Transgenic recombinant immune cell specifically targeting tumor and application thereof

Abstract

The invention discloses a transgenic recombinant immune cell for specifically targeting tumors. The recombinant immune cell comprises a chimeric polypeptide and a chimeric antigen receptor, the chimeric polypeptide comprises a first extracellular region, a first transmembrane region and a first intracellular region which have HLA-G protein binding activity, the N end of the first transmembrane region is connected with the C end of the first extracellular region, the N end of the first intracellular region is connected with the C end of the first transmembrane region, and the first transmembrane region comprises a transmembrane region of Notch receptor protein of Xenopus tropicalis; the antigen chimeric receptor does not have HLA-G protein binding activity. According to the recombinant immune cell, the accurate recognition of the immune cell on tumor cells is greatly improved, the off-target effect of a cell therapy is reduced, the tumor cells and normal cells are accurately and effectively distinguished, and an effective target spot and a targeting mode are provided for treatment of broad-spectrum tumors (especially solid tumors).

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and particularly to genetically modified recombinant immune cells specifically targeting tumors, and their preparation methods and applications. 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 their uses. 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, etc. Among them, in the field of adoptive cell therapy, 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 therapy in this field.

[0003] The principle of immune cell therapy represented by CAR-T is mainly to modify T cells extracted from the patient's own body by genetic engineering means to form CAR-T cells with chimeric antigen receptors. Through the modified chimeric antigen receptor, 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 therapies represented by CAR-T cells targeting CD19 and BCMA have significant curative effects in the treatment of hematological tumors such as B-lymphoma, and are considered to be one of the most promising tumor treatment methods.

[0004] However, due to the lack of a target that can well distinguish normal cells from tumor cells, CAR-T cell therapy usually inevitably kills some normal cells expressing the target protein, causing damage to normal tissues, or due to the widespread presence of the target, continuous activation of CAR-T cells occurs, and then a large amount of cytokines are released, leading to cytokine storm. At the same time, since CAR-T cell therapy usually can only recognize a single antigen, tumor cells can often escape by mutating to lose the antigen recognized by CAR-T cells, and then drug resistance occurs. Moreover, the high heterogeneity of tumors also makes the single target loaded by CAR-T cells unable to fully cover tumor cells, resulting in incomplete and incomplete tumor treatment. The above several key problems have become the main obstacles to the development and growth of the current 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 the target for CAR therapy may face great difficulties. Because broad-spectrum and specificity are like two sides of a coin, it is difficult for a single target to simultaneously possess these two characteristics, and no target molecule that perfectly combines the above two characteristics has been found in current cancer treatment research. Therefore, attempting to achieve the broad-spectrum and specificity of immunotherapy by combining specific target combinations and tools for identifying and activating target combinations is one of the new directions with great potential in current 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 a first aspect of the present invention, a chimeric polypeptide is provided. According to an embodiment of the present invention, the chimeric polypeptide includes: a first extracellular region having HLA-G protein binding activity; a first transmembrane region including the transmembrane region of the Notch receptor protein of Xenopus tropicalis or an amino acid sequence having at least 80% identity thereto, the N-terminus of the first transmembrane region being connected to the C-terminus of the first extracellular region; and a first intracellular region, the N-terminus of the first intracellular region being connected to the C-terminus of the first transmembrane region. The present invention selects the transmembrane region of the Notch receptor protein of Xenopus tropicalis 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, a first nucleic acid molecule is provided. 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 the embodiment of the present invention can encode the above-mentioned chimeric polypeptide.

[0009] In a third aspect of the present invention, a first expression vector is provided. According to an embodiment of the present invention, the first expression vector carries the first nucleic acid molecule described in the second aspect. When connecting the above first nucleic acid molecule to the expression vector, the first nucleic acid molecule can be directly or indirectly connected to the control elements on the expression vector as long as these control elements can control the translation and expression of the first nucleic acid molecule, etc. Of course, these control elements can directly come from the vector itself or be exogenous, that is, not from the vector itself. Of course, the first nucleic acid molecule and the control elements are operably connected.

[0010] In the fourth aspect of the present invention, the present invention provides a second nucleic acid molecule. According to an embodiment of the present invention, the second nucleic acid molecule includes a first nucleic acid fragment and a second nucleic acid fragment, and 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 for encoding an antigen chimeric receptor targeting a first molecule, and the first nucleic acid fragment is used for binding to a first intracellular region and inducing the expression of the antigen chimeric receptor; the first intracellular region is the same as 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 in the aforementioned chimeric polypeptide, thereby activating and inducing the expression of the antigen chimeric receptor.

[0011] In the fifth aspect of the present invention, 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 above-mentioned second nucleic acid molecule.

[0012] In the sixth aspect of the present invention, the present invention provides a recombinant immune cell. According to an embodiment of the present invention, the recombinant immune cell includes: 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. Under suitable conditions, the recombinant immune cell according to an embodiment of the present invention can express the aforementioned chimeric polypeptide on the surface of the recombinant immune cell, can recognize the HLA-G protein, and after binding to the HLA-G protein, it can achieve various different signal output types, such as activation of the expression of specific genes, etc., especially can activate the expression of chimeric antigen receptors containing factors with therapeutic effects (such as treating tumors, etc.), for treating diseases such as tumors.

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

[0014] In the eighth aspect of the present invention, the present invention provides 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.

[0015] Advantageous effects:

[0016] (1) The recombinant immune cells of the present invention greatly improve the precise recognition of tumor cells by immune cells, reduce the off-target effect of cell therapy, accurately and effectively distinguish tumor cells from normal cells, and provide effective targets and targeting methods for the treatment of a broad spectrum of tumors (especially solid tumors);

[0017] (2) The recombinant immune cells of the present invention can convert the inhibitory signals transmitted after HLA-G binds to ILT2 / 4 into activation signals, not only effectively killing HLA-G-positive tumor cells, but also being able to resist the inhibition from the tumor immune microenvironment and reverse immune cell exhaustion;

[0018] (3) The engineered multi-targeted recombinant immune cells of the present invention can both broadly recognize numerous different types of tumor cells and accurately distinguish normal cells, greatly improving the killing efficiency and accuracy of various tumor cells, thus providing a new means of tumor immunocyte therapy that is both broad-spectrum and specific and has broad application prospects.

[0019] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0021] Figure 1 is a schematic diagram of the structural composition of the SynNotch receptor in Example 1 of the present invention;

[0022] Figure 2 is a schematic diagram of the structural composition of the CAR receptor in Example 2 of the present invention;

[0023] Figure 3 is a graph showing the killing results of Syn-CAR-NK92 cells on double-target positive K562 cells in Example 3 of the present invention;

[0024] Figure 4 is a graph showing the killing results of Syn-CAR-NK92 cells on single-target positive Aspc-1 cells in Example 3 of the present invention;

[0025] Figure 5 is a graph showing the killing results of Syn-CAR-NK92 on non-target THLE3 cells in Example 3 of the present invention;

[0026] Figure 6 is a graph showing the treatment results of Syn-CAR-NK92 cells on subcutaneously transplanted tumors of colon cancer mice with double targets in Example 4 of the present invention;

[0027] Figure 7 This is the treatment result graph of Syn-CAR-NK92 cells on subcutaneous transplanted tumors of single-target positive pancreatic cancer mice in Example 4 of the present invention. Detailed implementation manners

[0028] The embodiments of the present invention will be described in detail below. The following described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention.

