Use of chimeric antigen receptors targeting non-classical HLA molecules for cancer therapy

Abstract

The invention relates to the technical field of biology, in particular to a chimeric antigen receptor of a targeted non-classical HLA molecule for cancer treatment, an immune cell modified by the chimeric antigen receptor, a preparation method and application of the chimeric antigen receptor. An extracellular domain of the chimeric antigen receptor specifically combined with the HLA-G in a targeted manner is an antigen binding structural domain of an ILT4 receptor, and compared with a CAR modified immune cell with an antigen binding region of an antibody as an extracellular domain, the HLA-G targeted immune response cell provided by the invention can recognize numerous tumor cells in a broad-spectrum manner on one hand, so that the HLA-G targeted immune response cell can be used for preparing an HLA-G targeted immune response cell; the killing efficiency and accuracy of various tumor cells are greatly improved; and on the other hand, the extracellular domain is an antigen binding domain of an ILT4 receptor, and the binding domain is a binding domain of a natural receptor, so that compared with an artificially synthesized antibody, the antigen binding domain is safe and reliable, and can truly reflect the target binding capacity in a natural state.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a chimeric antigen receptor targeting non-classical HLA molecules for cancer treatment, immune cells modified therefrom, a preparation method and applications thereof. Background Art

[0002] With the rapid development of biotechnology, immunotherapy has become one of the main therapies in the field of cancer treatment. Cancer immunotherapy mainly includes adoptive cell therapy, immunomodulators, tumor vaccines, antibody therapy, and immune checkpoint blockade therapy. Among them, in the field of adoptive cell therapy, chimeric antigen receptor-modified immune cell therapy, especially chimeric antigen receptor-modified T cell (Chimeric-Antigen Receptor T-Cell, CAR-T) therapy, is currently very popular and is a star therapy in this field.

[0003] The principle of CAR-T cell immunotherapy is to modify the T cells extracted from the patient's own body with chimeric antigen receptors through genetic engineering to form CAR-T cells. Through the modified chimeric antigen receptors, the CAR-T cells can specifically recognize tumor surface-related antigens (tumor cell markers) and thus target and kill tumors. Compared with ordinary immune cells, CAR-T cells have higher targeting, killing activity and durability. At present, modified immune cell therapy represented by CAR-T cells targeting CD19 and BCMA has a significant therapeutic effect in the treatment of hematological tumors such as lymphoma and multiple myeloma, and is considered to be one of the most promising tumor treatment methods.

[0004] However, since more than 90% of cancer patients suffer from solid tumors, more types of solid tumors and more tumor surface specific target antigens need to be further confirmed. One of the biggest difficulties in applying CAR-T immunotherapy to the treatment of solid tumors is that CAR-T cells have very high specificity requirements for antigen expression on tumor cells, otherwise it is easy to cause continuous activation of T cells to kill normal cells, or release a large number of cytokines to cause serious side effects. Although CAR-T cell immunotherapy has very high specificity requirements for tumor cell antigen expression, there are not many tumor-specific targeted antigens that can be selected, and most antigens expressed by tumors are not tumor-specific. CAR-T cell immunotherapy that targets tumor-associated antigens has problems such as "off-target" and drug resistance.

[0005] Therefore, finding a more broad-spectrum but specific, more efficient and safe molecule as a target for CAR therapy is an urgent problem to be solved in chimeric antigen receptor-modified immune cell therapy. The key to finding the above target is that the target antigen is highly expressed on the surface of tumor cells, but not expressed or low expressed on the surface of normal cells. Summary of the invention

[0006] Human leukocyte antigen G (HLA-G) is a non-classical HLA class I molecule. HLA-G molecules are widely expressed on the surface of many tumor cells (especially solid tumors). By interacting with the ILT4 receptor on the surface of immune cells, it activates the downstream pathway of immune cells to transmit inhibitory signals to immune cells, thereby causing immunosuppression to get rid of the attack of the immune system. Therefore, it is considered to be an important immune checkpoint molecule. In addition, HLA-G molecules are rarely expressed or only expressed briefly in healthy tissues. It is a tumor cell-specific change when a tumor occurs, and has become a precise target for immunotherapy of tumors, especially solid tumors, providing inspiration for the development of related new therapies and drugs.

[0007] CAR is usually composed of an extracellular targeting domain, a transmembrane spacer, and an intracellular signaling domain. The targeting domain of CAR is usually derived from the antigen binding region of the antibody, that is, the immunoglobulin heavy chain and light chain that form the antibody binding site. Common examples include the single-chain variable fragment scFv of the monoclonal antibody responsible for recognizing and binding to the antigen. CAR-modified immune cells with the antigen binding region of the antibody as the extracellular region have the advantages of strong specificity and high sensitivity, but at the same time, there are also the following problems: Due to the high specificity of the antibody, an antibody can usually only bind to one target, and it is easy to "off-target" when targeting tumor cells. For most tumors, the target molecules available for CAR immune cell therapy are scarce, and no target molecule can be applied to broad-spectrum tumor treatment.

[0008] The present invention aims to solve one of the technical problems in the above-mentioned technology at least to some extent.

[0009] To this end, the first aspect of the present invention provides a chimeric antigen receptor that specifically targets and binds to HLA-G, the chimeric antigen receptor comprising:

[0010] An extracellular domain, the extracellular domain comprising at least one binding domain for ILT4 targeting and binding to HLA-G;

[0011] a transmembrane domain, the transmembrane domain being connected to the extracellular domain;

[0012] An intracellular domain is connected to the transmembrane domain.

