] Genetic modification enabled anti-GPC3-CAR-iNKT cells to bind to CD8 + Anti-GPC3-CAR-iNKT cells and CD4 - CD8 - It is more advantageous to specifically express cytokines that differentiate anti-GPC3-CAR-iNKT cells, thereby enabling anti-GPC3-CAR-iNKT cells to exert their specific and non-specific killing functions against liver cancer cells.

[0014] By optimizing the combination of costimulatory domains, we can enhance the expansion and long-term survival of anti-GPC3-CAR-iNKT cells, reduce the apoptosis of anti-GPC3-CAR-iNKT cells, and fully exert the anti-tumor function of anti-GPC3-CAR-iNKT cells in patients.

[0015] By binding to downstream molecules such as RIP and TRADD, TRAF family molecules can ultimately activate the NF-κB pathway, promote cell proliferation, and prevent apoptosis. By referring to the motifs bound to TRAF family molecules, three intracellular signaling domains, ICD1, ICD2, and ICD3, are designed and constructed into CAR molecules, respectively, and the changes in the proliferation, cell subpopulations, and killing ability of CAR-iNKT cells against tumor cells are observed when the intracellular signaling domains are ICD1, ICD2, and ICD3, respectively.

[0016] IL-15 can promote T cell proliferation and differentiation into CD8+ T cell subpopulation, thereby improving the cytotoxic effect of T cells. However, for IL-15 to function, it must bind to IL-15Rα and then to IL-15Rγ, thereby stimulating cell proliferation and differentiation signals. In the present invention, IL-15-IL-15Rα fusion protein is constructed and co-expressed in CAR molecules to promote the differentiation of CAR-iNKT cells into CD8+ CAR-iNKT cell subpopulation, promote the proliferation of CAR-iNKT cells, and provide more favorable results for CAR-iNKT to kill tumor cells, meeting the needs of clinical application.

[0017] Compared with the prior art, the technical solution of the present application can improve the tumor killing efficiency, accelerate the proliferation rate of CAR-iNKT cells, shorten the in vitro cell culture cycle, increase the in vivo survival time of CAR-iNKT cells, greatly improve the therapeutic effect, reduce recurrence, and reduce toxicity and side effects. [Means for solving the problem]

[0018] In one aspect, the present application provides a chimeric antigen receptor comprising a GPC3 antigen-binding domain and an intracellular signal-stimulating domain.

[0019] Additionally, the chimeric antigen receptor further comprises an IL-15-IL-15α fusion protein.

[0020] Furthermore, the intracellular signal stimulatory domain is ICD1, ICD2 or ICD3, whose amino acid sequences are SEQ ID NOs. 29, 31 and 33, respectively.

[0021] Furthermore, the intracellular signal stimulatory domain is ICD3, the amino acid sequence of which is SEQ ID NO.33.

[0022] Furthermore, the amino acid sequence of said IL-15-IL-15α fusion protein is SEQ ID NO.7.

[0023] Furthermore, the GPC3 antigen-binding domain is GC33 ScFv comprising the amino acid sequences of SEQ ID NOs. 9 and 11.

[0024] Furthermore, the GPC3 antigen-binding domain is GC33 ScFv, whose amino acid sequence is SEQ ID NO.13.

[0025] Furthermore, the chimeric antigen receptor comprises, in sequence, GC33 ScFv, a hinge region, a transmembrane domain, a costimulatory signal domain, ICD1, ICD2 or ICD3, a CD3ζ signaling domain, and an IL-15-IL-15α fusion protein.

[0026] Furthermore, the amino acid sequences of the hinge region, transmembrane domain, costimulatory signal domain, and CD3ζ signaling domain are SEQ ID NOs. 21, 19, 23, and 35, respectively.

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

[0028] Furthermore, the immune cells are T cells, NK cells, or iNKT cells.

[0029] Furthermore, the immune cells are iNKT cells.

[0030] In another aspect, the present application provides a use of the chimeric antigen receptor or immune cell in the manufacture of a cancer therapeutic agent.

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

[0032] Furthermore, the cancer is a CPG3-overexpressing cancer.

[0033] Furthermore, the cancer is liver cancer.

[0034] In another aspect, the present application provides an expression vector for the chimeric antigen receptor.