[0029] It should be noted that the terms "first" and "second" are only used for descriptive purposes, and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise stated, the meaning of "a plurality" is two or more.

[0030] Definitions and general terms

[0031] To make the present invention easier to understand, 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 of ordinary skill in the art to which the present invention pertains.

[0032] In this article, the term "comprising" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0033] In this article, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where the events or conditions occur, as well as the cases where the events or conditions do not occur.

[0034] In this text, when the terms "identity", "homology", or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods. For example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.)). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997)); and the 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, 8th edition, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0035] 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%, 99.9% identity to each reference sequence.

[0036] As used herein, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replicating after insertion into a suitable host, which transfers the inserted nucleic acid molecule into host cells and / or between host cells. The expression vector may include vectors mainly for inserting DNA or RNA into cells, vectors mainly for replicating DNA or RNA, and expression vectors mainly for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having multiple of the above functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the expression vector, the expression vector can produce a desired expression product.

[0037] As used herein, the term "chimeric antigen receptor (CAR)" is a fusion protein that includes an extracellular domain capable of binding 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 "chimeric receptor", "T-body", or "chimeric immunoreceptor (CIR)". The "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide capable of binding a certain antigen. The "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain for transmitting signals to activate or inhibit intracellular biological processes.

[0038] As used herein, the term "recombinant immune cell" generally refers to a cell obtained by modifying or recombining the genetic material of a host cell using genetic engineering techniques or cell fusion techniques, and having unique traits with stable inheritance. Among them, the term "host cell" refers to a prokaryotic cell 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 nucleic acid (such as a vector) into a cell by various techniques known in the art. A suitable host cell 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.

[0039] 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 which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and intimately bringing into association the active chimeric polypeptide or recombinant immune cell with a liquid carrier, a finely divided solid carrier, or both.

[0040] As used herein, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the particular target dosage form. Their use is also contemplated within the scope of the present invention, except to the extent that any conventional excipient is incompatible with the chimeric polypeptide or recombinant immune cell of the present invention, such as any adverse biological effect produced or any interaction with any other component of the pharmaceutically acceptable composition in a detrimental manner.

[0041] As used herein, the term "administering" refers to introducing a predetermined amount of a substance into a patient by a suitable means. The recombinant immune cells or pharmaceutical compositions of the present invention can be administered by any common route as long as it can reach the intended tissue. Various modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, subcutaneous injection, etc., but the present invention is not limited to these exemplified modes of administration. Preferably, the compositions of the present invention are administered by intravenous injection or subcutaneous injection.

[0042] As used herein, the term "treatment" means to obtain the desired pharmacological and / or physiological effect. The effect can be prophylactic 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 adverse effects caused by the disease. "Treatment" as used herein covers diseases of mammals, particularly humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is predisposed to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, e.g., arresting the development of the disease; or (c) alleviating the disease, e.g., relieving the symptoms associated with the disease. "Treatment" as used herein covers any administration of recombinant immune cells, pharmaceutical compositions, or drugs to an individual to treat, cure, relieve, improve, mitigate, or inhibit a disease of the individual, 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 thereof.

[0043] The term "immune cell" generally refers to a cell capable of generating an immune response (e.g., an antigen-specific immune response). For example, the immune cell may or already contains an isolated nucleic acid of the present invention, and / or an expression vector, or is an individual cell, cell line, or cell culture capable of expressing the chimeric polypeptide and optionally the chimeric antigen receptor described in the present application. In the present invention, the immune cell may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, white blood cells, and / or peripheral blood mononuclear cells. In this article, "carbon terminus" and "C terminus" are synonymous; "nitrogen terminus" and "N terminus" are synonymous.

[0044] Detailed description of the transgenic recombinant immune cells specifically targeting tumors of the present invention, their preparation methods, and applications

[0045] 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 their uses, which will be described in detail below.

[0046] Chimeric polypeptide, first nucleic acid molecule, first expression vector

[0047] In the first aspect of the present invention, a chimeric polypeptide is provided. According to an embodiment of the present invention, the chimeric polypeptide includes: a first extracellular region having the activity of binding to HLA-G protein; a first transmembrane region including the transmembrane region of the Notch receptor protein of Xenopus tropicalis or an amino acid sequence having at least 80% identity therewith, the N-terminus of the first transmembrane region being connected to the C-terminus of the first extracellular region; and a first intracellular region, the N-terminus of the first intracellular region being connected to the C-terminus of the first transmembrane region. The present invention selects the transmembrane region of the Notch receptor protein of Xenopus tropicalis 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.

[0048] Human leukocyte antigen-G (HLA-G) is a group of closely linked gene clusters located on the short arm of human chromosome 6. It belongs to a non-classical class I molecule of the major histocompatibility complex (MHC) in humans, and has the characteristic 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, etc.

[0049] Furthermore, the above-mentioned chimeric polypeptide can be used to prepare multi-targeted recombinant immune cells, which enable the cells expressing the chimeric polypeptide to recognize HLA-G protein. After binding to HLA-G protein, it can achieve various different signal output types, such as activation of specific gene expression, etc. In particular, it can activate the expression of chimeric antigen receptors containing factors with therapeutic effects (such as treating tumors, etc.), and is used to treat diseases such as tumors.

[0050] In particular, recombinant immune cells for treating tumors can be prepared, which can broadly recognize numerous tumor cells, etc., greatly improving the killing efficiency and accuracy of various tumor cells, overcoming the problems of non-specific tumor recognition and incomplete tumor recognition coverage in the clinical application of existing cell therapy, thus providing a new means of tumor treatment with broad application prospects.

[0051] According to the embodiments of the present invention, the above-mentioned chimeric polypeptide may further include at least one of the following technical features:

[0052] According to the embodiments 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.

[0053] ILDYGFIGGLGKNITPPDNEEICENEQCAELADNKICNANCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFNDGKCDSQCNNSGCLYDGFDCQKVEVQCNPLYDQYCRDHFQDGHCDQGCNNAECEWDGLDCDNMPENLAEGTLLIVVLMPPEKLKNNSVNFLRELSRVLHTNVVFKKDSKGEYKIYPYYGNEEELKKHHIKKRSAASWSDAPTAIFSTMKESVLPGRRRRELDQMEVRGSIVYLEIDNRQCYKSSSQCFTSATDVAAFLGALATHGNLNIPYKIEAVKSEIVETAKPPPPLYAMFSMLVIPLLIIFVIMVVIVNKKRRR(SEQ ID NO:1).

[0054] According to the embodiments of the present invention, the transmembrane region further includes epidermal growth factor-like repeat sequences (EGF repeat) and / or RAM sequence. Thus, the background leakage activation of the SynNotch synthetic receptor can be further reduced.

[0055] 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 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.

[0056] According to an embodiment of the present invention, the epidermal growth factor-like repeat sequence has the amino acid sequence shown in SEQ ID NO: 23.

[0057] VVSPCASRPCYNGGTCQFSPEEPFFQCFCPTNFNGLFCH (SEQ ID NO: 23).