[0013] The chimeric antigen receptor specifically targeted to bind to HLA-G provided by the present invention, whose extracellular domain includes the antigen binding domain of the ILT4 receptor, is compared with the CAR-modified immune cells with the antigen binding region of the antibody as the extracellular region in the prior art. The engineered HLA-G targeted immune response cells of the present invention can, on the one hand, broadly identify numerous tumor cells, especially solid tumor cells, greatly improving the killing efficiency and accuracy of various tumor cells; on the other hand, the extracellular domain includes the antigen binding domain of the ILT4 receptor, which is the binding domain of the natural receptor, which is safe and reliable relative to the synthetic antibody, and can truly reflect the target binding ability in the natural state. Therefore, the present invention provides a new means of tumor treatment with application prospects.

[0014] According to a specific embodiment of the present invention, the ILT4 and HLA-G targeting binding domain has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 80% identity thereto.

[0015] According to a specific embodiment of the present invention, the extracellular domain further includes a signal peptide, and the signal peptide is connected to the binding domain of the ILT4 targeting and binding to HLA-G.

[0016] According to a specific embodiment of the present invention, the signal peptide includes at least one of a CD8α signal peptide, an IgG signal peptide, and a CD28 signal peptide.

[0017] According to a specific embodiment of the present invention, the signal peptide is derived from CD8α signal peptide.

[0018] According to a specific embodiment of the present invention, the signal peptide has an amino acid sequence as shown in SEQ ID NO:5.

[0019] According to a specific embodiment of the present invention, the extracellular domain is connected to the transmembrane domain via a hinge domain.

[0020] According to a specific embodiment of the present invention, the hinge domain includes a hinge domain derived from the CD8α hinge region, the IgG hinge region or at least a portion of an immunoglobulin, or the hinge domain is a variant of the CD8α hinge region, the IgG hinge region or at least a portion of an immunoglobulin hinge domain modified with one or more amino acids.

[0021] According to a specific embodiment of the present invention, the hinge domain is derived from the CD8α hinge region.

[0022] According to a specific embodiment of the present invention, the hinge domain has an amino acid sequence as shown in SEQ ID NO: 6, or an amino acid sequence having at least 80% identity thereto.

[0023] According to a specific embodiment of the present invention, the transmembrane domain includes any one of the transmembrane structural region derived from CD8α, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, and synthetic peptides, wherein the synthetic peptide does not contain proteins associated with immune response.

[0024] According to a specific embodiment of the present invention, the transmembrane domain is derived from the CD8α transmembrane region.

[0025] According to a specific embodiment of the present invention, the transmembrane domain has an amino acid sequence as shown in SEQ ID NO:7.

[0026] According to a specific embodiment of the present invention, the intracellular domain includes an immunoreceptor tyrosine-based activation motif and a co-stimulatory signal domain, and the co-stimulatory signal domain and the immunoreceptor tyrosine-based activation motif are connected via a connecting peptide.

[0027] According to a specific embodiment of the present invention, since HLA-G usually transmits inhibitory signals to immune cells after binding to immune cells and inhibits the function of immune cells, the chimeric antigen receptor extracellular segment designed by the present invention is the extracellular region of the inhibitory receptor ILT4 that recognizes HLA-G, and the intracellular segment of the inhibitory receptor is replaced with a co-stimulatory signal domain, so that the signal recognizing HLA-G can be converted into an activation signal to activate immune cells. The CAR-modified immune cells prepared by the present invention can resist the inhibition from the tumor immune microenvironment and reverse the exhaustion of immune cells.

[0028] According to a specific embodiment of the present invention, the immunoreceptor tyrosine-based activation motif is derived from the intracellular signaling domain of the CD3ζ chain or the FcεRIγ intracellular signaling domain.

[0029] According to a specific embodiment of the present invention, the immunoreceptor tyrosine-based activation motif is derived from the intracellular signaling domain of the CD3ζ chain.

[0030] According to a specific embodiment of the present invention, the immunoreceptor tyrosine activation motif has an amino acid sequence as shown in SEQ ID NO:9.

[0031] According to a specific embodiment of the present invention, the co-stimulatory signal domain includes at least one of a CD28 intracellular signaling domain, a 4-1BB intracellular signaling domain, an OX40 intracellular signaling domain, a CD40 or CD40L intracellular signaling domain, and an ICOS intracellular signaling domain.

[0032] According to a specific embodiment of the present invention, the co-stimulatory signal domain is derived from the 4-1BB intracellular signaling domain.

[0033] According to a specific embodiment of the present invention, the co-stimulatory signal domain has an amino acid sequence as shown in SEQ ID NO:8.

[0034] According to a specific embodiment of the present invention, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 17 or SEQ ID NO: 18.

[0035] The second aspect of the present invention provides an isolated polynucleotide encoding the chimeric antigen receptor of the first aspect.

[0036] The third aspect of the present invention provides an expression vector, which carries the polynucleotide described in the second aspect.

[0037] The fourth aspect of the present invention provides a recombinant virus, which carries the polynucleotide described in the second aspect or the expression vector described in the third aspect, and the recombinant virus can express the chimeric antigen receptor described in the first aspect.

[0038] The fifth aspect of the present invention provides a recombinant cell, which carries the polynucleotide described in the second aspect or the expression vector described in the third aspect or is infected by the recombinant virus described in the fourth aspect, and is capable of expressing the chimeric antigen receptor described in the first aspect.

[0039] According to a specific embodiment of the present invention, the recombinant cell is derived from an immune cell.

[0040] According to a specific embodiment of the present invention, the immune cells are obtained by separation from peripheral blood, umbilical cord blood, tissues, and organs, or the immune cells are obtained by differentiation of stem cells or immune cell progenitor cells.

[0041] According to a specific embodiment of the present invention, the stem cells include induced pluripotent stem cells, hematopoietic stem cells, or embryonic stem cells within 14 days of fertilization that have not undergone in vivo development.