[0035] Further, the expression vector comprises an ICD1, ICD2 or ICD3 nucleic acid sequence having the nucleotide sequence SEQ ID NO. 30, 32 or 34, respectively.

[0036] Additionally, the expression vector contains an IL-15-IL-15α fusion protein nucleic acid sequence, the nucleotide sequence of which is SEQ ID NO.8.

[0037] In another aspect, the present application provides a transduction system comprising the above-mentioned expression vector.

[0038] Furthermore, the transduction system may be a viral transduction system or a non-viral transduction system.

[0039] Furthermore, said transduction system is a lentiviral transduction system.

[0040] In another aspect, the present application provides an intracellular signal stimulatory molecule having an amino acid sequence of SEQ ID NO. 29, 31 or 33, respectively.

[0041] In another aspect, the present application provides a nucleic acid coding sequence of the intracellular signal stimulatory molecule, which is SEQ ID NO. 30, 32 or 34.

[0042] In another embodiment, the present application provides an IL-15-IL-15α fusion protein having an amino acid sequence of SEQ ID NO.7.

[0043] In another embodiment, the present application provides a nucleic acid coding sequence for the above-mentioned IL-15-IL-15α fusion protein, which is SEQ ID NO.8.

[0044] In another aspect, the present application provides a use of the intracellular signal stimulatory molecule or its nucleic acid coding sequence, or the IL-15-IL-15α fusion protein or its nucleic acid coding sequence, in the manufacture of a chimeric antigen receptor for treating cancer.

[0045] The types of CPG3-overexpressing cancers in the present invention can be known by those skilled in the art from the prior art prior to the filing date and subsequent research.

[0046] The optimized and modified intracellular signal stimulatory domains of the present invention (ICD1, ICD2, and ICD3 are used as examples in this invention) can increase the proliferation rate of CAR-iNKT, promote increased secretion of IFN-γ from CAR-iNKT, improve the tumoricidal effect of CAR-iNKT cells, alleviate the exhaustion state of CAR-iNKT cells, and extend the survival period of CAR-iNKT in vivo.

[0047] The IL-15-IL-15Rα fusion protein nucleic acid of the present invention can promote an increase in the proportion of CD8+CAR+iNKT cells, promote increased IFN-γ secretion from CAR-iNKT, improve the tumoricidal effect of CAR-iNKT cells, alleviate the exhaustion state of CAR-iNKT cells, and extend the in vivo survival period of CAR-iNKT.

[0048] The present invention includes a nucleic acid expression vector that contains one or more of the above elements and is used to construct a chimeric antigen receptor protein that is expressed on the surface of T cells, NK cells, or NKT cells. Various commercially available vectors may be selected as needed, or the vector may be constructed based on conventional techniques in the field of molecular biology. In one specific embodiment, the vector used by the present invention is a lentivirus plasmid vector pLV300. This plasmid is a fourth generation self-inactivating lentivirus vector system, which includes a total of four plasmids, namely, a packaging plasmid encoding Gag / Pol proteins, a packaging plasmid encoding Rev proteins, an envelope plasmid encoding VSV-G proteins, and an empty vector pLV300, and can be used to recombinantly transduce a target nucleic acid sequence, i.e., a nucleic acid sequence encoding a chimeric antigen receptor protein. In the vector pLV300, the expression of the chimeric antigen receptor protein is regulated by the pGK-300 promoter.

[0049] The present invention includes viruses comprising the above vectors, including but not limited to lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, etc. The viruses of the present invention include packaged infectious viruses, and also packaged viruses that contain the necessary components for packaged 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 invention. In one embodiment of the present invention, the virus is a lentivirus comprising the above pLV300-anti-GPC3 CAR recombinant vector.