[0058] According to an embodiment of the present invention, the RAM sequence has the amino acid sequence shown in SEQ ID NO: 24.

[0059] EHGQLWFP (SEQ ID NO: 24).

[0060] According to an embodiment of the present invention, 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 therewith.

[0061] According to an embodiment of the present invention, the first transmembrane region has the amino acid sequence shown in SEQ ID NO: 1 or 25.

[0062] VVSPCASRPCYNGGTCQFSPEEPFFQCFCPTNFNGLFCHVVSPCASRPCYNGGTCQFSPEEPFFQCFCPTNFNGLFCHILDYGFIGGLGKNITPPDNEEICENEQCAELADNKICNANCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFNDGKCDSQCNNSGCLYDGFDCQKVEVQCNPLYDQYCRDHFQDGHCDQGCNNAECEWDGLDCDNMPENLAEGTLLIVVLMPPEKLKNNSVNFLRELSRVLHTNVVFKKDSKGEYKIYPYYGNEEELKKHHIKKRSAASWSDAPTAIFSTMKESVLPGRRRRELDQMEVRGSIVYLEIDNRQCYKSSSQCFTSATDVAAFLGALATHGNLNIPYKIEAVKSEIVETAKPPPPLYAMFSMLVIPLLIIFVIMVVIVNKKRRREHGQLWFP EHGQLWFP (SEQ ID NO: 25).

[0063] According to an embodiment of the present invention, the first extracellular region comprises a first binding protein or a fragment thereof that binds to HLA-G protein.

[0064] In an alternative embodiment of the present invention, the first extracellular region may comprise one or more first binding proteins or fragments thereof.

[0065] According to an embodiment of the present invention, the first binding protein or a fragment thereof comprises at least one of an antibody or a functional fragment thereof, and a receptor.

[0066] According to an embodiment of the present invention, the first binding protein or a fragment thereof comprises a first binding fragment and / or a second binding fragment; the first binding fragment is an extracellular region of an ILT2 protein or an active fragment thereof; the second binding fragment is an extracellular region of an ILT4 protein or an active fragment thereof.

[0067] It should be noted that the extracellular regions (ECDs) of both ILT2 and ILT4 proteins can be divided into 4 Domains, named Domain 1 (D1), Domain 2 (D2), Domain 3 (D3), and Domain 4 (D4), respectively.

[0068] In an alternative embodiment of the present invention, when the first extracellular region comprises a plurality of 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.

[0069] 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 an amino acid sequence as shown in SEQ ID NO:2; the ILT2-D2 fragment has an amino acid sequence as shown in SEQ ID NO:3; the ILT2-D3 fragment has an amino acid sequence as shown in SEQ ID NO:4; the ILT2-D4 fragment has an amino acid sequence as shown in SEQ ID NO:5.

[0070] PKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKGQFPIPSITWEHTGRYRCYYGSDT AGRSESSDPLE (SEQ ID NO:2);

[0071] LVVTGAYIKPTLSAQPSPVVNSGGNVTLQCDSQVAFDGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRW WYRCYAYDSNSPYEWSLPSDLLELLVLG(SEQ ID NO:3);

[0072] VSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCY GAHNLSSEWSAPSD(SEQ ID NO:4);

[0073] PLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVT SAHAGTYRCYGSQSSKPYLLT(SEQ ID NO:5).

[0074] In an exemplary embodiment of the present invention, when the first extracellular region comprises a plurality of first binding proteins or fragments thereof, the plurality of first binding proteins or fragments thereof may all be ILT2-D1 fragments, or may be an ILT2-D1 fragment and an ILT2-D2 fragment respectively.

[0075] According to an embodiment of the present invention, the N-terminus to the C-terminus of the extracellular region of the ILT2 protein is successively an ILT2-D1 fragment, an ILT2-D2 fragment, an ILT2-D3 fragment and an ILT2-D4 fragment.

[0076] 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.

[0077] PKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKGQFPIPSITWEHTGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVTLQCDSQVAFDGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLT (SEQ ID NO:6).

[0078] According to an embodiment of the present invention, the first binding fragment is selected from at least one of the ILT2-D1 fragment, ILT2-D2 fragment, ILT2-D3 fragment, and ILT2-D4 fragment; or the extracellular region of the ILT2 protein.

[0079] 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.

[0080] 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.

[0081] According to an embodiment of the present invention, the first binding fragment is selected from the extracellular region of the ILT2 protein.

[0082] According to an embodiment of the present invention, the extracellular region of the ILT4 protein is composed of the ILT4-D1 fragment, ILT4-D2 fragment, ILT4-D3 fragment, and 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.

[0083] PKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRA RWSELS (SEQ ID NO:7);

[0084] DPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSP NRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSV (SEQ ID NO:8);

[0085] QPGPVMAPGESLTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSS ECSAPSDPLDILIT (SEQ ID NO:9);

[0086] GQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRC YGSLNSDPYLLSHPSEPLELVVS (SEQ ID NO:10).

[0087] In an exemplary embodiment of the present invention, when the first extracellular region comprises a plurality of first binding proteins or fragments thereof, the plurality of first binding proteins or fragments thereof may all be ILT4-D1 fragments, or may be an ILT4-D1 fragment and an ILT4-D2 fragment respectively.

[0088] According to an embodiment of the present invention, the extracellular region of the ILT4 protein has the amino acid sequence shown in SEQ ID NO:11.

[0089] PKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRA RWSELS(ILT4-D1)DPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEDEHPQCLNSQPHARGSSR AIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSV(ILT4-D2)QPGPVMAPGESLTLQCVSDVGY DRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLDILIT(ILT4-D3)GQIRGTP FISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPY LLSHPSEPLELVVS(SEQ ID NO:11).

[0090] In an exemplary embodiment of the present invention, when the first extracellular region includes a plurality of first binding proteins or fragments thereof, the plurality of 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, and the specific types are not limited and are all within the protection scope of the present invention.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] According to an embodiment of the present invention, the second binding fragment is selected from the extracellular region of the ILT4 protein.

[0095] 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 linker peptide, and any two of the first binding proteins or fragments thereof are connected with or without the first linker peptide.

[0096] As used herein, the term "any two of the first binding proteins or fragments thereof are connected with or without the first linker peptide" means that any two of the first binding proteins or fragments thereof may be connected by the first linker peptide, or any two of the first binding proteins or fragments thereof may not be connected by the first linker peptide. When there are more than three first binding proteins or fragments thereof, two of the first binding proteins or fragments thereof may be connected by the first linker peptide, and the other two may not be connected by the first linker peptide. The specific connection type is not limited and is within the protection scope of the present invention.

[0097] In an alternative 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, from the N-terminus to the C-terminus, the first binding protein or fragment thereof, the first binding protein or fragment thereof.

[0098] In an alternative 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, from the N-terminus to the C-terminus, the first binding protein or fragment thereof, the first linker peptide, the first binding protein or fragment thereof.

[0099] In an alternative embodiment of the present invention, when the first extracellular region comprises three of the first binding proteins or fragments thereof, the first extracellular region is, from the N-terminus to the C-terminus, the first binding protein or fragment thereof, the first binding protein or fragment thereof, the first binding protein or fragment thereof.

