Novel immune cell for treating tumors and preparation method therefor

Abstract

A novel immune cell for treating tumors and a preparation method therefor. Specifically, the novel immune cell carries an isolated nucleic acid. The isolated nucleic acid comprises: a first nucleic acid fragment, a second nucleic acid fragment and a third nucleic acid fragment. The first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are connected. The first nucleic acid fragment is used for encoding an antigen chimeric receptor targeting an NKG2D ligand. The second nucleic acid fragment is used for encoding a fusion protein, wherein the fusion protein comprises IL-15 and IL-15 Rα, and IL-15 is connected to IL-15 Rα. The third nucleic acid fragment is used for encoding a CXCR2 receptor. Therefore, the immune cell has good tumor specific recognition and killing capabilities, survival ability, proliferation ability and tumor internal infiltration ability, so that the clinical curative effect of the immune cell is further improved.

Description

Novel immune cells for treating tumors and preparation methods thereof Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular, to novel immune cells for treating tumors and a preparation method thereof. In particular, the present invention relates to an isolated nucleic acid, an expression vector, a transgenic immune cell, a kit, a pharmaceutical composition and uses thereof. Background Art

[0002] Natural killer (NK) cells are one of the main members of the innate immune system and can play a role in immune surveillance and immune regulation through various pathways. In clinical practice, NK cells are abundant in source, can be transfused allogeneically, have few side effects, and are highly safe. Therefore, NK cell therapy has huge application and development potential. However, NK cell therapy still faces many technical difficulties that need to be overcome or broken through, such as low cell activity after cryopreservation and thawing of NK cells, weak proliferation and persistence in the body, poor infiltration in tumors, and susceptibility to immunosuppression. These problems seriously limit the effectiveness and market value of NK cell therapy.

[0003] Most tumors possess a physical barrier of fibrous tissue, which harbors a microenvironment characterized by low oxygen, low pH, nutritional deficiencies, and high osmotic pressure. Furthermore, the microenvironment lacks mature blood vessels, making it highly unfavorable for immune cell localization, infiltration, survival, and proliferation within the tumor, and also prone to immunosuppression and exhaustion. Research and development of key technologies to enhance the tumor-killing activity and specificity of immune cells, strengthen their ability to infiltrate, survive, and proliferate within the tumor, and enhance their resistance to immunosuppression and exhaustion are promising breakthroughs in the field of immune cell therapy for tumor treatment.

[0004] Gene-modified cell therapy is a novel treatment approach that modifies the genome of a patient's cells to treat disease. The principle is to use genetic engineering techniques to introduce exogenous genes or regulatory factors into cells, thereby enabling them to acquire new functions or enhance therapeutic potential. Gene-modified cell technology has been extensively studied not only in T cells but also in NK cells, macrophages, hematopoietic stem cells, and non-hematopoietic stem cells.

[0005] Therefore, there is an urgent need to develop a new type of genetically modified immune cell that can survive in tumors for a long time, have good cell proliferation, resist immune exhaustion and improve tumor infiltration.

[0006] Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0008] The present invention is accomplished based on the following findings of the inventors:

[0009] NK cells are one of the main members of anti-tumor immunity and can participate in anti-tumor immune responses through various pathways. However, there are still certain problems in its current clinical application that limit its wider application. For example, the killing activity and specificity of NK cells need to be further improved, they are prone to immune exhaustion or immunosuppression in solid tumors, their expansion and persistence in vivo are weak, and their ability to infiltrate tumors is poor. Based on the above problems, the inventors have developed a multifunctional vector that expresses a chimeric antigen receptor with NK cell receptor as an extracellular recognition device, enhanced cytokines and chemokine receptors expressed on the cell membrane, wherein the chimeric antigen receptor targets NKG2D ligand; the enhanced cytokine expressed on the cell membrane is an IL15 / IL15Rα fusion protein formed by the fusion of IL15 and the IL-15 receptor α chain (IL15Rα); chemokine receptors include chemokine receptors such as CXCR2. This vector can be used to genetically modify immune cells such as NK cells, T cells, and macrophages to enhance the recognition and killing function of immune cells against tumors, increase the survival cycle and cell proliferation in the body, and improve the infiltration ability of immune cells into tumors.

