WO2024119768A1 - Method for preparing nk cells capable of reversing inhibitory signal from tumor microenvironment and application - Google Patents
Method for preparing nk cells capable of reversing inhibitory signal from tumor microenvironment and application Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Definitions
- the present invention belongs to the field of biomedicine. Specifically, the present invention relates to the preparation of a CAR-immune cell and its use in tumor treatment. More specifically, the present invention relates to a chimeric antigen receptor, expression vector, transgenic immune cell, and pharmaceutical composition for reversing inhibitory signals of the tumor microenvironment, and the preparation method and use thereof.
- T cell immunoglobulin and ITIM domain protein is 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 CD155 and CD112 recognized by TIGIT are mainly expressed on monocytes, macrophages, dendritic cells (DC), 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 competing receptors CD226 and CD96. After binding to its ligand, TIGIT transmits inhibitory signals to T cells or NK cells.
- TIGIT is highly expressed in T cells or NK cells of various malignancies (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).
- TIGIT's ligand CD155 is highly expressed in a variety of solid tumors and blood 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 patient's prognosis.
- the current treatment method is to develop monoclonal antibodies targeting TIGIT to effectively restore the function of T cells or NK cells, thereby playing a tumor-killing role.
- some biotechnology/pharmaceutical companies such as Roche, BeiGene, Fuhong Hanlin, etc.
- clinical trial results show that the treatment effect of TIGIT antibodies alone is not ideal, and it needs to be used in combination with PD-1 or PD-L1 antibodies to improve its effect.
- the anti-TIGIT monoclonal antibodies developed by Roche and BeiGene are combined with PD-L1 antibodies. The combined use for the treatment of non-small cell lung cancer has entered Phase III clinical trials.
- TIGIT and its ligands CD155 and CD112 are expected to become new targets for tumor immunotherapy, blocking the transmission of TIGIT inhibitory signals on the surface of immune cells, and have great application prospects in tumor immunotherapy.
- the present invention aims to solve one of the technical problems in the related art at least to a certain extent.
- the present invention provides a method for preparing immune cells that can selectively reverse the inhibitory signal of the immune checkpoint TIGIT and its application in the treatment of malignant tumors.
- the method for preparing immune cells for reversing the inhibitory signal of the immune checkpoint TIGIT is to construct a chimeric antigen receptor targeting the TIGIT ligand, whose extracellular segment is the TIGIT extracellular segment, whose transmembrane region is the TIGIT transmembrane region, and whose intracellular segment is the 4-1BB co-stimulatory signal domain, the IL18RB intracellular segment and the CD3 ⁇ intracellular region.
- This structure can convert the inhibitory signal of TIGIT recognizing its ligands CD155 and CD112 into a co-stimulatory activation signal mediated by 4-1BB, IL-18R and CD3, thereby converting the inhibitory signal received by NK cells from the tumor microenvironment into an activation signal, effectively enhancing the tumor-killing activity of immune cells, and can be applied to the treatment of various malignant tumors with high expression of TIGIT ligands.
- the inventors found that when the intracellular segment is the 4-1BB co-stimulatory signal domain, the IL18RB intracellular segment and the CD3 ⁇ intracellular region, the activated co-stimulatory signal is stronger than that when the intracellular segment is the 4-1BB co-stimulatory signal domain, the IL21R intracellular segment and the CD3 ⁇ intracellular region, and can stimulate NK cells to have stronger killing activity against TIGIT ligand-positive cells.
- the present invention proposes a chimeric antigen receptor.
- the chimeric antigen receptor includes: an extracellular region, the extracellular region includes the TIGIT extracellular segment; a transmembrane region, the transmembrane region includes the TIGIT transmembrane region, and is embedded in the cell membrane of the cell; an intracellular region, the intracellular region includes a 4-1BB co-stimulatory factor domain, an IL18RB intracellular segment and a CD3 ⁇ intracellular signal 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 described in the embodiment of the present invention is introduced into immune cells for expression. Among them, the immunosuppressive signals originally received by T cells or NK cells are converted into activation signals, which effectively improve the tumor killing effect
- the chimeric antigen receptor may further include at least one of the following additional technical features:
- the C-terminus of the 4-1BB co-stimulatory factor domain in the chimeric antigen receptor is connected to the N-terminus of the IL18RB intracellular segment, and the C-terminus of the IL18RB intracellular segment is connected to the N-terminus of the CD3 ⁇ intracellular signal segment.
- the immune activation effect of immune cells expressing the chimeric antigen receptor is stronger.
- the extracellular region is capable of binding to a ligand, and the ligand includes at least one of the PVR family members.
- the PVR family members include CD155 and CD112.
- the PVR family member is CD155.
- the TIGIT extracellular segment has an amino acid sequence as shown in SEQ ID NO.1.
- the TIGIT transmembrane region has an amino acid sequence as shown in SEQ ID NO.2.
- the 4-1BB co-stimulatory factor domain has an amino acid sequence as shown in SEQ ID NO.3.
- the intracellular segment of IL18RB has an amino acid sequence as shown in SEQ ID NO.4.
- the CD3 ⁇ intracellular signal segment has the amino acid sequence shown in SEQ ID NO:5.
- the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO:6.
- the present invention provides a nucleic acid molecule.
- the nucleic acid molecule encodes the chimeric antigen receptor described in the first aspect of the present invention.
- the above nucleic acid molecule according to an embodiment of the present invention is expressed in immune cells and can convert the inhibitory signal mediated by tumor cells into an activation signal.
- the above nucleic acid molecule may further include at least one of the following additional technical features:
- the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:7.
- the present invention provides an expression vector.
- the expression vector carries the nucleic acid molecule described in the second aspect of the present invention.
- the purpose of constructing the expression vector is to express the target gene sequence.
- the above expression vector may further include at least one of the following additional technical features:
- the expression vector further includes a promoter.
- the promoter is operably linked to the nucleic acid molecule described in the second aspect of the present invention.
- the promoter is selected from at least one of CMV, EF-1, and RSV.
- the expression vector is a non-pathogenic viral vector.
- the non-pathogenic virus is selected from retrovirus, lentivirus and adenovirus-associated virus.
- the non-pathogenic virus is a lentivirus.
- the present invention provides a lentiviral vector.
- the lentiviral vector has a nucleotide sequence shown in SEQ ID NO: 8. After the lentiviral vector is introduced into a recipient cell, it can assist in the expression of activation signals in immune cells.
- the present invention proposes a transgenic immune cell.
- the transgenic immune cell carries the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, and the lentiviral vector described in the fourth aspect of the present invention.
- the expression of the obtained transgenic immune cell can effectively enhance the killing ability of malignant tumors.
- the present invention provides a CAR-immune cell.
- the CAR-immune cell carries the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, and the lentiviral vector described in the fourth aspect of the present invention.
- 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- ⁇ T cells.
- the present invention proposes a pharmaceutical composition.
- the pharmaceutical composition comprises: the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the lentiviral vector described in the fourth aspect of the present invention, the transgenic immune cell described in the fifth aspect of the present invention, and the CAR-immune cell described in the sixth aspect of the present invention.
- the pharmaceutical composition further comprises: a pharmaceutically acceptable excipient.
- the present invention proposes a use of a pharmaceutical composition in the preparation of a drug.
- the chimeric antigen receptor, nucleic acid molecule, expression vector, lentiviral vector, transgenic immune cell, CAR-immune cell and pharmaceutical composition are used to treat or prevent solid tumors or hematological tumors.
- the solid tumor includes tangible tumors selected from those occurring in internal organs, including at least one of pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, prostate cancer, melanoma, etc.
- the blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, etc. in blood cells and hematopoietic system.
- the present invention provides a method for treating or preventing solid tumors or hematological tumors.
- the method comprises administering to a subject the chimeric antigen receptor of the first aspect of the present invention, the nucleic acid molecule of the second aspect, the At least one of the expression vector, the lentiviral vector of the fourth aspect, the transgenic immune cell of the fifth aspect, the CAR-immune cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect.
- a suitable dose of a chimeric antigen receptor, a nucleic acid molecule, an expression vector, a lentiviral vector, a transgenic cell, a CAR-immune cell, or a pharmaceutical composition is administered to a subject to inhibit the proliferation of solid tumor or hematological tumor cells.
- the present invention proposes a chimeric antigen receptor described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the lentiviral vector described in the fourth aspect, the transgenic immune cell described in the fifth aspect, the CAR-immune cell described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect for the treatment or prevention of solid tumors or hematological tumors.
- different treatment methods can be selected for different types of solid tumors or hematological tumors using the above-mentioned products, including one or more combinations of chimeric antigen receptors, nucleic acid molecules, expression vectors, lentiviral vectors, transgenic cells, CAR-immune cells or pharmaceutical compositions, to improve the pertinence and effectiveness of the treatment of solid tumors or hematological tumors.