[0042] According to a specific embodiment of the present invention, the immune cells include at least one selected from T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells and γδT cells.

[0043] The sixth aspect of the present invention provides a composition comprising at least one of the following:

[0044] The chimeric antigen receptor of the first aspect;

[0045] The polynucleotide of the second aspect;

[0046] The expression vector according to the third aspect;

[0047] The recombinant virus described in the fourth aspect;

[0048] The recombinant cell described in the fifth aspect.

[0049] The seventh aspect of the present invention provides use of the composition described in the sixth aspect in the preparation of anti-tumor drugs.

[0050] According to a specific embodiment of the present invention, HLA-G is expressed on the cell surface of the tumor.

[0051] In order to overcome the problem of scarcity of effective targets for most solid tumors in existing cell therapy clinical applications, the present invention provides a chimeric antigen receptor targeting human HLA-G and its gene and recombinant expression vector, an engineered HLA-G targeted chimeric antigen receptor modified immune response cell and its application.

[0052] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0054] Figure 1 The verification results of the expression of ILT4 receptor on CAR-NK cells in Example 3 of the present invention are shown;

[0055] Figure 2 The results of screening of HLA-G positive tumor cell lines in Example 4 of the present invention are shown;

[0056] Figure 3 The results of killing K562 cells by CAR-NK cells in Example 5 of the present invention are shown;

[0057] Figure 4 The results of killing NCI-H716 cells by CAR-NK cells in Example 5 of the present invention are shown;

[0058] Figure 5 The results of killing SKOV3 cells by CAR-NK cells in Example 5 of the present invention are shown;

[0059] Figure 6 The results of killing Huh7 cells by CAR-NK cells in Example 5 of the present invention are shown;

[0060] Figure 7The results of the treatment of subcutaneous transplanted tumors of mouse colon cancer by CAR-NK cells in Example 6 of the present invention are shown, wherein: Figure 7 A in the figure shows the images of tumors in each experimental group after the experiment was terminated. Figure 7 B in the figure shows the statistical results of the tumor weight of each experimental group after the experiment was terminated. DETAILED DESCRIPTION

[0061] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0062] It should be noted that 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 one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0063] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0064] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0065] 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.

[0066] 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.

[0067] In the present invention, the term "effector-target ratio" refers to the ratio of the number of effector cells (specific CAR-NK cells targeting HLA-G) to target cells (tumor cells).

[0068] In the present invention, the term "chimeric antigen receptor" is referred to as CAR. It is an artificial receptor molecule manufactured by genetic engineering technology, including an antigen binding domain (usually from a single-chain antibody variable region gene fragment, scFV; or from a single domain antibody), a hinge domain, a transmembrane domain, and an intracellular signal transduction domain.

[0069] In the present invention, the term "reduced expression" and "reduced expression" of gene expression refer to the elimination or reduction of the expression of one or more gene products encoded by the gene in the cell compared with the expression level of the gene mRNA or protein under normal circumstances. In some cases, this effect on gene expression is temporary or reversible, such as inhibiting the expression of the gene by small molecule compounds, RNAi, siRNA, shRNA and other nucleic acids or specific proteins. In some cases, this effect on gene expression is permanent, such as introducing base insertion or base deletion in the coding region of the target gene by gene editing, causing frameshift mutation, or inserting a DNA sequence in the coding region or non-coding region of the target gene by homologous recombination, causing the gene frameshift mutation or premature termination of transcription and / or translation, or translating a completely different polypeptide, which can make the gene permanently inactivated. The proportion of gene expression reduction caused by different methods can be 100%, more than 90%, more than 80%, more than 70%, more than 60%, more than 50%, more than 40%, more than 30%, or more than 20%.

[0070] In the present invention, the term "hinge region" refers to the region between the CH1 region and the CH2 region of the antibody heavy chain, which includes the inter-H chain disulfide bond, is rich in proline, does not form an α-helix, is prone to stretching and a certain degree of distortion, and is conducive to the complementary binding between the antibody antigen binding site and the antigen epitope. The heavy chain variable region in the heavy chain single-domain antibody of the present invention is connected to the Fc region through the hinge region.

[0071] In the present invention, the term "cell" refers to a eukaryotic cell, in particular a mammalian cell, such as a human cell.

[0072] In the present invention, the term "NK cell" refers to natural killer cells, which are generally CD56+CD45+ double positive and express multiple activation receptors, such as NKG2D, NKp44, NKp46, etc. NK cells can be differentiated from pluripotent stem cells (such as iPSC), or isolated from peripheral blood, or differentiated from CD34+ cells (such as umbilical cord blood or placental blood), or obtained from tumor cell lines (such as NK92).

[0073] In the present invention, the term "signal peptide" is also called a signal sequence, which is a localization tag for membrane proteins, secretory proteins, and lysosomal proteins, usually located at the amino terminus of the protein, and consists of 13-26 residues. Signal peptides do not have a conserved amino acid sequence, but have conserved structural features.

[0074] As used herein, the term "immune cell" refers to a cell from a human in the present invention. More specifically, the immune cells in the present invention can be derived from pluripotent stem cells by directed differentiation. Depending on the different differentiation conditions provided, these immune cells can be T cells, NK cells and macrophages. The immune cells in the present invention can also be derived from human hematopoietic stem cells. These hematopoietic stem cells can be separated from peripheral blood, umbilical cord blood, placental blood, or differentiated from pluripotent stem cells. Depending on the different differentiation conditions provided, these immune cells can be T cells, NK cells and macrophages. The immune cells in the present invention can also be separated from human blood, bone marrow, lymph or lymphoid organs, specific tissues (including but not limited to tumor tissue, abdominal cavity, liver, lungs and other organs, muscles, etc.). For example, cells of innate or adaptive immunity, for example, bone marrow or lymphocytes, are usually T cells, NK cells, macrophages, etc. Preferably, according to the present invention, immune cells include T cells, NK cells and macrophages. The immune cells of the present invention can also be differentiated from immune cell progenitor cells, such as lymphoid progenitor cells.

