Transgenic recombinant immune cells that specifically target tumors and their applications

Abstract

This invention discloses a transgenic recombinant immune cell that specifically targets tumors. The recombinant immune cell comprises a chimeric polypeptide and a chimeric antigen receptor; the chimeric polypeptide includes a first extracellular region, a first transmembrane region, and a first intracellular region with HLA-G protein binding activity, wherein the N-terminus of the first transmembrane region is connected to the C-terminus of the first extracellular region, and the N-terminus of the first intracellular region is connected to the C-terminus of the first transmembrane region; the first transmembrane region includes the transmembrane region of the Notch receptor protein from Xenopus laevis; the antigen-chimeric receptor does not have HLA-G protein binding activity. This recombinant immune cell significantly improves the precise recognition of tumor cells by immune cells, reduces off-target effects of cell therapy, and accurately and effectively distinguishes tumor cells from normal cells, providing an effective target and targeting method for the treatment of broad-spectrum tumors (especially solid tumors).

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, specifically to transgenic recombinant immune cells that specifically target tumors, their preparation methods, and applications. More specifically, this invention relates to a chimeric polypeptide, a first nucleic acid molecule, a first expression vector, a second nucleic acid molecule, a second expression vector, recombinant immune cells, a pharmaceutical composition, and their uses. Background Technology

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

[0003] CAR-T cell therapy, a representative of immunotherapy, works by modifying the patient's own T cells with chimeric antigen receptors through genetic engineering to create CAR-T cells. These modified chimeric receptors allow CAR-T cells to specifically recognize tumor-associated antigens (tumor cell markers) on the tumor surface, thereby targeting and killing the tumor. Compared to ordinary immune cells, CAR-T cells exhibit higher targeting specificity, killing activity, and persistence. Currently, modified immunotherapy, represented by CAR-T cells targeting CD19 and BCMA, has shown significant efficacy in treating hematologic malignancies such as B-cell lymphoma and is considered one of the most promising cancer treatment methods.

[0004] However, due to the lack of targets that can effectively distinguish between normal and tumor cells, CAR-T cell therapy often inevitably kills some normal cells expressing the target protein, causing damage to normal tissues. Alternatively, the widespread presence of the target can lead to continuous activation of CAR-T cells, resulting in a cytokine storm by releasing large amounts of cytokines. Furthermore, because CAR-T cell therapy typically recognizes only a single antigen, tumor cells can often escape by losing the antigens that CAR-T cells can recognize through gene mutations, leading to drug resistance. Moreover, the high heterogeneity of tumors means that the single target loaded on CAR-T cells cannot fully cover tumor cells, resulting in incomplete or ineffective tumor treatment. These key issues have become major obstacles to the development and expansion of the cell therapy field.

[0005] Recent studies have shown that attempting to address these challenges by finding a single molecule that is both broad-spectrum and specific, as well as more efficient and safer, as a target for CAR therapy may be extremely difficult. This is because broad-spectrum and specificity are two sides of the same coin; a single target cannot simultaneously possess both characteristics, and no target molecule has yet been found in current cancer treatment research that perfectly combines both of these properties. Therefore, attempting to achieve both broad-spectrum and specific immunocellular therapy through a combination of specific target combinations and tools for recognizing and activating these target combinations is one of the most promising new directions in cell therapy. Summary of the Invention

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

[0007] In a first aspect, the present invention provides a chimeric polypeptide. According to embodiments of the present invention, the chimeric polypeptide comprises: 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 laevis or an amino acid sequence having at least 80% identity with it, wherein the N-terminus of the first transmembrane region is linked to the C-terminus of the first extracellular region; and a first intracellular region, wherein the N-terminus of the first intracellular region is linked to the C-terminus of the first transmembrane region. The chimeric polypeptide of the present invention selects the transmembrane region of the Notch receptor protein of Xenopus laevis as the transmembrane region of the chimeric polypeptide (i.e., the SynNotch synthesis receptor), which can improve the ability and efficiency of the chimeric polypeptide to activate downstream gene transcription.

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

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

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

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

[0012] In a sixth aspect, the present invention provides a recombinant immune cell. According to embodiments of the present invention, the recombinant immune cell comprises: carrying the first nucleic acid molecule described in the second aspect or the first expression vector described in the third aspect; or expressing the chimeric polypeptide described in the first aspect. Under suitable conditions, the recombinant immune cell of the embodiments of the present invention can express the aforementioned chimeric polypeptide on its surface, recognize HLA-G protein, and, upon binding to HLA-G protein, can achieve various different signal output types, such as the activation of specific gene expression, especially the activation of the expression of chimeric antigen receptors containing factors with therapeutic effects (e.g., for treating tumors), for the treatment of diseases such as tumors.

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

[0014] In an eighth aspect of the invention, the invention provides for 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 medicament for the prevention and / or treatment of a disease.

[0015] Beneficial effects:

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

[0017] (2) The recombinant immune cells of the present invention can convert the inhibitory signal transmitted after HLA-G binds to ILT2 / 4 into an activation signal, which can not only effectively kill HLA-G positive tumor cells, but also resist the inhibition from the tumor immune microenvironment and reverse the depletion of immune cells.

[0018] (3) The engineered multi-target recombinant immune cells of this invention can not only broadly identify many different types of tumor cells, but also accurately distinguish normal cells, which greatly improves the killing efficiency and accuracy of various tumor cells, thus providing a new approach to tumor immune cell therapy with broad-spectrum and specific potential.

[0019] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

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

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

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

[0023] Figure 3 This is a diagram showing the killing effect of Syn-CAR-NK cells on dual-target positive K562 cells in Example 3 of the present invention;

[0024] Figure 4 This is a diagram showing the killing effect of Syn-CAR-NK cells on single-target positive Aspc-1 cells in Example 3 of the present invention.

[0025] Figure 5 This is a diagram showing the killing effect of Syn-CAR-NK on targetless THLE3 cells in Example 3 of the present invention;

[0026] Figure 6 This is a diagram showing the treatment results of Syn-CAR-NK cells on subcutaneous xenografts of colon cancer mice with dual-target positive tumors in Example 4 of the present invention;

[0027] Figure 7 This is a diagram showing the treatment results of Syn-CAR-NK cells on subcutaneous xenografts of pancreatic cancer mice with single-target positive tumors in Example 4 of the present invention. Detailed Implementation

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

[0029] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0030] Definitions and General Terms

[0031] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this invention, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0033] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0034] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, 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 procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and 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 utilizing these algorithms are also available, including but 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., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0035] In this document, the term "at least 80% identity" refers to an identity of at least 80% with each reference sequence, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0036] In this document, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule to host cells and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having multiple of the aforementioned functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the expression vector, the expression vector can produce the desired expression product.

[0037] In this document, the term "chimeric antigen receptor (CAR)" refers to a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a different polypeptide, and at least one intracellular domain. "Chimeric antigen receptor (CAR)" is also known as "chimeric receptor," "T-body," or "chimeric immune receptor (CIR)." The "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide capable of binding a particular antigen. The "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that transmits signals to activate or inhibit intracellular biological processes.

