Method for preparing nk cells to reverse tumor microenvironment inhibitory signals and applications thereof

Abstract

The application provides an immune cell preparation method capable of reversing tumor microenvironment immunosuppression signals and application thereof. The method for reversing the inhibitory signals is to replace the intracellular segment of a TIGIT receptor with a 4-1BB costimulatory domain and an IL-18R and a CD3 intracellular segment, and the immune cells expressing the chimeric antigen receptor recognize CD155 in a tumor microenvironment through TIGIT, avoid loss of function or exhaustion of the immune cells, and stimulate the immune cells to exert stronger tumor killing activity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and specifically relates to a preparation of CAR-immune cells and its use in tumor treatment, more specifically, the present application relates to a chimeric antigen receptor for reversing tumor microenvironment inhibitory signals, a preparation method of an expression vector, a transgenic immune cell and a pharmaceutical composition and its use. BACKGROUND

[0002] In recent years, immunotherapy has made remarkable progress in the field of tumor treatment, especially the immune checkpoint blocking therapy represented by anti-CTLA-4 and anti-PD-1 or PD-L1 antibodies. By blocking the binding of T cell surface inhibitory receptors to their ligands, blocking the transmission of inhibitory signals, correcting the immune suppression mediated by the immunosuppressive microenvironment, restoring the anti-tumor ability of T cells in the tumor microenvironment, and achieving a high response rate in the treatment of various metastatic advanced cancers (including metastatic melanoma, non-small cell lung cancer, renal cancer, etc.), making many advanced cancer patients who have lost the opportunity for treatment and are ineffective for radiotherapy and chemotherapy have the hope of re-treatment.

[0003] However, not all malignant tumor patients are effective for PD-1, PD-L1 or CTLA-4 blocking therapy, and only 10%-30% of patients treated with anti-PD-1 or PD-L1 antibodies show long-term and durable response, and most of the population lack response, and improving clinical response and overcoming drug resistance are the biggest challenges in this field. Exploring the mechanism of tumor non-response to immune checkpoint blocking therapy and finding other immune regulatory checkpoints affecting immune cell function have become urgent problems in the field of tumor immunotherapy. At present, more and more immune checkpoint molecules have been discovered and developed for application.

[0004] T cell immunoglobulin and ITIM domain protein (TIGIT) is an important immune checkpoint, mainly expressed on the surface of natural killer (NK) cells, activated CD8+ T and CD4+ T cells, regulatory T cells (Tregs) and follicular helper T cells (Tfh). The ligands recognized by TIGIT, CD155 and CD112, are mainly expressed on monocytes, macrophages, dendritic cells (DCs), T cells, B cells and many non-hematopoietic cells (including tumor cells of different histological types). The affinity of TIGIT binding to CD155 is significantly higher than that of its competitive receptors CD226 and CD96. After TIGIT binds to its ligand, it transmits an inhibitory signal to T cells or NK cells. Studies have found that TIGIT is highly expressed in T cells or NK cells of various malignant tumors such as non-small cell lung cancer, melanoma, head and neck squamous cell carcinoma, colorectal cancer, glioblastoma, gastric cancer, liver cancer, multiple myeloma, acute myeloid leukemia and follicular lymphoma. The ligand CD155 of TIGIT is highly expressed in various solid tumors and hematological tumors, including liver cancer, pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, ovarian cancer, head and neck cancer, breast cancer, lymphoma, leukemia, etc. The expression abundance of TIGIT and CD155 is closely related to the prognosis of patients. The current treatment method is to effectively restore the function of T cells or NK cells by developing TIGIT monoclonal antibodies, thereby exerting a killing effect on tumors. Some biotechnology / pharmaceutical companies (such as Roche, Beizhengzhizhou, Fuhonghanlin, etc.) are committed to developing anti-TIGIT antibodies, and related products are in different clinical development stages. However, clinical trial results show that the therapeutic effect of TIGIT antibody alone is not ideal, and needs to be combined with PD-1 or PD-L1 antibody to improve its effect. The anti-TIGIT monoclonal antibody developed by Roche and Beizhengzhizhou combined with PD-L1 antibody for the treatment of non-small cell lung cancer has entered the III phase of clinical trials.