[0010] Therefore, in its first aspect, the present invention provides an isolated nucleic acid. According to an embodiment of the present invention, the isolated nucleic acid comprises: a first nucleic acid fragment, a second nucleic acid fragment, and a third nucleic acid fragment, wherein the first, second, and third nucleic acid fragments are linked; wherein the first nucleic acid fragment encodes an antigen chimeric receptor targeting an NKG2D ligand; the second nucleic acid fragment encodes a fusion protein comprising IL-15 and IL-15Rα, wherein the IL-15 and IL-15Rα are linked; and the third nucleic acid fragment encodes a CXCR2 receptor. Through extensive inventive experiments, the inventors discovered that when immune cells carrying the isolated nucleic acid express the antigen chimeric receptor, the immune cells can bind to the NKG2D ligand, thereby effectively targeting and killing a variety of hematologic and solid tumor cells expressing NKG2D ligands. Furthermore, the immune cells carrying the isolated nucleic acid can also express the IL15 / IL15Rα fusion protein and the CXCR2 receptor, greatly enhancing their ability to survive and proliferate in vivo, and enabling them to infiltrate tumor tissue, more effectively exerting their tumor-killing function.

[0011] In its second aspect, the present invention provides an expression vector carrying the isolated nucleic acid described in the first aspect. Thus, using the expression vector described herein to prepare transgenic immune cells can enhance tumor recognition and killing capabilities, and can improve their survival time, proliferation capacity, and tumor infiltration ability in vitro and in vivo.

[0012] In its third aspect, the present invention provides a transgenic immune cell. According to an embodiment of the present invention, the transgenic immune cell expresses an antigen-targeting chimeric receptor targeting an NKG2D ligand, a fusion protein, and the CXCR2 receptor; wherein the fusion protein comprises IL-15Rα and IL-15, wherein the IL-15 and IL-15Rα are linked. The transgenic immune cell of the present invention has enhanced tumor recognition and killing capabilities, and can improve its survival, proliferation, and tumor infiltration capabilities in vitro and in vivo.

[0013] In its fourth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises: the isolated nucleic acid described in the first aspect, the expression vector described in the second aspect, or the transgenic immune cell described in the third aspect. The pharmaceutical composition of the present invention has high tumor killing efficiency and strong anti-tumor activity, and can be used for the prevention or treatment of a variety of tumor diseases.

[0014] In a fifth aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises: the isolated nucleic acid described in the first aspect or the expression vector described in the second aspect. The kit of the present invention can be used to prepare transgenic immune cells, which have enhanced tumor recognition and killing capabilities, and can improve their survival time, proliferation ability, and tumor infiltration ability in vitro and in vivo.

[0015] In its sixth aspect, the present invention provides a method for enhancing the killing, activation, proliferation, and chemotaxis of immune cells. According to an embodiment of the present invention, the method comprises: introducing the expression vector described in the second aspect into immune cells; and culturing the immune cells containing the expression vector. The method of the present invention can enhance the killing efficiency of immune cells, enhance the anti-tumor activity of immune cells, and improve the long-term survival and expansion ability, proliferation, and chemotaxis of immune cells in vitro and in vivo. In particular, immune cells with strong killing, activation, proliferation, and chemotaxis can be prepared in vitro for use in constructing desired immune cell models.

[0016] In the seventh aspect of the present invention, the present invention proposes the use of the isolated nucleic acid described in the first aspect, the expression vector described in the second aspect, the transgenic immune cell described in the third aspect or the pharmaceutical composition described in the fourth aspect in the preparation of a drug for treating or preventing tumors.