- FIG. 1 is a structural schematic diagram of a CAR activation signal targeting CD155 and CD112 according to Example 1 of the present invention, wherein the TIGIT extracellular segment represents a nucleotide sequence encoding a receptor binding to CD155 or CD112, 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 CD3 ⁇ intracellular segment represents a nucleotide sequence encoding a CD3 ⁇ intracellular segment; wherein TIGIT-CAR-2 is a structural schematic diagram of a CAR activation signal targeting CD155 and CD112 as a control, and its difference from the TIGIT-CAR structure of the embodiment of the present invention is that the intracellular segments are the 4-1BB intra
- Example 2 is a graph showing the expression level detection results of TIGIT ligand CD155 in tumor cells according to Example 2 of the present invention, wherein the shaded peak is the isotype antibody staining control group, and the black solid line is the CD155 antibody staining group;
- FIG3 is a graph showing the results of in vitro killing ability detection of TIGIT-CAR-NK cells according to Example 2 of the present invention.
- FIG4 is a graph showing the results of in vitro killing-related degranulation detection of NK cells according to Example 2 of the present invention.
- FIG5 is a graph showing the secretion level detection results of IFN- ⁇ and TNF- ⁇ of TIGIT-CAR-NK cells according to Example 2 of the present invention.
- FIG6 is a graph showing the results of a fluorescence experiment on tumor growth inhibition in the differentially treated groups according to Example 3 of the present invention.
- FIG7 is a statistical result diagram of fluorescence intensity of differentially treated groups according to Example 3 of the present invention.
- the terms “optionally”, “optional” or “optionally” generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
- operably linked means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended functions of regulating the transcription and translation of the exogenous gene.
- Commonly used vectors may be, for example, viral vectors, plasmids, bacteriophages, etc.
- chimeric antigen receptor 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 immunostimulatory factors contained in the intracellular region.
- the present application constructs a transgenic immune cell expressing a chimeric antigen receptor, wherein the chimeric antigen receptor targets at least one of the ligand PVR family members.
- the chimeric antigen receptor can express activation signals, enhance the tumor killing activity of CAR immune cells, and is applied to the treatment of solid tumors and hematological tumors.
- the TIGIT-CAR vector sequence designed by the present invention comprises the extracellular segment and transmembrane segment of the TIGIT receptor, the 4-1BB co-stimulatory signal domain, the IL18RB intracellular segment and the CD3 ⁇ intracellular region.
- the schematic diagram of the gene element structure is shown in Figure 1.
- the fully synthesized TIGIT-CAR fragment was inserted into the lentiviral vector pCDH-EF1-MSC-T2A-copGFP through the restriction sites XbaI and BamHI. After colony PCR identification and sequencing verification of the correct sequence, it was confirmed that the pCDH-EF1-TIGIT-CAR-T2A-copGFP plasmid was successfully constructed.
- lentiviral packaging system add 6 ⁇ g psPAX2 plasmid, 3 ⁇ g pMD2.G plasmid and 6 ⁇ g pCDH-EF1-TIGIT-CAR-T2A-copGFP plasmid to 250 ⁇ L serum-free DMEM medium, mix well, and prepare DNA mixture; add 15 ⁇ L PEIpro to 235 ⁇ L serum-free DMEM medium, mix well to prepare PEIpro mixture. Add PEIpro mixture to DNA mixture at one time, let stand to mix well, and incubate at room temperature for 15min. Add the mixture to 293T cell culture dish. After 24h of culture, change the medium and put the culture dish back to 37°C, 5% CO2 incubator.
- NK-92 cells purchased from ATCC
- 2mL ⁇ -MEM medium to resuspend the cells, and adjust the cell density to 5 ⁇ 10 5 /mL.
- NK-92, TIGIT-CAR-NK-92 and TIGIT-CAR2-NK-92 were used as effector cells, and the ovarian cancer cell line HO8910 was used as the target cell, and the effector-target ratio was set to 5:1, 2.5:1 and 1.25:1.
- the effector cells and target cells were co-incubated for 4 hours, and the killing efficiency of the effector cells on the target cells was detected by the LDH (lactate dehydrogenase) release method.
- LDH lactate dehydrogenase
- TIGIT-CAR gene modification can significantly improve the killing ability of NK cells against CD155-positive tumor cells, and the intracellular segment is the 4-1BB intracellular segment and CD3 ⁇
- the TIGIT-CAR structure of the intracellular segment is superior to the CAR structure whose intracellular segment is the 4-1BB intracellular segment, the IL-21 receptor (IL-21R, the amino acid sequence is shown in SEQ ID NO:9) intracellular segment and the CD3 ⁇ intracellular segment, and can stimulate NK cells to have a stronger killing ability against CD155-positive tumor cells.
- IL-21R the IL-21 receptor
- NK cells were collected in flow tubes, and the expression of CD107a, granzyme (Granzyme B) and perforin (Perforin) on NK cells was detected by flow cytometry.
- the results showed that after co-incubation with HO8910 cells, the expression levels of CD107a and secreted Granzyme B on TIGIT-CAR cells were significantly higher than those in the NK-92 group, and there was no statistical difference in the expression level of Perforin ( Figure 4). It was further verified that TIGIT-CAR modification can significantly improve the degranulation level (expression levels of CD107a, granzyme and perforin) and killing function of NK cells on CD155-positive tumor cells.
- TIGIT-CAR-NK-92 cells The changes in the secretion capacity of IFN- ⁇ and TNF- ⁇ of TIGIT-CAR-NK-92 cells were detected by flow cytometry. After NK cells were co-incubated with ovarian cancer HO8910 cells for 4 hours, NK cells were collected in flow tubes, and the secretion levels of IFN- ⁇ and TNF- ⁇ of NK cells were detected by flow cytometry after fixation and membrane permeabilization. The results showed that the levels of IFN- ⁇ and TNF- ⁇ secreted by TIGIT-CAR-NK-92 cells were significantly higher than those of the NK-92 group (Figure 5). This indicates that TIGIT-CAR can significantly improve the secretion capacity of IFN- ⁇ and TNF- ⁇ of NK cells when they come into contact with CD155-positive tumor cells.
- the ovarian cancer HO8910 cells labeled with luciferase were used for intraperitoneal tumor implantation to establish an ovarian cancer peritoneal metastasis model.
- Five-week-old female highly immunodeficient NCG mice were selected for intraperitoneal tumor implantation, and 2 ⁇ 10 5 luciferase-labeled HO8910 cells were injected intraperitoneally into each mouse.
- NK cell transfusion 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.
- mice in the treatment group were intraperitoneally injected with 5 ⁇ 10 6 NK cells/mouse, and the control group was injected with an equal volume of 1 ⁇ PBS solution, once every week, for a total of 3 treatments.
- IL-2 (50000 IU/mouse) was injected intraperitoneally every 3 days.
- the tumor size was observed by small animal in vivo imaging technology, and then the tumor growth curve was drawn.
- first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of “plurality” is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.
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Abstract
The present application provides a method for preparing immune cells capable of reversing an inhibitory signal from a tumor microenvironment and an application thereof. In a method for reversing an inhibitory signal, an intracellular segment of a TIGIT receptor is replaced with a 4-1BB costimulatory domain and IL-18R and CD3 intracellular segments, and immune cells expressing a chimeric antigen receptor recognize CD155 and CD112 in a tumor microenvironment by means of TIGIT, thereby avoiding function loss or depletion of immune cells, and exciting immune cells to exert higher tumor killing activity.
Description
本发明属于生物医药领域，具体地，本发明涉及一种CAR-免疫细胞的制备及其在肿瘤治疗中的用途，更具体地，本发明涉及一种逆转肿瘤微环境抑制性信号的嵌合抗原受体、表达载体、转基因免疫细胞、药物组合物的制备方法及其用途。The present invention belongs to the field of biomedicine. Specifically, the present invention relates to the preparation of a CAR-immune cell and its use in tumor treatment. More specifically, the present invention relates to a chimeric antigen receptor, expression vector, transgenic immune cell, and pharmaceutical composition for reversing inhibitory signals of the tumor microenvironment, and the preparation method and use thereof.
近年来，免疫疗法在肿瘤治疗领域取得了令人瞩目的进展，尤其是以抗CTLA-4和抗PD-1或PD-L1抗体为代表的免疫卡控点阻断疗法。通过阻断T细胞表面抑制性受体与其配体的结合，阻断抑制性信号的传递，纠正免疫抑制性微环境所介导的免疫抑制，恢复肿瘤微环境T细胞的抗肿瘤能力，在多种转移性晚期癌症(包括转移性黑色素瘤、非小细胞肺癌、肾癌等)治疗方面取得了较高的应答率，使众多原本已经失去治疗机会的对放化疗无效的晚期癌症患者有了重新治疗的希望。In recent years, immunotherapy has made remarkable progress in the field of tumor treatment, especially immune checkpoint blocking therapy represented by anti-CTLA-4 and anti-PD-1 or PD-L1 antibodies. By blocking the binding of inhibitory receptors on the surface of T cells to their ligands, blocking the transmission of inhibitory signals, correcting the immunosuppression mediated by the immunosuppressive microenvironment, and restoring the anti-tumor ability of T cells in the tumor microenvironment, a high response rate has been achieved in the treatment of various metastatic advanced cancers (including metastatic melanoma, non-small cell lung cancer, renal cancer, etc.), giving many advanced cancer patients who have lost the opportunity for treatment and are ineffective for radiotherapy and chemotherapy hope for re-treatment.