[0038] In this document, the term "recombinant immune cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this invention and can be used for the expression and / or secretion of target proteins.

[0039] In this document, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and adequately combining an active chimeric peptide or recombinant immune cell with a liquid carrier, a finely fragmented solid carrier, or both.

[0040] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except those incompatible with the chimeric peptides or recombinant immune cells of the present invention, for example, any adverse biological effects they may produce or interactions that may occur in a harmful manner with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0041] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The recombinant immune cells or pharmaceutical compositions of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous or subcutaneous injection.

[0042] In this document, the term "treatment" refers to the administration of a drug to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in individuals susceptible to the disease but not yet diagnosed with it; (b) suppression of a disease, such as inhibiting disease progression; or (c) alleviating a disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of recombinant immune cells, pharmaceutical compositions, or drugs to an individual to treat, cure, alleviate, improve, reduce, or suppress a disease in that individual, including but not limited to administration of drugs containing the chimeric peptides, recombinant immune cells, or pharmaceutical compositions described herein to an individual in need.

[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, said immune cell may contain, or already contains, nucleic acids isolated by this invention, and / or expression vectors, or individual cells, cell lines, or cell cultures capable of expressing the chimeric polypeptides described in this application and optionally chimeric antigen receptors. In this invention, said immune cell may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and / or peripheral blood mononuclear cells. In this document, "C-terminus" and "C-terminus" are synonymous; "N-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] This invention proposes a chimeric polypeptide, a first nucleic acid molecule, a first expression vector, a second nucleic acid molecule, a second expression vector, recombinant immune cells, 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 a first aspect, the present invention provides a chimeric polypeptide. According to embodiments of the present invention, the chimeric polypeptide comprises: 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 laevis or an amino acid sequence having at least 80% identity with it, wherein the N-terminus of the first transmembrane region is linked to the C-terminus of the first extracellular region; and a first intracellular region, wherein the N-terminus of the first intracellular region is linked to the C-terminus of the first transmembrane region. The chimeric polypeptide of the present invention selects the transmembrane region of the Notch receptor protein of Xenopus laevis as the transmembrane region of the chimeric polypeptide (i.e., the SynNotch synthesis 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 tightly linked genes located on the short arm of human chromosome 6. It belongs to class I molecules of the non-classical major histocompatibility complex (MHC) in humans and is characterized by selective tissue distribution. It is expressed in some tumor cell lines, tumor biopsy tissues, some infected tissue cells, and graft cells after heart transplantation.

[0049] Furthermore, the aforementioned chimeric peptide can be used to prepare multi-targeting recombinant immune cells. These recombinant immune cells enable cells expressing the chimeric peptide to recognize HLA-G proteins. After binding to HLA-G proteins, they can achieve various different signal output types, such as the expression activation of specific genes. In particular, they can activate the expression of chimeric antigen receptors containing factors with therapeutic effects (such as tumor treatment) for the treatment of diseases such as tumors.

[0050] In particular, it can prepare recombinant immune cells for the treatment of tumors, which can broadly recognize a wide range of tumor cells, greatly improving the killing efficiency and precision of various tumor cells, overcoming the problems of non-specific tumor recognition and incomplete tumor recognition coverage in existing cell therapy clinical applications, thus providing a new tumor treatment method with broad application prospects.

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

[0052] According to an embodiment of the present invention, the transmembrane region of the Notch receptor protein of the Xenopus tropicalalis has an amino acid sequence as shown in SEQ ID NO:1.

[0053] ILDYGFIGGLGKNITPPDNEEICENEQCAELADNKICNANCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFNDGKCDSQCNNSGCLYDGFDCQKVEVQCNPLYDQYCRDHFQDGHCDQGCNNAECEWDGLDCDNMPENLAEGTLLIVVLMPPEKLKNNSVNFLRE LSRVLHTNVVFKKDSKGEYKIYPYYGNEEELKKHHIKKRSAASWSDAPTAIFSTMKESVLPGRRRRELDQMEVRGSIVYLEIDNRQCYKSSSQCFTSATDVAAFLGALATHGNLNIPYKIEAVKSEIVETAKPPPPLYAMFSMLVIPLLIIFVIMVVIVNKKRRR(SEQ ID NO:1).

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

[0055] According to an embodiment of the present invention, the C-terminus of the epidermal growth factor-like repeat sequence is linked to the N-terminus of the transmembrane region of the Notch receptor protein of Xenopus laevis; and / or, the C-terminus of the transmembrane region of the Notch receptor protein of Xenopus laevis is linked to the N-terminus of the RAM sequence.

[0056] According to an embodiment of the present invention, the epidermal growth factor-like repeat sequence is 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 is 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 an amino acid sequence as shown in SEQ ID NO:1 or 25, or an amino acid sequence having at least 90% identity with it.

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

[0062] VVSPCASRPCYNGGTCQFSPEEPFFQCFCPTNFNGLFCHVVSPCASRPCYNGGTCQFSPEEPFFQCFCPTNFNGLFCHILDYGFIGLGLGKNITPPDNEEICENE QCAELADNKICNANCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFNDGKCDSQCNNSGCLYDGFDCQKVVQCNPLYDQYCRDHFQDGHCDQGCNNAECEWD GLDCDNMPENLAEGTLLIVVLMPPEKLKNNSVNFLRELSRVLHTNVVFKKDSKGEYKIYPYYGNEEELKKHHIKKRSAASWSDAPTAIFSTMKESVLPGRRRRELDQMEVRGSIVYLEIDNRQCYKSSSQCFTSATDVAAFLGALATHGNLNIPYKIEAVKSEIVETAKPPPPLYAMFSMLVIPLLIIFVIMVVIVNKKRRREHGQLWFP EHGQLWFP (SEQ ID NO:25).

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

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

[0065] According to embodiments of the present invention, the first binding protein or a fragment thereof includes 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 includes a first binding fragment and / or a second binding fragment; the first binding fragment is the extracellular region of the ILT2 protein or its active fragment; the second binding fragment is the extracellular region of the ILT4 protein or its active fragment.

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

[0068] In an optional embodiment of the present invention, when the first extracellular region includes a plurality of first binding proteins or fragments thereof, each first binding protein or fragment thereof is independently selected from a first binding fragment or a 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 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; and the ILT2-D4 fragment has the amino acid sequence shown in SEQ ID NO:5.

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

[0071] LVVTGAYIKPTLSAQPSPVVNSGGNVTLQCDSQVAFDGFILKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRW 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 includes 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 ILT2-D1 fragments and ILT2-D2 fragments respectively.

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

[0076] According to an embodiment of the present invention, the extracellular region of the ILT2 protein has an amino acid sequence as shown in SEQ ID NO:6.

[0077] PKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKGQFPIPSITWEHTGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVTLQCDSQVAFDGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLV LGVSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDI LIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLT(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 together.

[0080] According to an embodiment of the present invention, the C-terminus of the ILT2-D1 segment is connected to the N-terminus of the ILT2-D2 segment, or the N-terminus of the ILT2-D1 segment is connected to the C-terminus of the ILT2-D2 segment.

[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 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; and the ILT4-D4 fragment has the amino acid sequence shown in SEQ ID NO:10.