[0050] The present invention includes transgenic T lymphocytes, NK cells or iNKT cells transduced with the nucleic acid of the present invention, or the recombinant plasmid containing the nucleic acid, or the viral system of the present invention containing the plasmid. Any conventional nucleic acid transduction method in the field, including non-viral and viral, can be used in the present invention. Transduction methods using non-viral systems include electroporation and transposon. Recently, the nucleofector nuclear transfection device developed by Amaxa can directly transduce foreign genes into the cell nucleus, thereby achieving efficient transduction of target genes. In addition, the transduction efficiency of transposon subsystems such as Sleeping Beauty transposon (Sleeping Beauty system) or PiggyBac transposon is significantly improved compared to normal electroporation, and it has already been reported that the nucleofector transfection device is used in combination with the SB transposon subsystem, which not only has high transduction efficiency but also can realize integration of target genes into specific sites. In one embodiment of the present invention, the transduction method of iNKT cells that realizes chimeric antigen receptor gene modification is a lentivirus transduction method. This method has the advantages of high transduction efficiency, stable expression of foreign genes, and shortening the in vitro culture time required for iNKT lymphocytes to reach clinical levels. The nucleic acid introduced by lentiviral transfection is expressed on the membrane surface of iNKT cells by transcription and translation. The transgenic iNKT cells of the present invention expressing a chimeric antigen receptor on their surface have been proven to have a highly specific killing effect (also called cytotoxicity) against tumor cells by in vitro cytotoxicity experiments on various different cultured tumor cells. Therefore, the nucleic acid of the present invention encoding a 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.

[0051] When a chimeric antigen receptor protein is constructed by binding the intracellular signal domain of the present invention to an extracellular target binding region, the extracellular binding region includes a receptor (ligand), a specific single-chain antibody scFv, a TCRm, or a VLR (human GPC3 target is explored as an example in the present invention) that specifically recognizes the corresponding target (including but not limited to an existing known target). The receptor (ligand), the specific single-chain antibody scFv, the TCRm, or the VLR can be produced by genetic engineering or chemical synthesis methods according to the sequences disclosed in the above documents.

[0052] The nucleic acid of the present invention 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 a coding strand or a non-coding strand. The nucleic acid codons of the present invention that code for the amino acid sequence of the chimeric antigen receptor protein can be degenerate, i.e., multiple degenerate nucleic acid sequences that code for the same amino acid sequence are all within the scope of the present invention. Degenerate nucleic acid codons that code for the corresponding amino acids are known in the art. The present invention 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 of the present invention. 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 be substitutions, deletions, or insertions of one or more nucleotides, but do not substantially change the function of the polypeptide they encode.

[0053] In the current CAR-T and CAR-NK solid tumor treatment technology, normal T cells and NK cells cannot effectively infiltrate solid tumors, so that NK cells are limited in their ex vivo expansion, and the therapeutic effect of CAR-T and CAR-NK on solid tumors is limited. In the present invention, the optimization of the intracellular signal domain can promote the efficient expansion of CAR-iNKT, reduce the exhaustion state of CAR-iNKT, and extend the in vivo survival time of CAR-iNKT, and the co-expression of IL-15-IL-15Rα can differentiate CAR-iNKT into CD8+CAR-iNKT and CD4-CD8-CAR-iNKT phenotypes, which are more favorable for tumor killing, thereby enhancing the killing effect of CAR-iNKT against tumor cells. This is an advantage that is not currently available in the CAR-T and CAR-NK technologies used to treat solid tumors. The combination of the intracellular signal domains and the co-expression of the IL-15-IL-15Rα fusion protein of the present invention enables the use of CAR-iNKT technology in the treatment of solid tumors. [Brief description of the drawings]

[0054] [Figure 1] FIG. 1 is a curve diagram of total iNKT cell proliferation. [Figure 2(A)] Percentage of CD8+CAR+iNKT cell subpopulation at each time point in CAR-iNKT cell culture. [Figure 2(B)] Percentage of CD8+CAR+iNKT cell subpopulation at each time point in CAR-iNKT cell culture. [Figure 2(C)] Percentage of CD4+CAR+iNKT cell subpopulations at each time point in CAR-iNKT cell culture. [Figure 2(D)] Percentage of CD4-CD8-CAR+iNKT cell subpopulation at each time point in CAR-iNKT cell culture. [Figure 3(A)] This shows the killing effect of anti-GPC3 CAR-iNKT against hepatoma cells Huh-7 at each effector / target ratio. [Figure 3(B)]This is the fold increase in the killing effect of each group of anti-GPC3 CAR-iNKT against liver cancer cells Huh-7 at each effector / target ratio, compared to anti-GPC3 CAR-iNKT whose intracellular signal domain is 4-1BB. [Figure 4(A)] IFN-γ content in cell culture supernatant after co-culture of anti-GPC3 CAR-iNKT cells and hepatoma cells for 24 hours. [Figure 4(B)] Each group of anti-GPC3 CAR-iNKT cells was inoculated with hepatoma cells Huh-7 at a ratio of 3:1 and co-cultured for 24 hours. The IFN-γ content in the cell culture supernatant was then compared to the anti-GPC3-CAR-iNKT group with the intracellular signal domain 4-1BB. This is the fold change in IFN-γ. [Diagram 5] This shows the expression status of LAG-3 after co-culture of anti-GPC3 CAR-iNKT cells and hepatoma cells for 24 hours. [Figure 6] FIG. 1 is a schematic diagram of the structure of anti-GPC3 CAR. [Figure 7] FIG. 1 is a schematic diagram of the structure of a lentiviral vector. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] [Table 1]