然而，并非所有的恶性肿瘤患者对PD-1、PD-L1或CTLA-4阻断治疗有效，对PD-1或PD-L1抗体的治疗只有10％-30％的患者表现出长期持久的反应，大多数人群缺乏响应，改善临床响应及克服耐药是该领域面临的最大挑战。探究肿瘤对免疫卡控阻断治疗不应答的机制，寻找其他影响免疫细胞功能的免疫调控卡点，成为肿瘤免疫治疗领域亟待解决的问题。目前，越来越多的免疫卡控点分子被发现并开发应用。However, not all patients with malignant tumors respond to PD-1, PD-L1 or CTLA-4 blockade therapy. Only 10%-30% of patients show long-term and lasting responses to PD-1 or PD-L1 antibody treatment, and most people lack response. Improving clinical response and overcoming drug resistance are the biggest challenges facing this field. Exploring the mechanism of tumor non-response to immune checkpoint blockade therapy and finding other immune regulatory checkpoints that affect the function of immune cells have become urgent issues to be solved in the field of tumor immunotherapy. Currently, more and more immune checkpoint molecules are being discovered and developed for application.
T细胞免疫球蛋白和ITIM结构域蛋白(TIGIT)作为一个重要的免疫卡控点，主要表达在自然杀伤(NK)细胞、活化的CD8+T和CD4+T细胞、调节性T细胞(Tregs)和滤泡辅助性T细胞(Tfh)表面。TIGIT识别的配体CD155和CD112主要在单核细胞、巨噬细胞、树突状细胞(DC)、T细胞、B细胞和许多非造血细胞(包括不同组织学类型的肿瘤细胞)上表达。TIGIT结合CD155的亲和力显著高于其竞争受体CD226和CD96。TIGIT与其配体结合后向T细胞或NK细胞传递抑制性信号。研究发现TIGIT在多种恶性肿瘤(如非小细胞肺癌、黑色素瘤、头颈部鳞状细胞癌、结直肠癌、胶质母细胞瘤、胃癌、肝癌、多发性骨髓瘤、急性髓系白血病和滤泡性淋巴瘤)的T细胞或NK细胞高表达。TIGIT的配体CD155高表达于多种实体瘤和血液肿瘤，包括肝癌、胰腺癌、结直肠癌、胃癌、肺癌、卵巢癌、头颈癌、乳腺癌、淋巴瘤、白血病等。TIGIT与CD155的表达丰度与患者预后密切相关。目前的的治疗方法是通过开发靶向TIGIT单抗有效地恢复T细胞或NK细胞功能，进而发挥杀伤肿瘤作用。其中，一些生物技术/制药公司(如罗氏、百济神州、复宏汉霖等)正在致力于开发抗TIGIT的抗体，相关产品处于不同的临床开发阶段。但是，临床试验结果表明，单独使用TIGIT抗体治疗效果并不理想，需与PD-1或PD-L1抗体等联合应用才可改善其效果，罗氏和百济神州开发的抗TIGIT单抗与PD-L1抗体联
合用于非小细胞肺癌的治疗已经进入III期临床试验。As an important immune checkpoint, T cell immunoglobulin and ITIM domain protein (TIGIT) is 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 CD155 and CD112 recognized by TIGIT are mainly expressed on monocytes, macrophages, dendritic cells (DC), 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 competing receptors CD226 and CD96. After binding to its ligand, TIGIT transmits inhibitory signals to T cells or NK cells. Studies have found that TIGIT is highly expressed in T cells or NK cells of various malignancies (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). TIGIT's ligand CD155 is highly expressed in a variety of solid tumors and blood 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 patient's prognosis. The current treatment method is to develop monoclonal antibodies targeting TIGIT to effectively restore the function of T cells or NK cells, thereby playing a tumor-killing role. Among them, some biotechnology/pharmaceutical companies (such as Roche, BeiGene, Fuhong Hanlin, etc.) are committed to developing anti-TIGIT antibodies, and related products are in different stages of clinical development. However, clinical trial results show that the treatment effect of TIGIT antibodies alone is not ideal, and it needs to be used in combination with PD-1 or PD-L1 antibodies to improve its effect. The anti-TIGIT monoclonal antibodies developed by Roche and BeiGene are combined with PD-L1 antibodies. The combined use for the treatment of non-small cell lung cancer has entered Phase III clinical trials.
因此，TIGIT及其配体CD155、CD112有望成为肿瘤免疫治疗的新靶点，阻断免疫细胞表面TIGIT抑制性信号的传递，在肿瘤免疫治疗中具有巨大的应用前景。Therefore, TIGIT and its ligands CD155 and CD112 are expected to become new targets for tumor immunotherapy, blocking the transmission of TIGIT inhibitory signals on the surface of immune cells, and have great application prospects in tumor immunotherapy.
发明内容Summary of the invention
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。The present invention aims to solve one of the technical problems in the related art at least to a certain extent.
为了能够提高肿瘤的杀伤效果以及减少外用药物所带来的副作用，本发明提供了一种能够选择性逆转免疫卡控点TIGIT抑制性信号的免疫细胞制备方法及其在恶性肿瘤治疗中的应用。In order to improve the tumor killing effect and reduce the side effects of topical drugs, the present invention provides a method for preparing immune cells that can selectively reverse the inhibitory signal of the immune checkpoint TIGIT and its application in the treatment of malignant tumors.
本发明所提供的逆转免疫卡控点TIGIT抑制性信号的免疫细胞制备方法为，构建靶向TIGIT配体的嵌合抗原受体，其胞外段为TIGIT胞外段、其跨膜区为TIGIT跨膜区、其胞内段为4-1BB共刺激信号域、IL18RB胞内段与CD3ζ胞内区。该结构能够将TIGIT识别其配体CD155和CD112的抑制性信号转化为4-1BB、IL-18R和CD3介导的共刺激活化信号，从而将NK细胞接收到的来自肿瘤微环境的抑制性信号转换为激活信号，有效增强免疫细胞的杀伤肿瘤活性，可应用于TIGIT配体高表达的多种恶性肿瘤的治疗。发明人发现，当胞内段为4-1BB共刺激信号域、IL18RB胞内段与CD3ζ胞内区时，较胞内段为4-1BB共刺激信号域、IL21R胞内段与CD3ζ胞内区，其激活的共刺激信号更强，能够激发NK细胞对TIGIT配体阳性细胞更强的的杀伤活性。The method for preparing immune cells for reversing the inhibitory signal of the immune checkpoint TIGIT provided by the present invention is to construct a chimeric antigen receptor targeting the TIGIT ligand, whose extracellular segment is the TIGIT extracellular segment, whose transmembrane region is the TIGIT transmembrane region, and whose intracellular segment is the 4-1BB co-stimulatory signal domain, the IL18RB intracellular segment and the CD3ζ intracellular region. This structure can convert the inhibitory signal of TIGIT recognizing its ligands CD155 and CD112 into a co-stimulatory activation signal mediated by 4-1BB, IL-18R and CD3, thereby converting the inhibitory signal received by NK cells from the tumor microenvironment into an activation signal, effectively enhancing the tumor-killing activity of immune cells, and can be applied to the treatment of various malignant tumors with high expression of TIGIT ligands. The inventors found that when the intracellular segment is the 4-1BB co-stimulatory signal domain, the IL18RB intracellular segment and the CD3ζ intracellular region, the activated co-stimulatory signal is stronger than that when the intracellular segment is the 4-1BB co-stimulatory signal domain, the IL21R intracellular segment and the CD3ζ intracellular region, and can stimulate NK cells to have stronger killing activity against TIGIT ligand-positive cells.
因此，在本发明的第一方面，本发明提出了一种嵌合抗原受体。根据本发明的实施例，所述嵌合抗原受体包括：胞外区，所述胞外区包括TIGIT胞外段；跨膜区，所述跨膜区包括TIGIT跨膜区，并且嵌入到所述细胞细胞膜中；胞内区，所述胞内区包括4-1BB共刺激因子结构域、IL18RB胞内段与CD3ζ胞内信号段，其中，所述胞外区的C端与所述跨膜区的N端相连，所述跨膜区的C端与所述胞内区的N端相连。将本发明实施例中所述的嵌合抗原受体导入免疫细胞中表达。其中，所述原本T细胞或NK细胞收到的免疫抑制信号转换为激活信号，有效提高免疫细胞的杀伤肿瘤效果。Therefore, in the first aspect of the present invention, the present invention proposes a chimeric antigen receptor. According to an embodiment of the present invention, the chimeric antigen receptor includes: an extracellular region, the extracellular region includes the TIGIT extracellular segment; a transmembrane region, the transmembrane region includes the TIGIT transmembrane region, and is embedded in the cell membrane of the cell; an intracellular region, the intracellular region includes a 4-1BB co-stimulatory factor domain, an IL18RB intracellular segment and a CD3ζ intracellular signal 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 described in the embodiment of the present invention is introduced into immune cells for expression. Among them, the immunosuppressive signals originally received by T cells or NK cells are converted into activation signals, which effectively improve the tumor killing effect of immune cells.