[0005] Therefore, TIGIT and its ligand CD155 are expected to become a new target for tumor immunotherapy, and blocking the transmission of TIGIT inhibitory signals on the surface of immune cells has great application prospects in tumor immunotherapy. SUMMARY

[0006] The present application aims to at least partially solve one of the technical problems in the related art.

[0007] In order to improve the killing effect of tumors and reduce the side effects of external drugs, the present application provides an immune cell preparation method capable of selectively reversing the inhibitory signal of immune checkpoint TIGIT and its application in the treatment of malignant tumors.

[0008] The immune cell preparation method for reversing the inhibitory signal of the immune checkpoint TIGIT provided by the present application is to construct a chimeric antigen receptor targeting TIGIT ligand, the extracellular segment of which is a TIGIT extracellular segment, the transmembrane region of which is a TIGIT transmembrane region, and the intracellular segment of which is a 4-1BB co-stimulatory signal domain, an IL18RB intracellular segment and a CD3 intracellular region. The structure can convert the inhibitory signal of TIGIT recognizing its ligand CD155 into a 4-1BB, IL-18R and CD3 mediated co-stimulatory activation signal, thereby converting the inhibitory signal received by the NK cell from the tumor microenvironment into an activation signal, effectively enhancing the tumor killing activity of the immune cell, and can be applied to the treatment of various malignant tumors with high expression of TIGIT ligand. The inventors found that when the intracellular segment is a 4-1BB co-stimulatory signal domain, an IL18RB intracellular segment and a CD3 intracellular region, the activated co-stimulatory signal is stronger than that when the intracellular segment is a 4-1BB co-stimulatory signal domain, an IL21R intracellular segment and a CD3 intracellular region, and can stimulate the NK cell to have stronger killing activity on TIGIT ligand positive cells.

[0009] Therefore, in a first aspect of the present application, the present application provides a chimeric antigen receptor. According to an embodiment of the present application, the chimeric antigen receptor comprises: an extracellular region comprising a TIGIT extracellular segment; a transmembrane region comprising a TIGIT transmembrane region and embedded into the cell membrane; and an intracellular region comprising a 4-1BB co-stimulatory factor domain, an IL18RB intracellular segment and a CD3 intracellular signal segment, wherein the C-terminal of the extracellular region is connected to the N-terminal of the transmembrane region, and the C-terminal of the transmembrane region is connected to the N-terminal of the intracellular region. The chimeric antigen receptor described in the embodiments of the present application is introduced into immune cells for expression. The immune inhibitory signal originally received by the T cell or NK cell is converted into an activation signal, effectively improving the tumor killing effect of the immune cell.

[0010] According to an embodiment of the present application, the chimeric antigen receptor described above can further comprise at least one of the following additional technical features:

[0011] According to an embodiment of the present application, the C-terminal of the 4-1BB co-stimulatory factor domain in the chimeric antigen receptor is connected to the N-terminal of the IL18RB intracellular segment, and the C-terminal of the IL18RB intracellular segment is connected to the N-terminal of the CD3 intracellular signal segment. Furthermore, the immune activation effect of the immune cell expressing the chimeric antigen receptor is stronger.

[0012] According to an embodiment of the present application, the extracellular region can bind to a ligand, and the ligand comprises at least one of the PVR family members.

[0013] According to an embodiment of the present application, the PVR family members comprise CD155 and CD112.

[0014] According to an embodiment of the present application, the PVR family member is CD155.

[0015] According to an embodiment of the present application, the TIGIT extracellular segment has an amino acid sequence as shown in SEQ ID NO. 1.

