Transgenic recombinant immune cell specifically targeting tumor and use thereof

Abstract

Provided is a transgenic recombinant immune cell specifically targeting a tumor. The recombinant immune cell comprises a chimeric polypeptide and a chimeric antigen receptor. The chimeric polypeptide comprises: a first extracellular region having a HLA-G protein-binding activity, a first transmembrane region and a first intracellular region, wherein the N terminus of the first transmembrane region links to the C terminus of the first extracellular region, the N terminus of the first intracellular region links to the C terminus of the first transmembrane region, and the first transmembrane region comprises the transmembrane region of a Notch receptor protein of Xenopus tropicalis. The chimeric antigen receptor does not have a HLA-G protein-binding activity. The recombinant immune cell greatly improves the accurate recognition of tumor cells by immune cells, reduces the off-target effect of cell therapy, accurately and effectively distinguishes tumor cells from normal cells, and provides an effective target and targeting mode for the treatment of broad-spectrum tumors (especially solid tumors).

Description

Genetically modified recombinant immune cells specifically targeting tumors and their applications Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and more specifically to transgenic recombinant immune cells that specifically target tumors, as well as methods for preparing and using the cells. More specifically, the present invention relates to a chimeric polypeptide, a first nucleic acid molecule, a first expression vector, a second nucleic acid molecule, a second expression vector, a recombinant immune cell, a pharmaceutical composition, and uses 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, and immune checkpoint blockade therapy. Among them, chimeric antigen receptor-modified immune cell therapy, especially chimeric antigen receptor-modified T cell (CAR-T) therapy, is currently very popular and is a star treatment in this field.

[0003] The principle of immune cell therapy represented by CAR-T is to modify the patient's own T cells 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), thereby targeting and killing tumors. Compared with ordinary immune cells, CAR-T cells have higher targeting, killing activity and persistence. At present, modified immune cell therapy represented by CAR-T cells targeting CD19 and BCMA has a significant effect in the treatment of hematological tumors such as B lymphoma, and is considered one of the most promising tumor treatment methods.

[0004] However, due to the lack of targets that can clearly distinguish between normal cells and tumor cells, CAR-T cell therapy often inevitably kills some normal cells that express the target protein, causing damage to normal tissues. Alternatively, the widespread presence of the target leads to the continuous activation of CAR-T cells, which in turn releases a large amount of cytokines, causing a cytokine storm. At the same time, because CAR-T cell therapy can usually only recognize a single antigen, tumor cells can often escape through gene mutations that lose the antigens recognized by CAR-T cells, leading to drug resistance. In addition, the high heterogeneity of tumors also makes it impossible for the single target carried by CAR-T cells to fully cover tumor cells, resulting in incomplete and incomplete tumor treatment. The above-mentioned key issues have become the main obstacles to the development and growth of the cell therapy field.

[0005] However, recent studies have shown that attempting to solve the above problems by finding a single molecule that is more broad-spectrum but specific, more efficient and safe as a target for CAR therapy may be extremely difficult. Because broad-spectrum and specificity are like two sides of a coin, it is difficult for a single target to take into account both characteristics at the same time. In current tumor treatment research, no target molecule that perfectly combines the above two characteristics has been found. Therefore, trying to achieve the broad-spectrum and specificity of immune cell therapy through a combination of specific targets and tools for target combination identification and activation is one of the new directions with great potential in cell therapy. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0007] In the first aspect of the present invention, the present invention proposes a chimeric polypeptide. According to an embodiment of the present invention, the chimeric polypeptide includes: a first extracellular region, the first extracellular region has the activity of binding to the HLA-G protein; a first transmembrane region, the first transmembrane region includes the transmembrane region of the Notch receptor protein of tropical clawed frog or an amino acid sequence having at least 80% identity therewith, and the N-terminus of the first transmembrane region is connected to the C-terminus of the first extracellular region; a first intracellular region, the N-terminus of the first intracellular region is connected to the C-terminus of the first transmembrane region. The chimeric polypeptide of the present invention selects the transmembrane region of the Notch receptor protein of tropical clawed frog as the transmembrane region of the chimeric polypeptide (i.e., the SynNotch synthetic receptor), which can improve the ability and efficiency of the chimeric polypeptide to activate downstream gene transcription.

[0008] In a second aspect of the present invention, the present invention provides a first nucleic acid molecule. According to an embodiment of the present invention, the first nucleic acid molecule encodes the chimeric polypeptide described in the first aspect. The first nucleic acid molecule according to an embodiment of the present invention can encode the chimeric polypeptide described above.

[0009] In a third aspect, the present invention provides a first expression vector. According to an embodiment of the present invention, the first expression vector carries the first nucleic acid molecule described in the second aspect. When linking the first nucleic acid molecule to the expression vector, the first nucleic acid molecule can be directly or indirectly linked to control elements on the expression vector, as long as these control elements are capable of controlling translation and expression of the first nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself or exogenous, that is, not derived from the vector itself. Of course, it is sufficient that the first nucleic acid molecule and the control elements are operably linked.

[0010] In its fourth aspect, the present invention provides a second nucleic acid molecule. According to an embodiment of the present invention, the second nucleic acid molecule comprises a first nucleic acid fragment and a second nucleic acid fragment, wherein the 3' end of the first nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment; wherein the second nucleic acid fragment is used to encode an antigen chimeric receptor that targets the first molecule, and the first nucleic acid fragment is used to bind to a first intracellular region and induce expression of the antigen chimeric receptor; the first intracellular region is consistent with the first intracellular region defined in the chimeric polypeptide described in the first aspect. The second nucleic acid molecule of the present invention can bind to the first intracellular region of the aforementioned chimeric polypeptide, thereby activating and inducing expression of the antigen chimeric receptor.

[0011] In a fifth aspect, the present invention provides a second expression vector. According to an embodiment of the present invention, the second expression vector carries the second nucleic acid molecule described in the fourth aspect. The second expression vector according to an embodiment of the present invention can express the antigen chimeric receptor in the second nucleic acid molecule.

[0012] In the sixth aspect of the present invention, the present invention proposes a recombinant immune cell. According to an embodiment of the present invention, the recombinant immune cell comprises: carrying the first nucleic acid molecule described in the second aspect or the first expression vector described in the third aspect; or expressing the chimeric polypeptide described in the first aspect. The recombinant immune cell of the embodiment of the present invention can express the aforementioned chimeric polypeptide on the surface of the recombinant immune cell under suitable conditions, and can recognize the HLA-G protein. After binding to the HLA-G protein, it can achieve a variety of different signal output types, such as activation of expression of specific genes, etc., especially activating the expression of chimeric antigen receptors containing factors with therapeutic effects (such as treating tumors, etc.), for treating diseases such as tumors.

[0013] In a seventh aspect, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes the recombinant immune cells described in the sixth aspect. As mentioned above, cells expressing the chimeric polypeptide can recognize HLA-G protein, and after binding to HLA-G protein, they can achieve a variety of different signal output types, such as activation and inhibition of specific gene expression, or immune cells expressing the chimeric polypeptide can secrete factors with therapeutic effects for anti-tumor after contact with HLA-G protein. Therefore, a pharmaceutical composition containing the aforementioned recombinant immune cells can target HLA-G protein for the prevention and / or treatment of related diseases, such as cancer.

[0014] In the eighth aspect of the present invention, the present invention proposes the use of the recombinant immune cell described in the sixth aspect or the pharmaceutical composition described in the seventh aspect in the preparation of a drug for preventing and / or treating a disease. Beneficial effects:

[0015] (1) The recombinant immune cells of the present invention greatly improve the accurate recognition of tumor cells by immune cells, reduce the off-target effects of cell therapy, accurately and effectively distinguish tumor cells from normal cells, and provide effective targets and targeting methods for the treatment of a wide spectrum of tumors (especially solid tumors);

[0016] (2) The recombinant immune cells of the present invention can convert the inhibitory signal transmitted by HLA-G binding to ILT2 / 4 into an activation signal, which not only effectively kills HLA-G-positive tumor cells, but also resists the inhibition from the tumor immune microenvironment and reverses the exhaustion of immune cells;

[0017] (3) The engineered multi-targeted recombinant immune cells of the present invention can not only broadly identify many different types of tumor cells, but also accurately distinguish normal cells, greatly improving the killing efficiency and accuracy of various types of tumor cells, thereby providing a new method of tumor immune cell therapy that is both broad-spectrum and specific with broad application prospects.

[0018] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0020] FIG1 is a schematic diagram of the structural composition of the SynNotch receptor in Example 1 of the present invention;

[0021] FIG2 is a schematic diagram of the structural composition of the CAR receptor in Example 2 of the present invention;

[0022] FIG3 is a graph showing the killing results of Syn-CAR-NK cells against dual-target positive K562 cells in Example 3 of the present invention;

[0023] FIG4 is a graph showing the killing results of Syn-CAR-NK cells against single-target positive Aspc-1 cells in Example 3 of the present invention;

[0024] FIG5 is a graph showing the killing results of Syn-CAR-NK against target-free THLE3 cells in Example 3 of the present invention;

[0025] FIG6 is a diagram showing the therapeutic results of Syn-CAR-NK cells in Example 4 of the present invention on subcutaneous transplanted tumors in mice with dual-target positive colon cancer;

[0026] Figure 7 is a diagram showing the therapeutic results of Syn-CAR-NK cells in Example 4 of the present invention on subcutaneous transplanted tumors in mice with single-target positive pancreatic cancer. DETAILED DESCRIPTION

[0027] 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.

[0028] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0029] Definitions and General Terms

[0030] 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 the present invention, all other technical and scientific terms used in the present invention have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0031] 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.

[0032] 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.

[0033] As used herein, the terms "identity," "homology," or "similarity" are used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and 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. 10:106); the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 10:116; the similarity search method of Pearson et al. (1990) Proc. Natl. Acad. Sci. 10:117; the similarity search method of Pearson et al. (1990 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize 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, BLAST Altschul 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.

[0034] As used herein, the term "at least 80% identity" refers to at least 80%, and may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to the respective reference sequence.

[0035] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The expression vector may include a vector primarily used to insert DNA or RNA into a cell, a vector primarily used to replicate DNA or RNA, and a vector primarily used for expression of the transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0036] As used herein, the term "chimeric antigen receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a different polypeptide than the extracellular domain, and at least one intracellular domain. "Chimeric antigen receptor (CAR)" is also referred to as a "chimeric receptor," "T-body," or "chimeric immune receptor (CIR)." The "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to an antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to act as a domain that transmits signals to activate or inhibit biological processes within a cell.

[0037] As used herein, the term "recombinant immune cell" generally refers to a cell that has been modified or recombined using genetic engineering techniques or cell fusion techniques to modify or reorganize the genetic material of a host cell to obtain a cell with a unique trait of stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention and can be used for the expression and / or secretion of a target protein.

[0038] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and thoroughly combining the active chimeric polypeptide or recombinant immune cell with a liquid carrier, a finely divided solid carrier, or both.

[0039] As used herein, the term "pharmaceutically acceptable excipient" includes any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the particular intended dosage form. Except to the extent that any conventional excipient is incompatible with the chimeric polypeptide or recombinant immune cell of the present invention, such as by producing any adverse biological effect or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present invention.

[0040] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient via a suitable means. The recombinant immune cells or pharmaceutical compositions of the present invention can be administered via any common route, as long as they can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, and the like, but the present invention is not limited to these exemplified modes of administration. Preferably, the compositions of the present invention are administered via intravenous or subcutaneous injection.