[0075] In the present invention, the term "pluripotent stem cells" generally refers to human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC). Human ESCs are isolated from blastocysts during the development of human fertilized eggs, and can also be isolated from embryos obtained by nuclear transplantation (embryonic stem cells within 14 days of fertilization that have not undergone in vivo development); human iPSCs can be obtained by reprogramming human cells by overexpressing specific transcription factors (such as OCT4, SOX2, KLF4, MYC). Both human ESCs and iPSCs express OCT4, NANOG, Tra-1-60, SSEA-4, and do not express SSEA-1. A notable feature of pluripotent stem cells is that they can differentiate into three germ layer tissues or cells in vitro or in vivo.

[0076] In the present invention, the term "hematopoietic stem cells" refers to CD34+ cells. These cells can be isolated from peripheral blood, umbilical cord blood, placental blood, bone marrow, spleen, lymph, or other tissues, and can also be differentiated from pluripotent stem cells.

[0077] In the present invention, the term "progenitor cells" refers to cells that can differentiate into various types of immune cells, such as myeloid stem cells, lymphoid stem cells, and further, erythroid stem cells, granulocyte and monocytic stem cells, megakaryotic stem cells, precursor B cells, precursor T cells, NK progenitor cells, and macrophage progenitor cells.

[0078] In the present invention, the term "vector" refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) can be operatively inserted and the genetic element can be expressed, such as producing a protein, RNA or DNA encoded by the genetic element, or replicating the genetic element. The vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed in the host cell. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages such as lambda phage or M13 phage, and animal viruses, etc. The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist it in entering the cell, including, but not limited to, viral particles, liposomes or protein shells. The vector may be an expression vector or a cloning vector. In some embodiments, the vector provided by the present invention (e.g., expression vector) contains the nucleic acid sequence encoding the fusion protein described in the present invention, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0079] In the present invention, when the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs utilizing these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLASTAltschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0080] Chimeric Antigen Receptor

[0081] The present invention provides a chimeric antigen receptor that specifically targets and binds to HLA-G, the chimeric antigen receptor comprising:

[0082] An extracellular domain, wherein the extracellular domain includes a binding domain for ILT4 to target and bind to HLA-G;

[0083] a transmembrane domain, the transmembrane domain being connected to the extracellular domain;

[0084] An intracellular domain is connected to the transmembrane domain.

[0085] The ILT4 receptor is a receptor on the surface of immune cells that is responsible for binding to the HLA-G molecule. ILT4 is only expressed on immune cells of myeloid origin. The ILT4 receptor is divided into an extracellular region, a transmembrane region, and an intracellular region, of which the extracellular region is the functional region responsible for recognizing and binding to HLA-G. The extracellular region of the ILT4 receptor is divided into four domains, named Domain 1 (D1), Domain 2 (D2), Domain 3 (D3), and Domain 4 (D4). According to reports, D1 and D2 of the receptor are responsible for binding to HLA-G, while D3 and D4 are only responsible for supporting the external structure formed by the binding of D1 and D2 to HLA-G.

[0086] In a specific embodiment of the present invention, the present application provides a synthetic polypeptide that targets and binds to HLA-G, wherein the synthetic polypeptide sequence is derived from ILT4 protein. In general, the synthetic polypeptide includes but is not limited to the following composition: ILT4-D1D2, ILT4-ECD, ILT4-D1, ILT4-D2, ILT4-D1D1, ILT4-D2D1, ILT4-D2D2, preferably: ILT4-D1D2, ILT4-ECD.

[0087] According to a specific embodiment of the present invention, the ILT4 and HLA-G targeting binding domain has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 80% identity thereto.

[0088] According to a specific embodiment of the present invention, the extracellular domain further includes a signal peptide, and the signal peptide is connected to the binding domain of the ILT4 targeting and binding to HLA-G.

[0089] According to a specific embodiment of the present invention, the signal peptide includes at least one of a CD8α signal peptide, an IgG signal peptide, and a CD28 signal peptide.

[0090] According to a specific embodiment of the present invention, the signal peptide has an amino acid sequence as shown in SEQ ID NO:5.

[0091] According to a specific embodiment of the present invention, the extracellular domain is connected to the transmembrane domain via a hinge domain.

[0092] According to a specific embodiment of the present invention, the hinge domain includes a hinge domain derived from the CD8α hinge region, the IgG hinge region or at least a portion of an immunoglobulin, or the hinge domain is a variant of the CD8α hinge region, the IgG hinge region or at least a portion of an immunoglobulin hinge domain that has been modified with one or more amino acids.

[0093] According to a specific embodiment of the present invention, the hinge domain has an amino acid sequence as shown in SEQ ID NO: 6, or an amino acid sequence having at least 80% identity thereto.

[0094] According to a specific embodiment of the present invention, the transmembrane domain includes any one of the transmembrane structural region derived from CD8α, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, and synthetic peptides, wherein the synthetic peptide does not contain proteins associated with immune response.

[0095] According to a specific embodiment of the present invention, the transmembrane domain has an amino acid sequence as shown in SEQ ID NO:7.

[0096] According to a specific embodiment of the present invention, the intracellular domain includes an immunoreceptor tyrosine-based activation motif and a co-stimulatory signal domain, and the co-stimulatory signal domain and the immunoreceptor tyrosine-based activation motif are connected via a connecting peptide.