[0016] According to an embodiment of the present application, the TIGIT transmembrane region has an amino acid sequence as shown in SEQ ID NO. 2.

[0017] According to an embodiment of the present application, the 4-1BB co-stimulatory factor domain has an amino acid sequence as shown in SEQ ID NO. 3.

[0018] According to an embodiment of the present application, the IL18RB intracellular segment has an amino acid sequence as shown in SEQ ID NO. 4.

[0019] According to an embodiment of the present application, the CD3 zeta intracellular signal segment has an amino acid sequence as shown in SEQ ID NO: 5.

[0020] According to an embodiment of the present application, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 6.

[0021] MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQV NWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGT YTGRIFLEVLESSVAEHGARFQIPL (SEQ ID NO. 1)

[0022] LGAMAATLVVICTAVIVVVALTR (SEQ ID NO. 2)

[0023] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO. 3)

[0024] MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSALLYRHWIEIVLLYRTYQSKDQTLGDKKDFDAFVSYAKWSSFPSEATSSLSEEHLALSLFPDVLENKYGYSLCLLERDVAPGGVYAEDIVSIIKRSRRGIFILSPNYVNGPSIFELQAAVNLALDDQTLKLILIKFCYFQEPESLPHLVKKALRVLPTVTWRGLKSVPPNSRFWAKMRYHMPVKNSQGFTWNQLRITSRIFQWKGLSRTETTGRSSQPKEW (SEQ ID NO. 4)

[0025] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO. 5)

[0026] MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSALLYRHWIEIVLLYRTYQSKDQTLGDKKDFDAFVSYAKWSSFPSEATSSLSEEHLALSLFPDVLENKYGYSLCLLERDVAPGGVYAEDIVSIIKRSRRGIFILSPNYVNGPSIFELQAAVNLALDDQTLKLILIKFCYFQEPESLPHLVKKALRVLPTVTWRGLKSVPPNSRFWAKMRYHMPVKNSQGFTWNQLRITSRIFQWKGLSRTETTGRSSQPKEWRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO. 6)

[0027] In a second aspect, the present application provides a nucleic acid molecule. According to embodiments of the present application, the nucleic acid molecule encodes the chimeric antigen receptor of the first aspect of the present application. The nucleic acid molecule according to embodiments of the present application, when expressed in an immune cell, can convert an inhibitory signal mediated by tumor cells into an activating signal.

[0028] According to embodiments of the present application, the nucleic acid molecule can further comprise at least one of the following additional technical features:

[0029] According to embodiments of the present application, the nucleic acid molecule has a nucleotide sequence as set forth in SEQ ID NO: 7.

[0030]

[0031] In a third aspect, the present application provides an expression vector. According to an embodiment of the present application, the expression vector carries the nucleic acid molecule of the second aspect of the present application. Wherein, the purpose of constructing the expression vector is to express the target gene sequence.

[0032] According to an embodiment of the present application, the expression vector can further comprise at least one of the following additional technical features:

[0033] According to an embodiment of the present application, the expression vector further comprises a promoter.

[0034] According to an embodiment of the present application, the promoter is operably linked to the nucleic acid molecule of the second aspect of the present application.

[0035] According to an embodiment of the present application, the promoter is selected from at least one of CMV, EF-1, RSV.

[0036] According to an embodiment of the present application, the expression vector is a non-pathogenic viral vector.

[0037] According to an embodiment of the present application, the non-pathogenic virus is selected from retrovirus, lentivirus and adeno-associated virus, preferably, the non-pathogenic virus is lentivirus.

[0038] In a fourth aspect, the present application provides a lentivirus vector. According to an embodiment of the present application, the lentivirus vector has the nucleotide sequence shown in SEQ ID NO: 8. Wherein, after the lentivirus vector is introduced into the recipient cell, the expression of the auxiliary activation signal in the immune cell can be activated.