根据本发明的实施例，上述嵌合抗原受体还可以包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the chimeric antigen receptor may further include at least one of the following additional technical features:
根据本发明实施例，所述嵌合抗原受体中4-1BB共刺激因子结构域的C端与所述IL18RB胞内段的N端相连，所述IL18RB胞内段的C端与所述CD3ζ胞内信号段的N端相连。进而表达该嵌合抗原受体的免疫细胞的免疫激活效应更强。According to an embodiment of the present invention, the C-terminus of the 4-1BB co-stimulatory factor domain in the chimeric antigen receptor is connected to the N-terminus of the IL18RB intracellular segment, and the C-terminus of the IL18RB intracellular segment is connected to the N-terminus of the CD3ζ intracellular signal segment. Thus, the immune activation effect of immune cells expressing the chimeric antigen receptor is stronger.
根据本发明的实施例，所述胞外区能够结合配体，所述配体包括PVR家族成员中的至少之一。According to an embodiment of the present invention, the extracellular region is capable of binding to a ligand, and the ligand includes at least one of the PVR family members.
根据本发明的实施例，所述PVR家族成员包括CD155和CD112。According to an embodiment of the present invention, the PVR family members include CD155 and CD112.
根据本发明的实施例，所述PVR家族成员为CD155。According to an embodiment of the present invention, the PVR family member is CD155.
根据本发明的实施例，所述TIGIT胞外段具有如SEQ ID NO.1所示的氨基酸序列。According to an embodiment of the present invention, the TIGIT extracellular segment has an amino acid sequence as shown in SEQ ID NO.1.
根据本发明的实施例，所述TIGIT跨膜区具有如SEQ ID NO.2所示的氨基酸序列。
According to an embodiment of the present invention, the TIGIT transmembrane region has an amino acid sequence as shown in SEQ ID NO.2.
根据本发明的实施例，所述4-1BB共刺激因子结构域具有如SEQ ID NO.3所示的氨基酸序列。According to an embodiment of the present invention, the 4-1BB co-stimulatory factor domain has an amino acid sequence as shown in SEQ ID NO.3.
根据本发明的实施例，所述IL18RB胞内段具有如SEQ ID NO.4所示的氨基酸序列。According to an embodiment of the present invention, the intracellular segment of IL18RB has an amino acid sequence as shown in SEQ ID NO.4.
根据本发明的实施例，所述CD3ζ胞内信号段具有SEQ ID NO:5所示的氨基酸序列。According to an embodiment of the present invention, the CD3ζ intracellular signal segment has the amino acid sequence shown in SEQ ID NO:5.
根据本发明的实施例，所述嵌合抗原受体具有SEQ ID NO:6所示的氨基酸序列。
According to an embodiment of the present invention, the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO:6.
According to an embodiment of the present invention, the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO:6.
在本发明的第二方面，本发明提出了一种核酸分子。根据本发明的实施例，所述核酸分子编码本发明第一方面所述的嵌合抗原受体。根据本发明实施例的上述核酸分子在免疫细胞中表达，可将肿瘤细胞介导的抑制信号转换为激活信号。In a second aspect of the present invention, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the chimeric antigen receptor described in the first aspect of the present invention. The above nucleic acid molecule according to an embodiment of the present invention is expressed in immune cells and can convert the inhibitory signal mediated by tumor cells into an activation signal.
根据本发明的实施例，上述核酸分子还可以包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above nucleic acid molecule may further include at least one of the following additional technical features:
根据本发明的实施例，所述核酸分子具有SEQ ID NO:7所示的核苷酸序列。
According to an embodiment of the present invention, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:7.
According to an embodiment of the present invention, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:7.
在本发明的第三方面，本发明提出了一种表达载体。根据本发明的实施例，所述表达载体携带本发明第二方面所述的核酸分子。其中，构建表达载体的目的是为了表达目的基因序列。In the third aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the second aspect of the present invention. The purpose of constructing the expression vector is to express the target gene sequence.
根据本发明的实施例，上述表达载体还可以包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above expression vector may further include at least one of the following additional technical features:
根据本发明的实施例，所述表达载体进一步包括启动子。According to an embodiment of the present invention, the expression vector further includes a promoter.
根据本发明的实施例，所述启动子与本发明第二方面所述的核酸分子可操作地连接。According to an embodiment of the present invention, the promoter is operably linked to the nucleic acid molecule described in the second aspect of the present invention.
根据本发明的实施例，所述启动子选自CMV,EF-1,RSV的至少之一。According to an embodiment of the present invention, the promoter is selected from at least one of CMV, EF-1, and RSV.
根据本发明的实施例，所述表达载体是非致病性病毒载体。According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector.
根据本发明的实施例，所述非致病性病毒选自反转录病毒、慢病毒和腺病毒相关病毒，优选地，所
述非致病性病毒为慢病毒。According to an embodiment of the present invention, the non-pathogenic virus is selected from retrovirus, lentivirus and adenovirus-associated virus. The non-pathogenic virus is a lentivirus.
在本发明的第四方面，本发明提出了一种慢病毒载体。根据本发明的实施例，所述慢病毒载体具有SEQ ID NO：8所示的核苷酸序列。其中，在将慢病毒载体导入受体细胞后，可在辅助激活信号在免疫细胞中的表达。
In a fourth aspect of the present invention, the present invention provides a lentiviral vector. According to an embodiment of the present invention, the lentiviral vector has a nucleotide sequence shown in SEQ ID NO: 8. After the lentiviral vector is introduced into a recipient cell, it can assist in the expression of activation signals in immune cells.
In a fourth aspect of the present invention, the present invention provides a lentiviral vector. According to an embodiment of the present invention, the lentiviral vector has a nucleotide sequence shown in SEQ ID NO: 8. After the lentiviral vector is introduced into a recipient cell, it can assist in the expression of activation signals in immune cells.
在本发明的第五方面，本发明提出了一种转基因免疫细胞。根据本发明的实施例，所述转基因免疫细胞携带本发明第一方面所述的嵌合抗原受体、本发明第二方面所述的核酸分子、本发明第三方面所述的表达载体以及本发明第四方面所述的慢病毒载体。其中，所获得的转基因免疫细胞的表达可以有效增强对恶性肿瘤的杀伤能力。In the fifth aspect of the present invention, the present invention proposes a transgenic immune cell. According to an embodiment of the present invention, the transgenic immune cell carries the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, and the lentiviral vector described in the fourth aspect of the present invention. The expression of the obtained transgenic immune cell can effectively enhance the killing ability of malignant tumors.
在本发明的第六方面，本发明提出了一种CAR-免疫细胞。根据本发明的实施例，所述CAR-免疫细胞携带本发明第一方面所述的嵌合抗原受体、本发明第二方面所述的核酸分子、本发明第三方面所述的表达载体以及本发明第四方面所述的慢病毒载体。In a sixth aspect of the present invention, the present invention provides a CAR-immune cell. According to an embodiment of the present invention, the CAR-immune cell carries the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, and the lentiviral vector described in the fourth aspect of the present invention.
根据本发明的实施例，所述CAR-免疫细胞包括选自NK-92细胞，外周血NK细胞、脐带血NK细胞、iPSC、CAR-NK细胞、CAR-T细胞、CAR-NKT细胞、CAR-γδT细胞中的至少之一。According to an embodiment of the present invention, 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.
在本发明的第七方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括：本发明第一方面所述的嵌合抗原受体、本发明第二方面所述的核酸分子、本发明第三方面所述的表达载体、本发明第四方面所述的慢病毒载体、本发明第五方面所述的转基因免疫细胞以及本发明第六方面所述的CAR-免疫细胞。In the seventh aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the lentiviral vector described in the fourth aspect of the present invention, the transgenic immune cell described in the fifth aspect of the present invention, and the CAR-immune cell described in the sixth aspect of the present invention.
根据本发明的实施例，所述药物组合物进一步包括：药学上可接受的辅料。According to an embodiment of the present invention, the pharmaceutical composition further comprises: a pharmaceutically acceptable excipient.
在本发明的第八方面，本发明提出了一种药物组合物在制备药物中的用途。根据本发明的实施例，所述嵌合抗原受体、核酸分子、表达载体、慢病毒载体、转基因免疫细胞、CAR-免疫细胞以及药物组合物用于治疗或预防实体瘤或血液瘤。In an eighth aspect of the present invention, the present invention proposes a use of a pharmaceutical composition in the preparation of a drug. According to an embodiment of the present invention, the chimeric antigen receptor, nucleic acid molecule, expression vector, lentiviral vector, transgenic immune cell, CAR-immune cell and pharmaceutical composition are used to treat or prevent solid tumors or hematological tumors.
根据本发明的实施例，所述实体瘤包括选自发生在脏器中的有形瘤，包括胰腺癌、卵巢癌、间皮瘤、肝癌、胆管癌、胃癌、食管癌、结直肠癌、肺癌、头颈癌、***、脑胶质瘤、肾癌、乳腺癌、***癌、黑色素瘤等的至少之一。According to an embodiment of the present invention, the solid tumor includes tangible tumors selected from those occurring in internal organs, including at least one of pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, prostate cancer, melanoma, etc.