[0097] According to a specific embodiment of the present invention, the immunoreceptor tyrosine-based activation motif is derived from the intracellular signaling domain of the CD3ζ chain or the FcεRIγ intracellular signaling domain.

[0098] According to a specific embodiment of the present invention, the immunoreceptor tyrosine-based activation motif is derived from the intracellular signaling domain of the CD3ζ chain.

[0099] According to a specific embodiment of the present invention, the immunoreceptor tyrosine activation motif has an amino acid sequence as shown in SEQ ID NO:9.

[0100] According to a specific embodiment of the present invention, the co-stimulatory signal domain includes at least one of a CD28 intracellular signaling domain, a 4-1BB intracellular signaling domain, an OX40 intracellular signaling domain, a CD40 or CD40L intracellular signaling domain, and an ICOS intracellular signaling domain.

[0101] According to a specific embodiment of the present invention, the co-stimulatory signal domain is derived from the 4-1BB intracellular signaling domain.

[0102] According to a specific embodiment of the present invention, the co-stimulatory signal domain has an amino acid sequence as shown in SEQ ID NO:8.

[0103] According to a specific embodiment of the present invention, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 17 or SEQ ID NO: 18.

[0104] According to an embodiment of the present invention, from N-terminus to C-terminus, the CAR may sequentially comprise a synthetic polypeptide derived from ILT4 that binds to HLA-G, the hinge region, the transmembrane domain, the co-stimulatory signal domain, and the intracellular signaling domain.

[0105] According to a specific embodiment of the present invention, from N-terminus to C-terminus, the CAR may sequentially comprise a synthetic polypeptide binding to HLA-G derived from ILT4, a hinge region derived from CD8α, a transmembrane domain derived from CD8α, a co-stimulatory signaling domain derived from 4-1BB, and an intracellular signaling domain derived from CD3ζ.

[0106] According to a specific embodiment of the present invention, from N-terminus to C-terminus, the CAR may sequentially comprise a signal peptide, a synthetic polypeptide derived from ILT4 that binds to HLA-G, the hinge region, the transmembrane domain, the co-stimulatory signal domain, and the intracellular signaling domain.

[0107] According to a more specific embodiment of the present invention, the inventors designed two synthetic polypeptides that can target and bind to human HLA-G, as shown in Table 1, and the two synthetic polypeptides are composed of polypeptide sequences derived from ILT4. The corresponding amino acid sequence is shown in SEQ ID NO: 1-2, and the corresponding nucleotide sequence is shown in SEQ ID NO: 3-4. And any one of the two synthetic polypeptides is used as the extracellular region of the chimeric antigen receptor, and the signal peptide sequence derived from CD8α is connected to its N-terminus, and the hinge region sequence derived from CD8α, the transmembrane domain sequence derived from CD8α, the 4-1BB sequence and the CD3ζ sequence are sequentially connected to its C-terminus. Its amino acid sequence is shown in SEQ ID NO: 5-9, and its nucleotide sequence is shown in SEQ ID NO: 10-14. The two chimeric receptors finally formed include ILT4-D1D2 and ILT4-ECD, respectively, and are named CAR1 and CAR2, respectively.

[0108] Without substantially affecting the activity of the domain (retaining at least 95% of the activity), those skilled in the art may replace, add and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the sequence of the domain or its functional fragment. They are all deemed to be included in the scope of protection of the present invention. The variant sequence of the present invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. The amino acid sequences described in the present invention are all shown in an N-terminal to C-terminal manner.

[0109] Polynucleotide, expression vector, recombinant virus, cell, composition

[0110] The present invention also provides an isolated nucleic acid encoding CAR and an expression vector and a recombinant virus containing the nucleic acid. The nucleic acid molecule encodes the above-mentioned CAR, and the nucleic acid is preferably an expression cassette obtained by genetic engineering means.

[0111] The expression vector may refer to a cloning vector or a recombinant vector, and can be obtained by operably linking the nucleic acid to a commercially available vector (such as a plasmid or a viral vector). Commonly used plasmids include pSeTag2, PEE14, pMH3, pUC57, and the like.

[0112] According to an embodiment of the present invention, immune cells with enhanced functions can be obtained by using any one of the following methods or a combination thereof:

[0113] 1) Randomly integrate one or more circular or linear exogenous DNA fragments into target cells;

[0114] 2) Integrate one or more circular or linear exogenous DNA fragments into target cells. Site-specific integration can improve the efficiency of homologous recombination of exogenous DNA by introducing nucleases at the target site. The efficiency of homologous recombination can also be improved by other physical means, such as electroporation.

[0115] 3) one or more endonucleases that can recognize specific sites introduce one or more double-strand breaks into target cells at the specific sites, and the target cells generate base insertions or base deletions at the specific sites by non-homologous end joining;

[0116] 4) One or more nucleases that can recognize specific sites introduce one or more double-strand breaks into target cells at specific sites, and exogenous circular or linear DNA fragments are provided at the same time, so that the cells produce base insertions or base deletions at the selected sites through homologous recombination.

[0117] At this time, the target cell is the immune cell or stem cell or progenitor cell mentioned above, or a donor cell for reprogramming into iPSC, or any derivative cell in the process of differentiation of stem cells or progenitor cells into immune cells. The exogenous DNA fragment may contain a gene expression frame, that is, at least components such as a promoter and an open reading frame; or it may not contain a gene expression frame.

[0118] In some preferred embodiments, the nucleic acid molecule is species optimized and is more easily expressed in mammalian cells.