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

[0084] DPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSP 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 includes 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 ILT4-D1 fragments and ILT4-D2 fragments respectively.

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

[0089] PKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRA RWSELS(ILT4-D1)DPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGFILCKEGEDEHPQCLNSQPHARGSSR AIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSV(ILT4-D2)QPGPVMAPGESLTLQCVSDVGY DRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLDILIT(ILT4-D3)GQIRGTP FISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVS (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, or all be second binding fragments, or may be first binding fragments and second binding fragments respectively. The specific type is not limited and is 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, ILT4-D2 fragment, ILT4-D3 fragment and 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, the ILT4-D1 fragment and the ILT4-D2 fragment being connected.

[0093] According to an embodiment of the present invention, the C-terminus of the ILT4-D1 segment is connected to the N-terminus of the ILT4-D2 segment, or the N-terminus of the ILT4-D1 segment is connected to the C-terminus of the ILT4-D2 segment.

[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 contains a plurality of the first binding proteins or fragments thereof, the first extracellular region further includes a first linker peptide, wherein any two of the first binding proteins or fragments thereof are linked together, or not through the first linker peptide.

[0096] In this document, the term "any two first binding proteins or fragments thereof linked by or without the first linker peptide" means that any two first binding proteins or fragments thereof can be linked by the first linker peptide, or that any two first binding proteins or fragments thereof can be linked without the first linker peptide, or that when there are three or more first binding proteins or fragments thereof, two of the first binding proteins or fragments thereof are linked by the first linker peptide and the other two are not linked by the first linker peptide. The specific linking type is not limited and is within the scope of protection of this invention.

[0097] In an optional embodiment of the present invention, when the first extracellular region contains two of the first binding proteins or fragments thereof, the first extracellular region consists of, from the N-terminus to the C-terminus, the first binding protein or fragment thereof, and the first binding protein or fragment thereof.

[0098] In an optional embodiment of the present invention, when the first extracellular region contains two fragments of the first binding protein or thereof, the first extracellular region consists of, from the N-terminus to the C-terminus, the first binding protein or thereof, the first linker peptide, and the first binding protein or thereof.

[0099] In an optional embodiment of the present invention, when the first extracellular region contains three fragments of the first binding protein or thereof, the first extracellular region consists of, from the N-terminus to the C-terminus, the first binding protein or thereof fragment, the first binding protein or thereof fragment, and the first binding protein or thereof fragment.

[0100] In an optional embodiment of the present invention, when the first extracellular region contains three fragments of the first binding protein or thereof, the first extracellular region is, from the N-terminus to the C-terminus, sequentially consisting of the first binding protein or thereof, the first binding protein or thereof, the first linker peptide, and the first binding protein or thereof.

[0101] In an optional embodiment of the present invention, when the first extracellular region contains three fragments of the first binding protein or thereof, the first extracellular region is, from the N-terminus to the C-terminus, sequentially consisting of the first binding protein or thereof, the first linker peptide, the first binding protein or thereof, and the first binding protein or thereof.

[0102] In an optional embodiment of the present invention, when the first extracellular region contains three fragments of the first binding protein or thereof, the first extracellular region, from the N-terminus to the C-terminus, consists of the first binding protein or thereof, the first linker peptide, the first binding protein or thereof, the first linker peptide, and the first binding protein or thereof.

[0103] According to an embodiment of the present invention, the first binding protein or a fragment thereof includes a first binding fragment and a second binding fragment, the first binding fragment and the second binding fragment being linked together.

[0104] It should be noted that the term "linked" in this article can refer to direct or indirect linking, and the specific type is not limited, all of which are within the scope of protection of this invention. For example, "the first binding fragment and the second binding fragment are linked" means that the first binding fragment and the second binding fragment can be directly linked or indirectly linked (e.g., linked through a first linker peptide).

[0105] According to an embodiment of the present invention, the first binding protein or a fragment thereof includes a first binding fragment and a second binding fragment, the first binding fragment and the second binding fragment being linked 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 embodiments 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; LT2-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-D2 fragment; ILT2-D1 fragment + first linker peptide + extracellular region of ILT2 protein; ILT4-D1 fragment + first linker peptide + ILT4-D1 fragment T4-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-D2 fragment; ILT4-D1 fragment + first linker peptide + ILT2-D2 fragment; 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-D1 fragment T4-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 Extracellular region; 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 + ILT 4-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;Extracellular region of ILT2 protein + first linker peptide + ILT2-D1 fragment + ILT2-D2 fragment; Extracellular region of ILT2 protein + first linker peptide + extracellular region of ILT2 protein; Extracellular region of ILT4 protein + first linker peptide + ILT4-D1 fragment; Extracellular region of ILT4 protein + first linker peptide + ILT4-D2 fragment; Extracellular region of ILT4 protein + first linker peptide + ILT4-D1 fragment + ILT4-D2 fragment; Extracellular region of ILT4 protein + first linker peptide + ILT4 protein; Extracellular region of ILT4 protein + first linker peptide + ILT2-D1 fragment; Extracellular region of ILT4 protein + first linker peptide + ILT2-D2 fragment; Extracellular region of ILT4 protein + first linker peptide + ILT2-D2 fragment; Extracellular region of ILT4 protein + first linker peptide + ILT2-D2 fragment; Extracellular region of ILT4 protein + first linker peptide + extracellular region of ILT2 protein.

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

[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 sequentially from the N-terminus to the C-terminus; 4) the ILT4-D1 fragment and the ILT4-D2 fragment sequentially 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 sequentially 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 sequentially 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 transcriptional activating proteins, transcriptional repressor proteins, transcription factors, site-specific nucleases, recombinases, intracellular domains of activating immune receptors, and intracellular domains of inhibitory immune receptors.

[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, the present invention provides a first nucleic acid molecule. According to embodiments of the invention, the first nucleic acid molecule encodes the chimeric polypeptide described in the first aspect. The first nucleic acid molecule according to embodiments of the invention can encode the aforementioned 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, although only one strand is given in most cases herein, the other complementary strand is also disclosed. Furthermore, the molecular sequences in this invention include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0120] In a third aspect, the present invention provides a first expression vector. According to an embodiment of the present invention, the first expression vector carries the first nucleic acid molecule described in the second aspect. When the first nucleic acid molecule is linked to the expression vector, the first nucleic acid molecule can be directly or indirectly linked to control elements on the expression vector, as long as these control elements can control the translation and expression of the first nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself, or they can be exogenous, i.e., not derived from the vector itself. Naturally, the first nucleic acid molecule and the control elements need to be operably linked.

[0121] In this article, "operably ligated" refers to ligating a foreign gene to an expression vector, enabling the control elements within the expression vector, such as transcriptional control sequences and translational control sequences, to perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors include plasmids, bacteriophages, etc. After the vector according to some specific embodiments of the present invention is introduced into suitable recipient cells (also known as recipient cells or host cells), the aforementioned chimeric polypeptide can be effectively expressed 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 bacteriophage.

