WO2018058431A1 - Chimeric antigen receptor molecule and use thereof - Google Patents

Chimeric antigen receptor molecule and use thereof Download PDF

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Publication number
WO2018058431A1
WO2018058431A1 PCT/CN2016/100796 CN2016100796W WO2018058431A1 WO 2018058431 A1 WO2018058431 A1 WO 2018058431A1 CN 2016100796 W CN2016100796 W CN 2016100796W WO 2018058431 A1 WO2018058431 A1 WO 2018058431A1
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cells
peptide
chimeric antigen
cell
antigen receptor
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李华顺
韩昆昆
薛亚男
王保垒
任宝永
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李华顺
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to the field of molecular biology, in particular to a chimeric antigen receptor molecule and application thereof.
  • a malignant tumor is a disease that seriously threatens human life.
  • the pathogenesis of malignant tumors or cancers is diverse.
  • the common manifestation is that the mutant tumor cells are not cleared by the body's immune system, can propagate and spread without restriction, and destroy the normal cells and functions of surrounding tissues.
  • the medical profession has made a lot of efforts in trying to heal and control the course of tumor diseases, but the results have been minimal.
  • the clinical adjuvant treatment of mainstream medical circles is still radiation therapy, chemical drug therapy and antibody therapy.
  • the recognition and killing effect of immunocompetent cells on tumor cells depends on the expression of receptor molecules on the surface of tumor cell membranes. At least two factors lead to the inability of T lymphocytes in vivo to recognize cancer cells well: (1) Downregulation of antigens by cancer cells The expression of the presenting molecule, (2) the presented antigen has a weak affinity for the T cell receptor. Although there are highly specific T lymphocytes in cancer cells in cancer patients, the number is too small. Can not play a role in the treatment of cancer.
  • the chimeric antigen receptor is mainly composed of two parts, one end is located outside the cell and can specifically recognize an antigen on the surface of the cancer cell, and the other end is located in the cell containing a signal activation element (such as a Zeta chain of T cell receptor) to transmit a signal. Activate the role of T cells.
  • a signal activation element such as a Zeta chain of T cell receptor
  • CAR-T cell therapy developed with CD19 has achieved remarkable results in the clinical treatment of both primary and secondary hematological tumors, but the progress of CAR-T cell therapy in solid tumor research is slow. There is no significant breakthrough.
  • One of the reasons is that 95% of lymphocytic leukemia cells express the B cell antigen CD19, while the specific antigen expression of other solid tumor cells is between 40-70%; therefore, in the treatment of solid tumors, a single monoclonal antibody CAR - T cells are unlikely to kill cancer cells that express other tumor-specific antigens.
  • the second reason is the rate and number of immune killer cells infiltrating into the solid tumor through the blood/lymph circulation.
  • the third reason is the negative regulation of tumor tissue cells on immunocompetent cells.
  • T-regulated cells mainly controlled by Regulatory T cells (Treg).
  • T-regulated cells When the control of T-regulated cells is weakened, it may cause hyperimmune or autoimmune diseases. When the control is enhanced, the immune function will be low. , breeding tumors or other viral skin diseases.
  • CTL4 Cytotoxic T lymphocyte-associated antigen-4
  • programmed death protein 1 programmed yellow cell-associated antigen 1 (Programmed cell death protein) 1/Programmed cell death protein 1 ligand 1, PD-1/PD-L1) and other molecules.
  • Inhibitory T-regulated cells express CTLA4, which interacts with B7-type protein subunits on the surface of immune cells DC and T cells to inhibit immune cell function.
  • B7 protein family include B7-H1 (PD-L1), B7-H2 (PD-1L2) and the like.
  • B7-H1 (PD-L1) can also further inhibit the active function of target cells by binding to lymphocyte PD-1 molecules.
  • the inhibition of T cell function by PD-1 and PD-L1 interaction is a major obstacle to the treatment of tumors by immune cells. In most solid tumor tissues, cancer cells express an increased level of PD-L1/PD-1, which directly inhibits activated lymphocytes infiltrating into cancerous tissues.
  • the present invention prepares a high affinity chimeric antigen with higher specificity and lethality by modifying the PD-1 molecule of the extracellular domain of the chimeric antigen receptor molecule (CAR) and selecting a molecule having higher affinity than PD-1 and PDL1. Receptor molecule.
  • CAR chimeric antigen receptor molecule
  • an object of the present invention is to provide a chimeric antigen receptor molecule having a higher affinity for a PDL-1 molecule on a tumor surface and an application thereof.
  • a first aspect of the invention provides a chimeric antigen receptor molecule comprising a sequence of extracellular domain peptides, a transmembrane domain peptide and an intracellular domain peptide, wherein the extracellular domain peptide comprises a HAC peptide, Its sequence is shown in SEQ ID NO: 1. According to the article "Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging", the mutant HAC of PD-1 has higher affinity with the tumor surface PDL-1 molecule.
  • the transmembrane region peptide is a region spanning a cell membrane in a protein sequence, usually an ⁇ -helical structure, about 20-25 amino acid residues, and the amino acid is mostly a hydrophobic amino acid; wherein
  • the transmembrane region peptide includes, but is not limited to, a CD8 transmembrane region peptide or a PD-1 transmembrane region peptide, and when the transmembrane region peptide is a CD8 transmembrane region peptide, the HAC peptide segment and the CD8 transmembrane region The peptides are also linked by the hinge region of the CD8.
  • the sequence of the CD8 transmembrane region peptide is shown in SEQ ID NO: 2
  • the sequence of the hinge region of CD8 is shown in SEQ ID NO: 3
  • the sequence of the PD-1 transmembrane region peptide is as SEQ ID NO: 4. Shown.
  • the intracellular domain peptide is a costimulatory signal molecule selected from one or more of the intracellular domain peptides of 4-1BB (also known as CD137), CD28, and CD3 ⁇ .
  • the intracellular domain peptide is selected from the group consisting of interconnected 4-1BB and CD3 sputum intracellular domain peptides, the sequences of which are set forth in SEQ ID NOS: 5 and 6, respectively.
  • the chimeric antigen receptor molecule further comprises a signal peptide.
  • the signal peptide can enhance the secretion of the fusion protein of the chimeric antigen receptor molecule, and the signal peptide and the other amino acid sequence of the fusion protein After being expressed, it is finally excised by proteases.
  • the protease has a certain recognition sequence, and the signal peptide is fused with the peptide after it to form a new amino acid sequence. Therefore, if the selected signal peptide is improper, the protease may be miscut and the protein may be inactivated.
  • the signal peptide may be selected from a signal peptide of an immunoglobulin light chain or a signal peptide secreted by a PD-1 protein, the sequences of which are shown in SEQ ID NOS: 7 and 8, respectively.
  • the chimeric antigen receptor molecule of the present invention comprises a signal peptide of an immunoglobulin light chain, a HAC peptide, a hinge region peptide of CD8, a transmembrane region peptide of CD8, and a 4-1BB intracellular domain peptide.
  • the segment and the CD3 sputum intracellular domain peptide are sequentially ligated, and the sequence thereof is shown in SEQ ID NO: 9.
  • a second aspect of the invention provides a nucleotide encoding a chimeric antigen receptor molecule provided by the first aspect of the invention.
  • a third aspect of the invention provides a recombinant vector comprising the nucleotide provided by the second aspect of the invention.
  • the vector is a lentiviral vector, which can efficiently integrate a foreign gene or an exogenous shRNA into a host chromosome, thereby achieving the effect of persistently expressing the sequence of interest. It can effectively infect various types of cells such as neuron cells, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, stem cells, etc. in terms of infection ability, thereby achieving a good gene therapy effect.
  • the use of lentiviral vectors can greatly improve the transduction efficiency of the target gene or the target shRNA, and the target gene or the target shRNA is integrated into the host cell. The probability of genome is greatly increased, and the long-term and stable expression of the target gene or the target shRNA can be realized conveniently and quickly.
  • the lentiviral vector used in the present invention should include, but is not limited to, a pRRSLIN lentiviral expression vector and a pLVX vector, preferably a pRRSLIN lentiviral expression vector.
  • a fourth aspect of the invention provides a recombinant cell comprising the recombinant vector provided by the third aspect of the invention.
  • the recombinant cell is preferably a T cell or an NK cell.
  • the gene encoding the chimeric antigen receptor can be transferred to T cells or NK cells by the aforementioned vector, used to modify T cells or NK cells to become CAR-T or CAR-NK cells; and the chimeric antigen receptor is modified by the chimeric antigen receptor T cells or NK cells can kill tumor cells by identifying tissue factors on the surface of tumor cells for tumor treatment.
  • co-stimulating molecule refers to some adhesion molecules on the surface of immune cells, such as CD28, CD134/OX40, CD137/4-1BB, CD40, etc., which activate immune cells by binding to their ligands.
  • the second signal enhances the proliferative capacity of immune cells and the secretory function of cytokines, prolonging the survival time of activated immune cells.
  • extracellular domain refers to a segment of the membrane protein that is located outside the cell.