[0039]

[0040] In a fifth aspect of the present application, the present application provides a transgenic immune cell. According to embodiments of the present application, the transgenic immune cell carries the chimeric antigen receptor of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, and the lentiviral vector of the fourth aspect of the present application. Wherein, the expression of the obtained transgenic immune cell can effectively enhance the killing ability to malignant tumors.

[0041] In a sixth aspect of the present application, the present application provides a CAR- immune cell. According to embodiments of the present application, the CAR- immune cell carries the chimeric antigen receptor of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, and the lentiviral vector of the fourth aspect of the present application.

[0042] According to embodiments of the present application, the CAR- immune cell includes at least one selected from the group consisting of NK-92 cells, peripheral blood NK cells, umbilical cord blood NK cells, iPSCs, CAR-NK cells, CAR-T cells, CAR-NKT cells, and CAR-γδT cells.

[0043] In a seventh aspect of the present application, the present application provides a pharmaceutical composition. According to embodiments of the present application, the pharmaceutical composition includes the chimeric antigen receptor of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, the lentiviral vector of the fourth aspect of the present application, the transgenic immune cell of the fifth aspect of the present application, and the CAR- immune cell of the sixth aspect of the present application.

[0044] According to embodiments of the present application, the pharmaceutical composition further includes a pharmaceutically acceptable excipient.

[0045] In an eighth aspect of the present application, the present application provides a use of a pharmaceutical composition in the preparation of a medicament. According to embodiments of the present application, the chimeric antigen receptor, the nucleic acid molecule, the expression vector, the lentiviral vector, the transgenic immune cell, the CAR- immune cell, and the pharmaceutical composition are used for treating or preventing solid tumors or hematological tumors.

[0046] According to embodiments of the present application, the solid tumors include at least one selected from the group consisting of tangible tumors occurring in organs, including pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, cholangiocarcinoma, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, brain glioma, kidney cancer, breast cancer, prostate cancer, melanoma, etc.

[0047] According to an embodiment of the present application, the hematological tumor comprises at least one selected from the group consisting of acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, and the like in blood cells and hematopoietic system. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 is a CAR activation signal structure mode diagram targeting CD155 according to embodiment 1 of the present application, wherein the TIGIT extracellular segment represents a nucleotide sequence encoding a receptor that binds to CD155, the TIGIT transmembrane segment represents a nucleotide sequence encoding a TIGIT transmembrane segment domain, the 4-1BB intracellular segment represents a nucleotide sequence encoding a 4-1BB co-stimulatory factor domain, the IL18RB intracellular segment represents a nucleotide sequence encoding an IL18RB domain, and the CD3ζ intracellular segment represents a nucleotide sequence encoding a CD3ζ intracellular segment; wherein TIGIT-CAR-2 is a CAR activation signal structure mode diagram targeting CD155 as a control, which is different from the TIGIT-CAR structure of the present application in that the intracellular segment is a 4-1BB intracellular segment, an IL-21 receptor (IL-21R) intracellular segment, and a CD3ζ intracellular segment;

[0049] Figure 2 is a detection result chart of the expression level of TIGIT ligand CD155 in tumor cells according to embodiment 2 of the present application, wherein the shaded peak is the isotype antibody staining control group, and the black solid line is the CD155 antibody staining group;

[0050] Figure 3 is a detection result chart of the in vitro killing ability of TIGIT-CAR-NK cells according to embodiment 2 of the present application;

[0051] Figure 4 is a detection result chart of the in vitro killing-related degranulation of NK cells according to embodiment 2 of the present application;

[0052] Figure 5 is a detection result chart of the secretion level of IFN-γ and TNF-α of TIGIT-CAR-NK cells according to embodiment 2 of the present application;

[0053] Figure 6 is a detection result chart of the fluorescence experiment of the difference treatment group for inhibiting tumor growth according to embodiment 3 of the present application;

[0054] Figure 7 is a statistical result chart of the fluorescence intensity of the difference treatment group according to embodiment 3 of the present application; DETAILED DESCRIPTION

[0055] Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein like symbols generally indicate like items, or items with the same or similar functions throughout the figures. The embodiments described below are examples of implementations and are not intended to be limiting.