[0041] As used herein, the term "treatment" refers to any agent used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers recombinant immune cells, pharmaceutical compositions or drugs to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing the chimeric polypeptide, recombinant immune cell or pharmaceutical composition described herein to an individual in need.

[0042] The term "immune cell" generally refers to a cell that can produce an immune response (e.g., an antigen-specific immune response). For example, the immune cell may or may have contained a nucleic acid and / or expression vector comprising the present invention's separation, or an individual cell, cell line, or cell culture capable of expressing a chimeric polypeptide and optionally a chimeric antigen receptor as described herein. In the present invention, the immune cell may include a T cell, a B cell, a natural killer cell (NK cell), a macrophage, a NKT cell, a monocyte, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte, and / or a peripheral blood mononuclear cell. In this article, "carbon end" and "C end" are synonymous; "nitrogen end" and "N end" are synonymous.

[0043] Detailed description of the invention's transgenic recombinant immune cells specifically targeting tumors and their preparation method and application

[0044] The present invention provides a chimeric polypeptide, a first nucleic acid molecule, a first expression vector, a second nucleic acid molecule, a second expression vector, a recombinant immune cell, a pharmaceutical composition and uses thereof, which are described in detail below.

[0045] Chimeric polypeptide, first nucleic acid molecule, first expression vector

[0046] In the first aspect of the present invention, the present invention proposes a chimeric polypeptide. According to an embodiment of the present invention, the chimeric polypeptide includes: a first extracellular region, the first extracellular region has the activity of binding to the HLA-G protein; a first transmembrane region, the first transmembrane region includes the transmembrane region of the Notch receptor protein of tropical clawed frog or an amino acid sequence having at least 80% identity therewith, and the N-terminus of the first transmembrane region is connected to the C-terminus of the first extracellular region; a first intracellular region, the N-terminus of the first intracellular region is connected to the C-terminus of the first transmembrane region. The chimeric polypeptide of the present invention selects the transmembrane region of the Notch receptor protein of tropical clawed frog as the transmembrane region of the chimeric polypeptide (i.e., the SynNotch synthetic receptor), which can improve the ability and efficiency of the chimeric polypeptide to activate downstream gene transcription.

[0047] Human leukocyte antigen-G (HLA-G) is a group of tightly linked genes located on the short arm of human chromosome 6. It belongs to a non-classical major histocompatibility complex (MHC) class I molecule in humans and has the characteristics of selective tissue distribution. It is expressed in some tumor cell lines, tumor biopsy tissues, some infected tissue cells, and graft cells after heart transplantation.

[0048] Furthermore, the chimeric polypeptides can be used to prepare multi-targeted recombinant immune cells. These recombinant immune cells enable cells expressing the chimeric polypeptide to recognize HLA-G proteins. After binding to HLA-G proteins, they can achieve a variety of different signal output types, such as activation of expression of specific genes, and in particular, can activate the expression of chimeric antigen receptors containing factors with therapeutic effects (e.g., tumor treatment, etc.), thereby being used to treat diseases such as tumors.

[0049] In particular, recombinant immune cells can be prepared for tumor treatment, which can broadly identify numerous tumor cells, greatly improve the efficiency and accuracy of killing various types of tumor cells, overcome the problems of non-specific tumor recognition and incomplete tumor recognition coverage in existing cell therapy clinical applications, and thus provide a new means of tumor treatment with broad application prospects.

[0050] According to an embodiment of the present invention, the chimeric polypeptide may further include at least one of the following technical features:

[0051] According to an embodiment of the present invention, the transmembrane region of the Notch receptor protein of Xenopus tropicalis has the amino acid sequence shown in SEQ ID NO: 1.

[0052] According to an embodiment of the present invention, the transmembrane region further comprises an epidermal growth factor-like repeat sequence (EGF repeat) and / or a RAM sequence, thereby further reducing the background leakage activation of the SynNotch synthetic receptor.

[0053] According to an embodiment of the present invention, the C-terminus of the epidermal growth factor-like repeat sequence is connected to the N-terminus of the transmembrane region of the Notch receptor protein of the tropical clawed frog; and / or, the C-terminus of the transmembrane region of the Notch receptor protein of the tropical clawed frog is connected to the N-terminus of the RAM sequence.

[0054] According to an embodiment of the present invention, the epidermal growth factor-like repeat sequence has an amino acid sequence as shown in SEQ ID NO: 23.

[0055] According to an embodiment of the present invention, the RAM sequence is the amino acid sequence shown in SEQ ID NO: 24.

[0056] According to an embodiment of the present invention, the first transmembrane region has an amino acid sequence as shown in SEQ ID NO: 1 or 25 or an amino acid sequence having at least 90% identity thereto.

[0057] According to an embodiment of the present invention, the first transmembrane region has an amino acid sequence as shown in SEQ ID NO: 1 or 25.

[0058] According to an embodiment of the present invention, the first extracellular region includes a first binding protein or a fragment thereof that binds to the HLA-G protein.

[0059] In an optional embodiment of the present invention, the first extracellular region may include one or more first binding proteins or fragments thereof.

[0060] According to an embodiment of the present invention, the first binding protein or fragment thereof includes at least one of an antibody or a functional fragment thereof, and a receptor.

[0061] According to an embodiment of the present invention, the first binding protein or fragment thereof includes a first binding fragment and / or a second binding fragment; the first binding fragment is the extracellular region of the ILT2 protein or an active fragment thereof; the second binding fragment is the extracellular region of the ILT4 protein or an active fragment thereof.

[0062] It should be noted that the extracellular domains (ECDs) of ILT2 and ILT4 proteins can be divided into four domains, named Domain 1 (D1), Domain 2 (D2), Domain 3 (D3), and Domain 4 (D4).

[0063] In an optional embodiment of the present invention, when the first extracellular region includes multiple first binding proteins or fragments thereof, each first binding protein or fragment thereof is independently selected from the first binding fragment or the second binding fragment.

[0064] According to an embodiment of the present invention, the extracellular region of the ILT2 protein is composed of an ILT2-D1 fragment, an ILT2-D2 fragment, an ILT2-D3 fragment and an ILT2-D4 fragment; the ILT2-D1 fragment has the amino acid sequence shown in SEQ ID NO: 2; the ILT2-D2 fragment has the amino acid sequence shown in SEQ ID NO: 3; the ILT2-D3 fragment has the amino acid sequence shown in SEQ ID NO: 4; the ILT2-D4 fragment has the amino acid sequence shown in SEQ ID NO: 5.

[0065] In an exemplary embodiment of the present invention, when the first extracellular region includes multiple first binding proteins or fragments thereof, the multiple first binding proteins or fragments thereof may all be ILT2-D1 fragments, or may be ILT2-D1 fragments and ILT2-D2 fragments, respectively.

[0066] According to an embodiment of the present invention, the extracellular region of the ILT2 protein is sequentially divided from the N-terminus to the C-terminus into the ILT2-D1 fragment, the ILT2-D2 fragment, the ILT2-D3 fragment and the ILT2-D4 fragment.

[0067] According to an embodiment of the present invention, the extracellular region of the ILT2 protein has the amino acid sequence shown in SEQ ID NO:6.

[0068] According to an embodiment of the present invention, the first binding fragment is selected from: at least one of the ILT2-D1 fragment, the ILT2-D2 fragment, the ILT2-D3 fragment and the ILT2-D4 fragment; or the extracellular region of the ILT2 protein.

[0069] According to an embodiment of the present invention, the first binding fragment is selected from the ILT2-D1 fragment and the ILT2-D2 fragment, and the ILT2-D1 fragment and the ILT2-D2 fragment are connected.

[0070] According to an embodiment of the present invention, the C-terminus of the ILT2-D1 fragment is connected to the N-terminus of the ILT2-D2 fragment, or the N-terminus of the ILT2-D1 fragment is connected to the C-terminus of the ILT2-D2 fragment.

[0071] According to an embodiment of the present invention, the first binding fragment is selected from the extracellular region of the ILT2 protein.

[0072] According to an embodiment of the present invention, the extracellular region of the ILT4 protein is composed of an ILT4-D1 fragment, an ILT4-D2 fragment, an ILT4-D3 fragment and an ILT4-D4 fragment; the ILT4-D1 fragment has the amino acid sequence shown in SEQ ID NO: 7; the ILT4-D2 fragment has the amino acid sequence shown in SEQ ID NO: 8; the ILT4-D3 fragment has the amino acid sequence shown in SEQ ID NO: 9; the ILT4-D4 fragment has the amino acid sequence shown in SEQ ID NO: 10.

[0073] In an exemplary embodiment of the present invention, when the first extracellular region includes multiple first binding proteins or fragments thereof, the multiple first binding proteins or fragments thereof may all be ILT4-D1 fragments, or may be ILT4-D1 fragments and ILT4-D2 fragments, respectively.

[0074] According to an embodiment of the present invention, the extracellular region of the ILT4 protein has an amino acid sequence as shown in SEQ ID NO:11.

[0075] In an exemplary embodiment of the present invention, when the first extracellular region includes multiple first binding proteins or fragments thereof, the multiple first binding proteins or fragments thereof may all be first binding fragments, may all be second binding fragments, or may be first binding fragments and second binding fragments respectively. The specific types are not limited and are all within the scope of protection of the present invention.

[0076] According to an embodiment of the present invention, the second binding fragment is selected from: at least one of the ILT4-D1 fragment, the ILT4-D2 fragment, the ILT4-D3 fragment and the ILT4-D4 fragment; or the extracellular region of the ILT4 protein.

[0077] According to an embodiment of the present invention, the second binding fragment is selected from the ILT4-D1 fragment and the ILT4-D2 fragment, and the ILT4-D1 fragment and the ILT4-D2 fragment are connected.

[0078] According to an embodiment of the present invention, the C-terminus of the ILT4-D1 fragment is connected to the N-terminus of the ILT4-D2 fragment, or the N-terminus of the ILT4-D1 fragment is connected to the C-terminus of the ILT4-D2 fragment.

[0079] According to an embodiment of the present invention, the second binding fragment is selected from the extracellular region of the ILT4 protein.

[0080] According to an embodiment of the present invention, when the first extracellular region comprises a plurality of the first binding proteins or fragments thereof, the first extracellular region further comprises a first connecting peptide, and any two of the first binding proteins or fragments thereof are connected via or without the first connecting peptide.

[0081] In this article, the term "any two of the first binding proteins or fragments thereof are connected through or not through the first connecting peptide" means that any two of the first binding proteins or fragments thereof may be connected through the first connecting peptide, or any two of the first binding proteins or fragments thereof may not be connected through the first connecting peptide. It may also be that when there are three or more first binding proteins or fragments thereof, two of the first binding proteins or fragments thereof are connected through the first connecting peptide, and the other two are not connected through the first connecting peptide. The specific connection type is not limited and falls within the scope of protection of the present invention.

[0082] In an optional embodiment of the present invention, when the first extracellular region comprises two of the first binding proteins or fragments thereof, the first extracellular region is sequentially the first binding protein or fragment thereof, and the first binding protein or fragment thereof from the N-terminus to the C-terminus.

[0083] In an optional embodiment of the present invention, when the first extracellular region comprises two of the first binding proteins or fragments thereof, the first extracellular region is sequentially composed of the first binding protein or fragment thereof, the first connecting peptide, and the first binding protein or fragment thereof from the N-terminus to the C-terminus.