[0100] In an alternative embodiment of the present invention, when the first extracellular region comprises three of the first binding proteins or fragments thereof, the first extracellular region is, from the N-terminus to the C-terminus, the first binding protein or fragment thereof, the first binding protein or fragment thereof, the first linker peptide, the first binding protein or fragment thereof.

[0101] In an alternative embodiment of the present invention, when the first extracellular region comprises three of the first binding proteins or fragments thereof, the first extracellular region is, from the N-terminus to the C-terminus, the first binding protein or fragment thereof, the first linker peptide, the first binding protein or fragment thereof, the first binding protein or fragment thereof.

[0102] In an alternative embodiment of the present invention, when the first extracellular region comprises three of the first binding proteins or fragments thereof, the first extracellular region is, from the N-terminus to the C-terminus, the first binding protein or fragment thereof, the first linker peptide, the first binding protein or fragment thereof, the first linker peptide, the first binding protein or fragment thereof.

[0103] According to an embodiment of the present invention, 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.

[0104] It should be noted that the "connection" herein can be direct connection or indirect connection, and the specific type is not limited, and all are within the protection scope of the present invention. Exemplarily, "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 by a first linker peptide).

[0105] According to an embodiment of the present invention, 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 by the first linker peptide.

[0106] 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 linker peptide, the C-terminus of the first linker 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 linker peptide, and the C-terminus of the first linker peptide is connected to the N-terminus of the first binding fragment.

[0107] According to an embodiment of the present invention, the amino acid sequence of the first linker peptide is (GGGGS)n, where n is any integer between 1 and 10.

[0108] According to an embodiment of the present invention, n is 1, 2, 3 or 4.

[0109] According to an embodiment of the present invention, the amino acid sequence of the first linker peptide is GGGGS (SEQ ID NO: 22).

[0110] 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 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; ILT2-D2 fragment + first linker peptide + ILT4-D2 fragment; ILT2-D2 fragment + first linker peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT2-D2 fragment + first linker peptide + extracellular region of ILT4 protein; ILT2-D2 fragment + first linker peptide + ILT2-D1 fragment; ILT2-D2 fragment + first linker peptide + ILT2-D2 fragment; ILT2-D2 fragment + first linker peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT2-D2 fragment + first linker peptide + extracellular region of ILT2 protein; ILT4-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 the ILT4 protein + the first linker peptide + the ILT4-D2 fragment; the extracellular region of the ILT4 protein + the first linker peptide + the ILT4-D1 fragment + the ILT4-D2 fragment; the extracellular region of the ILT4 protein + the first linker peptide + the extracellular region of the ILT4 protein; the extracellular region of the ILT4 protein + the first linker peptide + the ILT2-D1 fragment; the extracellular region of the ILT4 protein + the first linker peptide + the ILT2-D2 fragment; the extracellular region of the ILT4 protein + the first linker peptide + the ILT2-D1 fragment + the ILT2-D2 fragment; the extracellular region of the ILT4 protein + the first linker peptide + the extracellular region of the ILT2 protein.

[0111] It should be noted that the above-mentioned first extracellular region refers to the connection mode from the N-terminus to the C-terminus. Exemplarily, "the first extracellular region is selected from the ILT2-D1 fragment + the ILT2-D2 fragment + the first linker peptide + the ILT4-D1 fragment + the ILT4-D2 fragment" means that the first extracellular region sequentially includes the ILT2-D1 fragment, the ILT2-D2 fragment, the first linker peptide, the ILT4-D1 fragment, and the ILT4-D2 fragment from the N-terminus to the C-terminus.

[0112] According to an embodiment of the present invention, the first extracellular region is selected from one of the following: 1) the extracellular region of the ILT2 protein; 2) the extracellular region of the ILT4 protein; 3) the ILT2-D1 fragment and the ILT2-D2 fragment in sequence from the N-terminus to the C-terminus; 4) the ILT4-D1 fragment and the ILT4-D2 fragment in sequence from the N-terminus to the C-terminus; 5) the ILT2-D1 fragment, the ILT2-D2 fragment, the first linker peptide, the ILT4-D1 fragment, and the ILT4-D2 fragment in sequence from the N-terminus to the C-terminus; 6) the ILT4-D1 fragment, the ILT4-D2 fragment, the first linker peptide, the ILT2-D1 fragment, and the ILT2-D2 fragment in sequence from the N-terminus to the C-terminus.

[0113] 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 intracellular domain of an activating immune receptor, and an intracellular domain of an inhibitory immune receptor.

[0114] 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.

[0115] According to an embodiment of the present invention, the Gal4-VP64 has an amino acid sequence as shown in SEQ ID NO: 12.

[0116] MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLD MILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGG SDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS(SEQ IDNO:12).

[0117] 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 and obtain the above-mentioned chimeric polypeptide.

[0118] According to an embodiment of the present invention, the first nucleic acid molecule is DNA.

[0119] It should be noted that for the first nucleic acid molecule mentioned herein, those skilled in the art should understand that it actually includes any one or both of the complementary double strands. For convenience, in this article, although only one strand is given in most cases, the other complementary strand is actually also disclosed. In addition, the molecular sequences in the present invention include DNA form or RNA form, and the disclosure of one means the disclosure of the other.

[0120] In a third aspect of the present invention, 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 the above-mentioned first nucleic acid molecule is ligated to the expression vector, the first nucleic acid molecule can be directly or indirectly connected to the control elements on the expression vector, as long as these control elements can control the translation and expression of the first nucleic acid molecule, etc. Of course, these control elements can directly come from the vector itself or be exogenous, that is, not from the vector itself. Of course, the first nucleic acid molecule and the control elements can be operably linked.

[0121] As used herein, "operatively linked" refers to the connection of a foreign gene to an expression vector such that control elements within the expression vector, such as transcriptional control sequences and translational control sequences, etc., can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors can be, for example, plasmids, phages, etc. After the vector according to some specific embodiments of the present invention is introduced into a suitable recipient cell (also referred to as a receptor cell or host cell), the expression of the aforementioned chimeric polypeptide can be effectively achieved under the mediation of a regulatory system.

[0122] 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 phage.

[0123] According to an embodiment of the present invention, the first expression vector is a plasmid expression vector.

[0124] Second nucleic acid molecule, second expression vector

[0125] In a fourth aspect of the present invention, 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 targeting a first molecule, and the first nucleic acid fragment is used to bind to a 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 described in the first aspect. The second nucleic acid molecule of the present invention can bind to the first intracellular region in the aforementioned chimeric polypeptide, thereby activating and inducing the expression of the antigen chimeric receptor.

[0126] Furthermore, the use of this 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 protein, and after binding to HLA-G protein, they can achieve various different types of signal outputs, such as the activation of the expression of specific genes, etc. In particular, they can activate the expression of chimeric antigen receptors containing factors with therapeutic effects (such as treating tumors, etc.), for the treatment of diseases such as tumors. In particular, recombinant immune cells for treating tumors can be prepared, which can broadly recognize numerous tumor cells, etc., greatly improving the killing efficiency and accuracy of various tumor cells, overcoming the problems of non-specific tumor recognition and incomplete tumor recognition coverage in the current clinical application of cell therapy, thereby providing a new means for tumor treatment with broad application prospects.