  • domain refers to a region of a protein biomacromolecule having a specific structure and an independent function.
  • the number of amino acid residues in a common domain is between 100 and 400, and the smallest domain is only 40 to 50 amino acid residues. Large domains can exceed 400 amino acid residues.
  • PD-1 refers to programmed cell death protein 1 and the gene name PDCD1_HUMAN.
  • the corresponding protein sequence number is UniProtKB-Q15116, which is a T cell immunosuppressive molecule whose extracellular domain resembles an immunoglobulin.
  • the variable region (V-section) of the protein has the property of specifically binding to its ligands PD-L1 and PD-L2 (Programmed cell death protein 1 ligand 1/2).
  • PD-1 is normally expressed in activated T lymphocytes and is also expressed in a variety of malignant cells.
  • P-L1 and P-L2 refer to the currently discovered human programmed death factor 1 ligand 1/2. Its extracellular domain domain has immunoglobulin-like V and C1 regions, which are combined with the V region of PD-1 via the V region (4zqk Structure 232341-2348, 2015). It is usually expressed in a small amount in dendritic cells DC, T regulatory cells and Th cells, macrophages, Mast cells and bone marrow, and is also expressed in various malignant cells.
  • the present invention has at least the following advantages: the chimeric antigen receptor molecule of the present invention has a HAC molecule having higher affinity with the PDL-1 molecule on the tumor surface, and is assembled in the extracellular segment of the CAR-T cell. To identify tumor cells with higher affinity and killing activity.
  • Figure 1 is a schematic diagram showing the construction of a lentiviral expression vector
  • Figure 2 is a flow chart of pRRSLIN-HAC infection for 3 days
  • Figure 3 is a graph showing the killing results of CAR-HAC against different target cells
  • Figure 4 is a graph showing the results of CAR-HAC detection of in vitro proliferation of different target cells
  • Figure 5 is a graph showing the results of detection of cytokines by different kinds of target cells by CAR-HAC;
  • Figure 6 is a graph showing the results of detection of four cell lines of MCF7/MCF7-PDL1/HeLa/SMC7721 by CAR-HAC;
  • Figure 7 is a graph comparing the killing effects of different cell lines CAR-PD-1 and CAR-HAC.
  • the invention provides a preparation method of a lentiviral expression vector for expressing a chimeric antigen receptor molecule, comprising the following steps:
  • HAC gene fragment is synthesized, and the known human CD8 cross is searched from the GenBank database.
  • the above gene sequences are sequentially linked according to human HAC gene, CD8 membrane region gene, human 4-1BB intracellular region gene and CD3 sputum intracellular region gene, and different enzyme cleavage sites are introduced at each sequence junction to form a complete HAC-CD8-4-1BB-CD3 ⁇ gene sequence information,
  • the gene sequence of HAC-CD8-4-1BB-CD3 ⁇ is ligated into the pRRSLIN vector by restriction enzyme transformation, and the upstream of the gene is the EP-1 ⁇ promoter.
  • the vector was transformed into Stbl3 Escherichia coli strain, then transferred to a solid medium containing ampicillin for propagation, screened, positive clones were obtained, plasmids were extracted, and clones were identified by restriction enzyme digestion. The vector was confirmed to be successfully constructed by sequencing, and pRRSLIN-HAC was obtained slowly.
  • FIG. 1 A schematic diagram of the construction of a viral expression vector and a lentiviral expression vector is shown in FIG.
  • the present invention provides a method for producing a lentivirus by expressing a lentiviral expression vector in Example 1, comprising the following step:
  • 293T cells were seeded into a 15 cm culture dish at approximately 8 x 10 6 per dish. Ensure that the cells are confluent at 80% and evenly distributed in the culture dish during transfection.
  • Solution B A mixture of the following plasmids was separately added: 112.5 ⁇ g pRRLSIN-HAC (target plasmid); 39.5 ⁇ g pMD2.G (VSV-G envelop); 73 ⁇ g pCMVR8.74 (gag, pol, tat, rev); 625 ⁇ L 2M calcium ion Solution.
  • the present invention provides a method for preparing CAR-T cells by lentiviral infection of cells in Example 2, comprising the steps of:
  • S1 take 0.5mL blood for rapid detection of pathogenic microorganisms, exclude microbial infections such as HBV, HCV, HDV and HEV, HIV-1/2, Treponema pallidum and parasites; under sterile conditions, use blood collection of heparin bottle 50mL (heparin antibiotic) Condensed), immediately (4 ° C, within 24 hours) to the cell preparation laboratory to ensure that this process is free of pathogenic microorganisms. After obtaining the patient's blood, in the GMP preparation room, the surface of the heparin bottle is wiped with an alcohol cotton ball for disinfection and then placed in a biological safety cabinet.
  • heparin bottle 50mL (heparin antibiotic) Condensed immediately (4 ° C, within 24 hours
  • PBMC peripheral blood mononuclear cells
  • V-VIVO15 added autologous AB (FBS) concentration of 5%, interleukin-2 (IL-2) concentration of 40 ng / mL, and the isolated PBMC was diluted to 2 ⁇ with the culture medium 10 6 /mL, 50 ⁇ L flow detection of the purity of T cells in PBMC.
  • FBS autologous AB
  • IL-2 interleukin-2
  • Example 2 S6 and Day 2 adjusted the cell density to 3-5 ⁇ 10 6 /mL, and the pRRSLIN-HAC lentiviral expression vector prepared in Example 1 was added at a ratio of viral carrier to cell 1:5, and polyglycolamine was added ( Polybrene) 4 ⁇ g/mL and 40 ng/mL IL-2. After 4 h, fresh complete medium was added to adjust the cell density to 1 ⁇ 10 6 /mL to continue the culture. All cells were centrifuged, fresh medium was added, and the culture was continued.
  • the CAR-T cells prepared in Example 3 were subjected to flow analysis, and the specific steps were as follows:
  • Figures A and B are the control group, which are T cells that do not infect the virus; FITC-conjugated antibodies for detecting CAR molecules do not detect CAR molecule expression; C and D maps are T transfected with PRRSLIN-HAC lentivirus Cells, after flow detection, compared with A and B, cells were successfully transfected; after infection of T cells by virus, the infection efficiency reached 53.26% after 3 days of flow detection, indicating successful preparation of HAC-CAR -T cells.
  • LDH release assay was used to detect the killing effect of HAC-CAR-T cells on engineered cell lines MCF-1/PDL1 and PDL1 high expressing cells. LDH release was detected by ELISA, including the following steps:
  • the target cells were adjusted to 5 ⁇ 10 4 /mL with RPMI-1640 medium containing 5% calf serum.
  • target cells were added to a 96-well cell culture plate, and 100 ⁇ L per well was added. Three wells were used as effector cells (HAC-CAR-T cells) to naturally release control wells, and no target cells were added, and only 100 ⁇ L of the culture solution was added.
  • HAC-CAR-T cells effector cells
  • effector cells 100 ⁇ L were added to each well, and the ratio of effector cells to target cells was 10:1; 5:1; 1:1.
  • the natural release well was added with only 100 ⁇ L of the culture medium without effector cells, and the effector cells were incubated with the target cells for 6 hours, and three replicate wells were placed for each experiment.
  • the optical density (OD value) of each well was measured on an enzyme-linked detector, and the detection wavelength was 490 nm or 492 nm, and the measurement was completed within 1 hour.
  • Killing rate experimental group LDH (OD) / maximum LDH release group (OD).
  • the cytokine secretion was measured by a CBA kit, and the proliferation of each group of CAR-T cells was calculated, and the ratio of CD8-positive T cells in the proliferating T cells was confirmed by staining with CD3 and CD8 antibodies.
  • HAC CAR-T can significantly kill SMCCC7721 tumor cells, and the killing effect on MCF-7 cells with high expression of PDL-1 is better than that of MCF-7 common tumor cells.
  • the abscissa indicates CAR-T cells. Different from tumor cells, the ordinate indicates the killing efficiency, and different types of histograms indicate different tumor cells.
  • the abscissa indicates the ratio of T cells to tumor or CAR-T cells to tumors
  • the ordinate indicates the number of cells
  • T represents T cells
  • HAC represents CAR-T cells
  • the abscissa indicates the different target ratio of CAR-T cells to tumor cells
  • the ordinate indicates the cytokine content
  • the cytokines in the culture supernatant were detected
  • the CAR-T killing experimental group IL-2 was found.
  • the secretion of Figure 5A) and TNF-[alpha] (Figure 5B) was significantly elevated.
  • the CD3/CD8 flow antibody was used to detect the ratio of CAR-T to CD8T cells after killing MCF7/MCF7-PDL1/HeLa/SMC7721 cell lines
  • the ordinate PE signal showed specific detection of CD3 molecule expression.
  • the abscissa FITC signal indicates that the expression of CD8 molecules is detected, and the specific proliferation of CD8 T cells is mainly detected by flow cytometry after activation of HAC CAR-T cells.