[0084] In an optional embodiment of the present invention, when the first extracellular region contains three of the first binding proteins or fragments thereof, the first extracellular region is, from N-terminus to C-terminus, the first binding protein or fragment thereof, the first binding protein or fragment thereof, and the first binding protein or fragment thereof.

[0085] In an optional embodiment of the present invention, when the first extracellular region contains three of the first binding proteins or fragments thereof, the first extracellular region is, from N-terminus to C-terminus, the first binding protein or fragment thereof, the first binding protein or fragment thereof, the first connecting peptide, and the first binding protein or fragment thereof.

[0086] In an optional embodiment of the present invention, when the first extracellular region contains three of the first binding proteins or fragments thereof, the first extracellular region, from N-terminus to C-terminus, is sequentially the first binding protein or fragment thereof, the first connecting peptide, the first binding protein or fragment thereof, and the first binding protein or fragment thereof.

[0087] In an optional embodiment of the present invention, when the first extracellular region contains three of the first binding proteins or fragments thereof, the first extracellular region, from N-terminus to C-terminus, is sequentially the first binding protein or fragment thereof, the first connecting peptide, the first binding protein or fragment thereof first connecting peptide, and the first binding protein or fragment thereof.

[0088] According to an embodiment of the present invention, the first binding protein or a fragment thereof includes a first binding fragment and a second binding fragment, and the first binding fragment and the second binding fragment are connected.

[0089] It should be noted that the term "connected" herein may refer to direct or indirect connection, and the specific type is not limited and falls within the scope of protection of the present invention. For example, "the first binding fragment and the second binding fragment are connected" means that the first binding fragment and the second binding fragment can be directly connected or indirectly connected (for example, connected via a first connecting peptide).

[0090] According to an embodiment of the present invention, the first binding protein or a fragment thereof includes a first binding fragment and a second binding fragment, and the first binding fragment and the second binding fragment are connected via the first connecting peptide.

[0091] According to an embodiment of the present invention, the C-terminus of the first binding fragment is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the second binding fragment, or the C-terminus of the second binding fragment is connected to the N-terminus of the first connecting peptide, and the C-terminus of the first connecting peptide is connected to the N-terminus of the first binding fragment.

[0092] According to an embodiment of the present invention, the amino acid sequence of the first connecting peptide is (GGGGS)n, wherein n is any integer between 1 and 10.

[0093] According to an embodiment of the present invention, n is 1, 2, 3 or 4.

[0094] According to an embodiment of the present invention, the amino acid sequence of the first connecting peptide is GGGGS (SEQ ID NO: 22).

[0095] According to an embodiment of the present invention, the first extracellular region is selected from one of the following: ILT2-D1 fragment + ILT2-D2 fragment + first connecting peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first connecting peptide + ILT2-D1 fragment + ILT2-D2 fragment; the extracellular region of ILT2 protein + first connecting peptide + the extracellular region of ILT4 protein; the extracellular region of ILT4 protein + first connecting peptide + the extracellular region of ILT2 protein; ILT2-D1 fragment + ILT2-D2 fragment + first connecting peptide + the extracellular region of ILT4 protein; ILT4-D1 fragment + ILT4-D2 fragment + first connecting peptide + the extracellular region of ILT2 protein; LT2-D1 fragment + first connecting peptide + ILT4-D1 fragment; ILT2-D1 fragment + first connecting peptide + ILT4-D2 fragment; ILT2-D1 fragment + first connecting peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT2-D1 fragment + first connecting peptide + extracellular region of ILT4 protein; ILT2-D1 fragment + first connecting peptide + ILT2-D1 fragment; ILT2-D1 fragment + first connecting peptide + ILT2-D2 fragment; ILT2-D1 fragment + first connecting peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT2-D1 fragment + first connecting peptide + extracellular region of ILT2 protein; ILT4-D1 fragment + first connecting peptide + ILT T4-D1 fragment; ILT4-D1 fragment + first connecting peptide + ILT4-D2 fragment; ILT4-D1 fragment + first connecting peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT4-D1 fragment + first connecting peptide + extracellular region of ILT4 protein; ILT4-D1 fragment + first connecting peptide + ILT2-D1 fragment; ILT4-D1 fragment + first connecting peptide + ILT2-D2 fragment; ILT4-D1 fragment + first connecting peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT4-D1 fragment + first connecting peptide + extracellular region of ILT2 protein; ILT2-D2 fragment + first connecting peptide + ILT4-D1 fragment; ILT2-D2 fragment + first connecting peptide + ILT4-D2 fragment; ILT2-D2 fragment + first connecting peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT2-D2 fragment + first connecting peptide + extracellular region of ILT4 protein; ILT2-D2 fragment + first connecting peptide + ILT2-D1 fragment; ILT2-D2 fragment + first connecting peptide + ILT2-D2 fragment; ILT2-D2 fragment + first connecting peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT2-D2 fragment + first connecting peptide + extracellular region of ILT2 protein; ILT4-D2 fragment + first connecting peptide + ILT4-D1 fragment; ILT4-D2 fragment + first connecting peptide + ILT4-D2 fragment;ILT4-D2 fragment + first connecting peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT4-D2 fragment + first connecting peptide + extracellular region of ILT4 protein; ILT4-D2 fragment + first connecting peptide + ILT2-D1 fragment; ILT4-D2 fragment + first connecting peptide + ILT2-D2 fragment; ILT4-D2 fragment + first connecting peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT4-D2 fragment + first connecting peptide + extracellular region of ILT2 protein; ILT2-D1 fragment + ILT2-D2 fragment + first connecting peptide + ILT4-D1 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first connecting peptide + ILT2-D2 fragment ILT4-D2 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first connecting peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first connecting peptide + extracellular region of ILT4 protein; ILT2-D1 fragment + ILT2-D2 fragment + first connecting peptide + ILT2-D1 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first connecting peptide + ILT2-D2 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first connecting peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first connecting peptide + extracellular region of ILT2 protein Extracellular region; ILT4-D1 fragment + ILT4-D2 fragment + first connecting peptide + ILT4-D1 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first connecting peptide + ILT4-D2 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first connecting peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first connecting peptide + extracellular region of ILT4 protein; ILT4-D1 fragment + ILT4-D2 fragment + first connecting peptide + ILT2-D1 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first connecting peptide + ILT2-D2 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first connecting peptide + ILT2-D2 fragment 4-D2 fragment + the first connecting peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT4-D1 fragment + ILT4-D2 fragment + the first connecting peptide + the extracellular region of ILT2 protein; the extracellular region of ILT2 protein + the first connecting peptide + ILT4-D1 fragment; the extracellular region of ILT2 protein + the first connecting peptide + ILT4-D2 fragment; the extracellular region of ILT2 protein + the first connecting peptide + ILT4-D1 fragment + ILT4-D2 fragment; the extracellular region of ILT2 protein + the first connecting peptide + the extracellular region of ILT4 protein; the extracellular region of ILT2 protein + the first connecting peptide + ILT2-D1 fragment; the extracellular region of ILT2 protein + the first connecting peptide + ILT2-D2 fragment;The extracellular region of ILT2 protein + the first connecting peptide + the ILT2-D1 fragment + the ILT2-D2 fragment; the extracellular region of ILT2 protein + the first connecting peptide + the extracellular region of ILT2 protein; the extracellular region of ILT4 protein + the first connecting peptide + the ILT4-D1 fragment; the extracellular region of ILT4 protein + the first connecting peptide + the ILT4-D2 fragment; the extracellular region of ILT4 protein + the first connecting peptide + the ILT4-D1 fragment + the ILT4-D2 fragment; the extracellular region of ILT4 protein + the first connecting peptide + the extracellular region of ILT4 protein; the extracellular region of ILT4 protein + the first connecting peptide + the ILT2-D1 fragment; the extracellular region of ILT4 protein + the first connecting peptide + the ILT2-D2 fragment; the extracellular region of ILT4 protein + the first connecting peptide + the ILT2-D1 fragment + the ILT2-D2 fragment; the extracellular region of ILT4 protein + the first connecting peptide + the extracellular region of ILT2 protein.

[0096] It should be noted that the above-mentioned first extracellular region refers to the connection mode from N-terminus to C-terminus. Exemplarily, "the first extracellular region is selected from the group consisting of ILT2-D1 fragment + ILT2-D2 fragment + first connecting peptide + ILT4-D1 fragment + ILT4-D2 fragment" means that the first extracellular region includes, from N-terminus to C-terminus, the ILT2-D1 fragment, the ILT2-D2 fragment, the first connecting peptide, the ILT4-D1 fragment, and the ILT4-D2 fragment.

[0097] According to an embodiment of the present invention, the first extracellular region is selected from one of the following: 1) the extracellular region of ILT2 protein; 2) the extracellular region of ILT4 protein; 3) from N-terminus to C-terminus, the ILT2-D1 fragment and the ILT2-D2 fragment; 4) from N-terminus to C-terminus, the ILT4-D1 fragment and the ILT4-D2 fragment; 5) from N-terminus to C-terminus, the ILT2-D1 fragment, the ILT2-D2 fragment, the first connecting peptide, the ILT4-D1 fragment and the ILT4-D2 fragment; 6) from N-terminus to C-terminus, the ILT4-D1 fragment, the ILT4-D2 fragment, the first connecting peptide, the ILT2-D1 fragment and the ILT2-D2 fragment.

[0098] According to an embodiment of the present invention, the first intracellular region includes at least one of a transcriptional activator protein, a transcriptional repressor protein, a transcription factor, a site-specific nuclease, a recombinase, an activating immune receptor intracellular domain and an inhibitory immune receptor intracellular domain.

[0099] According to an embodiment of the present invention, the first intracellular region includes at least one of GaL4-VP64, GaL4-VP16, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, HAP1-VP16 and LexA-VP64.

[0100] According to an embodiment of the present invention, the Gal4-VP64 has an amino acid sequence as shown in SEQ ID NO:12.

[0101] In a second aspect of the present invention, the present invention provides a first nucleic acid molecule. According to an embodiment of the present invention, the first nucleic acid molecule encodes the chimeric polypeptide described in the first aspect. The first nucleic acid molecule according to an embodiment of the present invention can encode the chimeric polypeptide described above.

[0102] According to an embodiment of the present invention, the first nucleic acid molecule is DNA.

[0103] It should be noted that, for the first nucleic acid molecule mentioned herein, those skilled in the art will understand that it actually includes any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases herein, the other strand complementary thereto is also disclosed. In addition, the molecular sequence in the present invention includes DNA or RNA forms, and disclosure of one of them means that the other is also disclosed.

[0104] In a third aspect, the present invention provides a first expression vector. According to an embodiment of the present invention, the first expression vector carries the first nucleic acid molecule described in the second aspect. When linking the first nucleic acid molecule to the expression vector, the first nucleic acid molecule can be directly or indirectly linked to control elements on the expression vector, as long as these control elements are capable of controlling translation and expression of the first nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself or exogenous, that is, not derived from the vector itself. Of course, it is sufficient that the first nucleic acid molecule and the control elements are operably linked.

[0105] As used herein, "operably linked" refers to linking an exogenous gene to an expression vector so that the control elements within the expression vector, such as transcriptional and translational control sequences, can function as intended to regulate the transcription and translation of the exogenous gene. Commonly used expression vectors include plasmids and bacteriophages. Following introduction of the vectors according to certain embodiments of the present invention into suitable recipient cells (also known as receptor cells or host cells), expression of the chimeric polypeptide described above can be effectively achieved under the guidance of a regulatory system.