[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, the present invention provides a second nucleic acid molecule. According to embodiments 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 and the 5' end of the second nucleic acid fragment are connected; wherein the second nucleic acid fragment is used to encode an antigen-chimeric receptor targeting the 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 consistent with the first intracellular region defined in the chimeric polypeptide described in the first aspect. The second nucleic acid molecule of the present invention can bind to the first intracellular region in the aforementioned chimeric polypeptide, thereby activating and inducing the expression of the antigen-chimeric receptor.

[0126] Furthermore, this second nucleic acid molecule and the aforementioned first nucleic acid molecule can be used to prepare multi-targeting recombinant immune cells. These recombinant immune cells enable cells expressing the chimeric polypeptide to recognize HLA-G proteins. After binding to HLA-G proteins, they can achieve various signal output types, such as the activation of specific gene expression, especially the activation of chimeric antigen receptors containing factors with therapeutic effects (e.g., tumor treatment), for the treatment of diseases such as tumors. In particular, recombinant immune cells for tumor treatment can be prepared, which can broadly recognize numerous tumor cells, greatly improving the killing efficiency and precision of various tumor cells, overcoming the problems of non-specific tumor recognition and incomplete tumor recognition coverage in existing cell therapy clinical applications, thus providing a new tumor treatment method with broad application prospects.

[0127] It should be noted that the term "inducing chimeric antigen receptor expression" refers to enabling the second nucleic acid fragment to encode an antigen chimeric receptor.

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

[0129] According to an embodiment of the present invention, the first molecule includes at least one of tumor antigen, virus, bacteria, endotoxin, antibody, cell receptor, and ligand of cell receptor.

[0130] In this article, the term "tumor antigen" generally refers to antigenic substances that newly emerge or are overexpressed during the occurrence and development of tumors. Based on the classification of tumor antigen specificity, they are divided into tumor-specific antigens and tumor-associated antigens (TAAs). Tumor-specific antigens (TSA) are novel antigens specific to tumor cells or present only in certain tumor cells and not in normal cells. Tumor-associated antigens (TAAs) are antigens not specific to tumor cells but also present in normal cells and other tissues, although their levels are significantly increased during cell carcinogenesis. These include, but are not limited to, PD-L1, PD-1, TGF-β, CEA, GD2, and GD3.

[0131] In this article, the term "cell receptor" or "receptor" should be interpreted broadly, referring to molecules located on the cell membrane that can recognize and bind to various extracellular signaling molecules (ligands), including but not limited to growth factor receptors (such as VEGF receptors), (NKG2D peptides (receptors for MICA, MICB, and ULBP1-6), cytokine receptors (such as IL-13 receptors, IL-2 receptors, etc.), epidermal growth factor (EGF) receptors, Her2, CD27, natural cytotoxic receptors (NCRs) (such as NKp30 (NCR3 / CD337) peptides (receptors for HLA-B-associated transcript 3 (BAT3) and B7-H6), T-cell antigen receptors, dihydrofolate receptors, chimeric cytokine receptors, Fc receptors, extracellular matrix receptors (such as integrin), cell adhesion receptors (such as cadherin), immunomodulatory receptors (including positive co-receptors (such as CD28) and negative (immunosuppressive) co-receptors (such as PD1)), and receptors for immunomodulatory molecules (such as TGFβ).

[0132] In this article, the term "ligand of cell receptor" should be interpreted broadly, and can refer to chemical substances that can bind to and interact with cell membrane receptors to produce specific biological effects, such as polypeptides, nucleic acids, glycoproteins, small molecules, carbohydrates, lipids, glycolipids, lipoproteins, lipopolysaccharides, etc., including but not limited to cytokines (e.g., IL-13), growth factors (e.g., heregulin, vascular endothelial growth factor (VEGF)), peptide hormones, integrin-binding peptides (e.g., peptides including the sequence Arg-Gly-Asp), N-glycans, etc.

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

[0134] In this article, the term "cytokine" should be interpreted broadly, referring to a class of proteins or small polypeptides that can transmit information between cells and have immunomodulatory and effector functions, such as IL-10. "Cytokine receptor" should also be interpreted broadly, referring to receptors on the cell surface that can bind to cytokines, such as IL-10R.

[0135] According to embodiments 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 MICA, MICB, ULBPs, B7H6, B7H3, GFP, eGFP, CD19, ALPPL2, BCMA, SIRPα, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44, CD44v6, CD45, CD48, CD51, CD52, and CD56. , CD59, CD66, CD70, CD71, CD72, CD73, CD74, CD79A, CD79B, CD80, CD86, CD94, CD95, CD123, CD133, CD134, CD140, CD152, CD154, CD158, CD178, CD1 81. CD182, CD183, CD200, CD210, CD221, CD246, CD252, CD253, CD261, CD262, CD269, CD273, CD274, CD276, CD279, CD295, CD339, CD340, EGFR, EGFR VIII, HER2, FGFR2, AFP, CA125, MSLN, GPC3, CEA, CLDN1, CLDN3, CLDN6, CLDN18.1, CLDN18.2, EpCAM, PSCA, GD2, GD3, IL-13, IL-13RA2, ROR1, MUC-1, PSMA, MAGEA1, 4-1BB, 5T4, BAFF, CA242, CA-IX, MET, CCR4, CNTO888, FAP, MORAb-009, EPHA2, VEGF-A, VEGFR-1, and VEGFR-2 are at least one of these.

[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 embodiments 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 receptor ligand, and a cell adhesion molecule.

[0140] According to an embodiment of the present invention, the second binding protein or a fragment thereof is the extracellular region of an activating receptor expressed on the surface of an immune cell or an antibody or a fragment thereof that binds to the first molecule.

[0141] According to an embodiment of the present invention, the antibody or 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 receptors on the surface of NK cells.

[0143] According to embodiments of the present invention, the activating receptor is selected from at least one of NKG2D, NKp30, NKp44, NKp46, DNAM-1, PD-1, TIGIT, NKG2A, NKG2B, NKG2C, NKG2E, NKG2H, CD16, NKp80, CD226, CD160, CD161, CD96, PVRIG, SLAM, CD200R, CD49a, TIM-3, LAG-3, CD112R, KIR2DS1, KIR2DS2, KIR2DS4, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, LIR1, LIR2, SIGLE3, SIGLE7, SIGLE9, and KLRG1.

[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 a fragment thereof includes a third binding fragment and / or a fourth binding fragment; the third binding fragment is the extracellular region of NKG2D or an active fragment thereof; the fourth binding fragment is the extracellular region of NKp30 or an active fragment thereof.

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

[0147] SLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWM GLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO: 13).

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

[0149] LWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIR DVRGHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLGAGTV (SEQ ID NO: 14).

[0150] According to an embodiment of the present invention, the third binding fragment has an amino acid sequence as shown in SEQ ID NO:13 or an amino acid sequence having at least 90% identity with it.

[0151] According to an embodiment of the present invention, the fourth binding fragment has an amino acid sequence as shown in SEQ ID NO:14 or an amino acid sequence having at least 90% identity with it.