根据本发明的实施例，所述血液瘤包括选自血细胞和造血***内的急性髓系白血病、急性淋巴细胞白血病、B细胞淋巴瘤、T细胞淋巴瘤、何杰金氏淋巴瘤、非何杰金氏淋巴瘤、多发性骨髓瘤等的至少之一。According to an embodiment of the present invention, the blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, etc. in blood cells and hematopoietic system.
在本发明的第九方面，本发明提出了一种治疗或预防实体瘤或血液瘤的方法。根据本发明的实施例，所述方法包括向受试者施用本发明第一方面所述的嵌合抗原受体、第二方面所述的核酸分子、第三方面
所述的表达载体、第四方面所述的慢病毒载体、第五方面所述的转基因免疫细胞、第六方面所述的CAR-免疫细胞或第七方面所述的药物组合物中的至少之一。根据本发明的一些具体实施例，通过向受试者施用合适剂量的嵌合抗原受体、核酸分子、表达载体、慢病毒载体、转基因细胞、CAR-免疫细胞或药物组合物用以抑制实体瘤或血液瘤细胞的增殖。In the ninth aspect of the present invention, the present invention provides a method for treating or preventing solid tumors or hematological tumors. According to an embodiment of the present invention, the method comprises administering to a subject the chimeric antigen receptor of the first aspect of the present invention, the nucleic acid molecule of the second aspect, the At least one of the expression vector, the lentiviral vector of the fourth aspect, the transgenic immune cell of the fifth aspect, the CAR-immune cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect. According to some specific embodiments of the present invention, a suitable dose of a chimeric antigen receptor, a nucleic acid molecule, an expression vector, a lentiviral vector, a transgenic cell, a CAR-immune cell, or a pharmaceutical composition is administered to a subject to inhibit the proliferation of solid tumor or hematological tumor cells.
在本发明的第十方面，本发明提出了一种第一方面所述的嵌合抗原受体、第二方面所述的核酸分子、第三方面所述的表达载体、第四方面所述的慢病毒载体、第五方面所述的转基因免疫细胞、第六方面所述的CAR-免疫细胞或第七方面所述的药物组合物在治疗或预防实体瘤或血液瘤中的用途。根据本发明的一些具体实施例，利用上述产品针对不同类型的实体瘤或血液瘤，可以选择使用不同的治疗手段，包括嵌合抗原受体、核酸分子、表达载体、慢病毒载体、转基因细胞、CAR-免疫细胞或药物组合物中的一种或多种组合，以提高实体瘤或血液瘤治疗的针对性和有效性。In the tenth aspect of the present invention, the present invention proposes a chimeric antigen receptor described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the lentiviral vector described in the fourth aspect, the transgenic immune cell described in the fifth aspect, the CAR-immune cell described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect for the treatment or prevention of solid tumors or hematological tumors. According to some specific embodiments of the present invention, different treatment methods can be selected for different types of solid tumors or hematological tumors using the above-mentioned products, including one or more combinations of chimeric antigen receptors, nucleic acid molecules, expression vectors, lentiviral vectors, transgenic cells, CAR-immune cells or pharmaceutical compositions, to improve the pertinence and effectiveness of the treatment of solid tumors or hematological tumors.
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention.
图1是根据本发明实施例1的靶向CD155和CD112的CAR激活信号结构模式图，其中，TIGIT胞外段表示编码与CD155或CD112结合的受体核苷酸序列，TIGIT跨膜段表示编码TIGIT跨膜段结构域的核苷酸序列，4-1BB胞内段表示编码4-1BB共刺激因子结构域的核苷酸序列，IL18RB胞内段表示编码IL18RB结构域的核苷酸序列，CD3ζ胞内段表示编码CD3ζ胞内段的核苷酸序列；其中TIGIT-CAR-2为作为对照的靶向CD155和CD112的CAR激活信号结构模式图，其与本发明实施例TIGIT-CAR结构不同之处在于，胞内段为4-1BB胞内段、IL-21受体(IL-21R)胞内段和CD3ζ胞内段；Figure 1 is a structural schematic diagram of a CAR activation signal targeting CD155 and CD112 according to Example 1 of the present invention, wherein the TIGIT extracellular segment represents a nucleotide sequence encoding a receptor binding to CD155 or CD112, 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 structural schematic diagram of a CAR activation signal targeting CD155 and CD112 as a control, and its difference from the TIGIT-CAR structure of the embodiment of the present invention is that the intracellular segments are the 4-1BB intracellular segment, the IL-21 receptor (IL-21R) intracellular segment and the CD3ζ intracellular segment;
图2是根据本发明实施例2的TIGIT配体CD155在肿瘤细胞中的表达水平检测结果图，其中，阴影峰为同型抗体染色对照组，黑色实线为CD155抗体染色组；2 is a graph showing the expression level detection results of TIGIT ligand CD155 in tumor cells according to Example 2 of the present invention, wherein the shaded peak is the isotype antibody staining control group, and the black solid line is the CD155 antibody staining group;
图3是根据本发明实施例2的TIGIT-CAR-NK细胞的体外杀伤能力检测结果图；FIG3 is a graph showing the results of in vitro killing ability detection of TIGIT-CAR-NK cells according to Example 2 of the present invention;
图4是根据本发明实施例2的NK细胞体外杀伤相关脱颗粒检测结果图；FIG4 is a graph showing the results of in vitro killing-related degranulation detection of NK cells according to Example 2 of the present invention;
图5是根据本发明实施例2的TIGIT-CAR-NK细胞的IFN-γ与TNF-α的分泌水平检测结果图；FIG5 is a graph showing the secretion level detection results of IFN-γ and TNF-α of TIGIT-CAR-NK cells according to Example 2 of the present invention;
图6是根据本发明实施例3的差异治疗组抑制肿瘤生长荧光实验检测结果图；FIG6 is a graph showing the results of a fluorescence experiment on tumor growth inhibition in the differentially treated groups according to Example 3 of the present invention;
图7是根据本发明实施例3的差异治疗组荧光强度统计结果图；FIG7 is a statistical result diagram of fluorescence intensity of differentially treated groups according to Example 3 of the present invention;
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。其中自始至终相同或类似的标号
表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be construed as limiting the present invention.
在对本发明描述的过程中，对于本文中有关的术语进行了解释和说明，这些解释和说明仅仅是为了方便对于方案的理解，并不能看做是对本发明保护方案的限制。In the process of describing the present invention, the relevant terms in this document are explained and illustrated. These explanations and illustrations are only for the convenience of understanding the scheme and cannot be regarded as limitations on the protection scheme of the present invention.
在本文中，术语“包含”或“包括”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.
在本文中，术语“任选地”、“任选的”或“任选”通常是指随后所述的事件或状况可以但未必发生，并且该描述包括其中发生该事件或状况的情况，以及其中未发生该事件或状况的情况。As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如转录控制序列和翻译控制序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。常用的载体例如可以为病毒载体、质粒、噬菌体等等。根据本发明的一些具体实施例的表达载体导入合适的受体细胞后，可在调控***的介导下，有效实现前面所述的核酸分子的表达，进而实现所述核酸分子编码的蛋白质的体外大量获得。Herein, "operably linked" means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors may be, for example, viral vectors, plasmids, bacteriophages, etc. After the expression vectors according to some specific embodiments of the present invention are introduced into suitable recipient cells, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of the regulatory system, thereby achieving the in vitro acquisition of a large amount of the protein encoded by the nucleic acid molecules.
本申请所述的“嵌合抗原受体”是指表达在细胞膜表面的人工受体片段，其包括胞外区、跨膜区和胞内区，所述胞外区能够特异性结合相应的配体或抗原，引起胞内区所包含的免疫刺激因子的激活。The "chimeric antigen receptor" described in this 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 immunostimulatory factors contained in the intracellular region.
本申请构建了一种表达嵌合抗原受体的转基因免疫细胞，其中，所述嵌合抗原受体靶向的配体PVR家族成员中的至少之一。所述嵌合抗原受体能够表达激活信号，增强CAR免疫细胞的杀伤肿瘤活性，并应用于实体瘤和血液瘤的治疗。The present application constructs a transgenic immune cell expressing a chimeric antigen receptor, wherein the chimeric antigen receptor targets at least one of the ligand PVR family members. The chimeric antigen receptor can express activation signals, enhance the tumor killing activity of CAR immune cells, and is applied to the treatment of solid tumors and hematological tumors.
下面将更详细地描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。Embodiments of the present invention will be described in more detail below, examples of which are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be construed as limiting the present invention.
需要说明的是，以下实施中所述的“质粒”与“载体”具有相同的意义，可互换使用。It should be noted that the "plasmid" and "vector" described in the following embodiments have the same meaning and can be used interchangeably.