[0127] It should be noted that the term "inducing the expression of an antigen chimeric receptor" means that the second nucleic acid fragment can encode the antigen chimeric receptor.

[0128] According to an embodiment of the present invention, the antigen chimeric receptor comprises: a second extracellular region having a first molecular binding activity, wherein the first molecule is not an HLA-G protein; a second transmembrane region, the N-terminus of which is connected to the C-terminus of the second extracellular region; and a second intracellular region, the N-terminus of which is connected to the C-terminus of the second transmembrane region.

[0129] According to an embodiment of the present invention, 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.

[0130] As used herein, the term "tumor antigen" generally refers to antigenic substances that newly appear or are overexpressed during tumorigenesis and development. According to the classification method based on tumor antigen specificity, it is divided into tumor specific antigen and tumor-associated antigen. Among them, tumor specific antigen (TSA) is a new antigen unique to tumor cells or only present in certain tumor cells but not in normal cells, and tumor-associated antigen (TAA) refers to an antigen that is not unique to tumor cells and is also present in normal cells and other tissues, but its content increases significantly during cell carcinogenesis. Among them, it includes but is not limited to PD-L1, PD-1, TGF-β, CEA, GD2, and GD3, etc.

[0131] As used herein, the term "cell receptor" or "receptor" should be understood in a broad sense and can refer to a molecule located on the cell membrane that can recognize various extracellular signal molecules (ligands) and bind to them, including but not limited to growth factor receptors (such as VEGF receptor), (NKG2D polypeptide (receptor of MICA, MICB, and ULBP1-6)), cytokine receptors (such as IL-13 receptor, IL-2 receptor, etc.), epidermal growth factor (EGF) receptor, Her2, CD27, natural cytotoxic receptor (NCR) (such as NKp30 (NCR3 / CD337) polypeptide (receptor of HLA-B associated transcript 3 (BAT3) and B7-H6)), T cell antigen receptor, dihydrofolate receptor, chimeric cytokine receptor, Fc receptor, extracellular matrix receptor (such as integrin), cell adhesion receptor (such as cadherin), immunomodulatory receptor (including positive co-receptor (such as CD28) and negative (immunosuppressive) co-receptor (such as PD1)), and receptor of immunomodulatory molecule (such as TGFβ), etc.

[0132] As used herein, the term "ligand of a cell receptor" should be understood in a broad sense and can refer to a chemical substance that can bind to, interact with, and produce specific biological effects with a cell membrane receptor, 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), etc.), peptide hormones, integrin-binding peptides (e.g., peptides including the sequence Arg-Gly-Asp), N-glycans, etc.

[0133] Exemplarily, the ligand is VEGF and the receptor is the VEGF receptor; or the ligand is heregulin and the receptor is HER2.

[0134] As used herein, the term "cytokine" should be understood in a broad sense and can refer to a class of proteins or small polypeptides that can transmit information between cells and have immunomodulatory and effector functions, such as IL-10. The term "cytokine receptor" should be understood in a broad sense and can refer to a receptor on the cell surface that can bind to a cytokine, such as IL-10R, etc.

[0135] According to an embodiment of the present invention, the tumor antigen includes at least one of tumor-associated antigens and tumor-specific antigens.

[0136] According to an embodiment of the present invention, the tumor antigen is a tumor-specific antigen.

[0137] According to an embodiment of the present invention, the first molecule includes 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.

[0138] 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.

[0139] According to an embodiment of the present invention, the second binding protein or a fragment thereof includes at least one of an antibody or a functional fragment thereof, a receptor, a ligand of the receptor, and a cell adhesion molecule.

[0140] According to an embodiment of the present invention, 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.

[0141] According to an embodiment of the present invention, the antibody or a fragment thereof that binds to the first molecule is a single-chain antibody.

[0142] According to an embodiment of the present invention, the activating receptor is selected from the receptors on the surface of NK cells.

[0143] 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, KLRG1.

[0144] According to an embodiment of the present invention, the activating receptor is selected from NKG2D and / or NKp30.

[0145] 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.

[0146] According to an embodiment of the present invention, the extracellular region of NKG2D has the amino acid sequence shown in SEQ ID NO: 13.

[0147] SLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV(SEQ IDNO:13).

[0148] According to an embodiment of the present invention, the extracellular region of NKp30 has the amino acid sequence shown in SEQ ID NO: 14.

[0149] LWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLGAGTV(SEQ ID NO:14).

[0150] According to an embodiment of the present invention, 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.

[0151] According to an embodiment of the present invention, 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.

[0152] According to an embodiment of the present invention, the second extracellular region further includes a second linker peptide, and the third binding fragment and the fourth binding fragment are connected by the second linker peptide.

[0153] 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 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.

[0154] According to an embodiment of the present invention, the amino acid sequence of the second linker peptide is (GGGGS)n, where n is any integer between 0 and 10.

[0155] According to an embodiment of the present invention, n is 0, 1, 2, 3 or 4.

[0156] According to an embodiment of the present invention, the amino acid sequence of the second linker peptide is GGGGS.

[0157] According to an embodiment of the present invention, the second extracellular region further includes a hinge region.

[0158] 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 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.

[0159] According to an embodiment of the present invention, 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.

[0160] As used herein, the term "immunoglobulin" refers to a globulin having antibody (Ab) activity or a chemical structure similar to that of an antibody molecule, and is a tetrapeptide chain structure formed by two identical light chains and two identical heavy chains linked by interchain disulfide bonds. It includes immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), and immunoglobulin E (IgE).

[0161] According to an embodiment of the present invention, the hinge region has the amino acid sequence shown in SEQ ID NO: 15.

[0162] GGGGSGGGGDESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 15).

[0163] 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.

[0164] According to an embodiment of the present invention, the second transmembrane region is selected from the transmembrane region of the CD8α molecule.

[0165] According to an embodiment of the present invention, the second transmembrane region has the amino acid sequence shown in SEQ ID NO: 16.

[0166] IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:16).

[0167] According to an embodiment of the present invention, the second intracellular region includes an intracellular signaling domain and a co-stimulatory domain.

[0168] According to an embodiment of the present invention, the intracellular signaling domain is selected from at least one of the intracellular signaling domains of CD3ζ molecule and the intracellular signaling domain of FcεRIγ molecule.

[0169] According to an embodiment of the present invention, the co-stimulatory domain is selected from at least one of the intracellular signaling domains of 4-1BB molecule, the intracellular signaling domain of CD28 molecule, the intracellular signaling domain of CD27 molecule, the intracellular signaling domain of CD40 molecule, the intracellular signaling domain of OX40 molecule, the intracellular signaling domain of ICOS molecule, the intracellular signaling domain of DAP10 molecule, the intracellular signaling domain of DAP12 molecule, and the intracellular signaling domain of DNAM-1 molecule.

[0170] According to an embodiment of the present invention, the second intracellular region has an amino acid sequence as shown in SEQ ID NO:17.

[0171] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R (SEQ ID NO:17).

[0172] According to an embodiment of the present invention, the second nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:18.

[0173]

[0174] According to an embodiment of the present invention, the second nucleic acid molecule further comprises 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.

[0175] According to an embodiment of the present invention, the signal peptide is selected from at least one of the signal peptides of CD8α molecule, IgG molecule, and CD28 molecule.