[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 linked 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, and 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, and the C-terminus of the fourth binding fragment is connected to the N-terminus of the hinge region; or the C-terminus of the fourth binding fragment is connected to the N-terminus of the second 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 embodiments of the present invention, the hinge region includes at least one of the hinge region of the CD8α molecule or a variant thereof, the hinge region of an immunoglobulin or a variant thereof.

[0160] In this article, the term "immunoglobulin" refers to a globulin with antibody (Ab) activity or chemical structure, similar to an antibody molecule, and is a tetrapeptide chain structure composed of 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 an amino acid sequence as shown in SEQ ID NO:15.

[0162] GGGGSGGGGDESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(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 regions of CD8α molecules, CD28 molecules, CD3ζ molecules, CD4 molecules, CD16 molecules, 4-1BB molecules, OX40 molecules, ICOS molecules, CTLA-4 molecules, PD-1 molecules, LAG-3 molecules, 2B4 molecules, NKG2D molecules, DNAM-1 molecules, NKp44 molecules, NKp46 molecules, KIR2DS1 molecules, KIR2DS2 molecules, KIR2DS4 molecules, and BTLA molecules.

[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 an amino acid sequence as 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 signal transduction domain and a co-stimulatory domain.

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

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

[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 includes a third nucleic acid fragment for encoding a signal peptide; the 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, and the 3' end of the third nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment.

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

[0176] According to an embodiment of the present invention, the signal peptide has an amino acid sequence as 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 a nucleotide sequence as shown in SEQ ID NO:20.

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

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

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

[0182] GGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCAC TGTCCTCCGAACGGAGCATGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGACTAGTTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGC (SEQ ID NO: 21).

[0183] In a fifth aspect, the present invention provides a second expression vector. According to embodiments of the invention, the second expression vector carries the second nucleic acid molecule described in the fourth aspect. The second expression vector according to embodiments of the invention can express the antigen-chimeric receptor in the aforementioned 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 bacteriophage.

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

[0186] Recombinant immune cells

[0187] In a sixth aspect, the present invention provides a recombinant immune cell. According to embodiments of the present invention, the recombinant immune cell comprises: carrying the first nucleic acid molecule described in the second aspect or the first expression vector described in the third aspect; or expressing the chimeric polypeptide described in the first aspect. Under suitable conditions, the recombinant immune cell of the embodiments of the present invention can express the aforementioned chimeric polypeptide on its surface, recognize HLA-G protein, and, upon binding to HLA-G protein, can achieve various different signal output types, such as the activation of specific gene expression, especially the activation of the expression of chimeric antigen receptors containing factors with therapeutic effects (e.g., for treating tumors), for the treatment of diseases such as tumors.

[0188] It should be noted that the "suitable conditions" mentioned in this invention refer to conditions suitable for the expression of the aforementioned chimeric peptides. Those skilled in the art will readily understand that suitable conditions for the expression of the aforementioned chimeric peptides include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for chimeric peptide expression based on the specific environment of their laboratory.

[0189] According to an embodiment of the present invention, the expression of the chimeric polypeptide in the recombinant immune cells is obtained by introducing the first expression vector described in the third aspect into the host cell.

[0190] According to embodiments of the present invention, the recombinant immune cells further include a chimeric antigen receptor, which is identical to the chimeric antigen receptor encoded by the second nucleic acid fragment defined in the second nucleic acid molecule described in the fourth aspect. Under suitable conditions, the recombinant immune cells of the present invention can simultaneously express the aforementioned chimeric polypeptide and the chimeric antigen receptor in the aforementioned second nucleic acid molecule on their surface. Therefore, the recombinant immune cells possess multi-targeting capabilities, recognizing HLA-G proteins. After binding to HLA-G proteins, they can achieve various different signal output types, such as the activation of specific gene expression, and especially the activation of the expression of chimeric antigen receptors containing factors with therapeutic effects (e.g., tumor treatment), for the treatment of diseases such as tumors. In particular, recombinant immune cells for tumor treatment can be prepared, which can broadly recognize numerous tumor cells, greatly improving the killing efficiency and precision of various tumor cells, overcoming the problems of non-specific tumor recognition and incomplete tumor recognition coverage in existing cell therapy clinical applications, thus providing a new tumor treatment method with broad application prospects.

[0191] In particular, when the chimeric peptide of the present invention is used in combination with a chimeric antigen receptor to prepare recombinant immune cells (such as NK cells and Jurkat cells), it is found that the immune cells prepared by combining the chimeric peptide with CAR / TCR can recognize the corresponding ligands and efficiently activate immune cells (such as NK cells and Jurkat cells) to perform the function of killing targets (such as tumor cells).

[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 described in the fifth aspect into the host cell.

[0193] According to embodiments 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 embodiments 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] Pharmaceutical Composition

[0196] In a seventh aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises the recombinant immune cells described in the sixth aspect. As is previously known, cells expressing the aforementioned chimeric polypeptide can recognize HLA-G protein, and upon binding to HLA-G protein, can achieve various different signal output types, such as activation and inhibition of specific gene expression, or, upon contact with HLA-G protein, immune cells expressing the chimeric polypeptide can secrete factors with therapeutic effects for anti-tumor purposes. Therefore, a pharmaceutical composition containing the aforementioned recombinant immune cells can target HLA-G protein for the prevention and / or treatment of related diseases, such as cancer.

[0197] According to embodiments of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0198] use

[0199] In an eighth aspect of the invention, the invention provides for 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 medicament for the prevention and / or treatment of a disease.

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

[0201] In this document, the terms "cancer" or "tumor" can refer to any unregulated cell growth. Examples include, but are not limited to, small cell lung cancer, non-small cell lung cancer, papillary thyroid carcinoma, glioblastoma multiforme, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer 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 carcinoma, oral squamous cell carcinoma, or gastric cancer, etc.

[0202] Methods of treating or preventing diseases

[0203] In a ninth aspect of the invention, a method for treating or preventing disease is provided. According to an embodiment of the invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the recombinant immune cells described in the sixth aspect or the pharmaceutical composition described in the seventh aspect. As is known prior art, cells expressing the aforementioned chimeric polypeptide can recognize HLA-G protein, and upon binding to HLA-G protein, can achieve various different signal output types, such as activation and inhibition of specific gene expression, etc. Alternatively, immune cells expressing the chimeric polypeptide, upon contact with HLA-G protein, can secrete factors with therapeutic effects for anti-tumor purposes. Therefore, a pharmaceutical composition containing the aforementioned recombinant immune cells can target HLA-G protein for the prevention and / or treatment of related diseases, such as cancer.

[0204] The effective amount of the recombinant protein or pharmaceutical composition described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

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

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

[0207] According to embodiments of the present invention, the diseases include cancer or tumors, immune-related diseases. According to embodiments of the present invention, the diseases include cancer or tumors, autoimmune diseases, inflammation, and diseases related to cellular senescence.

[0208] According to embodiments of the present invention, the tumors or cancers include, but are not limited to, small cell lung cancer, non-small cell lung cancer, papillary thyroid carcinoma, glioblastoma multiforme, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer 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 carcinoma, oral squamous cell carcinoma or gastric cancer, etc.