  • CAR-PD-1 indicates that the extracellular domain of the CAR molecule is a normal PD-1 molecule
  • CAR-PD-1-HAC indicates the HAC-CAR chimeric antigen receptor molecule constructed by the present invention
  • HAC-CART cells can specifically activate and proliferate after exposure to tumor cells, release cytokines and kill tumor cells, and CD8-positive T cells play a major role.
  • the killing effect of PD-1 CAR-T and HAC CAR-T on tumors was compared.
  • the results showed that HAC-CAR-T was significantly better than PD-1 CAR-T.

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Abstract

Provided are a chimeric antigen receptor molecule and the use thereof. The chimeric antigen receptor molecule comprises a peptide fragment in an extracellular region, a peptide fragment in a transmembrane region and a peptide fragment in an intracellular domain in successive tandem. The peptide fragment in the extracellular region is an HAC peptide fragment, and the sequence thereof is as shown in SEQ ID NO: 1. Also provided are a nucleotide, a recombinant vector and a recombinant cell using the chimeric antigen receptor molecule.

Description

一种嵌合抗原受体分子及其应用Chimeric antigen receptor molecule and application thereof
本申请要求了申请日为2016年9月28日，申请号为201610855798.7，发明名称为“一种嵌合抗原受体分子及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。The present application claims the priority of the Chinese Patent Application No. 2016. 008 579 8.7, the entire disclosure of which is hereby incorporated by reference. In this application.
技术领域Technical field
本发明涉及分子生物学领域，尤其涉及一种嵌合抗原受体分子及其应用。The invention relates to the field of molecular biology, in particular to a chimeric antigen receptor molecule and application thereof.
背景技术Background technique
恶性肿瘤是一种严重威胁人类生命的疾病。恶性肿瘤或癌症的发病机理多种多样，其共同的表现在于变异的肿瘤细胞不受机体免疫***的清除，能够无限制的繁殖和扩散，破坏周围组织的正常细胞和功能。长期以来，医药学界在尝试医治和控制肿瘤疾病的病程方面做了大量的努力，但收效甚微。目前，在恶性肿瘤的治疗上，除根除手术外，主流医学界的临床辅助治疗手段仍然是放射线治疗、化学药物治疗和抗体治疗。A malignant tumor is a disease that seriously threatens human life. The pathogenesis of malignant tumors or cancers is diverse. The common manifestation is that the mutant tumor cells are not cleared by the body's immune system, can propagate and spread without restriction, and destroy the normal cells and functions of surrounding tissues. For a long time, the medical profession has made a lot of efforts in trying to heal and control the course of tumor diseases, but the results have been minimal. At present, in the treatment of malignant tumors, in addition to eradication surgery, the clinical adjuvant treatment of mainstream medical circles is still radiation therapy, chemical drug therapy and antibody therapy.
1985年，美国科学家尝试分离病人的单个核细胞，并在体外用各种细胞因子诱导、激活、产生杀伤性T细胞(Cytokine-induced killer cells，CIK)，继而发现这些细胞通过静脉滴注回输给病人后，对肿瘤的生长有杀伤作用。经过近三十年的临床应用和技术发展，用免疫杀伤细胞***已经成为第四种公认的恶性肿瘤辅助治疗手段。常见的用于临床治疗的免疫杀伤细胞：自然杀伤细胞(Natural killer cells，NK)、γδT细胞、细胞毒性T淋巴细胞(Cytotoxic T lymphocytes，CTL)、NK样T淋巴细胞(Natural killer-like T cells，NKT)、Th1效应细胞(Effector cells)等。抗原提呈细胞之一树突状细胞(Dentritic cells，DC)也常用于免疫细胞治疗的应用，DC可以提高免疫杀伤细胞的特异性、加强免疫杀伤细胞的活力。In 1985, American scientists attempted to isolate the patient's mononuclear cells and induced, activated, and produced Cytokine-induced killer cells (CIK) with various cytokines in vitro, and then found that these cells were returned by intravenous drip. After giving the patient, it has a killing effect on the growth of the tumor. After nearly 30 years of clinical application and technological development, treatment of tumors with immune killer cells has become the fourth recognized adjuvant treatment for malignant tumors. Common immune killer cells for clinical treatment: natural killer cells (NK), γδT cells, Cytotoxic T lymphocytes (CTL), NK-like T lymphocytes (Natural killer-like T cells) , NKT), Th1 effector cells (Effector cells), and the like. One of the antigen-presenting cells, dendritic cells (DCs) are also commonly used in the treatment of immune cells. DC can increase the specificity of immune killer cells and enhance the viability of immune killer cells.
免疫活性细胞对肿瘤细胞的识别和杀伤效果，取决于肿瘤细胞膜表面的受体分子表达，至少有两方面的因素导致体内的T淋巴细胞不能很好地识别癌细胞：(1)癌细胞下调抗原呈递分子的表达，(2)被呈递的抗原与T细胞受体亲和力很弱。虽然癌症患者体内存在癌细胞高度特异性的T淋巴细胞，但是数量太少 起不到治疗癌症的作用。为了克服免疫杀伤细胞特异性低下的缺点，美国科学家发明一种转基因的方法：将识别肿瘤表面特异性抗原的单克隆抗体高变区序列在体外重组、亚克隆为单链抗体片段(Single chain antibody fragment of variable regions，scFv)，再与其他基因的跨膜蛋白片段和胞内信号肽融合形成人造的嵌合抗原受体(Chimeric antigen receptor，CAR)，转染至T细胞内而形成嵌合抗原受体T细胞(Chimeric antigen receptor，CAR-T)。嵌合抗原受体主要由两部分构成，一端位于细胞外能够特异性识别癌细胞表面的某一抗原，另一端位于胞内含有信号激活元件(如T细胞受体的Zeta链)，起传递信号激活T细胞的作用。The recognition and killing effect of immunocompetent cells on tumor cells depends on the expression of receptor molecules on the surface of tumor cell membranes. At least two factors lead to the inability of T lymphocytes in vivo to recognize cancer cells well: (1) Downregulation of antigens by cancer cells The expression of the presenting molecule, (2) the presented antigen has a weak affinity for the T cell receptor. Although there are highly specific T lymphocytes in cancer cells in cancer patients, the number is too small. Can not play a role in the treatment of cancer. In order to overcome the shortcomings of immunosuppressive cell specificity, American scientists have invented a transgenic method: recombining and subcloning a monoclonal antibody hypervariable region sequence recognizing a tumor surface-specific antigen into a single-chain antibody fragment (Single chain antibody) Fragment of variable regions (scFv), which are fused with other gene transmembrane protein fragments and intracellular signal peptides to form a chimeric antigen receptor (CAR), which is transfected into T cells to form a chimeric antigen. Chimeric antigen receptor (CAR-T). The chimeric antigen receptor is mainly composed of two parts, one end is located outside the cell and can specifically recognize an antigen on the surface of the cancer cell, and the other end is located in the cell containing a signal activation element (such as a Zeta chain of T cell receptor) to transmit a signal. Activate the role of T cells.
目前以CD19为靶点开发的CAR-T细胞疗法，在一期和二期血液***肿瘤临床治疗上都取得了明显的效果，但是CAR-T细胞疗法在实体瘤的研究中的进展缓慢，目前还没有显著性的突破。原因之一是淋巴性白血病癌细胞中95％都表达B细胞抗原CD19，而其他实体瘤细胞的特异性抗原表达在40-70％之间；所以在实体瘤的治疗中，单一的单抗CAR-T细胞不可能杀伤表达其他肿瘤特异性抗原的癌细胞。原因之二是免疫杀伤细胞通过血液/淋巴液循环浸润到实体肿瘤的速度和数目。原因之三是肿瘤组织细胞对免疫活性细胞的负调节作用。At present, CAR-T cell therapy developed with CD19 has achieved remarkable results in the clinical treatment of both primary and secondary hematological tumors, but the progress of CAR-T cell therapy in solid tumor research is slow. There is no significant breakthrough. One of the reasons is that 95% of lymphocytic leukemia cells express the B cell antigen CD19, while the specific antigen expression of other solid tumor cells is between 40-70%; therefore, in the treatment of solid tumors, a single monoclonal antibody CAR - T cells are unlikely to kill cancer cells that express other tumor-specific antigens. The second reason is the rate and number of immune killer cells infiltrating into the solid tumor through the blood/lymph circulation. The third reason is the negative regulation of tumor tissue cells on immunocompetent cells.