[0056] In the description of the present application, the explanations and descriptions of the terms related herein are only for the convenience of understanding the solutions and cannot be regarded as the limitation of the protection solutions of the present application.

[0057] In the present application, the term "comprising" or "including" is an open-ended expression, i.e., including the indicated content of the present application, but not excluding other aspects.

[0058] In the present application, the term "optionally", "optional" or "option" generally means that the event or condition described subsequently can but does not necessarily occur, and the description includes the case where the event or condition occurs and the case where the event or condition does not occur.

[0059] In the present application, "operably linked" means that the exogenous gene is linked to the vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, viral vectors, plasmids, bacteriophages, etc. After the expression vector according to some specific embodiments of the present application is introduced into a suitable recipient cell, the expression of the nucleic acid molecule described above can be effectively realized under the mediation of the regulation system, and then the in vitro mass acquisition of the protein encoded by the nucleic acid molecule is realized.

[0060] The "chimeric antigen receptor" described in the present application refers to an artificial receptor fragment expressed on the cell membrane surface, which includes an extracellular region, a transmembrane region and an intracellular region, the extracellular region can specifically bind to the corresponding ligand or antigen, causing the activation of the immune stimulating factor contained in the intracellular region.

[0061] The present application constructs a transgenic immune cell expressing a chimeric antigen receptor, wherein the chimeric antigen receptor targets at least one of the PVR family members. The chimeric antigen receptor can express an activation signal to enhance the tumor killing activity of CAR immune cells and be applied to the treatment of solid tumors and hematological tumors.

[0062] Embodiments of the present application will be described in more detail below, examples of which are shown in the attached drawings. The embodiments described below are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0063] It should be noted that the "plasmid" described in the following implementation has the same meaning as "vector" and can be used interchangeably.

[0064] Example 1: Preparation of TIGIT-CAR-NK cells

[0065] 1.1 Construction of pCDH-EF1-TIGIT-CAR-T2A-copGFP lentiviral plasmid

[0066] The TIGIT-CAR vector sequence designed by the application comprises an extracellular segment and a transmembrane segment of a TIGIT receptor, a 4-1BB costimulatory signal domain, an IL18RB intracellular segment, and a CD3 zeta intracellular region. The schematic diagram of the structure of the genetic element is shown in Figure 1 .

[0067] The full gene-synthesized TIGIT-CAR fragment is inserted into the lentiviral vector pCDH-EF1-MSC-T2A-copGFP vector through enzyme cutting sites XbaI and BamHI. After colony PCR identification and sequence verification, it is indicated that the pCDH-EF1-TIGIT-CAR-T2A-copGFP plasmid construction is successful.

[0068] 1.2 Packaging of lentivirus and concentration of virus liquid

[0069] 5×10 6 Inoculate in a 10 cm culture dish, add 10 mL of DMEM medium, and culture in a 37°C, 5% CO2 incubator overnight. When the cell density reaches 80%, replace 10 mL of fresh DMEM medium and continue to culture in the incubator.