[0056] Example 1: Production of lentiviruses containing chimeric antigen receptor molecules After passage of HEK-293T cells and growth until the cells reached 60-70% confluence, the expression vector containing the CAR molecule was transfected into HEK-293T cells together with the packaging plasmid using pEI reagent, and 4 hours after transfection, fresh medium was replaced. 48-72 hours after transfection, cell culture supernatant was collected. The lentivirus containing the CAR molecule after packaging was concentrated by ultracentrifugation of the supernatant. The concentrated lentivirus was measured for viral titer and frozen at -80°C until use. For the specific nucleotide and amino acid sequences of each portion of the chimeric antigen receptor, refer to the sequence listing in which the name of each portion is given.

[0057] Example 2: The effect of constructing anti-GPC3 CAR with different intracellular signaling domains and expressing IL-15-IL15Rα fusion protein on total iNKT cell proliferation iNKT cells were isolated from human peripheral blood mononuclear cells using anti-iNKT mircrobeads and then plated in a 24-well plate at 2 × 10 cells per well. 5 After inoculation, cells were added to each well in X-VIVO complete medium (containing 100IU / ml and 100ng / ml α-Galcer) and cultured for 48 hours, after which the cells were 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, and the cells were again inoculated into a 24-well plate, and each well was infected with different lentiviruses, including 4-1BB, ICD1, ICD2, ICD3, 4-1BB-IL-15, ICD1-IL-15, ICD2-IL-15, and ICD3-IL-15CAR. After 24 hours, the cells in each well were collected in 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. On the 7th, 14th, and 21st days of culture, samples were taken and counted and detected by flow cytometry to verify the expression status of the chimeric antigen receptor molecule. As a result, the intracellular signal stimulatory domains ICD1, ICD3 and IL-15-IL-15α fusion protein could obviously promote the proliferation of iNKT cells (see Figure 1) and promote the increase of CD8+CAR+iNKT cells. On the 7th day of culture, the percentage of CD8+CAR+iNKT cells was the highest in each group of CAR-iNKT cells, and in the CAR-iNKT group with the intracellular signal domain ICD3, the percentage of CD8+CAR+iNKT cells could reach 20%-30% (IL-15-IL-15Rα co-expression or no IL-15-IL-15Rα expression). Thereafter, as the culture time increased, the percentage of CD8+CAR+iNKT cells gradually decreased; however, compared with CAR-iNKT cells with an intracellular signal-stimulatory domain of 4-1BB, the percentage of CD8+CAR+iNKT cells at the same time point was 2- to 3-fold higher in CAR-iNKT cells with intracellular signal-stimulatory domains ICD1 and ICD3 (Figures 2(a) and 2(b)). During the culture, the percentage of CD4+CAR+iNKT cells in each group gradually increased (Figure 2(c)), but the increase in the percentage of CD4+CAR+iNKT cells was smaller in CAR-iNKTs with intracellular signaling stimulatory domains ICD1 and ICD3 than in CAR-iNKT cells with intracellular signaling stimulatory domains 4-1BB. On the other hand, in each group of cells, the percentage of CD4-CD8-CAR+iNKT cells decreased with the culture time, and in the CAR-iNKT group with the intracellular signaling domain ICD3, the percentage of CD4-CD8-CAR+iNKT cells was the lowest among the CAR-iNKTs in each group at the same time point (Figure 2(d)).