[0106] According to an embodiment of the present invention, the first expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0107] According to an embodiment of the present invention, the first expression vector is a plasmid expression vector.

[0108] Second nucleic acid molecule, second expression vector

[0109] In its fourth aspect, the present invention provides a second nucleic acid molecule. According to an embodiment of the present invention, the second nucleic acid molecule comprises a first nucleic acid fragment and a second nucleic acid fragment, wherein the 3' end of the first nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment; wherein the second nucleic acid fragment is used to encode an antigen chimeric receptor that targets the first molecule, and the first nucleic acid fragment is used to bind to a first intracellular region and induce expression of the antigen chimeric receptor; the first intracellular region is consistent with the first intracellular region defined in the chimeric polypeptide described in the first aspect. The second nucleic acid molecule of the present invention can bind to the first intracellular region of the aforementioned chimeric polypeptide, thereby activating and inducing expression of the antigen chimeric receptor.

[0110] Furthermore, the second nucleic acid molecule and the aforementioned first nucleic acid molecule can be used to prepare multi-targeted recombinant immune cells. These recombinant immune cells enable cells expressing the chimeric polypeptide to recognize HLA-G proteins. After binding to HLA-G proteins, they can achieve a variety of different signal output types, such as activation of specific gene expression, and in particular, activation of the expression of chimeric antigen receptors containing factors with therapeutic effects (e.g., tumor treatment), for the treatment of diseases such as tumors. In particular, recombinant immune cells for tumor treatment can be prepared, which can broadly recognize a wide range of tumor cells, greatly improving the efficiency and accuracy of killing various types of tumor cells, overcoming the problems of non-specific tumor recognition and incomplete tumor recognition coverage in existing cell therapy clinical applications, thereby providing a new tumor treatment method with broad application prospects.

[0111] It should be noted that the term "inducing expression of antigen chimeric receptor" means that the second nucleic acid fragment can encode an antigen chimeric receptor.

[0112] According to an embodiment of the present invention, the antigen chimeric receptor includes: a second extracellular region, the second extracellular region has a first molecule binding activity, and the first molecule is not an HLA-G protein; a second transmembrane region, the N-terminus of the second transmembrane region is connected to the C-terminus of the second extracellular region; and a second intracellular region, the N-terminus of the second intracellular region is connected to the C-terminus of the second transmembrane region.

[0113] According to an embodiment of the present invention, the first molecule includes at least one of a tumor antigen, a virus, a bacterium, an endotoxin, an antibody, a cell receptor, and a ligand of a cell receptor.

[0114] In this article, the term "tumor antigen" generally refers to antigenic substances that appear or are overexpressed during the occurrence and development of tumors. Tumor antigens are classified into tumor-specific antigens and tumor-associated antigens based on their specificity. Among them, tumor-specific antigens (TSAs) are neoantigens that are unique to tumor cells or only exist in certain tumor cells but not in normal cells. Tumor-associated antigens (TAAs) are antigens that are unique to non-tumor cells and also exist on normal cells and other tissues, but their content increases significantly when the cells become cancerous. These include but are not limited to PD-L1, PD-1, TGF-β, CEA, GD2, and GD3.

[0115] In this article, the term "cell receptor" or "receptor" should be understood in a broad sense and may refer to molecules located on the cell membrane that can recognize and bind to various extracellular signaling molecules (ligands), including but not limited to growth factor receptors (such as VEGF receptors), (NKG2D polypeptides (receptors for MICA, MICB and ULBP1-6), cytokine receptors (such as IL-13 receptor, IL-2 receptor, etc.), epidermal growth factor (EGF) receptor, Her2, CD27, natural cytotoxicity receptors (NCR) (such as NKp30 (NCR3 / CD337) polypeptide (receptor for HLA-B associated transcript 3 (BAT3) and B7-H6), etc.), T cell antigen receptors, dihydrofolate receptors, chimeric cytokine receptors, Fc receptors, extracellular matrix receptors (such as integrins), cell adhesion receptors (such as cadherins), immunomodulatory receptors (including positive co-receptors (such as CD28) and negative (immunosuppressive) co-receptors (such as PD1)) and receptors for immunomodulatory molecules (such as TGFβ), etc.

[0116] In this article, the term "ligand of a cell receptor" should be understood in a broad sense and may refer to chemical substances that can bind to and interact with cell membrane receptors and produce specific biological effects, such as polypeptides, nucleic acids, glycoproteins, small molecules, carbohydrates, lipids, glycolipids, lipoproteins, lipopolysaccharides, etc., including but not limited to cytokines (e.g., IL-13, etc.), growth factors (e.g., heregulin, vascular endothelial growth factor (VEGF)), peptide hormones, integrin-binding peptides (e.g., peptides including the sequence Arg-Gly-Asp), N-glycans, etc.

[0117] Illustratively, the ligand is VEGF and the receptor is VEGF receptor; or the ligand is heregulin and the receptor is HER2.

[0118] In this article, the term "cytokine" should be understood broadly to refer to a class of proteins or small peptides that can transmit information between cells and have immunomodulatory and effector functions, such as IL-10. The term "cytokine receptor" should be understood broadly to refer to receptors on the cell surface that can bind to cytokines, such as IL-10R.

[0119] According to an embodiment of the present invention, the tumor antigen includes at least one of a tumor-associated antigen and a tumor-specific antigen.

[0120] According to an embodiment of the present invention, the tumor antigen is a tumor-specific antigen.

[0121] According to an embodiment of the present invention, the first molecule includes MICA, MICB, ULBPs, B7H6, B7H3, GFP, eGFP, CD19, ALPPL2, BCMA, SIRPα, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44, CD44v6, CD45, CD48, CD51, CD52, and CD56. , CD59, CD66, CD70, CD71, CD72, CD73, CD74, CD79A, CD79B, CD80, CD86, CD94, CD95, CD123, CD133, CD134, CD140, CD152, CD154, CD158, CD178, CD1 81. CD182, CD183, CD200, CD210, CD221, CD246, CD252, CD253, CD261, CD262, CD269, CD273, CD274, CD276, CD279, CD295, CD339, CD340, EGFR, EGFR VIII, HER2, FGFR2, AFP, CA125, MSLN, GPC3, CEA, CLDN1, CLDN3, CLDN6, CLDN18.1, CLDN18.2, EpCAM, PSCA, GD2, GD3, IL-13, IL-13RA2, ROR1, MUC-1, PSMA, MAGEA1, 4-1BB, 5T4, BAFF, CA242, CA-IX, MET, CCR4, CNTO888, FAP, MORAb-009, EPHA2, VEGF-A, VEGFR-1, and VEGFR-2.

[0122] According to an embodiment of the present invention, the second extracellular region includes a second binding protein or a fragment thereof that binds to the first molecule.

[0123] According to an embodiment of the present invention, the second binding protein or fragment thereof includes at least one of an antibody or a functional fragment thereof, a receptor, a ligand of a receptor, and a cell adhesion molecule.

[0124] According to an embodiment of the present invention, the second binding protein or a fragment thereof is the extracellular region of an activating receptor expressed on the surface of an immune cell or an antibody or a fragment thereof that binds to the first molecule.

[0125] According to an embodiment of the present invention, the antibody or fragment thereof that binds to the first molecule is a single-chain antibody.

[0126] According to an embodiment of the present invention, the activating receptor is selected from receptors on the surface of NK cells.

[0127] According to an embodiment of the present invention, the activating receptor is selected from at least one of NKG2D, NKp30, NKp44, NKp46, DNAM-1, PD-1, TIGIT, NKG2A, NKG2B, NKG2C, NKG2E, NKG2H, CD16, NKp80, CD226, CD160, CD161, CD96, PVRIG, SLAM, CD200R, CD49a, TIM-3, LAG-3, CD112R, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, LIR1, LIR2, SIGLE3, SIGLE7, SIGLE9, and KLRG1.

[0128] According to an embodiment of the present invention, the activating receptor is selected from NKG2D and / or NKp30.

[0129] According to an embodiment of the present invention, the second binding protein or its fragment includes a third binding fragment and / or a fourth binding fragment; the third binding fragment is the extracellular region of NKG2D or its active fragment; the fourth binding fragment is the extracellular region of NKp30 or its active fragment.

[0130] According to an embodiment of the present invention, the extracellular region of NKG2D has an amino acid sequence as shown in SEQ ID NO:13.

[0131] According to an embodiment of the present invention, the extracellular region of NKp30 has an amino acid sequence as shown in SEQ ID NO:14.

[0132] According to an embodiment of the present invention, the third binding fragment has an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 90% identity thereto.

[0133] According to an embodiment of the present invention, the fourth binding fragment has an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% identity thereto.

[0134] According to an embodiment of the present invention, the second extracellular region further includes a second connecting peptide, and the third binding fragment and the fourth binding fragment are connected via the second connecting peptide.

[0135] According to an embodiment of the present invention, the C-terminus of the third binding fragment is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the fourth binding fragment, or the C-terminus of the fourth binding fragment is connected to the N-terminus of the second connecting peptide, and the C-terminus of the second connecting peptide is connected to the N-terminus of the third binding fragment.

[0136] According to an embodiment of the present invention, the amino acid sequence of the second connecting peptide is (GGGGS)n, wherein n is any integer between 0 and 10.

[0137] According to an embodiment of the present invention, n is 0, 1, 2, 3 or 4.

[0138] According to an embodiment of the present invention, the amino acid sequence of the second connecting peptide is GGGGS.

[0139] According to an embodiment of the present invention, the second extracellular region further includes a hinge region.

[0140] According to an embodiment of the present invention, the C-terminus of the third binding fragment is connected to the N-terminus of the second connecting peptide, the C-terminus of the second connecting peptide is connected to the N-terminus of the fourth binding fragment, and the C-terminus of the fourth binding fragment is connected to the N-terminus of the hinge region, or the C-terminus of the fourth binding fragment is connected to the N-terminus of the second connecting peptide, the C-terminus of the second connecting peptide is connected to the N-terminus of the third binding fragment, and the C-terminus of the third binding fragment is connected to the N-terminus of the hinge region.

[0141] According to an embodiment of the present invention, the hinge region includes at least one of the hinge region of a CD8α molecule or a variant thereof, and the hinge region of an immunoglobulin or a variant thereof.

[0142] As used herein, the term "immunoglobulin" refers to a globulin with antibody (Ab) activity or chemical structure, similar to an antibody molecule. It is a tetrapeptide chain structure consisting of two identical light chains and two identical heavy chains connected by interchain disulfide bonds. It includes immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), and immunoglobulin E (IgE).

[0143] According to an embodiment of the present invention, the hinge region has an amino acid sequence as shown in SEQ ID NO:15.

[0144] According to an embodiment of the present invention, the second transmembrane region is selected from at least one of the transmembrane region of the CD8α molecule, the transmembrane region of the CD28 molecule, the transmembrane region of the CD3ζ molecule, the transmembrane region of the CD4 molecule, the transmembrane region of the CD16 molecule, the transmembrane region of the 4-1BB molecule, the transmembrane region of the OX40 molecule, the transmembrane region of the ICOS molecule, the transmembrane region of the CTLA-4 molecule, the transmembrane region of the PD-1 molecule, the transmembrane region of the LAG-3 molecule, the transmembrane region of the 2B4 molecule, the transmembrane region of the NKG2D molecule, the transmembrane region of the DNAM-1 molecule, the transmembrane region of the NKp44 molecule, the transmembrane region of the NKp46 molecule, the transmembrane region of the KIR2DS1 molecule, the transmembrane region of the KIR2DS2 molecule, the transmembrane region of the KIR2DS4 molecule, and the transmembrane region of the BTLA molecule.