[0119] The present invention also provides an expression vector, which comprises the above-mentioned isolated nucleic acid molecule. When the above-mentioned isolated polynucleotide is connected to the vector, the polynucleotide can be directly or indirectly connected to the control element on the vector, as long as these control elements can control the translation and expression of the polynucleotide. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the polynucleotide can be operably connected to the control element. Herein, "operably connected" refers to connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, can play their expected functions of regulating the transcription and translation of the exogenous gene. Of course, the polynucleotides used to encode different domains of CAR can be inserted into different vectors separately and independently, and it is common to insert them into the same vector. Commonly used vectors can be, for example, plasmids, bacteriophages, and the like.

[0120] The present application provides a recombinant virus, which can express a specific chimeric antigen receptor targeting HLA-G and can infect immune response cells.

[0121] The present invention also provides a recombinant cell, which contains the expression vector. The expression vector can be introduced into mammalian cells to construct recombinant cells, and then these recombinant cells are used to express the chimeric antigen receptor provided by the present invention. The recombinant cells are derived from immune response cells, including at least one of T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells and γδT cells. The immune cells are obtained by separation from peripheral blood, umbilical cord blood or tissues and organs, or the immune cells are obtained by differentiation of stem cells or immune cell progenitor cells; the stem cells include induced pluripotent stem cells, hematopoietic stem cells or embryonic stem cells within 14 days of fertilization that have not undergone in vivo development.

[0122] The composition provided by the present invention contains at least one of the chimeric antigen receptor, polynucleotide, expression vector, recombinant virus or recombinant cell as described above, and further, the composition contains a pharmaceutically acceptable carrier. In certain embodiments, the composition includes a combination separated in time and / or space, as long as it can work together to achieve the purpose of the present invention. For example, the components contained in the composition can be applied to the subject as a whole, or separately. When the components contained in the composition are applied to the subject separately, each component can be applied to the subject simultaneously or sequentially.

[0123] In some embodiments, the drug carrier includes any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid binder, glidant or lubricant, etc., suitable for a specific target dosage form. Except for any conventional excipients incompatible with the compound of the present invention, such as any adverse biological effect produced or any other component of the pharmaceutically acceptable composition interacting in a harmful manner, their use is also within the scope of the present invention.

[0124] In a specific embodiment of the present invention, the present invention screens out a suitable target cell line for testing the killing ability of newly produced CAR-NK cells, wherein the screened target cells can be used for both in vitro experiments and in vivo experiments in mice.

[0125] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0126] Table 1 shows the nucleotide and amino acid sequences involved in the present invention. According to the sequences provided by the present invention, those skilled in the art can easily obtain the CAR-NK cells provided by the present invention.

[0127] Table 1

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] Example 1 Design of chimeric antigen receptor specifically targeting human HLA-G

[0135] The inventors designed two synthetic polypeptides that can target and bind to human HLA-G, as shown in Table 1. The two synthetic polypeptides are composed of polypeptide sequences derived from ILT4. The corresponding amino acid sequences are shown in SEQ ID NO: 1-2, and the corresponding nucleotide sequences are shown in SEQ ID NO: 3-4. The two synthetic polypeptides are used as the extracellular region of the chimeric antigen receptor, and the signal peptide sequence derived from CD8α is connected at its N-terminus, and the hinge region sequence derived from CD8α, the transmembrane domain sequence derived from CD8α, the 4-1BB sequence and the CD3ζ sequence are sequentially connected at its C-terminus. The amino acid sequence is shown in SEQ ID NO: 5-9, and the nucleotide sequence is shown in SEQ ID NO: 10-14. The two chimeric receptors finally formed are named CAR1 and CAR2, respectively.

[0136] Example 2 Construction of an expression plasmid vector containing a chimeric antigen receptor specifically targeting human HLA-G

[0137] The construction of expression plasmid vectors (pCDH-CAR1 and pCDH-CAR2) containing specific chimeric antigen receptors targeting human HLA-G specifically includes the following steps:

[0138] (1) GENEWIZ synthesized a DNA sequence of a chimeric antigen receptor (CAR1 and CAR2) targeting human HLA-G into a pUC57 vector. The DNA sequence is shown in SEQ ID NOs: 15-16, and the corresponding encoded amino acid sequence is shown in SEQ ID NOs: 17-18;

[0139] (2) The pCDH-EV vector was double-digested with EocR1 and BamH1 endonucleases, and the double-digested vector was recovered by gel electrophoresis using 1% agarose gel for 20 min;

[0140] (3) using primer pair 1 to obtain a nucleotide fragment of a chimeric antigen receptor that specifically targets and binds to human HLA-G by PCR from the pUC57 vector containing the DNA sequence of the chimeric antigen receptor that specifically targets and binds to human HLA-G synthesized in (1), wherein the nucleotide sequence of the primer pair (primer 1F, primer 1R) is as shown in SEQ ID NO: 19-20;

[0141] (4) homologously recombining the double-enzyme-cut vector obtained in (2) and (3) with a nucleotide fragment of a chimeric antigen receptor that specifically targets and binds to human HLA-G;

[0142] (5) The constructed expression plasmid vector (pCDH-CAR1 / 2) containing a chimeric antigen receptor that specifically targets human HLA-G was sequenced and verified, and the sequencing results confirmed that the construction was successful.

[0143] Example 3 Construction and identification of a cell line containing a chimeric antigen receptor that specifically targets human HLA-G

[0144] The expression plasmids (pCDH-CAR1 and pCDH-CAR2) were co-transfected with pMD2G and psPAX2 into a 15 cm culture dish containing HEK293-T cells at a density of 90%. Three days later, the cell supernatant was collected, the virus solution was filtered using a 0.45 μm filter, and the virus was concentrated to obtain lentivirus expressing plasmids (pCDH-CAR1 and pCDH-CAR2).