[0176] According to an embodiment of the present invention, the signal peptide has the amino acid sequence shown in SEQ ID NO:19.

[0177] MALPVTALLLPLALLLHAARP (SEQ ID NO:19).

[0178] According to an embodiment of the present invention, the third nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO:20.

[0179] ATGGCTCTGCCCGTGACAGCTCTGCTCCTGCCTCTCGCCCTCCTCCTGCACGCCGCTAGACCC (SEQ ID NO:20).

[0180] In an alternative embodiment of the present invention, the intracellular region is GaL4-VP64, and the first nucleic acid fragment is the UAS-minimal-CMV sequence. The UAS-minimal-CMV sequence has the nucleic acid sequence shown in SEQ ID NO:21.

[0181] According to an embodiment of the present invention, the first nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO:21.

[0182] GGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGGAGCATGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGACTAGTTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGC (SEQ ID NO:21).

[0183] In the fifth aspect of the present invention, a second expression vector is provided. 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 the embodiment of the present invention can express the antigen chimeric receptor in the above-mentioned second nucleic acid molecule.

[0184] 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 phage.

[0185] According to an embodiment of the present invention, the second expression vector is a plasmid expression vector.

[0186] Recombinant immune cell

[0187] In the sixth aspect of the present invention, a recombinant immune cell is provided. According to an embodiment of the present invention, the recombinant immune cell includes: 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 according to the embodiment of the present invention can, under suitable conditions, express the aforementioned chimeric polypeptide on the surface of the recombinant immune cell, and can recognize the HLA-G protein. After binding to the HLA-G protein, it can achieve various different signal output types, such as the activation of the expression of specific genes, etc. In particular, it can activate the expression of chimeric antigen receptors containing factors with therapeutic effects (such as treating tumors, etc.) for treating diseases such as tumors.

[0188] It should be noted that the "suitable conditions" described in the present invention refer to the conditions suitable for the expression of the aforementioned chimeric polypeptide. It is easily understood by those skilled in the art that the conditions suitable for the expression of the aforementioned chimeric polypeptide include but are not limited to suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell states, suitable cell densities, suitable cell culture environments, and suitable cell culture times. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the chimeric polypeptide according to the specific environment of the laboratory.

[0189] 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.

[0190] According to an embodiment of the present invention, the recombinant immune cell further comprises a chimeric antigen receptor, which is identical to the antigen chimeric receptor encoded by the second nucleic acid fragment defined in the second nucleic acid molecule of the fourth aspect. Under suitable conditions, the recombinant immune cell of the embodiment of the present invention can simultaneously express the aforementioned chimeric polypeptide and the chimeric antigen receptor in the second nucleic acid molecule on the surface of the recombinant immune cell. Thus, the recombinant immune cell has multi-targeting properties and can recognize HLA-G protein. After binding to HLA-G protein, it can achieve various different signal output types, such as activation of the expression of specific genes, etc. In particular, it can activate the expression of chimeric antigen receptors containing factors with therapeutic effects (such as treating tumors, etc.), and is used to treat diseases such as tumors. In particular, recombinant immune cells for treating tumors can be prepared, which can broadly recognize numerous tumor cells, etc., greatly improving the killing efficiency and accuracy of various tumor cells, overcoming the problems of non-specific tumor recognition and incomplete tumor recognition coverage in the clinical application of existing cell therapies, thereby providing a new means for tumor treatment with broad application prospects.

[0191] In particular, when the chimeric polypeptide of the present invention is used in combination with the chimeric antigen receptor, and recombinant immune cells (such as Jurkat cells and NK-92 cells) are co-transformed and prepared, it is found that the immune cells with the combination of chimeric polypeptide + CAR / TCR can recognize the corresponding ligands and efficiently activate the immune cells (such as Jurkat cells and NK-92 cells) to perform the function of killing targets (such as tumor cells, etc.).

[0192] 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 of the fifth aspect into a host cell.

[0193] According to an embodiment of the present invention, the host cell includes at least one of immune cells, neurons, progenitor cells or precursor cells, epithelial cells, endothelial cells, and stem cells.

[0194] 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.

[0195] Drug composition

[0196] In the seventh aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the recombinant immune cells described in the sixth aspect. As can be seen from the foregoing, the cells expressing the above chimeric polypeptide can recognize HLA-G protein, and after binding to HLA-G protein, they can achieve various different signal output types, such as activation and inhibition of the expression of specific genes, etc. Alternatively, after the immune cells expressing the chimeric polypeptide contact HLA-G protein, they can secrete therapeutic factors for anti-tumor treatment. Therefore, the pharmaceutical composition containing the aforementioned recombinant immune cells can target HLA-G protein and be used for the prevention and / or treatment of related diseases, such as cancer and the like.

[0197] According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0198] Use

[0199] In the eighth aspect of the present invention, the present invention provides the use of the recombinant immune cells described in the sixth aspect or the pharmaceutical composition described in the seventh aspect in the preparation of a drug for the prevention and / or treatment of diseases.

[0200] According to an embodiment of the present invention, the diseases include cancer or tumor, autoimmune diseases, inflammation, and related diseases caused by cellular senescence.

[0201] As used herein, the terms "cancer" or "tumor" can refer to any unregulated cell growth. Exemplarily, it can be 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, cholangiocarcinoma or sarcoma, acute myeloid leukemia, lymphoma, 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.

[0202] Method for treating or preventing diseases

[0203] In a ninth aspect of the present invention, the present invention provides a method for treating or preventing a disease. According to an embodiment of the present invention, the method comprises: administering to a subject a pharmaceutically acceptable amount of the recombinant immune cell described in the sixth aspect or the pharmaceutical composition described in the seventh aspect. As can be seen from the foregoing, cells expressing the above chimeric polypeptide can recognize HLA-G protein, and after binding to HLA-G protein, they can achieve various different signal output types, such as activation and inhibition of the expression of specific genes, etc., or immune cells expressing the chimeric polypeptide can secrete therapeutically active factors after contacting HLA-G protein for anti-tumor purposes. Therefore, a pharmaceutical composition containing the aforementioned recombinant immune cells can target HLA-G protein for preventing and / or treating related diseases, such as cancer and the like.

[0204] 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, etc. The selection of a preferred effective amount can be determined by those of ordinary skill in the art according to various factors (for example, through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's body weight, the patient's immune status, the route of administration, etc. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be proportionally reduced.

[0205] The recombinant protein or pharmaceutical composition of the present invention can be incorporated into a drug suitable for parenteral administration (such as intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. For example, liquid, semi-solid and solid dosage forms, etc., including but not limited to liquid solutions (such as injection solutions and infusion solutions) or lyophilized powders. A typical drug is in the form of an injection solution or an infusion solution. The aforementioned recombinant protein or pharmaceutical composition can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

[0206] According to an embodiment of the present invention, the route of administration of the method is subcutaneous injection or intravenous injection.

[0207] According to an embodiment of the present invention, the disease includes cancer or tumor, immune-related diseases. According to an embodiment of the present invention, the disease includes cancer or tumor, autoimmune diseases, inflammation and related diseases caused by cellular senescence.

[0208] According to embodiments 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, cholangiocarcinoma or sarcoma, acute myeloid leukemia, lymphoma, 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.