在正常情况下，人体的血液循环中存在少量的活性杀伤细胞，如NK，γδT细胞等，其作用在于清除衰老、变异的组织细胞或抵御病毒入侵。免疫细胞的活性主要受到T调节细胞(Regulatory T cells，Treg)的控制，当T调节细胞的控制减弱时，会造成免疫功能亢进或产生自身免疫性疾病；当控制增强时，会使免疫功能低下，滋生肿瘤或其他病毒性皮肤病。目前确认的参与负调节的蛋白因子有细胞毒T淋巴细胞相关抗原4(Cytotoxic T lymphocyte-associated antigen-4,CTLA4)和程序性死亡蛋白1/程序性死亡蛋白1配基1(Programmed cell death protein 1/Programmed cell death protein 1 ligand 1，PD-1/PD-L1)等分子。抑制性T调节细胞表达CTLA4，可以与免疫细胞DC和T细胞表面的B7族蛋白亚基相互作用，抑制免疫细胞功能。B7蛋白家族成员有B7-H1(PD-L1)，B7-H2(PD-1L2)等。B7-H1(PD-L1)也可以通过结合淋巴细胞PD-1分子，进一步抑制靶细胞的活性功能。PD-1和PD-L1相互作用对T细胞功能的抑制是免疫细胞***的一个主要障碍。在大多数实体瘤组织中，癌细胞表达PD-L1/PD-1的水平增高，对浸润到癌组织的活化淋巴细胞产生直接的抑制，这 也是肿瘤逃逸机体免疫监管的机制之一；活化T细胞表面PD-1的表达增加，更容易受到负调节的抑制。最新的抗体药物keytruda和opdivo已被美国FDA批准临床治疗恶性肿瘤，其作用原理是阻断CTLA4/B7以及PD-1/PD-L1的相互结合，从而解除体内T调节细胞或癌细胞对活性T杀伤细胞的抑制，让体内免疫活性细胞对肿瘤细胞进行杀伤、清除。本发明通过改变嵌合抗原受体分子(CAR)胞外段的PD-1分子，选用比PD-1与PDL1的亲和力更高的分子，制备特异性和杀伤性更高的高亲和力嵌合抗原受体分子。Under normal circumstances, there are a small number of active killer cells in the blood circulation of the human body, such as NK, γδT cells, etc., which function to remove aging, mutated tissue cells or resist viral invasion. The activity of immune cells is mainly controlled by Regulatory T cells (Treg). When the control of T-regulated cells is weakened, it may cause hyperimmune or autoimmune diseases. When the control is enhanced, the immune function will be low. , breeding tumors or other viral skin diseases. Cytotoxic T lymphocyte-associated antigen-4 (CTLA4) and programmed death protein 1 programmed yellow cell-associated antigen 1 (Programmed cell death protein) 1/Programmed cell death protein 1 ligand 1, PD-1/PD-L1) and other molecules. Inhibitory T-regulated cells express CTLA4, which interacts with B7-type protein subunits on the surface of immune cells DC and T cells to inhibit immune cell function. Members of the B7 protein family include B7-H1 (PD-L1), B7-H2 (PD-1L2) and the like. B7-H1 (PD-L1) can also further inhibit the active function of target cells by binding to lymphocyte PD-1 molecules. The inhibition of T cell function by PD-1 and PD-L1 interaction is a major obstacle to the treatment of tumors by immune cells. In most solid tumor tissues, cancer cells express an increased level of PD-L1/PD-1, which directly inhibits activated lymphocytes infiltrating into cancerous tissues. It is also one of the mechanisms of tumor immune immune regulation; the expression of PD-1 on activated T cells is increased, and it is more susceptible to negative regulation. The latest antibody drugs, keytruda and opdivo, have been approved by the US FDA for the clinical treatment of malignant tumors. The principle of action is to block the binding of CTLA4/B7 and PD-1/PD-L1, thereby releasing T-regulated cells or cancer cells in vivo. The inhibition of killer cells allows the immune-active cells in the body to kill and clear the tumor cells. The present invention prepares a high affinity chimeric antigen with higher specificity and lethality by modifying the PD-1 molecule of the extracellular domain of the chimeric antigen receptor molecule (CAR) and selecting a molecule having higher affinity than PD-1 and PDL1. Receptor molecule.
发明内容Summary of the invention
为解决上述技术问题，本发明的目的是提供一种具有与肿瘤表面PDL-1分子更高亲和力的嵌合抗原受体分子及其应用。In order to solve the above technical problems, an object of the present invention is to provide a chimeric antigen receptor molecule having a higher affinity for a PDL-1 molecule on a tumor surface and an application thereof.
本发明第一方面提供一种嵌合抗原受体分子，包括依次串联的胞外区肽段、跨膜区肽段和胞内结构域肽段，所述胞外区肽段包括HAC肽段，其序列如SEQ ID NO：1所示。据《Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging》文章报道，PD-1的突变体HAC具有与肿瘤表面PDL-1分子更高亲和力。A first aspect of the invention provides a chimeric antigen receptor molecule comprising a sequence of extracellular domain peptides, a transmembrane domain peptide and an intracellular domain peptide, wherein the extracellular domain peptide comprises a HAC peptide, Its sequence is shown in SEQ ID NO: 1. According to the article "Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging", the mutant HAC of PD-1 has higher affinity with the tumor surface PDL-1 molecule.
应当说明的是，所述跨膜区肽段为蛋白质序列中跨越细胞膜的区域，通常为α-螺旋结构，约20～25个氨基酸残基，其氨基酸大部分是疏水性氨基酸；其中，所述跨膜区肽段包括但不限于CD8跨膜区肽段或PD-1跨膜区肽段，当跨膜区肽段为CD8跨膜区肽段时，所述HAC肽段与CD8跨膜区肽段还通过CD8的hinge区肽段相连。CD8跨膜区肽段的序列如SEQ ID NO：2所示，CD8的hinge区肽段的序列如SEQ ID NO：3所示，PD-1跨膜区肽段的序列如SEQ ID NO：4所示。It should be noted that the transmembrane region peptide is a region spanning a cell membrane in a protein sequence, usually an α-helical structure, about 20-25 amino acid residues, and the amino acid is mostly a hydrophobic amino acid; wherein The transmembrane region peptide includes, but is not limited to, a CD8 transmembrane region peptide or a PD-1 transmembrane region peptide, and when the transmembrane region peptide is a CD8 transmembrane region peptide, the HAC peptide segment and the CD8 transmembrane region The peptides are also linked by the hinge region of the CD8. The sequence of the CD8 transmembrane region peptide is shown in SEQ ID NO: 2, the sequence of the hinge region of CD8 is shown in SEQ ID NO: 3, and the sequence of the PD-1 transmembrane region peptide is as SEQ ID NO: 4. Shown.
进一步的，所述胞内结构域肽段为共刺激信号分子，选自4-1BB(又称CD137)、CD28、CD3ζ的胞内结构域肽段中的一种或多种。优选的，所述胞内结构域肽段选自相互连接的4-1BB和CD3ζ胞内结构域肽段，其序列分别如SEQ ID NO：5和6所示。Further, the intracellular domain peptide is a costimulatory signal molecule selected from one or more of the intracellular domain peptides of 4-1BB (also known as CD137), CD28, and CD3ζ. Preferably, the intracellular domain peptide is selected from the group consisting of interconnected 4-1BB and CD3 sputum intracellular domain peptides, the sequences of which are set forth in SEQ ID NOS: 5 and 6, respectively.
进一步的，所述嵌合抗原受体分子还包括信号肽。信号肽可以提高嵌合抗原受体分子这种融合蛋白分泌的效果，在信号肽与融合蛋白其它氨基酸序列一 起被表达后，最终被蛋白酶切除。蛋白酶具有一定的识别序列，而信号肽与其后面的肽段融合后构成新的氨基酸序列，所以如果选择的信号肽不当，可能会导致蛋白酶的误切，蛋白失活。信号肽可选自免疫球蛋白轻链的信号肽、或是PD-1蛋白分泌的信号肽，其序列分别如SEQ ID NO：7和8所示。Further, the chimeric antigen receptor molecule further comprises a signal peptide. The signal peptide can enhance the secretion of the fusion protein of the chimeric antigen receptor molecule, and the signal peptide and the other amino acid sequence of the fusion protein After being expressed, it is finally excised by proteases. The protease has a certain recognition sequence, and the signal peptide is fused with the peptide after it to form a new amino acid sequence. Therefore, if the selected signal peptide is improper, the protease may be miscut and the protein may be inactivated. The signal peptide may be selected from a signal peptide of an immunoglobulin light chain or a signal peptide secreted by a PD-1 protein, the sequences of which are shown in SEQ ID NOS: 7 and 8, respectively.
优选的，本发明的嵌合抗原受体分子，由免疫球蛋白轻链的信号肽、HAC肽段、CD8的hinge区肽段、CD8的跨膜区肽段、4-1BB胞内结构域肽段和CD3ζ胞内结构域肽段依次连接而成，其序列如SEQ ID NO：9所示。Preferably, the chimeric antigen receptor molecule of the present invention comprises a signal peptide of an immunoglobulin light chain, a HAC peptide, a hinge region peptide of CD8, a transmembrane region peptide of CD8, and a 4-1BB intracellular domain peptide. The segment and the CD3 sputum intracellular domain peptide are sequentially ligated, and the sequence thereof is shown in SEQ ID NO: 9.
本发明第二方面提供一种核苷酸，其编码本发明第一方面提供的嵌合抗原受体分子。A second aspect of the invention provides a nucleotide encoding a chimeric antigen receptor molecule provided by the first aspect of the invention.