[0145] According to an embodiment of the present invention, the second transmembrane region is selected from the transmembrane region of the CD8α molecule.

[0146] According to an embodiment of the present invention, the second transmembrane region has an amino acid sequence as shown in SEQ ID NO:16.

[0147] According to an embodiment of the present invention, the second intracellular region includes an intracellular signaling domain and a costimulatory domain.

[0148] According to an embodiment of the present invention, the intracellular signaling domain is selected from at least one of the intracellular signaling domain of the CD3ζ molecule and the intracellular signaling structure of the FcεRIγ molecule.

[0149] According to an embodiment of the present invention, the costimulatory domain is selected from at least one of the intracellular signaling domain of the 4-1BB molecule, the intracellular signaling domain of the CD28 molecule, the intracellular signaling domain of the CD27 molecule, the intracellular signaling domain of the CD40 molecule, the intracellular signaling domain of the OX40 molecule, the intracellular signaling domain of the ICOS molecule, the intracellular signaling domain of the DAP10 molecule, the intracellular signaling domain of the DAP12 molecule, and the intracellular signaling domain of the DNAM-1 molecule.

[0150] According to an embodiment of the present invention, the second intracellular region has an amino acid sequence as shown in SEQ ID NO:17.

[0151] According to an embodiment of the present invention, the second nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:18.

[0152] According to an embodiment of the present invention, the second nucleic acid molecule further includes a third nucleic acid fragment, which is used to encode a signal peptide; the 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, and the 3' end of the third nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment.

[0153] According to an embodiment of the present invention, the signal peptide is selected from at least one of the signal peptide of CD8α molecule, the signal peptide of IgG molecule, and the signal peptide of CD28 molecule.

[0154] According to an embodiment of the present invention, the signal peptide has an amino acid sequence as shown in SEQ ID NO:19.

[0155] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO: 20.

[0156] In an optional embodiment of the present invention, the intracellular region is GaL4-VP64, and the first nucleic acid fragment is a UAS-minimal-CMV sequence. The UAS-minimal-CMV sequence has the nucleic acid sequence shown in SEQ ID NO: 21.

[0157] According to an embodiment of the present invention, the first nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO: 21.

[0158] In a fifth aspect, the present invention provides a second expression vector. According to an embodiment of the present invention, the second expression vector carries the second nucleic acid molecule described in the fourth aspect. The second expression vector according to an embodiment of the present invention can express the antigen chimeric receptor in the second nucleic acid molecule.

[0159] According to an embodiment of the present invention, the second expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0160] According to an embodiment of the present invention, the second expression vector is a plasmid expression vector.

[0161] Recombinant immune cells

[0162] In the sixth aspect of the present invention, the present invention proposes a recombinant immune cell. According to an embodiment of the present invention, the recombinant immune cell comprises: carrying the first nucleic acid molecule described in the second aspect or the first expression vector described in the third aspect; or expressing the chimeric polypeptide described in the first aspect. The recombinant immune cell of the embodiment of the present invention can express the aforementioned chimeric polypeptide on the surface of the recombinant immune cell under suitable conditions, and can recognize the HLA-G protein. After binding to the HLA-G protein, it can achieve a variety of different signal output types, such as activation of expression of specific genes, etc., especially activating the expression of chimeric antigen receptors containing factors with therapeutic effects (such as treating tumors, etc.), for treating diseases such as tumors.

[0163] It should be noted that the "suitable conditions" referred to herein refer to conditions suitable for the expression of the aforementioned chimeric polypeptide. Those skilled in the art will readily appreciate that suitable conditions for the expression of the aforementioned chimeric polypeptide include, but are not limited to, a suitable transformation or transfection method, suitable transformation or transfection conditions, healthy cells, suitable cell density, a suitable cell culture environment, and a suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for chimeric polypeptide expression based on the specific laboratory environment.

[0164] According to an embodiment of the present invention, the expression of the chimeric polypeptide in the recombinant immune cell is obtained by introducing the first expression vector described in the third aspect into a host cell.

[0165] According to an embodiment of the present invention, the recombinant immune cell further includes a chimeric antigen receptor, which is consistent with the antigen chimeric receptor encoded by the second nucleic acid fragment defined in the second nucleic acid molecule described in the fourth aspect. The recombinant immune cell of the embodiment of the present invention can simultaneously express the chimeric polypeptide and the chimeric antigen receptor in the second nucleic acid molecule on the surface of the recombinant immune cell under suitable conditions. Thus, the recombinant immune cell has multi-targeting and can recognize HLA-G protein, which can achieve a variety of different signal output types after binding to HLA-G protein, such as expression activation of specific genes, etc., especially activating the expression of chimeric antigen receptors containing factors with therapeutic effects (such as treating tumors, etc.), for treating diseases such as tumors. In particular, recombinant immune cells for treating tumors can be prepared, which can broadly identify numerous tumor cells, etc., greatly improving the killing efficiency and accuracy of various types of tumor cells, overcoming the problems of non-specific tumor recognition and incomplete tumor recognition coverage in existing cell therapy clinical applications, thereby providing a new means of tumor treatment with broad application prospects.

[0166] In particular, the chimeric polypeptide of the present invention is combined with a chimeric antigen receptor to jointly transform and prepare recombinant immune cells (such as Jurkat cells and NK cells). It is found that the immune cells combined with the chimeric polypeptide + CAR / TCR can recognize the corresponding ligands and efficiently activate immune cells (such as Jurkat cells and NK cells) to exercise the function of killing targets (such as tumor cells, etc.).

[0167] According to an embodiment of the present invention, the expression of the chimeric antigen receptor in the recombinant immune cell is obtained by introducing the second expression vector described in the fifth aspect into the host cell.

[0168] According to an embodiment of the present invention, the host cell includes at least one of an immune cell, a neuron, a progenitor cell or a precursor cell, an epithelial cell, an endothelial cell and a stem cell.

[0169] According to an embodiment of the present invention, the host cell includes 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.

[0170] Pharmaceutical composition

[0171] In a seventh aspect, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes the recombinant immune cells described in the sixth aspect. As mentioned above, cells expressing the chimeric polypeptide can recognize HLA-G protein, and after binding to HLA-G protein, they can achieve a variety of different signal output types, such as activation and inhibition of specific gene expression, or immune cells expressing the chimeric polypeptide can secrete factors with therapeutic effects for anti-tumor after contact with HLA-G protein. Therefore, a pharmaceutical composition containing the aforementioned recombinant immune cells can target HLA-G protein for the prevention and / or treatment of related diseases, such as cancer.

[0172] According to an embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable excipient.

[0173] use

[0174] In the eighth aspect of the present invention, the present invention proposes the use of the recombinant immune cell described in the sixth aspect or the pharmaceutical composition described in the seventh aspect in the preparation of a drug for preventing and / or treating a disease.

[0175] According to an embodiment of the present invention, the disease includes cancer or tumor, autoimmune disease, inflammation and related diseases caused by cell aging.

[0176] As used herein, the term "cancer" or "tumor" may be any unregulated cell growth. Exemplary cancers include small cell lung cancer, non-small cell lung cancer, papillary thyroid cancer, glioblastoma multiforme, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer, sarcoma, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasms, large cell neuroendocrine carcinoma, neuroblastoma, prostate cancer, fibroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, glioma, esophageal cancer, nasopharyngeal cancer, oral squamous cell carcinoma, or gastric cancer, among others.

[0177] Methods of treating or preventing disease

[0178] In the ninth aspect of the present invention, a method for treating or preventing a disease is provided. According to an embodiment of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the recombinant immune cells of the sixth aspect or the pharmaceutical composition of the seventh aspect. As previously mentioned, cells expressing the chimeric polypeptide can recognize HLA-G protein, and after binding to the HLA-G protein, they can achieve a variety of different signal output types, such as activation and inhibition of expression of specific genes, or immune cells expressing the chimeric polypeptide can secrete factors with therapeutic effects for anti-tumor purposes after contacting the HLA-G protein. Therefore, a pharmaceutical composition containing the aforementioned recombinant immune cells can target the HLA-G protein for the prevention and / or treatment of related diseases, such as cancer.

[0179] The effective amount of the recombinant protein or pharmaceutical composition of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to, pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, and the like. For example, depending on the urgency of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0180] The recombinant protein or pharmaceutical composition of the present invention can be incorporated into a drug suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. For example, liquid, semisolid and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical drugs are in the form of injection solutions or infusion solutions. The aforementioned recombinant protein or pharmaceutical composition can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

[0181] According to an embodiment of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0182] According to an embodiment of the present invention, the disease includes cancer or tumor, immune-related disease. According to an embodiment of the present invention, the disease includes cancer or tumor, autoimmune disease, inflammation and related diseases caused by cell senescence.

[0183] According to an embodiment of the present invention, the tumor or cancer includes but is not limited to small cell lung cancer, non-small cell lung cancer, papillary thyroid cancer, glioblastoma multiforme, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer, sarcoma, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome, myeloproliferative tumors, large cell neuroendocrine carcinoma, neuroblastoma, prostate cancer, fibroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, glioma, esophageal cancer, nasopharyngeal cancer, oral squamous cell carcinoma or gastric cancer, etc.

[0184] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0185] Example 1: Design and construction of a SynNotch receptor specifically targeting human HLA-G

[0186] ILT2 and ILT4 receptors are two receptors on the surface of immune cells that are responsible for binding to HLA-G molecules. The ILT4 receptor is only expressed on myeloid-derived immune cells, while the ILT2 receptor is expressed on both lymphoid-derived and myeloid-derived immune cells. Both ILT2 and ILT4 receptors are divided into extracellular, transmembrane, and intracellular regions, of which the extracellular region is the functional region responsible for recognizing and binding to HLA-G. In addition, the extracellular regions of both ILT2 and ILT4 receptors are divided into four domains, named Domain 1 (D1), Domain 2 (D2), Domain 3 (D3), and Domain 4 (D4).

[0187] Based on the binding properties of ILT2 and ILT4 to HLA-G, the inventors designed and synthesized a series of synthetic peptides that can target and bind to human HLA-G. These synthetic peptides are composed of peptide sequences derived from ILT2 and ILT4 receptors. Since the peptide sequences derived from ILT2 and ILT4 both have the ability to bind to HLA-G, they can maximize the binding ability of our designed synthetic peptides to HLA-G. The synthetic polypeptide is used as the extracellular region of the SynNotch receptor (the amino acid sequence of ILT2-ECD in 1-Syn-CAR is shown in SEQ ID NO: 6; the amino acid sequence of ILT4-ECD in 2-Syn-CAR is shown in SEQ ID NO: 11; the amino acid sequence of ILT2-D1D2 in 3-Syn-CAR is shown in SEQ ID NO: 2 and 3; the amino acid sequence of ILT4-D1D2 in 4-Syn-CAR is shown in SEQ ID NO: 7 and 8; the amino acid sequence of ILT2-D1D2 in 5-Syn-CAR is shown in SEQ ID NO: 2 and 3, the amino acid sequence of Linker is GGGGS, and the amino acid sequence of ILT4-D1D2 is shown in SEQ ID NO: 7 and 8; the amino acid sequence of ILT4-D1D2 in 6-Syn-CAR is shown in SEQ ID NO: 7 and 8, the amino acid sequence of Linker is GGGGS, and the amino acid sequence of ILT2-D1D2 is shown in SEQ ID NO: NO:2 and 3), a CD8α-derived signal peptide sequence (amino acid sequence shown in SEQ ID NO:19) was linked to its N-terminus, and a tropical Xenopus Notch transmembrane domain sequence (amino acid sequence shown in SEQ ID NO:1) and a Gal4-VP64 artificial transcription factor sequence (amino acid sequence shown in SEQ ID NO:12) were sequentially linked to its C-terminus, ultimately forming a series of SynNotch receptor sequences, as shown in Figure 1. These sequences were cloned into the pCDH vector, viruses were produced, and NK cells or Jurkat cells were infected, and stable cell lines were established.