[0209] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0210] Example 1: Design and construction of a SynNotch receptor specifically targeting and binding to human HLA-G

[0211] ILT2 and ILT4 receptors are two receptors on the surface of immune cells responsible for binding to HLA-G molecules. Among them, the ILT4 receptor is only expressed in immune cells derived from myeloid lineage, while the ILT2 receptor is expressed on both immune cells derived from lymphoid lineage and myeloid lineage. Both ILT2 and ILT4 receptors are divided into an extracellular region, a transmembrane region, and an intracellular region, where the extracellular region is the functional region responsible for recognizing and binding to HLA-G. Moreover, the extracellular regions of both ILT2 and ILT4 receptors are divided into 4 Domains, named Domain 1 (D1), Domain 2 (D2), Domain 3 (D3), and Domain 4 (D4) respectively.

[0212] Based on the characteristics of the binding of ILT2 and ILT4 to HLA-G, the inventors designed and synthesized a series of synthetic polypeptides that can specifically bind to human HLA-G. The synthetic polypeptides are composed of polypeptide sequences derived from the ILT2 and ILT4 receptors. Since the polypeptide sequences derived from ILT2 and ILT4 both have the ability to bind to HLA-G, the binding ability of the synthetic polypeptides designed and synthesized by us can be maximally enhanced. Taking this synthetic polypeptide 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:2 and 3), a signal peptide sequence derived from CD8α (the amino acid sequence is shown in SEQ ID NO:19) is linked to its N-terminus, and a Xenopus tropicalis Notch transmembrane domain sequence (the amino acid sequence is shown in SEQ ID NO:1) and a Gal4-VP64 artificial transcription factor sequence (the amino acid sequence is shown in SEQ ID NO:12) are sequentially linked to its C-terminus, finally forming a series of SynNotch receptor sequences, see Figure 1 These series of sequences were respectively cloned into the pCDH vector, viruses were respectively produced, and NK92 cells or Jurkat cells were infected, and stable transfected cell lines were respectively established.

[0213] Example 2: Design and construction of a CAR receptor that specifically binds to human NKG2D ligand and NKp30 ligand

[0214] The 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. The ligands of NKG2D and NKp30 are widely expressed on the surface of many tumor cells and are not expressed or are expressed at low levels in most normal tissues. Both the NKG2D and NKp30 receptors are divided into an extracellular region, a transmembrane region, and an intracellular region, and their extracellular regions are functional regions responsible for recognizing and binding to the corresponding ligands.

[0215] Based on the property that NKG2D and NKp30 can bind to the above ligands, the inventors designed and synthesized synthetic polypeptides that can target and bind to human NKG2D ligands and NKp30 ligands. These synthetic polypeptides are composed of the extracellular polypeptide sequences of NKp30 and NKG2D in combination, and can bind to the ligands of NKG2D and NKp30 simultaneously. Using this synthetic polypeptide as the extracellular region of the CAR receptor (the C-terminus of the extracellular region of the NKp30 receptor is connected to the N-terminus of the extracellular region of the NKG2D receptor, where the amino acid sequence of the extracellular region of the NKp30 receptor is shown in SEQ ID NO:14 and the amino acid sequence of the extracellular region of the NKG2D receptor is shown in SEQ ID NO:13), a signal peptide sequence derived from CD8α (amino acid sequence shown in SEQ ID NO:19, nucleotide sequence shown in SEQ ID NO:20) is connected to its N-terminus, and a hinge region sequence derived from CD8α (amino acid sequence shown in SEQ ID NO:15), a transmembrane domain sequence derived from CD8α (amino acid sequence shown in SEQ ID NO:16), a 4-1BB sequence, and a CD3ζ sequence (the amino acid sequences of 4-1BB and CD3ζ are shown in SEQ ID NO:17) are sequentially connected to its C-terminus, ultimately forming the CAR receptor sequence. Meanwhile, a UAS-mini-CMV sequence (nucleotide sequence shown in SEQ ID NO:21) is added before the nucleotide sequence encoding the CAR receptor, see Figure 2 This sequence was cloned into the pCDH vector with the promoter removed to produce the virus, and the stably transfected NK92 cells established in Example 1 were infected respectively to establish stably transfected cell lines transfected with the two receptor nucleic acid sequences, named the corresponding Syn-CAR-NK92 cell line and Syn-CAR-Jurkat cell line. Meanwhile, the CAR receptor without the UAS-mini-CMV sequence was cloned into the pCDH vector with a promoter, the virus was produced and used to infect NK92 and Jurkat cells to establish stably transfected cell lines as the control of Syn-CAR-NK92 / Jurkat cells, named CAR-NK92 cells and CAR-Jurkat cells.

[0216] Example 3: In vitro experimental verification of the precise and efficient killing of target cells by Syn-CAR-NK cells

[0217] Next, the in vitro killing activities of different types of Syn-CAR-NK92 cells obtained in Example 2 were tested by PI and CFSE staining. Specifically, for the tumor cell line K562 (which has all the targets required for the activation of Syn-CAR-NK92), after this target cell line was stained with CSFE fluorescence, at 2×10 per well 4The inoculation concentration of cells / ml was spread on the 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 the cell suspension of Syn-CAR-NK92 cells obtained in Example 2 that needed to simultaneously target HLA-G, NKG2D ligands and NKp30 ligands; the blank control group was added with NK cells infected with empty vector virus. In the above-mentioned experimental group, Syn-CAR-NK92 cells were mixed with target cells for 48 hours according to a 1:2 effector-target ratio (here, the term "effector-target ratio" refers to the number ratio of effector cells, i.e., Syn-CAR-NK92 cells that simultaneously target HLA-G, NKG2D ligands and NKp30 ligands, and target cells, i.e., tumor cells). After 48 hours of culture, the supernatant was removed by centrifugation, the cell precipitate was washed and stained with PI, the stained cells were obtained by flow cytometry, and the results were analyzed by FlowJo software. The tumor cell killing rate test results with leukemia cells K562 as target cells are shown in the following figure. Figure 3 As shown. Figure 3 It can be seen that Syn-CAR-NK92 cells have a significant killing effect on leukemia cells K562 (significantly higher than the control group), among which 5-Syn-CAR-NK92 and 6-Syn-CAR-NK92 have the highest killing efficiency.

[0218] For the tumor cell line Aspc-1 (only with the target of activating SynNotch) and the immortalized normal cell line THLE3 (without any target of 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 then the growth of the target cells was detected by RTAC instrument. The results showed that the experimental group cells of the present invention had no obvious killing effect on pancreatic cancer cells Aspc-1 and Aspc-1 that were negative for NKG2D ligand and NKp30 ligand (similar to the killing effect of the control group). Among them, the results of 6-Syn-CAR-NK92 are exemplified in this example. The tumor cell killing rate test results with pancreatic cancer Aspc-1 as the target cell are as follows Figure 4 As shown, the killing rate test results using immortalized liver cell lines as target cells are as follows Figure 5 shown.