[0209] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

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

[0211] ILT2 and ILT4 receptors are two receptors on the surface of immune cells responsible for binding to HLA-G molecules. ILT4 is expressed only on myeloid-derived immune cells, while ILT2 is expressed on both lymphoid and myeloid-derived immune cells. Both ILT2 and ILT4 receptors are divided into extracellular, transmembrane, and intracellular regions, with the extracellular region being the functional area responsible for recognizing and binding HLA-G. Furthermore, the extracellular regions of both ILT2 and ILT4 receptors are divided into four domains, named Domain 1 (D1), Domain 2 (D2), Domain 3 (D3), and Domain 4 (D4).

[0212] Based on the binding properties of ILT2 and ILT4 with HLA-G, the inventors designed and synthesized a series of synthetic peptides that can target and bind to human HLA-G. These synthetic peptides are composed of peptide sequences derived from ILT2 and ILT4 receptors. Since the peptide sequences derived from ILT2 and ILT4 both have the ability to bind to HLA-G, the binding ability of our designed synthetic peptides to HLA-G can be maximized. The synthesized polypeptide was used as the extracellular region of the SynNotch receptor (the amino acid sequence of ILT2-ECD in 1-Syn-CAR is shown in SEQ ID NO:6; the amino acid sequence of ILT4-ECD in 2-Syn-CAR is shown in SEQ ID NO:11; the amino acid sequence of ILT2-D1D2 in 3-Syn-CAR is shown in SEQ ID NO:2 and 3; the amino acid sequence of ILT4-D1D2 in 4-Syn-CAR is shown in SEQ ID NO:7 and 8; the amino acid sequence of ILT2-D1D2 in 5-Syn-CAR is shown in SEQ ID NO:2 and 3, the amino acid sequence of the 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 the Linker is GGGGS, and the amino acid sequence of ILT2-D1D2 is shown in SEQ ID NO:6). As shown in NO:2 and 3), a signal peptide sequence derived from CD8α (amino acid sequence shown in SEQ ID NO:19) is linked to its N-terminus, and a Notch transmembrane domain sequence from Xenopus laevis (amino acid sequence shown in SEQ ID NO:1) and a Gal4-VP64 artificial transcription factor sequence (amino acid sequence shown in SEQ ID NO:12) are sequentially linked to its C-terminus, ultimately forming a series of SynNotch receptor sequences, see [SEQ ID NO:12]. Figure 1 The sequences were cloned into pCDH vectors to produce viruses, which were then used to infect NK cells or Jurkat cells, and stable cell lines were established for each virus.

[0213] Example 2: Design and construction of CAR receptors that specifically target and bind to human NKG2D and NKp30 ligands

[0214] NKG2D and NKp30 receptors are two receptors on the surface of immune cells responsible for binding to molecular ligands such as MICA / B, ULBPs, and B7H6. NKG2D and NKp30 ligands are widely expressed on the surface of numerous tumor cells, and are not expressed or are expressed at low levels in most normal tissues. Both NKG2D and NKp30 receptors are divided into extracellular, transmembrane, and intracellular regions, with the extracellular region being the functional area responsible for recognizing and binding the corresponding ligands.

[0215] Based on the properties that NKG2D and NKp30 can bind to the aforementioned ligands, the inventors designed and synthesized synthetic peptides that can target and bind to human NKG2D and NKp30 ligands. These synthetic peptides are composed of extracellular polypeptide sequences of NKp30 and NKG2D and can bind to ligands of NKG2D and NKp30 simultaneously. The synthesized polypeptide was used as the extracellular region of the CAR receptor (the C-terminus of the NKp30 receptor extracellular region is linked to the N-terminus of the NKG2D receptor extracellular region, where the amino acid sequence of the NKp30 receptor extracellular region is shown in SEQ ID NO:14 and the amino acid sequence of the NKG2D receptor extracellular region is shown in SEQ ID NO:13). A CD8α-derived signal peptide sequence (amino acid sequence shown in SEQ ID NO:19, nucleotide sequence shown in SEQ ID NO:20) was attached to its N-terminus. Then, a CD8α-derived hinge region sequence (amino acid sequence shown in SEQ ID NO:15), a CD8α-derived transmembrane domain sequence (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) were sequentially attached to its C-terminus, ultimately forming the CAR receptor sequence. Simultaneously, a UAS-mini-CMV sequence (nucleotide sequence shown in SEQ ID NO:21) was added before the nucleotide sequence encoding the CAR receptor. Figure 2 The sequence was cloned into a pCDH vector with the promoter removed to produce the virus, which was then used to infect the stable NK cells and stable Jurkat cells established in Example 1, respectively, to establish stable cell lines transfected with the two receptor nucleic acid sequences, named the corresponding Syn-CAR-NK cell line and Syn-CAR-Jurkat cell line. Simultaneously, the CAR receptor without the added UAS-mini-CMV sequence was cloned into a pCDH vector with the promoter, used to produce the virus, and then infecting NK and Jurkat cells to establish stable cell lines, serving as controls for Syn-CAR-NK / Jurkat cells, named CAR-NK cells and CAR-Jurkat cells.

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

[0217] Next, the in vitro killing activity of different types of Syn-CAR-NK cells obtained in Example 2 was tested using PI and CFSE staining. Specifically, for the tumor cell line K562 (which possesses all the targets required for Syn-CAR-NK activation), this target cell line was stained with CSFE fluorescence and then sampled at 2 × 10⁶ cells per well. 4The cells were seeded at a concentration of [number] cells / ml in culture plates. Six experimental groups and one control group were set up for the target cell line. The experimental groups were supplemented with a suspension of Syn-CAR-NK cells (obtained in Example 2) that simultaneously target HLA-G, NKG2D ligands, and NKp30 ligands; the blank control group was supplemented with NK cells infected with an empty vector virus. In the experimental groups, Syn-CAR-NK cells and target cells were mixed at a 1:2 effector-to-target ratio (here, the term "effector-to-target ratio" refers to the ratio of effector cells, i.e., Syn-CAR-NK cells simultaneously targeting HLA-G, NKG2D ligands, and NKp30 ligands, to target cells, i.e., the number of tumor cells), for 48 hours. After 48 hours of culture, the cells were centrifuged to remove the supernatant. The cell pellet was washed and stained with PI. Stained cells were obtained by flow cytometry, and the results were analyzed using FlowJo software. The tumor cell killing rate test results using K562 leukemia cells as the target cells are shown below. Figure 3 As shown. From Figure 3 It can be seen that Syn-CAR-NK cells have a significant killing effect on K562 leukemia cells (significantly higher than the control group), with 5-Syn-CAR-NK and 6-Syn-CAR-NK having the highest killing efficiency.

[0218] For the tumor cell line Aspc-1 (which only possesses a target for activating SynNotch) and the immortalized normal cell line THLE3 (which does not possess a target for activating either SynNotch or CAR), 5×10 3 After the target cells adhered to the E-plate, the experimental group cells and control group cells from Example 2 were added, and the growth of the target cells was then detected using a RTAC instrument. The results showed that the experimental group cells of this invention had no significant killing effect on NKG2D and NKp30 ligand-negative pancreatic cancer cells Aspc-1 (the killing effect was similar to that of the control group). This example exemplifies the results of 6-Syn-CAR-NK, with the tumor cell killing rate test results using pancreatic cancer Aspc-1 as the target cells as shown below. Figure 4 As shown, the results of the killing rate test using immortalized hepatocyte lines as target cells are as follows: Figure 5 As shown.