[0017] In the eighth aspect of the present invention, the present invention proposes the use of the isolated nucleic acid described in the first aspect, the expression vector described in the second aspect, the transgenic immune cell described in the third aspect or the pharmaceutical composition described in the fourth aspect in preventing and / or treating tumors.

[0018] In a ninth aspect, the present invention provides a method for treating or preventing tumors. According to an embodiment of the present invention, the method comprises administering a pharmaceutically acceptable amount of the transgenic immune cells of the third aspect or the pharmaceutical composition of the fourth aspect to a subject.

[0019] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] FIG1 is a schematic diagram of the gene element structure of the multifunctional vector of Example 1 of the present invention;

[0022] FIG2 is a flow cytometry diagram showing the expression of various elements of the multifunctional vector of Example 2 of the present invention; wherein,

[0023] Figure A is a flow cytometry analysis of the expression of mbIL15RF and CXCR2 elements on NK cells in the uninfected group;

[0024] Figure B is a flow cytometry analysis of the expression of mbIL15RF and CXCR2 elements on NK cells in the multifunctional vector lentivirus infection group;

[0025] Figure C shows the MFI results of NKG2D expression on NK cells in the uninfected and infected groups;

[0026] FIG3 is a graph showing the investigation of the killing activity of multifunctional NK cells on tumor cells according to Example 3 of the present invention; wherein,

[0027] Figure A shows the killing efficiency of NCI-H716 cells;

[0028] Figure B is the detection of killing efficiency of HCT-116 cells;

[0029] FIG4 is a graph showing the survival rate changes of NK cells and multifunctional NK cells in Example 3 of the present invention at different IL-2 culture concentrations;

[0030] FIG5 is a graph showing the results of detecting the chemotactic ability of NK cells and multifunctional NK cells at different CXCL8 concentrations according to Example 3 of the present invention;

[0031] FIG6 is a diagram showing the tumor inhibition effect of peripheral blood-derived multifunctional primary NK cells according to Example 4 of the present invention on a mouse model bearing human colorectal cancer NCI-H716 cells;

[0032] FIG7 is a graph showing the killing efficiency of NK cells and multifunctional NK cells of Example 5 of the present invention against human chronic myeloid leukemia K562 cells;

[0033] FIG8 is a graph showing the chemotactic ability of various transgenic immune cells expressing different chemokine receptors according to Example 6 of the present invention. DETAILED DESCRIPTION

[0034] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0035] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly indicate the number of technical features indicated, nor should they be understood to indicate a sequential order. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0036] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0037] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in the present invention, all other technical and scientific terms used in the present invention have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0038] In the present invention, the term "comprise" or "include" is an open expression, that is, it includes the content specified in the present invention, but does not exclude other aspects of the content.

[0039] In the present invention, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0040] As used herein, the term "vector" or "expression vector" generally refers to a nucleic acid molecule capable of being inserted into a suitable host and replicating itself, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector primarily used to insert DNA or RNA into a cell, a vector primarily used to replicate DNA or RNA, and a vector primarily used for expression by transcription and / or translation of DNA or RNA. The vector also includes vectors having a variety of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0041] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with a liquid carrier, a solid carrier, or both.

[0042] As used herein, the term "treatment" refers to a method for obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or genetically modified immune cell to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease.

[0043] The term "immune cell" generally refers to a cell that can produce an immune response (e.g., an antigen-specific immune response). For example, the immune cell can or has contained an isolated nucleic acid and / or vector comprising the present application, or can express an individual cell, cell line or cell culture comprising an antigen chimeric receptor, a fusion protein and a CXCR2 receptor as described herein. In the present application, the immune cell can include a T cell, a B cell, a natural killer cell (NK cell), a macrophage, an NKT cell, a monocyte, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte and / or a peripheral blood mononuclear cell.

[0044] As used herein, "carbon terminus" and "C-terminus" are synonymous; "nitrogen terminus" and "N-terminus" are synonymous.