[0188] Example 2: Design and construction of CAR receptors that specifically target and bind to human NKG2D ligands and NKp30 ligands

[0189] NKG2D and NKp30 receptors are two receptors on the surface of immune cells responsible for binding to molecular ligands such as MICA / B, ULBPs, and B7H6. NKG2D and NKp30 ligands are widely expressed on the surfaces of numerous tumor cells and are absent or expressed at low levels in most normal tissues. Both NKG2D and NKp30 receptors are divided into extracellular, transmembrane, and intracellular regions, with the extracellular region being the functional region responsible for recognizing and binding the corresponding ligand.

[0190] Based on the properties of NKG2D and NKp30 that can bind to the above-mentioned ligands, the inventors designed and synthesized synthetic polypeptides that can target and bind to human NKG2D ligands and NKp30 ligands. The synthetic polypeptides are composed of the extracellular segment polypeptide sequences of NKp30 and NKG2D and can bind to the ligands of NKG2D and NKp30 at the same time. The synthetic polypeptide is used as the extracellular region of the CAR receptor (the C-terminus of the NKp30 receptor extracellular region and the N-terminus of the NKG2D receptor extracellular region are connected, wherein the amino acid sequence of the NKp30 receptor extracellular region is shown in SEQ ID NO: 14, and the amino acid sequence of the NKG2D receptor extracellular region is shown in SEQ ID NO: 13), and the signal peptide sequence derived from CD8α is connected at its N-terminus (amino acid sequence as shown in SEQ ID NO: 19, nucleotide sequence as shown in SEQ ID NO: 20), and the hinge region sequence derived from CD8α (amino acid sequence as shown in SEQ ID NO: 15), the transmembrane domain sequence derived from CD8α (amino acid sequence as shown in SEQ ID NO: 16), 4-1BB sequence and CD3ζ sequence (amino acid sequence of 4-1BB and CD3ζ as shown in SEQ ID NO: 17) are sequentially connected at its C-terminus to finally form the CAR receptor sequence. At the same time, the UAS-mini-CMV sequence (nucleotide sequence as shown in SEQ ID NO: 21) is added before the nucleotide sequence encoding the CAR receptor, as shown in Figure 2. The sequence was cloned into the pCDH vector with the promoter removed to produce virus, and the stably transfected NK cells established in Example 1 were infected respectively to establish stable cell lines transfected with two receptor nucleic acid sequences, named the corresponding Syn-CAR-NK cell line and Syn-CAR-Jurkat cell line. At the same time, the CAR receptor without the UAS-mini-CMV sequence was cloned into the pCDH vector with a promoter, and the virus was produced and infected with NK and Jurkat cells to establish stable cell lines as controls for Syn-CAR-NK / Jurkat cells, named CAR-NK cells and CAR-Jurkat cells.

[0191] Example 3: In vitro experimental verification of Syn-CAR-NK cells' precise and efficient killing of target cells

[0192] Next, the in vitro killing activity of different types of Syn-CAR-NK cells obtained in Example 2 was tested by PI and CFSE staining. Specifically, for the tumor cell line K562 (which has all the targets required for Syn-CAR-NK activation), the target cell line was stained with CSFE fluorescence and the cells were plated at 2×10 4The inoculation concentration of cells / ml was plated on a culture plate. Six experimental groups and one control group were set up correspondingly for the target cell line, wherein the experimental group was added with a cell suspension of Syn-CAR-NK cells that needed to simultaneously target HLA-G, NKG2D ligands, and NKp30 ligands obtained in Example 2; the blank control group was added with NK cells infected with an empty vector virus. In the above-mentioned experimental group, Syn-CAR-NK cells were mixed with target cells for 48 hours according to a 1:2 effector-target ratio (herein, the term "effector-target ratio" refers to the number ratio of effector cells, i.e., Syn-CAR-NK cells that simultaneously target HLA-G, NKG2D ligands, and NKp30 ligands, i.e., tumor cells). After 48 hours of culture, the supernatant was centrifuged and 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. The tumor cell killing rate test results using leukemia cells K562 as target cells are shown in Figure 3. As can be seen from Figure 3, Syn-CAR-NK cells have a significant killing effect on leukemia cells K562 (significantly higher than the control group), among which 5-Syn-CAR-NK and 6-Syn-CAR-NK have the highest killing efficiency.

[0193] For the tumor cell line Aspc-1 (which only has the target for activating SynNotch) and the immortalized normal cell line THLE3 (which does not have any target for activating SynNotch and CAR), 5×10 3 After the target cells were attached to the E-plate, the experimental group cells and control group cells in Example 2 were added, and the growth of the target cells was detected by RTAC instrument. It was found that the experimental group cells of the present invention had no obvious killing effect on the pancreatic cancer cells Aspc-1 and negative for NKG2D ligands and NKp30 ligands (similar to the killing effect of the control group). Among them, the results of 6-Syn-CAR-NK are exemplified in this embodiment. The tumor cell killing rate test results with pancreatic cancer Aspc-1 as the target cell are shown in Figure 4, and the killing rate test results with immortalized liver cell line as the target cell are shown in Figure 5.

[0194] As can be seen from Figure 4, 6-Syn-CAR-NK cells had no significant killing effect on pancreatic cancer cells Aspc-1, which are negative for NKG2D ligands and NKp30 ligands (the killing effect is similar to that of the control group). As can be seen from Figure 5, 6-Syn-CAR-NK cells had no significant killing enhancement effect on immortalized liver cells THLE3, which are negative for HLA-G and negative for NKG2D ligands and NKp30 ligands (the killing effect is similar to that of the control group). The above results show that the Syn-CAR system can work well, has no killing effect on cells that only express a single target or do not express the target, and can more accurately distinguish normal cells from tumor cells.

[0195] Example 4: In vivo experimental verification of Syn-CAR-NK cells' precise and efficient killing of target cells

[0196] 1. Take 1×10 7 NCI-H716 cells were subcutaneously injected into 5-week-old NCG mice. After 10 days of tumor growth, the tumor size was measured with a vernier caliper. Mice with similar tumor formation were randomly divided into 9 groups, six of which were experimental groups (i.e., different types of Syn-CAR-NK cells obtained in Example 2), and the remaining three groups were control groups (one group was untreated control, one group was treated with ordinary NK cells, and one group was treated with CAR-NK cells). Afterwards, 1×10 7 A control group of mice was injected with 1×10 6-Syn-CAR-NK cells into the tail vein. 7 A control group of mice were injected with 1×10 7 CAR-NK cells were injected into the tail vein of a control group of mice with equal volumes of normal saline. Each group was treated / treated with the same treatment method once a week, and the size of the subcutaneous transplanted tumor was measured. IL2 was injected intraperitoneally every 3 days, with 5×10 per mouse each time. 4 U, a total of 3 treatments. It was found that the Syn-CAR-NK cells of the present invention had a significant killing effect on the target cells-colon cancer cells NCI-H716 that simultaneously expressed HLA-G and NKG2D ligands and NKp30 ligands (significantly higher than the NK control group), and their killing efficiency was not significantly different from that of the CAR-NK group alone. Among them, the results of 6-Syn-CAR-NK are exemplified in this embodiment, specifically referring to Figure 6 (after colon cancer cells NCI-H716 were used as target cells for tumor loading, the killing rate test results of 6-Syn-CAR-NK cells were obtained).

[0197] As can be seen from Figure 6, the 6-Syn-CAR-NK cells of Example 4 have a significant killing effect on the target cells - colon cancer cells NCI-H716, which simultaneously express HLA-G, NKG2D ligands and NKp30 ligands in vivo (significantly higher than the NK control group), and their killing efficiency is not significantly different from that of the CAR-NK group alone, indicating that the SynNotch receptor does not reduce its killing efficiency.

[0198] 2. Further, take 1×10 7Aspc-1 cells were subcutaneously injected into 5-week-old NCG mice. After 10 days of tumor growth, the tumor size was measured with a vernier caliper. Mice with similar tumor formation were randomly divided into three groups. One group was the experimental group with 6-Syn-CAR-NK cells, and the remaining two groups were the control groups (one group was the untreated control group, and the other group was the ordinary NK treatment control group). Afterwards, 1×10 7 A control group of mice was injected with 1×10 6-Syn-CAR-NK cells into the tail vein. 7 The same volume of normal saline was injected into the tail vein of a control group of mice. Each group was treated / treated once a week with the same treatment method. The size of the subcutaneous transplanted tumor was measured at the same time. IL2 was injected intraperitoneally every 3 days, with 5×10 per mouse each time. 4 U, a total of 3 treatments. The results of the killing rate test of 6-Syn-CAR-NK cells on pancreatic cancer cells Aspc-1 after tumor loading are shown in Figure 7.

[0199] As can be seen from Figure 7, the 6-Syn-CAR-NK cells of Example 4 had no significant killing enhancement effect on the target cells expressing only HLA-G - pancreatic cancer cells Aspc-1 in vivo (no improvement in killing efficiency compared with the NK control group), indicating that the Syn-CAR system can work well, has no killing effect on cells expressing only a single target, and can more accurately distinguish normal cells from tumor cells.

[0200] Example 5: In vitro experimental verification of Syn-CAR-Jurkat cells' precise and efficient killing of target cells

[0201] Next, flow cytometry was used to detect the in vitro killing activity of different types of 6-Syn-CAR-Jurkat cells obtained in Example 2. Specifically, for the tumor cell line K562 (which has all the targets required for Syn-CAR-Jurkat activation), the target cell line was inoculated with 2×10 4The cells were plated on a culture plate at an inoculation concentration of 100 / ml. One experimental group and one control group were set up correspondingly for the target cell line, wherein the experimental group was added with a cell suspension of 6-Syn-CAR-Jurkat cells obtained in Example 2 that simultaneously targeted HLA-G, NKG2D ligands, and NKp30 ligands; the blank control group was added with Jurkat cells infected with an empty vector virus. In the above-mentioned experimental group, 6-Syn-CAR-Jurkat cells were mixed with target cells for 48 hours according to a 1:2 effector-target ratio (herein, the term "effector-target ratio" refers to the ratio of the number of effector cells, i.e., 6-Syn-CAR-Jurkat cells that simultaneously targeted HLA-G, NKG2D ligands, and NKp30 ligands, to target cells, i.e., tumor cells). After 48 hours of culture, the supernatant was removed by centrifugation, the cell pellet was washed and stained with TNF-α, IFN-γ, and CD69 antibodies. The stained cells were obtained by flow cytometry, and the results were analyzed using FlowJo software. The activation effect of Jurkat cells was tested using leukemia cells K562 as target cells. The results showed that 6-Syn-CAR-Jurkat cells were significantly activated by leukemia cells K562, and the expression of TNF-α, IFN-γ, and CD69 was significantly increased (significantly higher than the control group). This shows that the Syn-CAR system designed by the present invention can also play a good role in the CAR-T cell system.