进一步的，包含或为SEQ ID NO：10所示的核苷酸序列。Further, it comprises or is the nucleotide sequence shown in SEQ ID NO: 10.
本发明第三方面提供一种重组载体，其包含本发明第二方面提供的核苷酸。A third aspect of the invention provides a recombinant vector comprising the nucleotide provided by the second aspect of the invention.
进一步的，所述载体为慢病毒载体，其可以将外源基因或外源的shRNA有效地整合到宿主染色体上，从而达到持久性表达目的序列的效果。在感染能力方面可有效地感染神经元细胞、肝细胞、心肌细胞、肿瘤细胞、内皮细胞、干细胞等多种类型的细胞，从而达到良好的的基因治疗效果。对于一些较难转染的细胞，如原代细胞、干细胞、不分化的细胞等，使用慢病毒载体，能大大提高目的基因或目的shRNA的转导效率，且目的基因或目的shRNA整合到宿主细胞基因组的几率大大增加，能够比较方便快捷地实现目的基因或目的shRNA的长期、稳定表达。Further, the vector is a lentiviral vector, which can efficiently integrate a foreign gene or an exogenous shRNA into a host chromosome, thereby achieving the effect of persistently expressing the sequence of interest. It can effectively infect various types of cells such as neuron cells, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, stem cells, etc. in terms of infection ability, thereby achieving a good gene therapy effect. For some cells that are difficult to transfect, such as primary cells, stem cells, undifferentiated cells, etc., the use of lentiviral vectors can greatly improve the transduction efficiency of the target gene or the target shRNA, and the target gene or the target shRNA is integrated into the host cell. The probability of genome is greatly increased, and the long-term and stable expression of the target gene or the target shRNA can be realized conveniently and quickly.
应当说明的是，本发明中所用的慢病毒载体，应当包括但不限于pRRSLIN慢病毒表达载体和pLVX载体，优选为pRRSLIN慢病毒表达载体。It should be noted that the lentiviral vector used in the present invention should include, but is not limited to, a pRRSLIN lentiviral expression vector and a pLVX vector, preferably a pRRSLIN lentiviral expression vector.
本发明第四方面提供一种重组细胞，其包含本发明第三方面提供的重组载体。所述重组细胞优选为T细胞或NK细胞。该嵌合抗原受体的编码基因能够通过前述载体被转移至T细胞或NK细胞内，用于修饰T细胞或NK细胞，成为CAR-T或CAR-NK细胞；利用该嵌合抗原受体修饰的T细胞或NK细胞，能够通过识别肿瘤细胞表面的组织因子，杀死肿瘤细胞，进行肿瘤治疗。 A fourth aspect of the invention provides a recombinant cell comprising the recombinant vector provided by the third aspect of the invention. The recombinant cell is preferably a T cell or an NK cell. The gene encoding the chimeric antigen receptor can be transferred to T cells or NK cells by the aforementioned vector, used to modify T cells or NK cells to become CAR-T or CAR-NK cells; and the chimeric antigen receptor is modified by the chimeric antigen receptor T cells or NK cells can kill tumor cells by identifying tissue factors on the surface of tumor cells for tumor treatment.
在本发明中：In the present invention:
术语“共刺激信号分子”(Co-stimulating molecule)是指免疫细胞表面的一些粘附分子，如CD28、CD134/OX40、CD137/4-1BB、CD40等，通过与其配体结合，激活免疫细胞的第二信号，增强免疫细胞的增殖能力及细胞因子的分泌功能，延长活化免疫细胞的存活时间。The term "co-stimulating molecule" refers to some adhesion molecules on the surface of immune cells, such as CD28, CD134/OX40, CD137/4-1BB, CD40, etc., which activate immune cells by binding to their ligands. The second signal enhances the proliferative capacity of immune cells and the secretory function of cytokines, prolonging the survival time of activated immune cells.
术语“胞外区”是指膜蛋白位于细胞外的区段。The term "extracellular domain" refers to a segment of the membrane protein that is located outside the cell.
术语“结构域”是指蛋白质生物大分子中具有特异结构和独立功能的区域，常见结构域的氨基酸残基数在100～400个之间，最小的结构域只有40～50个氨基酸残基，大的结构域可超过400个氨基酸残基。The term "domain" refers to a region of a protein biomacromolecule having a specific structure and an independent function. The number of amino acid residues in a common domain is between 100 and 400, and the smallest domain is only 40 to 50 amino acid residues. Large domains can exceed 400 amino acid residues.
术语“PD-1”是指人类程序性死亡因子1(programmed cell death protein1)，基因名称PDCD1_HUMAN，对应蛋白序列编号有UniProtKB-Q15116，是T细胞免疫抑制分子，其胞外区结构域类似免疫球蛋白的可变区(V-section)，具有特异性结合其配体PD-L1和PD-L2(Programmed cell death protein 1 ligand 1/2)的特性。PD-1通常在活化的T淋巴细胞中表达，在多种恶性肿瘤细胞中也有表达。The term "PD-1" refers to programmed cell death protein 1 and the gene name PDCD1_HUMAN. The corresponding protein sequence number is UniProtKB-Q15116, which is a T cell immunosuppressive molecule whose extracellular domain resembles an immunoglobulin. The variable region (V-section) of the protein has the property of specifically binding to its ligands PD-L1 and PD-L2 (Programmed cell death protein 1 ligand 1/2). PD-1 is normally expressed in activated T lymphocytes and is also expressed in a variety of malignant cells.
术语“PD-L1”、“PD-L2”是指目前发现的人类程序性死亡因子1配基1和配基2(programmed cell death protein 1 ligand 1/2)。其胞外区结构域具有类似免疫球蛋白的V和C1区，通过V区与PD-1的V区相结合(4zqk Structure 232341-2348，2015)。通常在树突状细胞DC、T调节细胞和Th细胞、巨噬细胞、Mast细胞和骨髓中少量表达，在多种恶性肿瘤细胞中也有表达。The terms "PD-L1" and "PD-L2" refer to the currently discovered human programmed death factor 1 ligand 1/2. Its extracellular domain domain has immunoglobulin-like V and C1 regions, which are combined with the V region of PD-1 via the V region (4zqk Structure 232341-2348, 2015). It is usually expressed in a small amount in dendritic cells DC, T regulatory cells and Th cells, macrophages, Mast cells and bone marrow, and is also expressed in various malignant cells.
借由上述方案，本发明至少具有以下优点：本发明的嵌合抗原受体分子，具有与肿瘤表面PDL-1分子更高亲和力的HAC分子，将其组装在CAR-T细胞胞外段，用来识别肿瘤细胞，具有更高的亲和力和杀伤活性。By the above scheme, the present invention has at least the following advantages: the chimeric antigen receptor molecule of the present invention has a HAC molecule having higher affinity with the PDL-1 molecule on the tumor surface, and is assembled in the extracellular segment of the CAR-T cell. To identify tumor cells with higher affinity and killing activity.
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明如后。The above description is only an overview of the technical solutions of the present invention, and the technical means of the present invention can be more clearly understood and can be implemented in accordance with the contents of the specification. Hereinafter, the preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.
附图说明DRAWINGS
图1是慢病毒表达载体构建示意图；Figure 1 is a schematic diagram showing the construction of a lentiviral expression vector;
图2是pRRSLIN-HAC侵染3天的流式结果图； Figure 2 is a flow chart of pRRSLIN-HAC infection for 3 days;
图3是CAR-HAC对不同种靶细胞的杀伤结果图；Figure 3 is a graph showing the killing results of CAR-HAC against different target cells;
图4是CAR-HAC对不同种靶细胞的体外增殖的检测结果图；Figure 4 is a graph showing the results of CAR-HAC detection of in vitro proliferation of different target cells;
图5是CAR-HAC对不同种靶细胞的细胞因子的检测结果图；Figure 5 is a graph showing the results of detection of cytokines by different kinds of target cells by CAR-HAC;
图6是CAR-HAC对MCF7/MCF7-PDL1/HeLa/SMC7721 4种细胞株的检测结果图；Figure 6 is a graph showing the results of detection of four cell lines of MCF7/MCF7-PDL1/HeLa/SMC7721 by CAR-HAC;
图7是针对不同细胞株CAR-PD-1和CAR-HAC的杀伤效果对比图。Figure 7 is a graph comparing the killing effects of different cell lines CAR-PD-1 and CAR-HAC.