[0058] Example 3: Effects of the presence or absence of expression of each intracellular signal domain and IL-15-IL-15Rα fusion protein on the tumor-killing ability of CAR-iNKT When the CAR-iNKT cells in each group were cultured for 21 days, the effector cell to target cell ratios were set at 1:3, 1:1, and 3:1, respectively, and 0.33 × 10 5 CAR-iNKT cells, 1 x 10 5 CAR-iNKT cells, 3 × 10 5 CAR-iNKT cells, 1 x 10 5After co-culturing 1000 Huh-7 cells for 6 hours, the fluorescence intensity in the culture supernatant of each group (the higher the killing ability of CAR-iNKT cells, the more CFSE released into Huh-7 cells and the higher the fluorescence intensity) was measured using a microplate reader, and the killing efficiency of CAR-iNKT cells of each group was calculated (Figure 3). CAR-iNKT cells with intracellular signal domains ICD1 and ICD3 clearly had improved killing effect against liver cancer cells Huh-7, and co-expression of IL-15-IL-15Rα clearly improved the killing effect of CAR-iNKT cells with intracellular signal domains ICD1 and ICD3 (Figure 3(a) and Figure 3(b)).

[0059] Example 4: Effect of the presence or absence of expression of each intracellular signal domain and IL-15-IL-15Rα fusion protein on IFN-γ secretion from CAR-iNKT cells After culturing the CAR-iNKT cells of each group until day 21, the effector cell to target cell ratio was set to 3:1, and the CAR-iNKT cells of each group (3 × 10 5 CAR-iNKT cells) at 1 × 10 5 After culturing with 1000 Huh-7 cells in 0.5 ml of X-VIVO (without IL-2 and α-GalCer) for 24 hours, the cells were collected and centrifuged at 400xg for 5 minutes, and the supernatant was taken and the IFN-γ content in the supernatant was measured by ELISA. As can be seen from the above, when the intracellular signal domain was ICD1 or ICD3, the IFN-γ secretion amount of anti-GPC3CAR-iNKT cells was obviously increased (Figure 4(A)), and the IFN-γ secretion ability of each group of CAR-iNKT cells could be improved by co-expressing IL-15-IL-15Rα fusion protein. In particular, when the intracellular signal domain of CAR-iNKT cells was ICD3, its IFN-γ secretion ability was about twice that of CAR-iNKT with the intracellular signal domain 4-1BB (Figure 4(B)).

[0060] Example 5: Effect of the presence or absence of expression of each intracellular signal domain and IL-15-IL-15Rα fusion protein on the expression of immune checkpoint LAG-3 in CAR-iNKT cells Studies have shown that in iNKT cells, increased expression of LAG3 (but not PD-1) impairs the ability of iNKT cells to secrete IFN-γ. After culturing each group of CAR-iNKT cells for 21 days, the effector cell to target cell ratio was set to 1:1, and each group of CAR-iNKT cells (1 × 10 5 CAR-iNKT cells) at 1 × 10 5 After culturing with Huh-7 cells in 0.5 ml of X-VIVO (without IL-2 and α-GalCer) for 48 hours, the cells were centrifuged at 400xg for 5 minutes, the supernatant was discarded, the cells were collected, and the LAG-3 antibody was labeled, and the LAG3 expression status on the surface of CAR-iNKT in each group was detected by flow cytometry. When the intracellular signal domain was ICD1 or ICD3, the percentage of cells expressing LAG-3 decreased, and when IL-15-IL-15Rα was co-expressed, the percentage of cells expressing LAG-3 decreased (Figure 5). This indicates that the expression of the co-inhibitory molecule LAG3 can be reduced by co-expressing the IL-15-IL-15Rα fusion protein using ICD1 and ICD3 as intracellular signaling domains, which may improve the IFN-γ expression ability of CAR-iNKT cells. This is consistent with the fact that the IFN-γ secretion of CAR-iNKT cells can be increased by co-expressing the IL-15-IL-15Rα fusion protein using ICD1 and ICD3 as intracellular signaling domains as mentioned above (Figure 4(A)), thereby improving the killing ability of CAR-iNKT cells against tumor cells (Figure 3(A)).