[0202] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean 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 can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0203] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A chimeric polypeptide, characterized in that, Comprising: A first extracellular region having the activity of binding to HLA-G protein; A first transmembrane region, which comprises the transmembrane region of the Notch receptor protein of Xenopus tropicalis or an amino acid sequence having at least 80% identity thereto, and the N-terminus of the first transmembrane region is connected to the C-terminus of the first extracellular region; A first intracellular region, the N-terminus of which is connected to the C-terminus of the first transmembrane region.

2. The chimeric polypeptide according to claim 1, wherein The transmembrane region of the Notch receptor protein of Xenopus tropicalis has the amino acid sequence shown in SEQ ID NO:1; Optionally, the transmembrane region further comprises epidermal growth factor-like repeats and / or RAM sequences; Optionally, the C-terminus of the epidermal growth factor-like repeats is connected to the N-terminus of the transmembrane region of the Notch receptor protein of Xenopus tropicalis; and / or, the C-terminus of the transmembrane region of the Notch receptor protein of Xenopus tropicalis is connected to the N-terminus of the RAM sequence; Optionally, the first transmembrane region has the amino acid sequence shown in SEQ ID NO:1 or 25 or an amino acid sequence having at least 90% identity thereto; Optionally, the first transmembrane region has the amino acid sequence shown in SEQ ID NO:1 or 25.

3. The chimeric polypeptide according to claim 1, characterized in that, The first extracellular region comprises a first binding protein or a fragment thereof that binds to HLA-G protein; Optionally, the first binding protein or a fragment thereof comprises at least one of an antibody or a functional fragment thereof, a receptor; Optionally, the first binding protein or a fragment thereof comprises a first binding fragment and / or a second binding fragment; The first binding fragment is the extracellular region of the ILT2 protein or an active fragment thereof; The second binding fragment is the extracellular region of the ILT4 protein or an active fragment thereof; Optionally, the first binding protein or a fragment thereof comprises a first binding fragment and a second binding fragment, and the first binding fragment and the second binding fragment are connected; Optionally, the extracellular region of the ILT2 protein consists of the ILT2-D1 fragment, the ILT2-D2 fragment, the ILT2-D3 fragment and the ILT2-D4 fragment; The ILT2-D1 fragment has the amino acid sequence shown in SEQ ID NO:2; The ILT2-D2 fragment has the amino acid sequence shown in SEQ ID NO:3; The ILT2-D3 fragment has the amino acid sequence shown in SEQ ID NO:4; The ILT2-D4 fragment has the amino acid sequence shown in SEQ ID NO:5; Optionally, the extracellular region of the ILT2 protein has the amino acid sequence shown in SEQ ID NO:6; Optionally, the first binding fragment is selected from: At least one of the ILT2-D1 fragment, the ILT2-D2 fragment, the ILT2-D3 fragment and the ILT2-D4 fragment; or The extracellular region of the ILT2 protein; Optionally, the first binding fragment is selected from the ILT2-D1 fragment and the ILT2-D2 fragment, and the ILT2-D1 fragment and the ILT2-D2 fragment are connected; Optionally, the C-terminus of the ILT2-D1 fragment is linked to the N-terminus of the ILT2-D2 fragment, or the N-terminus of the ILT2-D1 fragment is linked to the C-terminus of the ILT2-D2 fragment; Optionally, the first binding fragment is selected from the extracellular region of the ILT2 protein; Optionally, the extracellular region of the ILT4 protein consists of an ILT4-D1 fragment, an ILT4-D2 fragment, an ILT4-D3 fragment, and an ILT4-D4 fragment; The ILT4-D1 fragment has the amino acid sequence shown in SEQ ID NO:7; The ILT4-D2 fragment has the amino acid sequence shown in SEQ ID NO:8; The ILT4-D3 fragment has the amino acid sequence shown in SEQ ID NO:9; The ILT4-D4 fragment has the amino acid sequence shown in SEQ ID NO:10; Optionally, the extracellular region of the ILT4 protein has the amino acid sequence shown in SEQ ID NO:11; Optionally, the second binding fragment is selected from: At least one of the ILT4-D1 fragment, the ILT4-D2 fragment, the ILT4-D3 fragment, and the ILT4-D4 fragment; or The extracellular region of the ILT4 protein; Optionally, the second binding fragment is selected from the ILT4-D1 fragment and the ILT4-D2 fragment, and the ILT4-D1 fragment and the ILT4-D2 fragment are linked; Optionally, the C-terminus of the ILT4-D1 fragment is linked to the N-terminus of the ILT4-D2 fragment, or the N-terminus of the ILT4-D1 fragment is linked to the C-terminus of the ILT4-D2 fragment; Optionally, the second binding fragment is selected from the extracellular region of the ILT4 protein; Optionally, when the first extracellular region contains multiple copies of the first binding protein or its fragment, the first extracellular region further includes a first linker peptide, and any two of the first binding proteins or their fragments are linked with or without the first linker peptide; Optionally, the first binding protein or its fragment includes a first binding fragment and a second binding fragment, and the first binding fragment and the second binding fragment are linked by the first linker peptide; Optionally, the C-terminus of the first binding fragment is linked to the N-terminus of the first linker peptide, the C-terminus of the first linker peptide is linked to the N-terminus of the second binding fragment, or the C-terminus of the second binding fragment is linked to the N-terminus of the first linker peptide, and the C-terminus of the first linker peptide is linked to the N-terminus of the first binding fragment; Optionally, the amino acid sequence of the first linker peptide is (GGGGS)n, where n is any integer between 1 and 10; Optionally, n is 1, 2, 3, or 4; Optionally, the amino acid sequence of the first linker peptide is GGGGS; Optionally, the first extracellular region is selected from one of the following: ILT2-D1 fragment + ILT2-D2 fragment + first linker peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first linker peptide + ILT2-D1 fragment + ILT2-D2 fragment; extracellular region of ILT2 protein + first linker peptide + extracellular region of ILT4 protein; extracellular region of ILT4 protein + first linker peptide + extracellular region of ILT2 protein; ILT2-D1 fragment + ILT2-D2 fragment + first linker peptide + extracellular region of ILT4 protein; ILT4-D1 fragment + ILT4-D2 fragment + first linker peptide + extracellular region of ILT2 protein; ILT2-D1 fragment + first linker peptide + ILT4-D1 fragment; ILT2-D1 fragment + first linker peptide + ILT4-D2 fragment; ILT2-D1 fragment + first linker peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT2-D1 fragment + first linker peptide + extracellular region of ILT4 protein; ILT2-D1 fragment + first linker peptide + ILT2-D1 fragment; ILT2-D1 fragment + first linker peptide + ILT2-D2 fragment; ILT2-D1 fragment + first linker peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT2-D1 fragment + first linker peptide + extracellular region of ILT2 protein; ILT4-D1 fragment + first linker peptide + ILT4-D1 fragment; ILT4-D1 fragment + first linker peptide + ILT4-D2 fragment; ILT4-D1 fragment + first linker peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT4-D1 fragment + first linker peptide + extracellular region of ILT4 protein; ILT4-D1 fragment + first linker peptide + ILT2-D1 fragment; ILT4-D1 fragment + first linker peptide + ILT2-D2 fragment; ILT4-D1 fragment + first linker peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT4-D1 fragment + first linker peptide + extracellular region of ILT2 protein; ILT2-D2 fragment + first linker peptide + ILT4-D1 fragment; ILT4-D2 fragment + first linker peptide + ILT4-D2 fragment; ILT4-D2 fragment + first linker peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT4-D2 fragment + first linker peptide + extracellular region of ILT4 protein; ILT4-D2 fragment + first linker peptide + ILT2-D1 fragment; ILT4-D2 fragment + first linker peptide + ILT2-D2 fragment; ILT4-D2 fragment + first linker peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT4-D2 fragment + first linker peptide + extracellular region of ILT2 protein; ILT2-D1 fragment + ILT2-D2 fragment + first linker peptide + ILT4-D1 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first linker peptide + ILT4-D2 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first linker peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first linker peptide + extracellular region of ILT4 protein; ILT2-D1 fragment + ILT2-D2 fragment + first linker peptide + ILT2-D1 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first linker peptide + ILT2-D2 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first linker peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT2-D1 fragment + ILT2-D2 fragment + first linker peptide + extracellular region of ILT2 protein; ILT4-D1 fragment + ILT4-D2 fragment + first linker peptide + ILT4-D1 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first linker peptide + ILT4-D2 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first linker peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first linker peptide + extracellular region of ILT4 protein; ILT4-D1 fragment + ILT4-D2 fragment + first linker peptide + ILT2-D1 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first linker peptide + ILT2-D2 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first linker peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT4-D1 fragment + ILT4-D2 fragment + first linker peptide + extracellular region of ILT2 protein; extracellular region of ILT2 protein + first linker peptide + ILT4-D1 fragment; extracellular region of ILT2 protein + first linker peptide + ILT4-D2 fragment; extracellular region of ILT2 protein + first linker peptide + ILT4-D1 fragment + ILT4-D2 fragment; extracellular region of ILT2 protein + first linker peptide + extracellular region of ILT4 protein; extracellular region of ILT2 protein + first linker peptide + ILT2-D1 fragment; extracellular region of ILT2 protein + first linker peptide + ILT2-D2 fragment;The extracellular region of the ILT2 protein + the first linker peptide + the ILT2-D1 fragment + the ILT2-D2 fragment; the extracellular region of the ILT2 protein + the first linker peptide + the extracellular region of the ILT2 protein; the extracellular region of the ILT4 protein + the first linker peptide + the ILT4-D1 fragment; The extracellular region of ILT4 protein + the first linker peptide + ILT4-D2 fragment; the extracellular region of ILT4 protein + the first linker peptide + ILT4-D1 fragment + ILT4-D2 fragment; the extracellular region of ILT4 protein + the first linker peptide + the extracellular region of ILT4 protein; the extracellular region of ILT4 protein + the first linker peptide + ILT2-D1 fragment; the extracellular region of ILT4 protein + the first linker peptide + ILT2-D2 fragment; the extracellular region of ILT4 protein + the first linker peptide + ILT2-D1 fragment + ILT2-D2 fragment; the extracellular region of ILT4 protein + the first linker peptide + the extracellular region of ILT2 protein; Optionally, the first extracellular region is selected from one of the following: 1) The extracellular region of ILT2 protein; 2) The extracellular region of ILT4 protein; 3) Sequentially from the N-terminus to the C-terminus are ILT2-D1 fragment and ILT2-D2 fragment; 4) Sequentially from the N-terminus to the C-terminus are ILT4-D1 fragment and ILT4-D2 fragment; 5) Sequentially from the N-terminus to the C-terminus are ILT2-D1 fragment, ILT2-D2 fragment, the first linker peptide, ILT4-D1 fragment and ILT4-D2 fragment; 6) Sequentially from the N-terminus to the C-terminus are ILT4-D1 fragment, ILT4-D2 fragment, the first linker peptide, ILT2-D1 fragment and ILT2-D2 fragment.