[0070] Preparation of lentivirus packaging system: 6 μg psPAX2 plasmid, 3 μg pMD2.G plasmid and 6 μg pCDH-EF1-TIGIT-CAR-T2A-copGFP plasmid were added to 250 μL of serum-free DMEM medium, mixed uniformly to prepare the DNA mixture; 15 μL PEIpro was added to 235 μL of serum-free DMEM medium, mixed uniformly to prepare the PEIpro mixture. The PEIpro mixture was added to the DNA mixture at one time, and the mixture was left to mix uniformly, incubated at room temperature for 15 min. The mixture was added to the 293T cell culture dish. After 24 h of culture, the medium was changed, and the culture dish was placed back in a 37°C, 5% CO2 incubator. The cell supernatant was collected after 48 h, centrifuged at 400 x g for 5 min to remove cell debris, and the supernatant was filtered with a 0.45 μm filter into a new 50 ml centrifuge tube. Add 5x PEG8000 solution, mix well by inverting the centrifuge tube, and place in a 4°C refrigerator overnight. Centrifuge at 4°C, 4000 x g for 20 min, discard the supernatant, resuspend the virus precipitate with an appropriate amount of serum-free DMEM medium, and aliquot into EP tubes and store in a -80°C refrigerator.

[0071] 1.3 Lentivirus infection of human NK cells

[0072] Take NK-92 cells in the logarithmic growth phase (purchased from ATCC), resuspend the cells in 2 mL of α-MEM medium, and adjust the cell density to 5 x 10 5 cells / mL. In a 24-well plate, inoculate 5 x 10 5 NK-92 cells, 1 mL of virus concentrate, and 1 μL of protamine (purchased from Solabio, final concentration 8 μg / mL). Incubate in a 37°C, 5% CO2 incubator. After 24 h, observe the cell state, change the medium, transfer the infected cells to an EP tube, centrifuge at 100 x g for 5 min, resuspend the cells with a small amount of fresh α-MEM medium, and transfer the cells to a cell culture bottle with 10 mL of fresh α-MEM medium and IL-2 (final concentration 200 IU / mL) for further culture. After the cells are expanded, transfer the cells to a flow tube, resuspend the cells with 3 mL of 1x PBS solution, centrifuge at 100 x g for 5 min, discard the supernatant, vortex the cell pellet, and repeat once. The infected NK-92 cells are sorted by flow cytometry to obtain GFP-positive TIGIT-CAR-NK cells for subsequent experiments.

[0073] Example 2: Biological function identification of TIGIT-CAR-NK cells

[0074] 2.1 Expression of TIGIT ligand CD155 on tumor cells

[0075] It has been shown that TIGIT ligand CD155 is highly expressed on the surface of ovarian cancer tissues and various ovarian cancer cells. The inventors detected the expression of CD155 on the surface of human ovarian cancer cell lines HO8910 and SKOV-3 by flow cytometry. The results showed that both HO8910 and SKOV-3 cells highly expressed CD155 molecules Figure 2

[0076] 2.2 In vitro killing ability of TIGIT-CAR-NK cells

[0077] NK-92, TIGIT-CAR-NK-92 and TIGIT-CAR2-NK-92 were used as effector cells, and ovarian cancer cell line HO8910 was used as target cells. The effector-to-target ratio was set at 5:1, 2.5:1 and 1.25:1. The effector cells and target cells were co-incubated for 4 h, and the LDH (lactate dehydrogenase) release method was used to detect the killing efficiency of effector cells on target cells. The results showed that the killing efficiency of TIGIT-CAR cells on H08910 cells was significantly higher than that of NK-92 and TIGIT-CAR2 (amino acid sequence as shown in SEQ ID NO: 10) cells Figure 3 ) groups. The above results show that TIGIT-CAR gene modification can significantly improve the killing ability of NK cells on CD155 positive tumor cells, and the TIGIT-CAR structure with 4-1BB intracellular segment and CD3ζ intracellular segment is superior to the CAR structure with 4-1BB intracellular segment, IL-21 receptor (IL-21R, amino acid sequence as shown in SEQ ID NO: 9) intracellular segment and CD3ζ intracellular segment, which can stimulate NK cells to have stronger killing ability on CD155 positive tumor cells.