[0061] References: 1. https: / / gco.iarc.fr / today / data / factsheets / populations / 900-world-fact-sheets.pdf 2. https: / / gco.iarc.fr / today / data / factsheets / populations / 160-china-fact-sheets.pdf 3. Park, J.W. et al. Global patterns of hepatocellular carcinoma management from diagnosis to death: the bridge study. Liver Int. 35, 2155-2166 (2015). 4. https: / / www.fda.gov / media / 106989 / download 5. https: / / www.fda.gov / media / 108458 / download 6. Baumhoer D, Tornillo L, Stadlmann S, et al. Glypican 3expression in human nonneoplastic, preneoplastic, and neoplastic tissues: a tissue microarray analysis of 4,387tissue samples. Am J Clin Pathol. (2008) 129:899.doi:10.1309 / HCQWPWD50XHD2DW6. 7. Li N, Gao W, Zhang YF, Ho M. Glypicans as Cancer Therapeutic Targets. Trends Cancer. 2018Nov;4(11):741-754.doi:10.1016 / j.trecan.2018.09.004. 8. Guo M, Zhang H, Zheng J, et al. Glypican-3: A New Target for Diagnosis and Treatment of Hepatocellular Carcinoma. J Cancer. 2020 Feb 3; 11(8):2008-2021.doi:10.7150 / jca.39972. 9. Crosby, C.M. and M. Kronenberg, Tissue-specific functions of invariant natural killer T cells. Nat Rev Immunol, 2018.18(9):p.559-574. 10. Bennstein, S.B., Unraveling Natural Killer T-Cells Development. Front Immunol, 2017.8:p.1950. 11. Georgiev, H., et al., Distinct gene expression patterns correlate with developmental and functional traits of iNKT subsets. Nat Commun, 2016.7:p.13116. 12. 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. 13. 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. 14. Verykokakis, M. and B.L. Kee, Transcriptional and epigenetic regulation of innate-like T lymphocyte development. Curr Opin Immunol, 2018. 51: p. 39-45. 15. 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. 16. Lee, Y.J., et al., Tissue-Specific Distribution of iNKT Cells Impacts Their Cytokine Response. Immunity, 2015. 43(3): p. 566-78. 17. Gaya, M., et al., Initiation of Antiviral B Cell Immunity Relies on InnateSignals from Spatially Positioned NKT Cells. Cell, 2018. 172(3): p. 517-533 e20.

Claims

1. A polynucleotide encoding a chimeric antigen receptor and an IL-15-IL-15Rα fusion protein, comprising: The chimeric antigen receptor comprises a GPC3 antigen binding domain and an intracellular signal domain, and the intracellular signal domain comprises a CD28 costimulatory signal domain, an ICD1 having an amino acid sequence of SEQ ID NO. 29 or an ICD3 having an amino acid sequence of SEQ ID NO. 33, and a CD3 zeta signaling domain.

2. The polynucleotide of claim 1, wherein the intracellular signaling domain is ICD3, the amino acid sequence of which is SEQ ID NO.

3. The polynucleotide of claim 1, wherein the amino acid sequence of the IL-15-IL-15Rα fusion protein is SEQ ID NO.

4. The polynucleotide of claim 1, wherein the GPC3 antigen-binding domain is GC33 ScFv comprising the amino acid sequence of SEQ ID NO.

5. The polynucleotide of claim 4, wherein the GPC3 antigen-binding domain is GC33 ScFv having an amino acid sequence comprising SEQ ID NO.

13.

6. The polynucleotide of any one of claims 1 to 5, wherein the chimeric antigen receptor comprises, in sequence, GC33 ScFv, a hinge region, a transmembrane domain, a costimulatory signal domain, ICD1 or ICD3, and a CD3ζ signaling domain.

7. The amino acid sequences of the hinge region, the transmembrane domain, the costimulatory signal domain, and the CD3ζ signaling domain are SEQ ID NOs. 21, 19, 23, and 35, respectively.

8. An immune cell transduced with the polynucleotide according to any one of claims 1 to 7, wherein the immune cell is an iNKT.

9. Use of the polynucleotide according to any one of claims 1 to 7 or the immune cell according to claim 8 in the manufacture of a cancer therapeutic agent, wherein the cancer is liver cancer.

10. The use according to claim 9, wherein the cancer is a GPC3-overexpressing cancer.

11. An expression vector for the chimeric antigen receptor according to any one of claims 1 to 7, An expression vector comprising a nucleic acid sequence of ICD1, the nucleotide sequence of which is SEQ ID NO. 30, or a nucleic acid sequence of ICD3, the nucleotide sequence of which is SEQ ID NO.

34.

12. 12. The expression vector of claim 11, further comprising a nucleic acid sequence for an IL-15-IL-15Rα fusion protein, the nucleotide sequence of which is SEQ ID NO.

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