[0219] from Figure 4 It can be seen that 6-Syn-CAR-NK cells have no obvious killing effect on pancreatic cancer cells Aspc-1 that are negative for NKG2D ligands and NKp30 ligands (the killing effect is similar to that of the control group). Figure 5It can be seen that 6-Syn-CAR-NK cells have no obvious killing enhancement effect on immortalized hepatocyte THLE3 cells that are negative for HLA-G, NKG2D ligands and NKp30 ligands (similar to the killing effect 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 a target, and can more accurately distinguish normal cells from tumor cells.

[0220] Example 4: In vivo experimental verification of Syn-CAR-NK cells' precise and efficient killing of target cells

[0221] 1. Take 1×10 7 NCI-H716 cells were subcutaneously injected into 5-week-old NCG mice. After 10 days of tumor bearing, the tumor size was measured by vernier calipers. Mice with similar tumor formation were randomly divided into 9 groups, six of which were experimental groups (i.e., different types of Syn-CAR-NK92 cells obtained in Example 2), and the remaining three groups were control groups (one group was untreated control, one group was ordinary NK92 treatment control, and one group was CAR-NK92 treatment control). Afterwards, 1×10 7 A control group of mice was injected with 1×10 6-Syn-CAR-NK92 cells into the tail vein. 7 A control group of mice were injected with 1×10 7 CAR-NK92 cells were injected into the tail vein of a control group of mice with the same volume of saline. Each group was treated / treated once a week with the same treatment method, and the size of the subcutaneous transplanted tumor was measured. During this period, IL2 was injected intraperitoneally once every 3 days, with 5×10 per mouse each time. 4 U, a total of 3 treatments. The results showed that the Syn-CAR-NK92 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 NK92 control group), and its killing efficiency was not significantly different from that of the single CAR-NK92 group. Among them, the results of 6-Syn-CAR-NK92 are exemplified in this example, see Figure 6 (Results of the test on the killing rate of 6-Syn-CAR-NK92 cells on colon cancer cells NCI-H716 after the cells were used as target cells for tumor loading).

[0222] from Figure 6It can be seen that the 6-Syn-CAR-NK92 cells of Example 4 have obvious killing effects on target cells - colon cancer cells NCI-H716 that simultaneously express HLA-G, NKG2D ligands, and NKp30 ligands in vivo (significantly higher than the NK92 control group), and their killing efficiency has no significant difference from that of the single CAR-NK92 group, indicating that the SynNotch receptor does not reduce its killing efficiency.

[0223] 2. Further, 1×10 7 Aspc-1 cells were subcutaneously injected into 5-week-old NCG mice. Ten days after tumor bearing, the tumor size was measured with a vernier caliper. Mice with similar tumor formation degrees were randomly divided into 3 groups. One group was the experimental group of 6-Syn-CAR-NK92 cells, and the remaining two groups were control groups (one was the untreated control group, and the other was the ordinary NK92 treatment control group). Then, 1×10 7 6-Syn-CAR-NK92 cells were injected into the tail vein of the experimental group mice, 1×10 7 ordinary NK92 cells were injected into the tail vein of one group of control group mice, and the same volume of normal saline as the above three groups was injected into the tail vein of one group of control group mice. Then, each group was treated / treated in the same way once a week, and the size of subcutaneous transplanted tumors was measured at the same time. During this period, IL2 was intraperitoneally injected once every 3 days, 5×10 4 U per mouse, for a total of 3 treatments. After tumor bearing with pancreatic cancer cells Aspc-1 as target cells, the test results of the killing rate of 6-Syn-CAR-NK92 cells against them are as Figure 7 shown.

[0224] From Figure 7 it can be seen that the 6-Syn-CAR-NK92 cells of Example 4 have no obvious enhanced killing effect on target cells - pancreatic cancer cells Aspc-1 that only express HLA-G in vivo (there is no improvement in killing efficiency compared with the NK92 control group), indicating that the Syn-CAR system can function well, has no killing effect on cells that only express a single target, and can more accurately distinguish normal cells from tumor cells.

[0225] Example 5: In vitro experimental verification of the precise and efficient killing of target cells by Syn-CAR-Jurkat cells

[0226] Next, the in vitro killing activities of different types of 6-Syn-CAR-Jurkat cells obtained in Example 2 were detected by flow cytometry. Specifically, for the tumor cell line K562 (which has all the targets required for Syn-CAR-Jurkat activation), this target cell line was seeded at 2×10 per well 4Inoculate at a concentration of cells / ml in a culture plate. For the target cell line, 1 experimental group and 1 control group are set correspondingly. Among them, the cell suspension of 6-Syn-CAR-Jurkat cells that need to target HLA-G, NKG2D ligand, and NKp30 ligand obtained in Example 2 is added to the experimental group; the Jurkat cells infected with the empty vector virus are added to the blank control group. In the above experimental group, according to an effector-to-target ratio of 1:2 (here, the term "effector-to-target ratio" refers to the ratio of the number of effector cells, that is, 6-Syn-CAR-Jurkat cells that target HLA-G, NKG2D ligand, and NKp30 ligand simultaneously, to the target cells, that is, tumor cells), mix 6-Syn-CAR-Jurkat cells with the target cells for 48 hours. After culturing for 48 hours, centrifuge to remove the supernatant, wash the cell pellet, and then stain with TNF-α, IFN-γ, and CD69 antibody. Use a flow cytometer to obtain the stained cells, and use FlowJo software to analyze the results. Use leukemia cell K562 as the target cell to test the activation effect of Jurkat cells. The results show that 6-Syn-CAR-Jurkat cells are significantly activated by leukemia cell K562, and the expressions of TNF-α, IFN-γ, and CD69 are significantly increased (significantly higher than those of the control group). It shows that the designed Syn-CAR system of the present invention can also play a good role in the CAR-T cell system.

[0227] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions 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 a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0228] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

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

2. The chimeric polypeptide according to claim 1, characterized in that, 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 repeat 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 that binds to HLA-G protein or a fragment thereof; 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; ILT2-D2 fragment + first linker peptide + ILT4-D2 fragment; ILT2-D2 fragment + first linker peptide + ILT4-D1 fragment + ILT4-D2 fragment; ILT2-D2 fragment + first linker peptide + extracellular region of ILT4 protein; ILT2-D2 fragment + first linker peptide + ILT2-D1 fragment; ILT2-D2 fragment + first linker peptide + ILT2-D2 fragment; ILT2-D2 fragment + first linker peptide + ILT2-D1 fragment + ILT2-D2 fragment; ILT2-D2 fragment + first linker peptide + extracellular region of ILT2 protein; ILT4-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 ILT2 + the first linker peptide + ILT2-D1 fragment + ILT2-D2 fragment; the extracellular region of ILT2 + the first linker peptide + the extracellular region of ILT2; the extracellular region of ILT4 + the first linker peptide + 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 a 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, wherein the N-terminus of the second intracellular region is 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 its fragment comprises 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; 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 therewith; 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 therewith; 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, 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 comprises 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 comprises an intracellular signaling domain and a co-stimulatory domain; Optionally, the intracellular signaling domain is selected from at least one of the intracellular signaling domain of the CD3ζ molecule and the intracellular signaling domain 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, wherein The second nucleic acid molecule further comprises 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, which 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 in the preparation of a drug for preventing and / or treating a disease; Optionally, the disease includes cancer or tumor, autoimmune disease, inflammation and related diseases caused by cellular senescence.

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