[0219] from Figure 4 It can be seen that 6-Syn-CAR-NK cells did not have a significant killing effect on NKG2D and NKp30 ligand-negative pancreatic cancer cells Aspc-1 (the killing effect was similar to that of the control group). From Figure 5It can be seen that 6-Syn-CAR-NK cells did not significantly enhance the killing effect on HLA-G negative, and also negative for NKG2D and NKp30 ligands immortalized hepatocytes THLE3 (the killing effect was similar to that of the control group). These results indicate that the Syn-CAR system works effectively, has no killing effect on cells expressing only a single target or not expressing any target, and can more accurately distinguish between normal cells and 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. Ten days after tumor formation, tumor size was measured using calipers. Mice with similar tumor formation were randomly divided into 9 groups: six groups were experimental groups (containing different types of Syn-CAR-NK cells obtained in Example 2), and the remaining three groups were control groups (one untreated control, one conventional NK cell treatment control, and one CAR-NK cell treatment control). Subsequently, 1×10⁶ NCI-H716 cells were injected into the tail vein of the experimental group mice. 7 1 × 10⁶ 6-Syn-CAR-NK cells were injected via the tail vein into a control group of mice. 7 One × 10⁶ normal NK cells were injected into the tail vein of a control group of mice. 7 CAR-NK cells were injected intravenously into a control group of mice with equal volumes of saline from the three groups mentioned above. Subsequently, each group underwent the same treatment / treatment once a week, with subcutaneous xenograft size measured. During this period, IL-2 was injected intraperitoneally every 3 days, with 5 × 10⁶ cells per mouse per injection. 4 U, a total of 3 treatments were administered. Results showed that the Syn-CAR-NK cells of this invention exhibited significant killing activity against NCI-H716 colon cancer cells, a target cell line simultaneously expressing HLA-G, NKG2D ligands, and NKp30 ligands (significantly higher than the NK control group), and its killing efficiency was not significantly different from the CAR-NK group alone. This embodiment exemplarily demonstrates the results of 6-Syn-CAR-NK; see details below. Figure 6 (Results of the killing rate test of 6-Syn-CAR-NK cells after NCI-H716 colon cancer cells were used as target cells).

[0222] from Figure 6As can be seen, the 6-Syn-CAR-NK cells of Example 4 showed significant killing effect on NCI-H716 colon cancer cells, which simultaneously expressed HLA-G and NKG2D ligands and NKp30 ligands, in vivo (significantly higher than the NK control group), and their killing efficiency was not significantly different from that of the CAR-NK group alone, indicating that the SynNotch receptor did not reduce its killing efficiency.

[0223] 2. Further, take 1×10 7 Aspc-1 cells were subcutaneously injected into 5-week-old NCG mice. Ten days after tumor formation, tumor size was measured using calipers. Mice with similar tumor formation were randomly divided into three groups: one experimental group (6-Syn-CAR-NK cells), and the other two control groups (one untreated control and one standard NK cell treatment control). Subsequently, 1×10n Aspc-1 cells were injected intravenously into the tail vein of the experimental group mice. 7 1 × 10⁶ 6-Syn-CAR-NK cells were injected via the tail vein into a control group of mice. 7 Ordinary NK cells were injected intravenously into the tail vein of a control group of mice with the same volume of physiological saline as the three groups mentioned above. Subsequently, each group was treated / monitored weekly using the same method, and the size of the subcutaneous xenograft was measured. During this period, IL-2 was injected intraperitoneally every 3 days, with 5 × 10⁶ cells per mouse each time. 4 U, a total of 3 treatments were administered. The results of the 6-Syn-CAR-NK cell killing rate against pancreatic cancer cells (Aspc-1) after tumor bearing were as follows: Figure 7 As shown.

[0224] from Figure 7 As can be seen, the 6-Syn-CAR-NK cells in Example 4 did not significantly enhance the killing effect on pancreatic cancer cells Aspc-1, which only express HLA-G, in vivo (there was no increase in killing efficiency compared with the NK control group). This indicates that the Syn-CAR system can work well and has no killing effect on cells that only express a single target, and can more accurately distinguish between normal cells and tumor cells.

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

[0226] Next, the in vitro killing activity of different types of 6-Syn-CAR-Jurkat cells obtained in Example 2 was 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 used in a flow cytometry assay at 2 × 10⁶ cells per well. 4The cells were seeded at a concentration of [number] cells / ml in culture plates. One experimental group and one control group were set up for the target cell lines. The experimental group contained a cell suspension of 6-Syn-CAR-Jurkat cells obtained in Example 2, which simultaneously target HLA-G, NKG2D ligands, and NKp30 ligands. The blank control group contained Jurkat cells infected with an empty vector virus. In the experimental groups, the 6-Syn-CAR-Jurkat cells and target cells were mixed at a 1:2 effector-to-target ratio (here, the term "effector-to-target ratio" refers to the ratio of effector cells, i.e., the number of 6-Syn-CAR-Jurkat cells simultaneously targeting HLA-G, NKG2D ligands, and NKp30 ligands, to target cells, i.e., tumor cells), for 48 hours. After 48 hours of culture, the cells were centrifuged to remove the supernatant. The cell pellet was washed and stained with TNF-α, IFN-γ, and CD69 antibodies. Stained cells were obtained using flow cytometry, and the results were analyzed using FlowJo software. The activation effect of Jurkat cells was tested using K562 leukemia cells as the target cells. The results showed that 6-Syn-CAR-Jurkat cells were significantly activated by K562 leukemia cells, and the expression of TNF-α, IFN-γ, and CD69 was significantly increased (significantly higher than the control group). This indicates that the Syn-CAR system designed in this invention can also function well in the CAR-T cell system.

[0227] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0228] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled 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, include: The first extracellular region is a fragment that binds to the HLA-G protein; the first extracellular region is selected from one of the following: From the N-terminus to the C-terminus, the fragments are ILT2-D1, ILT2-D2, the first linker peptide, ILT4-D1, and ILT4-D2. Or, from the N-terminus to the C-terminus, the sequence is: ILT4-D1 fragment, ILT4-D2 fragment, first linker peptide, ILT2-D1 fragment, and ILT2-D2 fragment. The first transmembrane region includes the amino acid sequence of the transmembrane region of the Notch receptor protein of Xenopus laevis, wherein the N-terminus of the first transmembrane region is connected to the C-terminus of the first extracellular region; the amino acid sequence of the transmembrane region of the Notch receptor protein of Xenopus laevis is shown in SEQ ID NO:

1. The first intracellular region has its N-terminus connected to the C-terminus of the first transmembrane region; the first intracellular region is GaL4-VP64, which has the amino acid sequence shown in SEQ ID NO:12; the amino acid sequence of the ILT2-D1 fragment is shown in SEQ ID NO:2; The amino acid sequence of the ILT2-D2 fragment is shown in SEQ ID NO:3; The amino acid sequence of the ILT4-D1 fragment is shown in SEQ ID NO:7; The amino acid sequence of the ILT4-D2 fragment is shown in SEQ ID NO:

8.