具体实施方式detailed description
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。The specific embodiments of the present invention are further described in detail below with reference to the drawings and embodiments. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
实施例1慢病毒表达载体制备Example 1 Preparation of lentiviral expression vector
本发明提供一种用于表达嵌合抗原受体分子的慢病毒表达载体的制备方法，包括以下步骤：The invention provides a preparation method of a lentiviral expression vector for expressing a chimeric antigen receptor molecule, comprising the following steps:
S1、根据《Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging》文章报道的PD-1分子突变体HAC序列信息，合成HAC基因片段，从GenBank数据库中搜寻已知的人CD8跨膜区基因序列、人4-1BB胞内区基因序列和CD3ζ胞内区基因序列；S1. According to the PD-1 molecular mutant HAC sequence information reported in the article "Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging", the HAC gene fragment is synthesized, and the known human CD8 cross is searched from the GenBank database. Membrane region gene sequence, human 4-1BB intracellular region gene sequence and CD3 intracellular region gene sequence;
S2、将上述基因序列依次按人HAC基因、CD8膜区基因、人4-1BB胞内区基因和CD3ζ胞内区基因进行连接，在各序列连接处引入不同的酶切位点，形成完整的HAC-CD8-4-1BB-CD3ζ基因序列信息，S2, the above gene sequences are sequentially linked according to human HAC gene, CD8 membrane region gene, human 4-1BB intracellular region gene and CD3 sputum intracellular region gene, and different enzyme cleavage sites are introduced at each sequence junction to form a complete HAC-CD8-4-1BB-CD3ζ gene sequence information,
S3、将HAC-CD8-4-1BB-CD3ζ的基因序列通过酶切转化连接到pRRSLIN载体中，基因上游为EP-1α启动子。将载体转化到Stbl3大肠杆菌菌株后，转种到含有氨苄青霉素的固体培养基中进行繁殖，筛选，获得阳性克隆，提取质粒，酶切鉴定克隆，通过测序确认载体构建成功，获得pRRSLIN-HAC慢病毒表达载体，慢病毒表达载体构建示意图如图1所示。S3. The gene sequence of HAC-CD8-4-1BB-CD3ζ is ligated into the pRRSLIN vector by restriction enzyme transformation, and the upstream of the gene is the EP-1α promoter. The vector was transformed into Stbl3 Escherichia coli strain, then transferred to a solid medium containing ampicillin for propagation, screened, positive clones were obtained, plasmids were extracted, and clones were identified by restriction enzyme digestion. The vector was confirmed to be successfully constructed by sequencing, and pRRSLIN-HAC was obtained slowly. A schematic diagram of the construction of a viral expression vector and a lentiviral expression vector is shown in FIG.
实施例2慢病毒制备Example 2 lentivirus preparation
本发明提供实施例1中慢病毒表达载体表达制备慢病毒的方法，包括以下 步骤：The present invention provides a method for producing a lentivirus by expressing a lentiviral expression vector in Example 1, comprising the following step:
S1、转染前24小时，以每皿约8×106将293T细胞接种至15cm培养皿中。确保转染时细胞在80％左右的汇合度且均匀分布于培养皿中。S1, 24 hours before transfection, 293T cells were seeded into a 15 cm culture dish at approximately 8 x 10 6 per dish. Ensure that the cells are confluent at 80% and evenly distributed in the culture dish during transfection.
S2、准备溶液A和溶液BS2, preparing solution A and solution B
溶液A：6.25mL 2×HEPES buffer缓冲液Solution A: 6.25 mL 2 x HEPES buffer buffer
溶液B：分别加入以下质粒的混合物：112.5μg pRRLSIN-HAC(target plasmid)；39.5μg pMD2.G(VSV-G envelop)；73μg pCMVR8.74(gag，pol，tat，rev)；625μL 2M钙离子溶液。Solution B: A mixture of the following plasmids was separately added: 112.5 μg pRRLSIN-HAC (target plasmid); 39.5 μg pMD2.G (VSV-G envelop); 73 μg pCMVR8.74 (gag, pol, tat, rev); 625 μL 2M calcium ion Solution.
S3、充分混匀溶液B，轻轻涡旋溶液A的同时，逐滴加入溶液B，静置2-3分钟。轻轻涡旋上述A和B的混合溶液，逐滴加入含293T细胞的培养皿中，轻轻前后晃动培养皿使DNA与钙离子的混合物均匀分布。放置于培养箱中培养16-18小时。更换新鲜培养基，继续培养，在转速500g，温度25℃下离心10min，使用PES膜(0.45μm)过滤；以70％乙醇消毒离心管，并置于紫外灯下消毒30min；将已过滤的含慢病毒的上清液转移至离心管中，在离心管底部小心铺上一层20％蔗糖(每8mL上清液加1mL蔗糖)，以PBS平衡离心管，在转速25000rpm、温度4℃下离心2h；小心取出离心管，倒掉上清液，倒置离心管去掉残余液体；加入100μL PBS，密封离心管，在4℃放置2h，每20min轻轻涡旋一次，500g离心1min(25℃)，收集病毒上清；冰上冷却后，置于-80℃保存。S3, thoroughly mix the solution B, while gently swirling the solution A, add the solution B dropwise, and let stand for 2-3 minutes. Gently swirl the above mixed solution of A and B, add dropwise to the Petri dish containing 293T cells, and gently shake the Petri dish to spread the mixture of DNA and calcium ions evenly. Place in an incubator for 16-18 hours. Replace the fresh medium, continue the culture, centrifuge at a speed of 500g, temperature 25 ° C for 10min, filter with PES membrane (0.45μm); disinfect the centrifuge tube with 70% ethanol, and sterilize it under UV lamp for 30min; The supernatant of the lentivirus was transferred to a centrifuge tube, and a layer of 20% sucrose (8 mL of sucrose per 8 mL of supernatant) was carefully placed on the bottom of the tube, and the tube was equilibrated with PBS, and centrifuged at a speed of 25,000 rpm and a temperature of 4 ° C. 2h; carefully remove the centrifuge tube, pour off the supernatant, invert the centrifuge tube to remove residual liquid; add 100 μL PBS, seal the centrifuge tube, place at 4 ° C for 2 h, gently vortex every 20 min, centrifuge at 500 g for 1 min (25 ° C), The virus supernatant was collected; after cooling on ice, it was stored at -80 °C.
实施例3 CAR-T细胞制备Example 3 Preparation of CAR-T cells
本发明提供实施例2中慢病毒侵染细胞制备CAR-T细胞的方法，包括以下步骤：The present invention provides a method for preparing CAR-T cells by lentiviral infection of cells in Example 2, comprising the steps of:
S1、取0.5mL血进行快速的病原微生物检测，排除HBV、HCV、HDV和HEV、HIV-1/2、***及寄生虫等微生物感染；无菌条件下，用肝素瓶采血50mL(肝素抗凝)，立即(4℃，24小时内)送至细胞制备实验室，保证此过程无病原微生物污染。得到患者血液后，在GMP制备室，用酒精棉球擦拭肝素瓶表面进行消毒后放入生物安全柜。S1, take 0.5mL blood for rapid detection of pathogenic microorganisms, exclude microbial infections such as HBV, HCV, HDV and HEV, HIV-1/2, Treponema pallidum and parasites; under sterile conditions, use blood collection of heparin bottle 50mL (heparin antibiotic) Condensed), immediately (4 ° C, within 24 hours) to the cell preparation laboratory to ensure that this process is free of pathogenic microorganisms. After obtaining the patient's blood, in the GMP preparation room, the surface of the heparin bottle is wiped with an alcohol cotton ball for disinfection and then placed in a biological safety cabinet.
S2、预先打开2个50mL离心管，将血液转入两个50mL离心管中，旋紧；将上述装好血液的两个50mL离心管放入离心机离心，2000rpm离心10min， 室温离心后收集上层血浆，留下沉淀层；收集的自体血浆经56℃，30min灭活，4℃放置15min后，900g，离心30min(4℃)，取上清备用。S2, pre-open two 50mL centrifuge tubes, transfer the blood into two 50mL centrifuge tubes, and tighten; put the two 50mL centrifuge tubes filled with blood into the centrifuge for centrifugation, centrifuge at 2000rpm for 10min, The supernatant plasma was collected after centrifugation at room temperature, leaving a sediment layer; the collected autologous plasma was inactivated at 56 ° C for 30 min, placed at 4 ° C for 15 min, 900 g, centrifuged for 30 min (4 ° C), and the supernatant was taken for use.
S3、将上述富集的血细胞用生理盐水稀释至30mL/管，打开2个新的50mL离心管，每个离心管分别加入15mL人淋巴细胞分离液，用移液管把稀释后的血细胞液缓缓加入到盛有人淋巴分离液的离心管中，旋紧。注意血液要加到淋巴分离液的上层，勿打破人淋巴分离液的界面。将加好的血细胞液放入离心机，调至最小的升降速率，常温2000rpm离心20min。收集两管的中层白细胞层于一支15mL无菌离心管中，加入5mL生理盐水，洗两次(2000rpm离心10min)，得外周血单核细胞(PBMC)。S3, diluting the above enriched blood cells to 30 mL/tube with physiological saline, opening two new 50 mL centrifuge tubes, adding 15 mL of human lymphocyte separation liquid to each centrifuge tube, and pipetting the diluted blood cell liquid with a pipette. Slowly add to the centrifuge tube containing the human lymphatic separation solution and tighten. Note that the blood should be added to the upper layer of the lymphatic separation solution, and the interface of the human lymphatic separation solution should not be broken. Put the added blood cell solution into the centrifuge, adjust to the minimum lifting rate, and centrifuge at normal temperature 2000 rpm for 20 min. Two tubes of the middle white blood cell layer were collected in a 15 mL sterile centrifuge tube, and 5 mL of physiological saline was added thereto, and washed twice (2000 rpm for 10 min) to obtain peripheral blood mononuclear cells (PBMC).