4. The chimeric polypeptide according to claim 1, wherein The first intracellular region includes at least one of a transcriptional activator protein, a transcriptional repressor protein, a transcription factor, a site-specific nuclease, a recombinase, an intracellular domain of an activating immune receptor, and an intracellular domain of an inhibitory immune receptor; Optionally, the first intracellular region includes at least one of GaL4-VP64, GaL4-VP16, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, HAP1-VP16, and LexA-VP64; Optionally, the Gal4-VP64 has the amino acid sequence shown in SEQ ID NO:

12.

5. A first nucleic acid molecule, characterized in that, The first nucleic acid molecule encodes the chimeric polypeptide according to any one of claims 1 to 4; Optionally, the first nucleic acid molecule is DNA.

6. A first expression vector, characterized in that, Carrying the first nucleic acid molecule according to claim 5; Optionally, the first expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a phage; Preferably, the first expression vector is a plasmid expression vector.

7. A second nucleic acid molecule, characterized in that, Comprising a first nucleic acid fragment and a second nucleic acid fragment, the 3'-end of the first nucleic acid fragment is connected to the 5'-end of the second nucleic acid fragment; Wherein, the second nucleic acid fragment is used to encode an antigen chimeric receptor targeting a first molecule, and the first nucleic acid fragment is used to bind to the first intracellular region and induce the expression of the antigen chimeric receptor; The first intracellular region is the same as the first intracellular region defined in the chimeric polypeptide according to any one of claims 1 to 4.

8. The second nucleic acid molecule according to claim 7, wherein The antigen chimeric receptor includes: A second extracellular region, the second extracellular region has first molecule binding activity, and the first molecule is not HLA-G protein; A second transmembrane region, the N-terminus of the second transmembrane region is connected to the C-terminus of the second extracellular region; The second intracellular region, the N-terminus of the second intracellular region being connected to the C-terminus of the second transmembrane region; Optionally, the first molecule comprises at least one of a tumor antigen, a virus, a bacterium, an endotoxin, an antibody, a cell receptor, and a ligand of a cell receptor; Optionally, the tumor antigen comprises at least one of a tumor-associated antigen and a tumor-specific antigen, preferably a tumor-specific antigen; Optionally, the first molecule comprises at least one of MICA, MICB, ULBPs, B7H6, B7H3, GFP, eGFP, CD19, ALPPL2, BCMA, SIRPα, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44, CD44v6, CD45, CD48, CD51, CD52, CD56, CD59, CD66, CD70, CD71, CD72, CD73, CD74, CD79A, CD79B, CD80, CD86, CD94, CD95, CD123, CD133, CD134, CD140, CD152, CD154, CD158, CD178, CD181, CD182, CD183, CD200, CD210, CD221, CD246, CD252, CD253, CD261, CD262, CD269, CD273, CD274, CD276, CD279, CD295, CD339, CD340, EGFR, EGFR VIII, HER2, FGFR2, AFP, CA125, MSLN, GPC3, CEA, CLDN1, CLDN3, CLDN6, CLDN18.1, CLDN18.2, EpCAM, PSCA, GD2, GD3, IL-13, IL-13RA2, ROR1, MUC-1, PSMA, MAGEA1, 4-1BB, 5T4, BAFF, CA242, CA-IX, MET, CCR4, CNTO888, FAP, MORAb-009, EPHA2, VEGF-A, VEGFR-1, and VEGFR-2; Optionally, the second extracellular region comprises a second binding protein or a fragment thereof that binds to the first molecule; Optionally, the second binding protein or a fragment thereof comprises at least one of an antibody or a functional fragment thereof, a receptor, a ligand of a receptor, and a cell adhesion molecule; Optionally, the second binding protein or a fragment thereof is an extracellular region of an activating receptor expressed on the surface of an immune cell or an antibody or a fragment thereof that binds to the first molecule; Optionally, the antibody or a fragment thereof that binds to the first molecule is a single-chain antibody; Optionally, the activating receptor is selected from the receptors on the surface of NK cells; Optionally, the activating receptor is selected from at least one of NKG2D, NKp30, NKp44, NKp46, DNAM-1, PD-1, TIGIT, NKG2A, NKG2B, NKG2C, NKG2E, NKG2H, CD16, NKp80, CD226, CD160, CD161, CD96, PVRIG, SLAM, CD200R, CD49a, TIM-3, LAG-3, CD112R, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, LIR1, LIR2, SIGLE3, SIGLE7, SIGLE9, KLRG1; Optionally, the activating receptor is selected from NKG2D and / or NKp30; Optionally, the second binding protein or a fragment thereof comprises a third binding fragment and / or a fourth binding fragment; The third binding fragment is the extracellular region of NKG2D or an active fragment thereof; The fourth binding fragment is the extracellular region of NKp30 or an active fragment thereof; Optionally, the extracellular region of NKG2D has the amino acid sequence shown in SEQ ID NO: 13; Optionally, the extracellular region of NKp30 has the amino acid sequence shown in SEQ ID NO: 14; Optionally, the third binding fragment has the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 90% identity thereto; Optionally, the fourth binding fragment has the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% identity thereto; Optionally, the second extracellular region further comprises a second linker peptide, and the third binding fragment and the fourth binding fragment are connected by the second linker peptide; Optionally, the C-terminus of the third binding fragment is connected to the N-terminus of the second linker peptide, the C-terminus of the second linker peptide is connected to the N-terminus of the fourth binding fragment, or the C-terminus of the fourth binding fragment is connected to the N-terminus of the second linker peptide, and the C-terminus of the second linker peptide is connected to the N-terminus of the third binding fragment; Optionally, the amino acid sequence of the second linker peptide is (GGGGS)n, where n is any integer between 0 and 10; Optionally, n is 0, 1, 2, 3 or 4; Optionally, the amino acid sequence of the second linker peptide is GGGGS; Optionally, the second extracellular region further comprises a hinge region; The C-terminus of the third binding fragment is connected to the N-terminus of the second linker peptide, the C-terminus of the second linker peptide is connected to the N-terminus of the fourth binding fragment, the C-terminus of the fourth binding fragment is connected to the N-terminus of the hinge region, or the C-terminus of the fourth binding fragment is connected to the N-terminus of the second linker peptide, the C-terminus of the second linker peptide is connected to the N-terminus of the third binding fragment, and the C-terminus of the third binding fragment is connected to the N-terminus of the hinge region; Optionally, the hinge region includes at least one of the hinge region of the CD8α molecule or a variant thereof, and the hinge region of an immunoglobulin or a variant thereof; Optionally, the hinge region has the amino acid sequence shown in SEQ ID NO:

15.

9. The second nucleic acid molecule according to claim 8, wherein The second transmembrane region is selected from at least one of the transmembrane region of the CD8α molecule, the transmembrane region of the CD28 molecule, the transmembrane region of the CD3ζ molecule, the transmembrane region of the CD4 molecule, the transmembrane region of the CD16 molecule, the transmembrane region of the 4-1BB molecule, the transmembrane region of the OX40 molecule, the transmembrane region of the ICOS molecule, the transmembrane region of the CTLA-4 molecule, the transmembrane region of the PD-1 molecule, the transmembrane region of the LAG-3 molecule, the transmembrane region of the 2B4 molecule, the transmembrane region of the NKG2D molecule, the transmembrane region of the DNAM-1 molecule, the transmembrane region of the NKp44 molecule, the transmembrane region of the NKp46 molecule, the transmembrane region of the KIR2DS1 molecule, the transmembrane region of the KIR2DS2 molecule, the transmembrane region of the KIR2DS4 molecule, and the transmembrane region of the BTLA molecule; Optionally, the second transmembrane region is selected from the transmembrane region of the CD8α molecule; Optionally, the second transmembrane region has the amino acid sequence shown in SEQ ID NO:16; Optionally, the second intracellular region includes an intracellular signaling domain and a co-stimulatory domain; Optionally, the intracellular signaling domain is selected from at least one of the intracellular signaling structure of the CD3ζ molecule and the intracellular signaling structure of the FcεRIγ molecule; Optionally, the co-stimulatory domain is selected from at least one of the intracellular signaling domain of the 4-1BB molecule, the intracellular signaling domain of the CD28 molecule, the intracellular signaling domain of the CD27 molecule, the intracellular signaling domain of the CD40 molecule, the intracellular signaling domain of the OX40 molecule, the intracellular signaling domain of the ICOS molecule, the intracellular signaling domain of the DAP10 molecule, the intracellular signaling domain of the DAP12 molecule, and the intracellular signaling domain of the DNAM-1 molecule; Optionally, the second intracellular region has the amino acid sequence shown in SEQ ID NO:17; Optionally, the second nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO:

18.

10. The second nucleic acid molecule according to any one of claims 7 to 9, characterized in that The second nucleic acid molecule further includes a third nucleic acid fragment for encoding a signal peptide; The 3'-end of the first nucleic acid fragment is connected to the 5'-end of the third nucleic acid fragment, and the 3'-end of the third nucleic acid fragment is connected to the 5'-end of the second nucleic acid fragment; Optionally, the signal peptide is selected from at least one of the signal peptide of the CD8α molecule, the signal peptide of the IgG molecule, and the signal peptide of the CD28 molecule; Optionally, the signal peptide has the amino acid sequence shown in SEQ ID NO:19; Optionally, the third nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO:20; Optionally, the first nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO:

21.

11. A second expression vector, characterized in that, Carrying the second nucleic acid molecule according to any one of claims 7 to 10; Optionally, the second expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a phage; Preferably, the second expression vector is a plasmid expression vector.

12. A recombinant immune cell, characterized in that, Comprising: Carrying the first nucleic acid molecule according to claim 5 or the first expression vector according to claim 6; Or, Expressing the chimeric polypeptide according to any one of claims 1 to 4.

13. The recombinant immune cell according to claim 12, wherein The expression of the chimeric polypeptide in the recombinant immune cell is obtained by introducing the first expression vector according to claim 6 into a host cell; Optionally, the recombinant immune cell further comprises a chimeric antigen receptor, and the chimeric antigen receptor is identical to the antigen chimeric receptor encoded by the second nucleic acid fragment defined in the second nucleic acid molecule according to any one of claims 7 to 10; Optionally, the expression of the chimeric antigen receptor in the recombinant immune cell is obtained by introducing the second expression vector according to claim 11 into a host cell.

14. The recombinant immune cell according to claim 13, wherein The host cell comprises at least one of immune cells, neurons, progenitor cells or precursor cells, epithelial cells, endothelial cells and stem cells; Optionally, the host cell comprises 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 γδ T cells.

15. A pharmaceutical composition, characterized in that, Comprising the recombinant immune cell according to any one of claims 12 to 14; Optionally, further comprising a pharmaceutically acceptable excipient.

16. Use of the recombinant immune cell according to any one of claims 12 to 14 or the pharmaceutical composition according to claim 15, the use being for: Use in the preparation of a drug for preventing and / or treating a disease, Preventing and / or treating a disease; Optionally, the disease includes cancer or tumor, autoimmune disease, inflammation and related diseases caused by cellular senescence.

17. The recombinant immune cell according to any one of claims 12 to 14 or the pharmaceutical composition according to claim 15, for preventing and / or treating a disease; Optionally, the disease includes cancer or tumor, autoimmune disease, inflammation and related diseases caused by cellular senescence.

18. A method for treating or preventing a disease, characterized in that, Comprising: Administering to a subject a pharmaceutically acceptable amount of the recombinant immune cell according to any one of claims 12 to 14 or the pharmaceutical composition according to claim 15.

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