实施例1：TIGIT-CAR-NK细胞的制备Example 1: Preparation of TIGIT-CAR-NK cells
1.1pCDH-EF1-TIGIT-CAR-T2A-copGFP慢病毒质粒的构建1.1 Construction of pCDH-EF1-TIGIT-CAR-T2A-copGFP lentiviral plasmid
本发明设计的TIGIT-CAR载体序列，包含TIGIT受体的胞外段与跨膜段、4-1BB共刺激信号域、IL18RB胞内段与CD3ζ胞内区。基因元件结构示意图见图1。The TIGIT-CAR vector sequence designed by the present invention comprises the extracellular segment and transmembrane segment of the TIGIT receptor, the 4-1BB co-stimulatory signal domain, the IL18RB intracellular segment and the CD3ζ intracellular region. The schematic diagram of the gene element structure is shown in Figure 1.
将全基因合成的TIGIT-CAR片段通过酶切位点XbaI与BamHI***到慢病毒载体pCDH-EF1-MSC-T2A-copGFP载体中。经菌落PCR鉴定以及测序验证序列正确后，表示pCDH-EF1-TIGIT-CAR-T2A-copGFP质粒构建成功。The fully synthesized TIGIT-CAR fragment was inserted into the lentiviral vector pCDH-EF1-MSC-T2A-copGFP through the restriction sites XbaI and BamHI. After colony PCR identification and sequencing verification of the correct sequence, it was confirmed that the pCDH-EF1-TIGIT-CAR-T2A-copGFP plasmid was successfully constructed.
1.2慢病毒的包装及病毒液浓缩1.2 Lentivirus packaging and virus liquid concentration
取处于对数生长期的293T细胞5×106接种于10cm的培养皿中，加入10mL DMEM培养基，37℃、
5％CO2培养箱中培养过夜。待细胞密度达到80％时，更换10mL新鲜的DMEM培养基，继续置于培养箱中培养。5×10 6 293T cells in the logarithmic growth phase were inoculated into a 10 cm culture dish, 10 mL of DMEM medium was added, and the culture was incubated at 37°C. Culture overnight in a 5% CO 2 incubator. When the cell density reaches 80%, replace with 10 mL of fresh DMEM medium and continue to culture in the incubator.
配制慢病毒包装体系：将6μg psPAX2质粒，3μg pMD2.G质粒与6μg pCDH-EF1-TIGIT-CAR-T2A-copGFP质粒加入至体积为250μL无血清DMEM培养基中，混合均匀，配制DNA混合液；将15μL PEIpro加到体积为235μL的无血清DMEM培养基中，混合均匀配制PEIpro混合液。将PEIpro混合液一次性加入到DNA混合液中，静置混匀，室温孵育15min。将混合液加入293T细胞培养皿中。培养24h后进行换液，将培养皿放回37℃，5％CO2培养箱中。在48h后收取细胞上清，400×g离心5min，去除细胞碎片，将上清用0.45μm的滤头过滤至新的50ml离心管中。加入5×PEG8000溶液，上下颠倒离心管混合均匀，放于4℃冰箱中过夜。4℃，4000×g离心20min，弃上清，加适量无血清DMEM培养基重悬病毒沉淀，分装至EP管中，放于-80℃冰箱中保存。Prepare lentiviral packaging system: add 6μg psPAX2 plasmid, 3μg pMD2.G plasmid and 6μg pCDH-EF1-TIGIT-CAR-T2A-copGFP plasmid to 250μL serum-free DMEM medium, mix well, and prepare DNA mixture; add 15μL PEIpro to 235μL serum-free DMEM medium, mix well to prepare PEIpro mixture. Add PEIpro mixture to DNA mixture at one time, let stand to mix well, and incubate at room temperature for 15min. Add the mixture to 293T cell culture dish. After 24h of culture, change the medium and put the culture dish back to 37℃, 5% CO2 incubator. After 48h, collect the cell supernatant, centrifuge at 400×g for 5min to remove cell debris, and filter the supernatant with a 0.45μm filter into a new 50ml centrifuge tube. Add 5×PEG8000 solution, mix well by inverting the centrifuge tube, and place in a 4°C refrigerator overnight. Centrifuge at 4°C, 4000×g for 20 min, discard the supernatant, add an appropriate amount of serum-free DMEM medium to resuspend the virus precipitate, divide into EP tubes, and store in a -80°C refrigerator.
1.3慢病毒感染人NK细胞1.3 Lentiviral infection of human NK cells
取处于生长对数期的NK-92细胞(购自ATCC)，加入2mLα-MEM培养基重悬细胞，调整细胞密度为5×105个/mL。在24孔板中接入5×105个NK-92细胞，1mL病毒浓缩液，1μL鱼精蛋白(购自索莱宝，终浓度8μg/mL)。置于37℃、5％CO2培养箱中培养。24h后，观察细胞状态，换液，将感染细胞转移入EP管中，100×g离心5min，加入少量新鲜α-MEM培养基重悬细胞，将细胞转入细胞培养瓶中，加入10mL新鲜α-MEM培养基和IL-2(终浓度为200IU/mL)继续培养。待细胞扩增后，将细胞转移入流式管中，加入3mL 1×PBS溶液重悬细胞，100×g离心5min，弃上清，涡旋起细胞沉淀，重复一次。将感染后的NK-92细胞通过流式仪分选GFP阳性的经流式分选GFP阳性的TIGIT-CAR-NK细胞用于后续实验。Take NK-92 cells (purchased from ATCC) in the logarithmic growth phase, add 2mL α-MEM medium to resuspend the cells, and adjust the cell density to 5×10 5 /mL. Inoculate 5×10 5 NK-92 cells, 1mL virus concentrate, and 1μL protamine (purchased from Solebo, final concentration 8μg/mL) in a 24-well plate. Culture in a 37°C, 5% CO 2 incubator. After 24h, observe the cell state, change the medium, transfer the infected cells to an EP tube, centrifuge at 100×g for 5min, add a small amount of fresh α-MEM medium to resuspend the cells, transfer the cells to a cell culture flask, add 10mL fresh α-MEM medium and IL-2 (final concentration of 200IU/mL) to continue culturing. After cell amplification, transfer the cells to a flow tube, add 3mL 1×PBS solution to resuspend the cells, centrifuge at 100×g for 5min, discard the supernatant, vortex the cell pellet, and repeat once. The infected NK-92 cells were sorted by flow cytometry for GFP-positive TIGIT-CAR-NK cells, which were used for subsequent experiments.
实施例2：TIGIT-CAR-NK细胞的生物学功能鉴定Example 2: Identification of biological functions of TIGIT-CAR-NK cells
2.1 TIGIT配体CD155在肿瘤细胞的表达2.1 Expression of TIGIT ligand CD155 in tumor cells
已有研究表明卵巢癌组织以及多种卵巢癌细胞表面高表达TIGIT配体CD155。发明人通过流式细胞术检测了人卵巢癌细胞系HO8910与SKOV-3细胞表面CD155的表达情况。结果显示，HO8910与SKOV-3细胞均高表达CD155分子(图2)。Studies have shown that ovarian cancer tissues and various ovarian cancer cells highly express TIGIT ligand CD155. The inventors detected the expression of CD155 on the surface of human ovarian cancer cell lines HO8910 and SKOV-3 cells by flow cytometry. The results showed that both HO8910 and SKOV-3 cells highly expressed CD155 molecules (Figure 2).
2.2 TIGIT-CAR-NK细胞的体外杀伤能力2.2 In vitro killing ability of TIGIT-CAR-NK cells
以NK-92、TIGIT-CAR-NK-92和TIGIT-CAR2-NK-92为效应细胞，以卵巢癌细胞系HO8910作为靶细胞，设置效靶比为5:1、2.5:1与1.25:1。将效应细胞与靶细胞共孵育4h，LDH(乳酸脱氢酶)释放法检测效应细胞对靶细胞的杀伤效率。结果表明，TIGIT-CAR细胞对H08910细胞的杀伤效率均明显高于NK-92与TIGIT-CAR2(氨基酸序列如SEQ ID NO:10所示)细胞组(图3)。以上结果说明经TIGIT-CAR基因修饰可明显提高NK细胞对CD155阳性肿瘤细胞的杀伤能力，且胞内段为4-1BB胞内段和CD3ζ
胞内段的TIGIT-CAR结构优于胞内段为4-1BB胞内段、IL-21受体(IL-21R，氨基酸序列如SEQ ID NO:9所示)胞内段和CD3ζ胞内段的CAR结构，能够激发NK细胞更强的对CD155阳性肿瘤细胞的杀伤能力。
NK-92, TIGIT-CAR-NK-92 and TIGIT-CAR2-NK-92 were used as effector cells, and the ovarian cancer cell line HO8910 was used as the target cell, and the effector-target ratio was set to 5:1, 2.5:1 and 1.25:1. The effector cells and target cells were co-incubated for 4 hours, and the killing efficiency of the effector cells on the target cells was detected by the LDH (lactate dehydrogenase) release method. The results showed that the killing efficiency of TIGIT-CAR cells on H08910 cells was significantly higher than that of the NK-92 and TIGIT-CAR2 (amino acid sequence as shown in SEQ ID NO: 10) cell groups (Figure 3). The above results show that TIGIT-CAR gene modification can significantly improve the killing ability of NK cells against CD155-positive tumor cells, and the intracellular segment is the 4-1BB intracellular segment and CD3ζ The TIGIT-CAR structure of the intracellular segment is superior to the CAR structure whose intracellular segment is the 4-1BB intracellular segment, the IL-21 receptor (IL-21R, the amino acid sequence is shown in SEQ ID NO:9) intracellular segment and the CD3ζ intracellular segment, and can stimulate NK cells to have a stronger killing ability against CD155-positive tumor cells.