S4、配置完全生长培养基，V-VIVO15添加自体AB(FBS)浓度为5％，白细胞介素-2(IL-2)浓度为40ng/mL，将分离得到的PBMC用培养基稀释成2×106/mL，取50μL流式检测PBMC中T细胞的纯度。S4, configured complete growth medium, V-VIVO15 added autologous AB (FBS) concentration of 5%, interleukin-2 (IL-2) concentration of 40 ng / mL, and the isolated PBMC was diluted to 2 × with the culture medium 10 6 /mL, 50 μL flow detection of the purity of T cells in PBMC.
S5、Day 0，配置缓冲液(在PBS缓冲液中添加1％的胎牛血清(FBS))，选用微珠作为细胞培养载体，将微珠振荡30s或手动上下摇匀5min，按照微珠与T细胞的用量比为3：1取CD3/CD28微珠置于1.5mL EP管中，添加1mL缓冲液清洗微珠，之后使用磁铁从EP管向外吸微珠1min，弃洗液，重复两次，再使用培养基将微珠重悬到原体积，将细胞和微珠混合后按2×106PBMC/mL加到合适的培养瓶中。S5, Day 0, configure buffer (add 1% fetal bovine serum (FBS) in PBS buffer), use microbeads as cell culture carrier, shake the beads for 30s or shake them up and down for 5min manually, according to the microbeads The ratio of T cells was 3:1, and the CD3/CD28 microbeads were placed in a 1.5 mL EP tube. The beads were washed with 1 mL of buffer, and then the beads were sucked from the EP tube for 1 min using a magnet, and the washing solution was discarded. Then, the beads were resuspended to the original volume using the medium, and the cells and the beads were mixed and added to a suitable flask at 2 × 10 6 PBMC/mL.
S6、Day 2将细胞密度调整至3-5×106/mL，按病毒载体与细胞的比例为1:5添加实施例1制备得到的pRRSLIN-HAC慢病毒表达载体，同时添加聚凝胺(polybrene)4μg/mL和40ng/mL IL-2。4h之后，补加新鲜的完全培养基将细胞密度调整至1×106/mL继续培养。将所有的细胞离心，加入新鲜的培养基，继续培养。S6 and Day 2 adjusted the cell density to 3-5×10 6 /mL, and the pRRSLIN-HAC lentiviral expression vector prepared in Example 1 was added at a ratio of viral carrier to cell 1:5, and polyglycolamine was added ( Polybrene) 4 μg/mL and 40 ng/mL IL-2. After 4 h, fresh complete medium was added to adjust the cell density to 1 × 10 6 /mL to continue the culture. All cells were centrifuged, fresh medium was added, and the culture was continued.
S7、每隔2-3天进行半量换液，维持细胞密度在0.5-1×106/mL。S7, half a change of liquid every 2-3 days, maintaining a cell density of 0.5-1 × 10 6 /mL.
S8、Day 10-12，细胞数量达到109级别，在400g下离心5min得到免疫细胞，再用预冷的PBS洗涤两遍。S8, Day 10-12, the number of cells reached 10 9 grades, and the cells were centrifuged at 400 g for 5 min to obtain immune cells, and then washed twice with pre-cooled PBS.
S9、用血球计数板计数，流式细胞仪检测细胞类群，CAR-T细胞比例。每天观察培养基的颜色变化、细胞密度、细胞形态并作相应记录。逐步扩大培养过程中，加入总体积所需的白细胞介素-2。 S9, counting with a blood cell counting plate, and detecting the cell group and the ratio of CAR-T cells by flow cytometry. The color change, cell density, and cell morphology of the medium were observed daily and recorded accordingly. Gradually expand the culture process and add the required interleukin-2 to the total volume.
实施例4 CAR-T细胞流式分析Example 4 CAR-T Cell Flow Analysis
对实施例3制备的CAR-T细胞进行流式分析，其具体步骤如下：The CAR-T cells prepared in Example 3 were subjected to flow analysis, and the specific steps were as follows:
S1、取5×104细胞(包括T细胞、CAR-T细胞)用于染色；S1, taking 5×10 4 cells (including T cells, CAR-T cells) for staining;
S2、细胞与抗体(抗体可与HAC分子识别结合，偶联FITC荧光分子)共孵育45min，50μl，置于冰上；S2, cells and antibodies (antibodies can be combined with HAC molecule recognition, coupled with FITC fluorescent molecules) for 45 min, 50 μl, placed on ice;
S3、PBS洗脱两次；S3, PBS eluted twice;
S4、用120μl FACS试剂重悬细胞；S4, resuspending the cells with 120 μl of FACS reagent;
S5、流式细胞仪器测量FITC荧光信号，如果与对照T细胞对比，CAR细胞FITC荧光信号增强，表面CAR细胞构建成功。S5, flow cytometry measured FITC fluorescence signal, if compared with control T cells, CAR cells FITC fluorescence signal enhanced, surface CAR cells were successfully constructed.
CAR-T细胞流式浸染效果如图2所示，图中，纵坐标为流式SSC-H侧向角散射信号，横坐标为FITC荧光信号，该信号值越强，表明HAC分子在膜上表达的越多，CAR-T细胞转染成功的比例越高。A图和B图为对照组，为不侵染病毒的T细胞；FITC偶联的检测CAR分子的抗体检测不到CAR分子表达；C图和D图，为转染PRRSLIN-HAC慢病毒的T细胞，经流式检测，与A图和B图对比，有细胞成功转染；病毒侵染T细胞后，流式检测侵染3天后，侵染效率可以达到53.26％，说明成功制备HAC-CAR-T细胞。The effect of CAR-T cell flow dip staining is shown in Figure 2. In the figure, the ordinate is the flow SSC-H lateral scatter signal, and the abscissa is the FITC fluorescence signal. The stronger the signal value, the HAC molecule is on the membrane. The more expressed, the higher the proportion of successful CAR-T cell transfection. Figures A and B are the control group, which are T cells that do not infect the virus; FITC-conjugated antibodies for detecting CAR molecules do not detect CAR molecule expression; C and D maps are T transfected with PRRSLIN-HAC lentivirus Cells, after flow detection, compared with A and B, cells were successfully transfected; after infection of T cells by virus, the infection efficiency reached 53.26% after 3 days of flow detection, indicating successful preparation of HAC-CAR -T cells.
实施例5 HAC-CAR-T细胞体外活性检测Example 5 Detection of in vitro activity of HAC-CAR-T cells
采用LDH释放法检测HAC-CAR-T细胞对工程细胞株MCF-1/PDL1和高表达PDL1的肿瘤细胞的杀伤效应，通过ELISA方法检测LDH释放，包括以下步骤：LDH release assay was used to detect the killing effect of HAC-CAR-T cells on engineered cell lines MCF-1/PDL1 and PDL1 high expressing cells. LDH release was detected by ELISA, including the following steps:
S1、用含5％小牛血清的RPMI-1640培养液将靶细胞调整到5×104/mL。S1, the target cells were adjusted to 5 × 10 4 /mL with RPMI-1640 medium containing 5% calf serum.
S2、在96孔细胞培养板中加入靶细胞，每孔加100μL。取3个孔作为效应细胞(HAC-CAR-T细胞)自然释放对照孔，不加靶细胞，仅加100μL培养液。S2, target cells were added to a 96-well cell culture plate, and 100 μL per well was added. Three wells were used as effector cells (HAC-CAR-T cells) to naturally release control wells, and no target cells were added, and only 100 μL of the culture solution was added.
S3、向各孔加100μL效应细胞，效应细胞与靶细胞的比例10:1；5:1；1:1。自然释放孔不加效应细胞只加100μL培养液，效应细胞与靶细胞共孵育6小时，每个实验置三个复孔。 S3, 100 μL of effector cells were added to each well, and the ratio of effector cells to target cells was 10:1; 5:1; 1:1. The natural release well was added with only 100 μL of the culture medium without effector cells, and the effector cells were incubated with the target cells for 6 hours, and three replicate wells were placed for each experiment.
S4、最大释放孔中(阳性对照)加10μL Lysis Solution(10×)，孵育45min-60min，每个实验置三个复孔。S4, the largest release well (positive control) plus 10 μL Lysis Solution (10×), incubate for 45 min-60 min, and place three replicate wells for each experiment.
S5、取上述3和4中待测样品和对照样品各50μL，加入新鲜的96孔酶标板中，再加入反应液和底物，避光30min。S5, taking 50 μL of the sample to be tested and the control sample in the above 3 and 4, adding the fresh 96-well microtiter plate, and then adding the reaction solution and the substrate, and avoiding the light for 30 min.