2. The chimeric polypeptide according to claim 1, characterized in that, The transmembrane region further includes epidermal growth factor-like repeat sequences and / or RAM sequences.

3. The chimeric polypeptide according to claim 2, characterized in that, The C-terminus of the epidermal growth factor-like repeat sequence is linked to the N-terminus of the transmembrane region of the Notch receptor protein of Xenopus laevis; and / or, the C-terminus of the transmembrane region of the Notch receptor protein of Xenopus laevis is linked to the N-terminus of the RAM sequence.

4. The chimeric polypeptide according to claim 1, characterized in that, The amino acid sequence of the first linker peptide is (GGGGS)n, where n is any integer between 1 and 10.

5. The chimeric polypeptide according to claim 4, characterized in that, The n is 1, 2, 3 or 4.

6. The chimeric polypeptide according to claim 4, characterized in that, The amino acid sequence of the first linker peptide is GGGGS.

7. 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 6.

8. The first nucleic acid molecule according to claim 7, characterized in that, The first nucleic acid molecule is DNA.

9. A first expression vector, characterized in that, Carrying the first nucleic acid molecule as described in claim 7 or 8.

10. The first expression vector according to claim 9, characterized in that, The first expression vector is a eukaryotic expression vector, a prokaryotic expression vector, or a virus.

11. The first expression vector according to claim 9, characterized in that, The first expression vector is a plasmid expression vector.

12. A recombinant NK cell, characterized in that, include: Carrying the first nucleic acid molecule as described in claim 7 or 8 or the first expression vector as described in any one of claims 9 to 11; or, The chimeric polypeptide according to any one of claims 1 to 6 is expressed.

13. The recombinant NK cells according to claim 12, characterized in that, The expression of the chimeric polypeptide in the recombinant NK cells is obtained by introducing the first expression vector according to any one of claims 9 to 11 into the NK cells.

14. The recombinant NK cells according to claim 12, characterized in that, The recombinant NK cells further include a chimeric antigen receptor encoded by a second nucleic acid fragment, the chimeric antigen receptor comprising: The second extracellular region has a first molecule binding activity, wherein the first molecule is not an HLA-G protein; the second extracellular region includes a second binding protein or a fragment thereof that binds to the first molecule; the second binding protein or a fragment thereof includes a third binding fragment and a fourth binding fragment. The second transmembrane region, the N-terminus of which is connected to the C-terminus of the second extracellular region; The second intracellular region, the N-terminus of which is connected to the C-terminus of the second transmembrane region; in, The nucleotide sequence of the second nucleic acid fragment is shown in SEQ ID NO:18; The amino acid sequence of the third binding fragment is shown in SEQ ID NO:13; The amino acid sequence of the fourth binding fragment is shown in SEQ ID NO:

14.

15. The recombinant NK cells according to claim 14, characterized in that, The second extracellular region further includes a second linker peptide, through which the third and fourth binding fragments are linked.

16. The recombinant NK cells according to claim 15, characterized in that, The C-terminus of the third 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 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.

17. The recombinant NK cells according to claim 15, characterized in that, The amino acid sequence of the second linker peptide is (GGGGS)n, where n is any integer between 0 and 10.

18. The recombinant NK cells according to claim 17, characterized in that, The value of n is 0, 1, 2, 3 or 4.

19. The recombinant NK cells according to claim 15, characterized in that, The amino acid sequence of the second linker peptide is GGGGS.

20. The recombinant NK cells according to claim 15, characterized in that, The second extracellular region further includes 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, and the C-terminus of the fourth binding fragment is connected to the N-terminus of the hinge region; or the C-terminus of the fourth binding fragment is connected to the N-terminus of the second 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.

21. The recombinant NK cells according to claim 20, characterized in that, The hinge region includes at least one of the hinge region of the CD8α molecule or a variant thereof, or the hinge region of an immunoglobulin or a variant thereof.

22. The recombinant NK cells according to claim 20, characterized in that, The amino acid sequence of the hinge region is shown in SEQ ID NO:

15.

23. The recombinant NK cells according to claim 14, characterized in that, The second transmembrane region is selected from the transmembrane region of the CD8α molecule.

24. The recombinant NK cells according to claim 14, characterized in that, The amino acid sequence of the second transmembrane region is shown in SEQ ID NO:

16.

25. The recombinant NK cells according to claim 14, characterized in that, The second intracellular region includes an intracellular signal transduction domain and a co-stimulatory domain.

26. The recombinant NK cells according to claim 14, characterized in that, The amino acid sequence of the second intracellular region is shown in SEQ ID NO:

17.

27. The recombinant NK cells according to claim 14, characterized in that, The expression of the chimeric antigen receptor in the recombinant NK cells is obtained by introducing a second expression vector into the NK cells; the second expression vector carries a second nucleic acid molecule, the second nucleic acid molecule comprising a first nucleic acid fragment and a second nucleic acid fragment, wherein the 3' end of the first nucleic acid fragment and the 5' end of the second nucleic acid fragment are linked together; The second nucleic acid fragment is used to encode an antigen-chimeric receptor that targets the first molecule, and the first nucleic acid fragment is used to bind to the first intracellular region and induce the expression of the antigen-chimeric receptor. The first intracellular region is consistent with the first intracellular region defined in the chimeric polypeptide according to any one of claims 1 to 6; The antigen chimeric receptor is identical to the antigen chimeric receptor defined in any one of claims 14 to 26.

28. The recombinant NK cells according to claim 27, characterized in that, The second nucleic acid molecule further includes a third nucleic acid fragment for encoding a signal peptide; The 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, and the 3' end of the third nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment.

29. The recombinant NK cells according to claim 28, characterized in that, The signal peptide is selected from at least one of the signal peptides of CD8α molecules, IgG molecules, and CD28 molecules.

30. The recombinant NK cells according to claim 28, characterized in that, The amino acid sequence of the signal peptide is shown in SEQ ID NO:

19.

31. The recombinant NK cells according to claim 28, characterized in that, The nucleotide sequence of the third nucleic acid fragment is shown in SEQ ID NO:

20.

32. The recombinant NK cells according to claim 28, characterized in that, The nucleotide sequence of the first nucleic acid fragment is shown in SEQ ID NO:

21.

33. The recombinant NK cells according to claim 27, characterized in that, The second expression vector is a eukaryotic expression vector, a prokaryotic expression vector, or a virus.

34. The recombinant NK cells according to claim 33, characterized in that, The second expression vector is a plasmid expression vector.

35. A pharmaceutical composition, characterized in that, Includes the recombinant NK cells as described in any one of claims 27 to 34.

36. The pharmaceutical composition according to claim 35, characterized in that, This further includes pharmaceutically acceptable excipients.

37. Use of the recombinant NK cells according to any one of claims 27 to 34 or the pharmaceutical composition according to claim 35 or 36 in the preparation of a medicament for treating diseases, including leukemia and colon cancer.

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