[0145] Human recombinant IL-2 and 20 ng / mL human IL-21 were added to NK92 cells at a final concentration of 1000 U / mL and cultured for 24 hours. Then, the above-mentioned lentivirus and the infection-promoting reagent polybrene were added at an MOI of 1 and infected for 24 hours. Then, an appropriate amount of culture medium was added, and the infection efficiency of NK92 cells was detected by flow cytometry after 48 hours. The specific operation is as follows.

[0146] (1) Blank control group: NK cells not infected with virus fluid;

[0147] (2) Experimental group: 2 NK cells infected with different CAR1 or CAR2 viruses.

[0148] The experimental group and control group were washed twice with PBS and then resuspended in FACS solution (PBS containing 0.1% sodium azide and 0.4% BSA). According to the antibody instructions, APC-labeled anti-human ILT4 antibody was added to the cell suspension of the cell group to be tested and the control group, and incubated at 4°C for 30 minutes. The stained cells were obtained by flow cytometry, and the results were analyzed using FlowJo software. The flow cytometry results are shown in Figure 2. Figure 1 As shown in the figure, the expression of CAR molecules was almost undetectable in the control group, and the expression rate of CAR molecules in each experimental group reached more than 90%. Figure 1 It can be seen from the flow cytometry results that the cells to be detected collected in this example expressed a specific chimeric antigen receptor targeting HLA-G.

[0149] Example 4 Screening of target cell lines

[0150] In order to screen suitable target cell lines for testing the killing ability of newly produced CAR-NK cells, the inventors detected and analyzed the expression of HLA-G in tumor cell lines such as ovarian cancer cell SKOV3, colon cancer cell NCI-H716 and leukemia cell K562. The tumor cells for testing were collected, washed twice with PBS, and resuspended in FACS solution (PBS containing 0.1% sodium azide and 0.4% BSA), counted, and the cell concentration was adjusted to 1×10 6 / ml; PE-HLA-G and isotype control antibodies were added respectively and incubated at 4°C for 30 minutes; after washing twice with PBS, the cells were resuspended in FACS solution and the cell fluorescence was analyzed by flow cytometry and the results were analyzed by FlowJo software. Figure 2 As shown, K562, NCI-H716, and SKOV3 cells in the tested tumor cell groups all showed high expression of HLA-G antigen, while the liver cancer cell line Huh7 did not express HLA-G.

[0151] Example 5 In vitro verification of the killing of target cells by chimeric antigen receptor cells that specifically target and bind to human HLA-G

[0152] Next, the in vitro killing activity of CAR-NK cells was tested by PI and CFSE staining. Specifically, for the tumor cell lines K562, NCI-H716, and Huh7, the three target cell lines were stained with CSFE fluorescence at 2×10 4 The inoculation concentration of cells / ml was spread on the culture plate. Four experimental groups and one control group were set up for each target cell line, wherein the experimental group was added with the cell suspension of the specific CAR-NK cells targeting HLA-G obtained in Example 3; the blank control group was added with NK cells infected with empty vector virus. In the above experimental group, the specific CAR-NK cells targeting HLA-G in Example 3 were mixed with the target cells at a 1:1 effector-target ratio.

[0153] For the tumor cell line SKOV3, 5 × 10 3 After the target cells adhered to the E-plate, the experimental group cells and the control group cells were added, and then the growth of the target cells was detected by RTCA instrument.

[0154] Similarly, the blank control group also mixed NK cells and target cells at a 1:1 effector-target ratio. After 4 hours of culture, the supernatant was removed by centrifugation, the cell pellet was washed and stained with PI, the stained cells were obtained by flow cytometry, and the results were analyzed using FlowJo software. Figure 3 The results are shown for the tumor cell killing rate test using leukemia cells K562 as target cells. Figure 3It can be seen that the specific CAR-NK cells targeting HLA-G in Example 3 have a significant killing effect on leukemia cells K562 (significantly higher than the control group). Figure 4 The results are shown for the tumor cell killing rate test using colon cancer cell NCI-H716 as target cells. Figure 4 It can be seen that the specific CAR-NK cells targeting HLA-G in Example 3 have a significant killing effect on colon cancer cells NCI-H716 (significantly higher than the control group). Figure 5 The results of the tumor cell killing rate test using ovarian cancer cell SKOV3 as the target cell are shown. Figure 5 It can be seen that the specific CAR-NK cells targeting HLA-G in Example 3 have a significant killing effect on ovarian cancer SKOV3 (significantly higher than the control group). Figure 6 It can be seen that the specific CAR-NK cells targeting HLA-G in Example 3 have no obvious killing enhancement effect on HLA-G negative Huh7 (the killing effect is similar to that of the control group).

[0155] From the above results, it can be seen that the CAR-NK cells provided by the present invention have the ability to specifically recognize and kill HLA-G positive tumor cells.

[0156] Example 6 In vivo experimental verification of chimeric antigen receptor NK cells targeting human HLA-G to effectively kill target cells

[0157] Take 1×10 7 NCI-H716 cells were subcutaneously injected into 5-week-old NCG mice. After 10 days of tumor bearing, the tumor size was measured with a vernier caliper. Mice with similar tumor formation were randomly divided into 4 groups, two of which were experimental groups and the remaining two groups were control groups (one group was untreated control and the other group was treated with ordinary NK92). Afterwards, 1×10 7 A control group of mice was injected with 1×10 CAR1 or CAR2-NK92 cells into the tail vein. 7 Ordinary NK92 cells without genetic modification were injected into the tail vein of a group of control mice with an equal volume of PBS. After treatment once a week, the size of the subcutaneous transplanted tumor was measured at the same time. During this period, IL2 was injected intraperitoneally every 3 days, with 5×10 per mouse each time. 4 U, 3 treatments in total. Figure 7 The results show the killing rate test results of CAR1 and CAR2-NK cells on colon cancer cells NCI-H716 after the target cells were used for tumor loading. Figure 7 It can be seen that specific CAR-NK cells targeting HLA-G have a significant tumor-suppressing effect on colon cancer cells NCI-H716 in vivo (significantly higher than the control group).