S6、加入50μL终止液。S6, 50 μL of stop solution was added.
S7、在酶联检测仪上测定各孔的光密度(OD值)，检测波长490nm或492nm，在1小时内测完。S7. The optical density (OD value) of each well was measured on an enzyme-linked detector, and the detection wavelength was 490 nm or 492 nm, and the measurement was completed within 1 hour.
S8、特异性杀伤效率计算S8, specific killing efficiency calculation
杀伤率＝实验组LDH(OD)/最大LDH释放组(OD)。Killing rate = experimental group LDH (OD) / maximum LDH release group (OD).
计算公式：杀伤效率＝(实验组-效应自然释放-靶自然释放)/(靶最大释放-靶自然释放)×100％。Calculation formula: killing efficiency = (experimental group - effect natural release - target natural release) / (target maximum release - target natural release) × 100%.
S9、通过CBA试剂盒测定细胞因子分泌情况，同时计算CAR-T细胞各组中的增殖情况，并利用CD3和CD8抗体染色，确认增殖的T细胞中CD8阳性的T细胞的比例。S9, the cytokine secretion was measured by a CBA kit, and the proliferation of each group of CAR-T cells was calculated, and the ratio of CD8-positive T cells in the proliferating T cells was confirmed by staining with CD3 and CD8 antibodies.
如图3所示，HAC CAR-T可以显著杀伤SMCCC7721肿瘤细胞，对高表达PDL-1的MCF-7细胞的杀伤效果好于MCF-7普通肿瘤细胞，图中，横坐标表示CAR-T细胞与肿瘤细胞不同的效靶比，纵坐标表示杀伤效率，不同类型的柱状图表示不同肿瘤细胞。相应的，如图4所示，横坐标表示T细胞与肿瘤或CAR-T细胞与肿瘤的效靶比，纵坐标表示细胞数目，T代表T细胞，HAC代表CAR-T细胞，可见与普通T细胞相比，CAR-T细胞在接触到肿瘤细胞刺激后，可发生特异性激活和增殖，且高效靶比情况下更为显著。As shown in Figure 3, HAC CAR-T can significantly kill SMCCC7721 tumor cells, and the killing effect on MCF-7 cells with high expression of PDL-1 is better than that of MCF-7 common tumor cells. In the figure, the abscissa indicates CAR-T cells. Different from tumor cells, the ordinate indicates the killing efficiency, and different types of histograms indicate different tumor cells. Correspondingly, as shown in Figure 4, the abscissa indicates the ratio of T cells to tumor or CAR-T cells to tumors, the ordinate indicates the number of cells, T represents T cells, and HAC represents CAR-T cells, visible and normal T Compared with cells, CAR-T cells can undergo specific activation and proliferation after exposure to tumor cells, and the high-efficiency target is more significant than the case.
如图5杀伤实验中，横坐标表示CAR-T细胞与肿瘤细胞不同的效靶比，纵坐标表示细胞因子含量，检测培养上清中的细胞因子，发现CAR-T杀伤实验组IL-2(图5A)和TNF-α(图5B)的分泌显著升高。如图6所示，为CD3/CD8流式抗体检测CAR-T对MCF7/MCF7-PDL1/HeLa/SMC7721 4种细胞株杀伤后CD8T细胞的比例，纵坐标PE信号表示特异性检测CD3分子表达，横坐标FITC信号表示检测CD8分子表达，通过流式细胞仪检测，HAC CAR-T细胞激活后，主要是CD8T细胞发生特异性增殖。In the killing experiment in Figure 5, the abscissa indicates the different target ratio of CAR-T cells to tumor cells, the ordinate indicates the cytokine content, the cytokines in the culture supernatant were detected, and the CAR-T killing experimental group IL-2 was found. The secretion of Figure 5A) and TNF-[alpha] (Figure 5B) was significantly elevated. As shown in Figure 6, the CD3/CD8 flow antibody was used to detect the ratio of CAR-T to CD8T cells after killing MCF7/MCF7-PDL1/HeLa/SMC7721 cell lines, and the ordinate PE signal showed specific detection of CD3 molecule expression. The abscissa FITC signal indicates that the expression of CD8 molecules is detected, and the specific proliferation of CD8 T cells is mainly detected by flow cytometry after activation of HAC CAR-T cells.
如图7所示，对不同细胞MCF7(图7A)、MCF7-PD-L1(图7B)和SMC7721 (图7C)，CAR-PD-1表示CAR分子的胞外段为普通PD-1分子，CAR-PD-1-HAC表示本发明构建的HAC-CAR嵌合抗原受体分子，与PD-1 CAR-T相比，HAC CAR-T效果更好。As shown in Figure 7, for different cells MCF7 (Figure 7A), MCF7-PD-L1 (Figure 7B) and SMC7721 (Fig. 7C), CAR-PD-1 indicates that the extracellular domain of the CAR molecule is a normal PD-1 molecule, and CAR-PD-1-HAC indicates the HAC-CAR chimeric antigen receptor molecule constructed by the present invention, and PD-1 Compared with CAR-T, HAC CAR-T works better.
上述结果证明，HAC-CART细胞在接触肿瘤细胞后，可以特异性的发生活化和增殖，释放细胞因子，杀伤肿瘤细胞，其中CD8阳性T细胞起到主要作用。同时比较了PD-1 CAR-T和HAC CAR-T对肿瘤的杀伤效果，结果显示HAC-CAR-T明显优于PD-1 CAR-T.The above results demonstrate that HAC-CART cells can specifically activate and proliferate after exposure to tumor cells, release cytokines and kill tumor cells, and CD8-positive T cells play a major role. At the same time, the killing effect of PD-1 CAR-T and HAC CAR-T on tumors was compared. The results showed that HAC-CAR-T was significantly better than PD-1 CAR-T.
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。 The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. It should be noted that those skilled in the art can make some improvements without departing from the technical principles of the present invention. And modifications and variations are also considered to be within the scope of the invention.
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Claims (10)

  1. 一种嵌合抗原受体分子，其特征在于：包括依次串联的胞外区肽段、跨膜区肽段和胞内结构域肽段，所述胞外区肽段包括HAC肽段，其序列如SEQ ID NO：1所示。A chimeric antigen receptor molecule comprising: an extracellular domain peptide, a transmembrane domain peptide and an intracellular domain peptide in tandem, the extracellular domain peptide comprising a HAC peptide, the sequence thereof As shown in SEQ ID NO: 1.
  2. 根据权利要求1所述的嵌合抗原受体分子，其特征在于：所述跨膜区肽段包括但不限于CD8跨膜区肽段或PD-1跨膜区肽段，当跨膜区肽段为CD8跨膜区肽段时，所述HAC肽段与CD8跨膜区肽段还通过CD8的hinge区肽段相连。The chimeric antigen receptor molecule according to claim 1, wherein the transmembrane region peptide comprises, but is not limited to, a CD8 transmembrane region peptide or a PD-1 transmembrane region peptide, when a transmembrane region peptide When the segment is a CD8 transmembrane region peptide, the HAC peptide segment and the CD8 transmembrane region peptide are also linked by the hinge region of CD8.
  3. 根据权利要求1所述的嵌合抗原受体分子，其特征在于：所述胞内结构域肽段选自4-1BB、CD28、CD3ζ的胞内结构域肽段中的一种或多种。The chimeric antigen receptor molecule according to claim 1, wherein the intracellular domain peptide is selected from one or more of the intracellular domain peptides of 4-1BB, CD28, and CD3ζ.
  4. 根据权利要求1所述的嵌合抗原受体分子，其特征在于：所述嵌合抗原受体分子还包括信号肽。The chimeric antigen receptor molecule according to claim 1, wherein the chimeric antigen receptor molecule further comprises a signal peptide.
  5. 一种核苷酸，其特征在于：其编码根据权利要求1至4任一项所述的嵌合抗原受体分子。A nucleotide characterized by encoding a chimeric antigen receptor molecule according to any one of claims 1 to 4.
  6. 根据权利要求5所述的核苷酸，其特征在于：包含或为SEQ ID NO：10所示的核苷酸序列。The nucleotide according to claim 5, which comprises or is the nucleotide sequence shown by SEQ ID NO: 10.
  7. 一种重组载体，其特征在于：其包含根据权利要求5或6所述的的核苷酸。A recombinant vector comprising the nucleotide according to claim 5 or 6.
  8. 根据权利要求7所述的重组载体，其特征在于：所述重组载体为pRRSLIN慢病毒表达载体。The recombinant vector according to claim 7, wherein the recombinant vector is a pRRSLIN lentiviral expression vector.
  9. 一种重组细胞，其特征在于：其包含根据权利要求7或8任一项所述的重组载体。A recombinant cell comprising the recombinant vector of any one of claims 7 or 8.
  10. 根据权利要求9所述的重组细胞，其特征在于：所述重组细胞优选为T细胞或NK细胞。 The recombinant cell according to claim 9, wherein the recombinant cell is preferably a T cell or an NK cell.
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