NK-92, TIGIT-CAR-NK-92 and TIGIT-CAR2-NK-92 were used as effector cells, and the ovarian cancer cell line HO8910 was used as the target cell, and the effector-target ratio was set to 5:1, 2.5:1 and 1.25:1. The effector cells and target cells were co-incubated for 4 hours, and the killing efficiency of the effector cells on the target cells was detected by the LDH (lactate dehydrogenase) release method. The results showed that the killing efficiency of TIGIT-CAR cells on H08910 cells was significantly higher than that of the NK-92 and TIGIT-CAR2 (amino acid sequence as shown in SEQ ID NO: 10) cell groups (Figure 3). The above results show that TIGIT-CAR gene modification can significantly improve the killing ability of NK cells against CD155-positive tumor cells, and the intracellular segment is the 4-1BB intracellular segment and CD3ζ The TIGIT-CAR structure of the intracellular segment is superior to the CAR structure whose intracellular segment is the 4-1BB intracellular segment, the IL-21 receptor (IL-21R, the amino acid sequence is shown in SEQ ID NO:9) intracellular segment and the CD3ζ intracellular segment, and can stimulate NK cells to have a stronger killing ability against CD155-positive tumor cells.
另外，检测了NK细胞杀伤相关脱颗粒的情况，将不同效应细胞分别与HO8910细胞共孵育4h后，收集NK细胞于流式管中，分别通过流式细胞术检测NK细胞上CD107a、颗粒酶(Granzyme B)和穿孔素(Perforin)的表达情况。结果可见，与HO8910细胞共孵育后，TIGIT-CAR细胞上CD107a与分泌Granzyme B的表达水平明显高于NK-92组，Perforin的表达水平无统计学差异(图4)。进一步验证了经TIGIT-CAR修饰可明显提高NK细胞对CD155阳性肿瘤细胞的脱颗粒水平(CD107a、颗粒酶和穿孔素的表达水平)和杀伤功能。In addition, the degranulation related to NK cell killing was detected. After different effector cells were co-incubated with HO8910 cells for 4 hours, NK cells were collected in flow tubes, and the expression of CD107a, granzyme (Granzyme B) and perforin (Perforin) on NK cells was detected by flow cytometry. The results showed that after co-incubation with HO8910 cells, the expression levels of CD107a and secreted Granzyme B on TIGIT-CAR cells were significantly higher than those in the NK-92 group, and there was no statistical difference in the expression level of Perforin (Figure 4). It was further verified that TIGIT-CAR modification can significantly improve the degranulation level (expression levels of CD107a, granzyme and perforin) and killing function of NK cells on CD155-positive tumor cells.
2.3 TIGIT-CAR-NK细胞的IFN-γ与TNF-α的分泌水平2.3 Secretion levels of IFN-γ and TNF-α by TIGIT-CAR-NK cells
通过流式细胞术检测TIGIT-CAR-NK-92细胞的IFN-γ与TNF-α的分泌能力变化。将NK细胞与卵巢癌HO8910细胞共孵育4h后，收集NK细胞于流式管中，通过固定破膜处理，流式细胞术检测NK细胞IFN-γ与TNF-α的分泌水平。结果可见，TIGIT-CAR-NK-92细胞分泌IFN-γ与TNF-α的水平均显著高于NK-92组(图5)。说明TIGIT-CAR可明显提高NK细胞与CD155阳性肿瘤细胞接触时IFN-γ与TNF-α的分泌能力。
The changes in the secretion capacity of IFN-γ and TNF-α of TIGIT-CAR-NK-92 cells were detected by flow cytometry. After NK cells were co-incubated with ovarian cancer HO8910 cells for 4 hours, NK cells were collected in flow tubes, and the secretion levels of IFN-γ and TNF-α of NK cells were detected by flow cytometry after fixation and membrane permeabilization. The results showed that the levels of IFN-γ and TNF-α secreted by TIGIT-CAR-NK-92 cells were significantly higher than those of the NK-92 group (Figure 5). This indicates that TIGIT-CAR can significantly improve the secretion capacity of IFN-γ and TNF-α of NK cells when they come into contact with CD155-positive tumor cells.
实施例3：TIGIT-CAR-NK细胞体内抗肿瘤能力Example 3: Anti-tumor ability of TIGIT-CAR-NK cells in vivo
以荧光素酶标记的卵巢癌HO8910细胞进行腹腔荷瘤，建立卵巢癌腹腔转移模型。选择5周龄雌性高度免疫缺陷NCG鼠进行腹腔荷瘤，每只小鼠腹腔注射2×105个荧光素酶标记的HO8910细胞。荷瘤后第2天，进行NK细胞回输治疗。将小鼠随机分为对照组、NK-92治疗组和TIGIT-CAR-NK-92治疗组。治疗组小鼠采用腹腔注射NK细胞5×106个/只，对照组注射等体积的1×PBS溶液，每隔一周注射一次，共治疗3次。每隔3天腹腔注射IL-2(50000IU/只)。通过小动物活体成像技术观察肿瘤大小，进而绘制肿瘤生长曲线。The ovarian cancer HO8910 cells labeled with luciferase were used for intraperitoneal tumor implantation to establish an ovarian cancer peritoneal metastasis model. Five-week-old female highly immunodeficient NCG mice were selected for intraperitoneal tumor implantation, and 2×10 5 luciferase-labeled HO8910 cells were injected intraperitoneally into each mouse. On the second day after tumor implantation, NK cell transfusion 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 mice in the treatment group were intraperitoneally injected with 5×10 6 NK cells/mouse, and the control group was injected with an equal volume of 1×PBS solution, once every week, for a total of 3 treatments. IL-2 (50000 IU/mouse) was injected intraperitoneally every 3 days. The tumor size was observed by small animal in vivo imaging technology, and then the tumor growth curve was drawn.
结果显示，与荷瘤对照组相比，NK-92和TIGIT-CAR-NK-92治疗组均能明显抑制肿瘤的生长，而以TIGIT-CAR-NK-92为治疗组效果最佳，活体成像技术观测的肿瘤荧光信号强度明显小于NK-92治疗组(图6与图7)。说明TIGIT-CAR基因修饰后表达激活信号，激活NK细胞的杀伤肿瘤作用。The results showed that compared with the tumor-bearing control group, both the NK-92 and TIGIT-CAR-NK-92 treatment groups could significantly inhibit tumor growth, and the TIGIT-CAR-NK-92 treatment group had the best effect. The tumor fluorescence signal intensity observed by in vivo imaging technology was significantly lower than that of the NK-92 treatment group (Figure 6 and Figure 7). This indicates that the TIGIT-CAR gene expresses activation signals after modification, activating the tumor-killing effect of NK cells.
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.
Claims (25)
- 一种嵌合抗原受体，其特征在于，包括：A chimeric antigen receptor, comprising:胞外区，所述胞外区包括TIGIT胞外段；An extracellular region, wherein the extracellular region includes the TIGIT extracellular segment;跨膜区，所述跨膜区包括TIGIT跨膜区，并且嵌入到细胞膜中；以及a transmembrane region, which includes a TIGIT transmembrane region and is embedded in the cell membrane; and胞内区，所述胞内区包括4-1BB共刺激因子结构域、IL18RB胞内段与CD3ζ胞内信号段；The intracellular region includes a 4-1BB co-stimulatory factor domain, an IL18RB intracellular segment, and a CD3ζ intracellular signal segment;其中，所述胞外区的C端与所述跨膜区的N端相连，所述跨膜区的C端与所述胞内区的N端相连。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.
- 根据权利要求1所述的嵌合抗原受体，其特征在于，所述4-1BB共刺激因子结构域的C端与所述IL18RB胞内段的N端相连，所述IL18RB胞内段的C端与所述CD3ζ胞内信号段的N端相连。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 IL18RB intracellular segment, and the C-terminus of the IL18RB intracellular segment is connected to the N-terminus of the CD3ζ intracellular signal segment.
- 根据权利要求1所述的嵌合抗原受体，其特征在于，所述胞外区能够结合配体，所述配体包括PVR家族成员中的至少之一。The chimeric antigen receptor according to claim 1, characterized in that the extracellular region is capable of binding to a ligand, and the ligand includes at least one of the PVR family members.
- 根据权利要求3所述的嵌合抗原受体，其特征在于，所述PVR家族成员包括CD155和CD112。The chimeric antigen receptor according to claim 3, characterized in that the PVR family members include CD155 and CD112.
- 根据权利要求4所述的嵌合抗原受体，其特征在于，所述PVR家族成员为CD155。The chimeric antigen receptor according to claim 4, characterized in that the PVR family member is CD155.