[0078] SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS (SEQ ID NO: 9)

[0079] ​MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGADFQIPLLGAMAATLVVICTAVIVVVALTRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQASRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 10)

[0080] In addition, the degranulation related to NK cell killing was detected. After different effector cells were co-incubated with HO8910 cells for 4 h, NK cells were collected in a flow tube, and the expression of CD107a, granzyme B and perforin on NK cells was detected by flow cytometry, respectively. The results showed that the expression levels of CD107a and secreted granzyme B on TIGIT-CAR cells were significantly higher than those of NK-92 group after co-incubation with HO8910 cells, and the expression level of perforin had no statistical difference. Figure 4 Further verification showed that TIGIT-CAR modification could significantly improve the degranulation level (expression level of CD107a, granzyme and perforin) and killing function of NK cells on CD155 positive tumor cells.

[0081] 2.3 Secretion level of IFN-γ and TNF-α of TIGIT-CAR-NK cells

[0082] The secretion ability of IFN-γ and TNF-α of TIGIT-CAR-NK-92 cells was detected by flow cytometry. After the NK cells were co-incubated with ovarian cancer HO8910 cells for 4h, the NK cells were collected in a flow tube, and the secretion level of IFN-γ and TNF-α of the NK cells was detected by flow cytometry after fixation and membrane rupture treatment. The results showed that the secretion levels of IFN-γ and TNF-α of TIGIT-CAR-NK-92 cells were significantly higher than those of NK-92 cells Figure 5 ), indicating that TIGIT-CAR can significantly improve the secretion ability of IFN-γ and TNF-α of NK cells when contacting with CD155 positive tumor cells.

[0083] Example 3: Anti-tumor ability of TIGIT-CAR-NK cells in vivo

[0084] Luciferase-labeled ovarian cancer HO8910 cells were used to establish an ovarian cancer peritoneal metastasis model. Five-week-old female highly immunodeficient NCG mice were selected for peritoneal tumor injection, and each mouse was injected with 2x10 5 luciferase-labeled HO8910 cells into the peritoneal cavity. On the second day after tumor inoculation, NK cell infusion therapy was performed. The mice were randomly divided into a control group, an NK-92 treatment group, and a TIGIT-CAR-NK-92 treatment group. The treatment group mice were injected with 5x10 6 NK cells into the peritoneal cavity, and the control group was injected with the same volume of 1xPBS solution, once every other week, for a total of 3 times. IL-2 (50000IU / mouse) was injected into the peritoneal cavity every 3 days. The tumor size was observed by small animal live imaging technology, and then the tumor growth curve was drawn.

[0085] The results showed that compared with the tumor control group, the NK-92 and TIGIT-CAR-NK-92 treatment groups could significantly inhibit the growth of tumors, and the TIGIT-CAR-NK-92 treatment group had the best effect, with a significantly smaller tumor fluorescence signal intensity observed by live imaging technology than the NK-92 treatment group Figure 6 and Figure 7 ), indicating that the TIGIT-CAR gene modification expresses an activation signal, which activates the killing effect of NK cells on tumors.

[0086] In addition, the terms "first", "second", "third", etc. are only used for descriptive purposes and cannot be understood to indicate or imply relative importance or imply the number of the technical features indicated. Therefore, the features defined as "first", "second", etc. can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is at least two, such as two, three, etc., unless otherwise specifically limited.

[0087] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples, without contradiction.

[0088] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A chimeric antigen receptor, characterized in that, include: Extracellular region, wherein the extracellular region includes the TIGIT extracellular segment; The transmembrane region includes the TIGIT transmembrane region and is embedded in the cell membrane; as well as Intracellular region, which includes the 4-1BB costimulatory factor domain, the IL18RB intracellular segment, and the CD3ζ intracellular signaling segment; Wherein, the C-terminus of the extracellular region is connected to the N-terminus of the transmembrane region, and the C-terminus of the transmembrane region is connected to the N-terminus of the intracellular region; The chimeric antigen receptor is shown in the amino acid sequence of SEQ ID NO:

6.