[0158] From the above results, it can be seen that the CAR-NK cells provided by the present invention have the ability to specifically recognize and kill HLA-G positive tumor cells both in vitro and in vivo.

[0159] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

[0160] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" 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, unless they are contradictory.

[0161] 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

1. A chimeric antigen receptor that specifically targets and binds to HLA-G, characterized in that: The chimeric antigen receptor comprises: An extracellular domain, the extracellular domain comprising at least one binding domain for ILT4 targeting and binding to HLA-G; a transmembrane domain, the transmembrane domain being connected to the extracellular domain; An intracellular domain is connected to the transmembrane domain.

2. The chimeric antigen receptor according to claim 1, characterized in that The ILT4 and HLA-G targeted binding domain has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 80% identity thereto.

3. The chimeric antigen receptor according to claim 1, characterized in that The extracellular domain further includes a signal peptide, which is connected to the binding domain of the ILT4 and HLA-G targeted binding; Optionally, the signal peptide comprises at least one of a CD8α signal peptide, an IgG signal peptide, and a CD28 signal peptide; Optionally, the signal peptide is derived from a CD8α signal peptide; Optionally, the signal peptide has the amino acid sequence shown in SEQ ID NO:

5.

4. The chimeric antigen receptor according to claim 1, characterized in that The extracellular domain is connected to the transmembrane domain via a hinge domain; Optionally, the hinge domain comprises a hinge domain derived from a CD8α hinge region, an IgG hinge region, or at least a portion of an immunoglobulin; Optionally, the hinge domain is a CD8α hinge region, an IgG hinge region, or a variant of at least a portion of an immunoglobulin hinge domain with one or more amino acid modifications; Optionally, the hinge domain is derived from the CD8α hinge region; Optionally, the hinge domain has an amino acid sequence as shown in SEQ ID NO: 6, or an amino acid sequence having at least 80% identity thereto.

5. The chimeric antigen receptor according to claim 1, characterized in that The transmembrane domain includes any one of the transmembrane structural region derived from CD8α, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, and synthetic peptides, wherein the synthetic peptide does not contain proteins associated with immune response; Optionally, the transmembrane domain is derived from the CD8α transmembrane region; Optionally, the transmembrane domain has the amino acid sequence shown in SEQ ID NO:

7.

6. The chimeric antigen receptor according to claim 1, characterized in that The intracellular domain includes an immunoreceptor tyrosine-based activation motif and a co-stimulatory signal domain, wherein the co-stimulatory signal domain and the immunoreceptor tyrosine-based activation motif are connected via a connecting peptide; Optionally, the immunoreceptor tyrosine-based activation motif is derived from the intracellular signaling domain of the CD3ζ chain or the FcεRIγ intracellular signaling domain; Optionally, the immunoreceptor tyrosine-based activation motif is derived from the intracellular signaling domain of the CD3ζ chain; Optionally, the immunoreceptor tyrosine-based activation motif has an amino acid sequence as shown in SEQ ID NO: 9; Optionally, the co-stimulatory signaling domain comprises at least one of a CD28 intracellular signaling domain, a 4-1BB intracellular signaling domain, an OX40 intracellular signaling domain, a CD40 or CD40L intracellular signaling domain, and an ICOS intracellular signaling domain; Optionally, the co-stimulatory signaling domain is derived from the 4-1BB intracellular signaling domain; Optionally, the co-stimulatory signal domain has an amino acid sequence as shown in SEQ ID NO:

8.

7. The chimeric antigen receptor according to claim 1, characterized in that The chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 17 or SEQ ID NO:

18.

8. An isolated polynucleotide, characterized in that The polynucleotide encodes the chimeric antigen receptor according to any one of claims 1 to 7.

9. An expression vector, characterized in that The expression vector carries the polynucleotide according to claim 8.

10. A recombinant virus, characterized in that The recombinant virus carries the polynucleotide of claim 8 or the expression vector of claim 9, and the recombinant virus is capable of expressing the chimeric antigen receptor of any one of claims 1-7.

11. A recombinant cell, characterized in that The recombinant cell carries the polynucleotide of claim 8 or the expression vector of claim 9 or is infected by the recombinant virus of claim 10, and the recombinant cell is capable of expressing the chimeric antigen receptor of any one of claims 1-7.

12. The recombinant cell according to claim 11, characterized in that The recombinant cell is derived from an immune cell; Optionally, the immune cells are obtained by separation from peripheral blood, umbilical cord blood, tissues, or organs, or the immune cells are obtained by differentiation of stem cells or immune cell progenitor cells; Optionally, the stem cells include induced pluripotent stem cells, hematopoietic stem cells, or embryonic stem cells within 14 days of fertilization that have not undergone in vivo development; Optionally, the immune cells include at least one selected from T cells, B cells, monocytes, NK cells, dendritic cells, macrophages, regulatory T cells, helper T cells, cytotoxic T cells, NKT cells and γδT cells.

13. A composition, characterized in that Contains at least one of the following: The chimeric antigen receptor according to any one of claims 1 to 7; The polynucleotide of claim 8; The expression vector according to claim 9; The recombinant virus according to claim 10; The recombinant cell according to claim 11 or 12.

14. Use of the composition of claim 13 in the preparation of an anti-tumor drug, wherein HLA-G is expressed on the cell surface of the tumor.

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