- 根据权利要求1所述的嵌合抗原受体，其特征在于，所述TIGIT胞外段具有如SEQ ID NO.1所示的氨基酸序列；The chimeric antigen receptor according to claim 1, characterized in that the TIGIT extracellular segment has an amino acid sequence as shown in SEQ ID NO.1;所述TIGIT跨膜区具有如SEQ ID NO.2所示的氨基酸序列；The TIGIT transmembrane region has an amino acid sequence as shown in SEQ ID NO.2;所述4-1BB共刺激因子结构域具有如SEQ ID NO.3所示的氨基酸序列；The 4-1BB co-stimulatory factor domain has an amino acid sequence as shown in SEQ ID NO.3;所述IL18RB胞内段具有如SEQ ID NO.4所示的氨基酸序列；The IL18RB intracellular segment has an amino acid sequence as shown in SEQ ID NO.4;所述CD3ζ胞内信号段具有SEQ ID NO:5所示的氨基酸序列；The CD3ζ intracellular signal segment has an amino acid sequence shown in SEQ ID NO:5;所述嵌合抗原受体具有SEQ ID NO:6所示的氨基酸序列。The chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO:6.
- 一种核酸分子，其特征在于，编码权利要求1～6任一项所述的嵌合抗原受体。A nucleic acid molecule, characterized in that it encodes the chimeric antigen receptor according to any one of claims 1 to 6.
- 根据权利要求7所述的核酸分子，其特征在于，所述核酸分子具有SEQ ID NO:7所示的核苷酸序列。The nucleic acid molecule according to claim 7 is characterized in that the nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:7.
- 一种表达载体，其特征在于，携带权利要求7或8所述的核酸分子。An expression vector, characterized in that it carries the nucleic acid molecule according to claim 7 or 8.
- 根据权利要求9所述的表达载体，其特征在于，进一步包括：启动子，所述启动子与权利要求7或8所述的核酸分子可操作地连接。The expression vector according to claim 9 is characterized in that it further comprises: a promoter, wherein the promoter is operably linked to the nucleic acid molecule according to claim 7 or 8.
- 根据权利要求10所述的表达载体，其特征在于，所述启动子选自CMV,EF-1,RSV的至少之一。The expression vector according to claim 10, characterized in that the promoter is selected from at least one of CMV, EF-1, and RSV.
- 根据权利要求9所述的表达载体，其特征在于，所述表达载体是非致病性病毒载体。The expression vector according to claim 9, characterized in that the expression vector is a non-pathogenic viral vector.
- 根据权利要求12所述的表达载体，其特征在于，所述非致病性病毒选自反转录病毒、慢病毒和腺病毒相关病毒。The expression vector according to claim 12, characterized in that the non-pathogenic virus is selected from retrovirus, lentivirus and adenovirus-associated virus.
- 根据权利要求13所述的表达载体，其特征在于，所述非致病性病毒为慢病毒。 The expression vector according to claim 13, characterized in that the non-pathogenic virus is a lentivirus.
- 一种慢病毒载体，其特征在于，具有SEQ ID NO：8所示的核苷酸序列。A lentiviral vector, characterized in that it has a nucleotide sequence shown in SEQ ID NO: 8.
- 一种转基因免疫细胞，其特征在于，所述转基因免疫细胞表达权利要求1～6任一项所述的嵌合抗原受体、携带权利要求7～8任一项所述的核酸分子、权利要求9～14任一项所述的表达载体或权利要求15所述的慢病毒载体。A transgenic immune cell, characterized in that the transgenic immune cell expresses the chimeric antigen receptor described in any one of claims 1 to 6, carries the nucleic acid molecule described in any one of claims 7 to 8, the expression vector described in any one of claims 9 to 14, or the lentiviral vector described in claim 15.
- 一种CAR-免疫细胞，其特征在于，所述CAR-免疫细胞表达权利要求1～6任一项所述的嵌合抗原受体、携带权利要求7～8任一项所述的核酸分子、权利要求9～14任一项所述的表达载体或权利要求15所述的慢病毒载体。A CAR-immune cell, characterized in that the CAR-immune cell expresses the chimeric antigen receptor according to any one of claims 1 to 6, carries the nucleic acid molecule according to any one of claims 7 to 8, the expression vector according to any one of claims 9 to 14, or the lentiviral vector according to claim 15.
- 根据权利要求17所述的CAR-免疫细胞，其特征在于，所述CAR-免疫细胞包括选自NK-92细胞，外周血NK细胞、脐带血NK细胞、iPSC、CAR-NK细胞、CAR-T细胞、CAR-NKT细胞、CAR-γδT细胞中的至少之一。The CAR-immune cell according to claim 17, characterized in that the CAR-immune cell comprises 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.
- 一种药物组合物，其特征在于，包括：A pharmaceutical composition, characterized in that it comprises:表达权利要求1～6任一项所述的嵌合抗原受体、携带权利要求7～8任一项所述的核酸分子、权利要求9～14任一项所述的表达载体或权利要求15所述的慢病毒载体、权利要求16所述的转基因免疫细胞或权利要求17～18任一项所述的CAR-免疫细胞。A chimeric antigen receptor expressing any one of claims 1 to 6, a nucleic acid molecule carrying any one of claims 7 to 8, an expression vector according to any one of claims 9 to 14 or a lentiviral vector according to claim 15, a transgenic immune cell according to claim 16 or a CAR-immune cell according to any one of claims 17 to 18.
- 根据权利要求19所述的药物组合物，其特征在于，所述药物组合物进一步包括：药学上可接受的辅料。The pharmaceutical composition according to claim 19, characterized in that the pharmaceutical composition further comprises: a pharmaceutically acceptable excipient.
- 表达权利要求1～6任一项所述的嵌合抗原受体、携带权利要求7～8任一项所述的核酸分子、权利要求9～14任一项所述的表达载体或权利要求15所述的慢病毒载体、权利要求16所述的转基因免疫细胞或权利要求17～18任一项所述的CAR-免疫细胞在制备药物中的用途，所述药物用于治疗或预防实体瘤或血液瘤。Use of a chimeric antigen receptor expressing any one of claims 1 to 6, a nucleic acid molecule carrying any one of claims 7 to 8, an expression vector according to any one of claims 9 to 14 or a lentiviral vector according to claim 15, a transgenic immune cell according to claim 16 or a CAR-immune cell according to any one of claims 17 to 18 in the preparation of a drug for treating or preventing solid tumors or hematological tumors.
- 根据权利要求21所述的用途，其特征在于，所述实体瘤包括选自发生在脏器中的有形瘤，包括胰腺癌、卵巢癌、间皮瘤、肝癌、胆管癌、胃癌、结直肠癌、食管癌、肺癌、头颈癌、***、脑胶质瘤、肾癌、乳腺癌、***癌、黑色素瘤中的至少之一。The use according to claim 21 is characterized in that the solid tumor includes a tangible tumor selected from those occurring in internal organs, including at least one of pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, colorectal cancer, esophageal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, prostate cancer, and melanoma.
- 根据权利要求21所述的用途，其特征在于，所述血液瘤包括选自血细胞和造血***内的急性髓系白血病、急性淋巴细胞性白血病、B细胞淋巴瘤、T细胞淋巴瘤、何杰金氏淋巴瘤、非何杰金氏淋巴瘤、多发性骨髓瘤中的至少之一。The use according to claim 21 is characterized in that the blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma in blood cells and hematopoietic system.
- 一种治疗或预防实体瘤或血液瘤的方法，其特征在于，包括向受试者施用权利要求1～6任一项所述的嵌合抗原受体、权利要求7～8任一项所述的核酸分子、权利要求9～14任一项所述的表达载体、权利要求15所述的慢病毒载体、权利要求16所述的转基因免疫细胞、权利要求17～18任一项所述的CAR-免疫细胞或权利要求19～20任一项所述的药物组合物中的至少之一。 A method for treating or preventing solid tumors or hematological tumors, characterized in that it comprises administering to a subject at least one of the chimeric antigen receptor described in any one of claims 1 to 6, the nucleic acid molecule described in any one of claims 7 to 8, the expression vector described in any one of claims 9 to 14, the lentiviral vector described in claim 15, the transgenic immune cell described in claim 16, the CAR-immune cell described in any one of claims 17 to 18, or the pharmaceutical composition described in any one of claims 19 to 20.
- 权利要求1～6任一项所述的嵌合抗原受体、权利要求7～8任一项所述的核酸分子、权利要求9～14任一项所述的表达载体、权利要求15所述的慢病毒载体、权利要求16所述的转基因免疫细胞、权利要求17～18任一项所述的CAR-免疫细胞或权利要求19～20任一项所述的药物组合物在治疗或预防实体瘤或血液瘤中的用途。 Use of the chimeric antigen receptor according to any one of claims 1 to 6, the nucleic acid molecule according to any one of claims 7 to 8, the expression vector according to any one of claims 9 to 14, the lentiviral vector according to claim 15, the transgenic immune cell according to claim 16, the CAR-immune cell according to any one of claims 17 to 18, or the pharmaceutical composition according to any one of claims 19 to 20 in the treatment or prevention of solid tumors or hematological tumors.
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