2. The chimeric antigen receptor according to claim 1, characterized in that, The C-terminus of the 4-1BB co-stimulatory factor domain is connected to the N-terminus of the intracellular segment of IL18RB, and the C-terminus of the intracellular segment of IL18RB is connected to the N-terminus of the intracellular signal segment of CD3ζ.

3. The chimeric antigen receptor according to claim 1, characterized in that, The extracellular region is capable of binding ligands, which include at least one member of the PVR family.

4. The chimeric antigen receptor according to claim 3, characterized in that, The PVR family members include CD155 and CD112.

5. The chimeric antigen receptor according to claim 4, characterized in that, The PVR family member is CD155.

6. The chimeric antigen receptor according to claim 1, characterized in that, The extracellular segment of TIGIT is shown in the amino acid sequence of SEQ ID NO.1; The TIGIT transmembrane region is shown in the amino acid sequence of SEQ ID NO.2; The 4-1BB co-stimulatory factor domain is shown in the amino acid sequence of SEQ ID NO.3; The intracellular segment of IL18RB is shown in the amino acid sequence of SEQ ID NO.4; The intracellular signal segment of CD3ζ is shown in the amino acid sequence of SEQ ID NO:

5.

7. A nucleic acid molecule, characterized in that, The chimeric antigen receptor as described in any one of claims 1 to 6 is encoded.

8. The nucleic acid molecule according to claim 7, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

7.

9. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 7 or 8.

10. The expression vector according to claim 9, characterized in that, Further includes: A promoter operatively linked to the nucleic acid molecule of claim 7 or 8.

11. The expression vector according to claim 10, characterized in that, The promoter is selected from at least one of CMV, EF-1, and RSV.

12. The expression vector according to claim 9, characterized in that, The expression vector is a non-pathogenic viral vector.

13. The expression vector according to claim 12, characterized in that, The non-pathogenic viruses are selected from retroviruses, lentiviruses, and adenovirus-associated viruses.

14. The expression vector according to claim 13, characterized in that, The non-pathogenic virus is a lentivirus.

15. A lentiviral vector, characterized in that, The nucleotide sequence of the lentiviral vector is shown in SEQ ID NO:

8.

16. A transgenic immune cell, characterized in that, The transgenic immune cells express the chimeric antigen receptor as described in any one of claims 1 to 6, carry the nucleic acid molecule as described in any one of claims 7 to 8, the expression vector as described in any one of claims 9 to 14, or the lentiviral vector as described in claim 15.

17. A CAR-immune cell, characterized in that, The CAR-immune cells express the chimeric antigen receptor as described in any one of claims 1 to 6, carry the nucleic acid molecule as described in any one of claims 7 to 8, the expression vector as described in any one of claims 9 to 14, or the lentiviral vector as described in claim 15.

18. The CAR-immune cells according to claim 17, characterized in that, The CAR-immune cells include at least one selected from NK-92 cells, peripheral blood NK cells, umbilical cord blood NK cells, iPSCs, CAR-NK cells, CAR-T cells, CAR-NKT cells, and CAR-γδT cells.

19. A pharmaceutical composition, characterized in that, include: The expression vector of any one of claims 1 to 6, the expression vector of any one of claims 7 to 8, the expression vector of any one of claims 9 to 14, or the lentiviral vector of claim 15, the transgenic immune cell of claim 16, or the CAR-immune cell of claim 17 or 18.

20. The pharmaceutical composition according to claim 19, characterized in that, Further includes: Pharmaceutically acceptable excipients.

21. Use of a chimeric antigen receptor as described in any one of claims 1 to 6, a nucleic acid molecule as described in any one of claims 7 to 8, an expression vector as described in any one of claims 9 to 14, or a lentiviral vector as described in claim 15, a transgenic immune cell as described in claim 16, or a CAR-immune cell as described in claim 17 or 18, in the preparation of a medicament for the treatment or prevention of ovarian cancer.

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