[0045] The inventors have constructed a multifunctional vector that expresses a chimeric antigen receptor (NKG2D-NKR) with NKG2D as its extracellular recognition receptor, a cell membrane-expressed IL15 / IL15Rα fusion protein, and the chemokine receptor CXCR2. This vector can be used to prepare multifunctional gene-modified NK cells. The inventors discovered that, after gene transduction into NK cells, the proposed vector exhibits the following beneficial effects: 1) expression of NKG2D-NKR, enhancing NK cell recognition and cytotoxicity against various NKG2D ligand-positive tumor cells; 2) expression of the IL15 / IL15Rα fusion protein on the NK cell membrane, improving the long-term survival and proliferation of NK cells in vivo; and 3) expression of the chemokine receptor CXCR2, enhancing the ability of NK cells to infiltrate tumors. These three aspects collectively enhance the anti-tumor activity of NK cells in vivo.

[0046] Specifically, the present invention provides an isolated nucleic acid, an expression vector, a transgenic immune cell, a pharmaceutical composition, a kit, a method for enhancing immune cell killing, activation, proliferation and chemotaxis, and uses thereof, which are described in detail below.

[0047] Isolated nucleic acids

[0048] The present invention provides an isolated nucleic acid, which includes: a first nucleic acid fragment, a second nucleic acid fragment and a third nucleic acid fragment, wherein the first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are connected; wherein the first nucleic acid fragment is used to encode an antigen chimeric receptor targeting an NKG2D ligand; the second nucleic acid fragment is used to encode a fusion protein, wherein the fusion protein includes IL-15 and IL-15Rα, and the IL-15 and IL-15Rα are connected; and the third nucleic acid fragment is used to encode a CXCR2 receptor.

[0049] After a large number of creative experiments, the inventors discovered that when the immune cells carrying the isolated nucleic acid express antigen chimeric receptors, the immune cells can better bind to the NKG2D ligand, thereby effectively targeting and killing a variety of blood tumors and solid tumor cells expressing NKG2D ligands; in addition, the immune cells carrying the isolated nucleic acid can also express IL15 / IL15Rα fusion protein and CXCR2 receptor, which greatly improves their ability to survive and proliferate in the body, and can infiltrate into the tumor tissue, more effectively exerting their tumor-killing function.

[0050] It should be noted that the above three nucleic acid fragments of the present invention are intended to be expressed independently. Therefore, the connection order of the three nucleic acid fragments in the above gene of the present invention can be selected according to actual needs and does not need to be specifically limited.

[0051] In some embodiments, the isolated nucleic acid may further include at least one of the following additional technical features:

[0052] In some embodiments, the antigen chimeric receptor comprises: a first fragment and a second fragment, the first fragment and the second fragment are connected; the first fragment comprises an extracellular region, a transmembrane region and an intracellular region, the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region, and the extracellular region and the transmembrane region are selected from the NKG2D receptor; the second fragment comprises the full-length sequence of DAP10.

[0053] In some embodiments, the NKG2D receptor has the amino acid sequence shown in SEQ ID NO: 1.

[0054] In some embodiments, the intracellular region is selected from the intracellular segment of the CD3ζ molecule.

[0055] In some embodiments, the intracellular segment of the CD3ζ molecule has the amino acid sequence shown in SEQ ID NO:2.

[0056] In some embodiments, the full-length DAP10 sequence has the amino acid sequence shown in SEQ ID NO:12.

[0057] In some embodiments, the full-length sequence encoding DAP10 has a nucleotide sequence as shown in SEQ ID NO:13.

[0058] In some embodiments, the antigen chimeric receptor further includes a third fragment, the N-terminus of the third fragment is connected to the C-terminus of the first fragment, and the C-terminus of the third fragment is connected to the N-terminus of the second fragment, or the C-terminus of the third fragment is connected to the N-terminus of the first fragment, and the N-terminus of the third fragment is connected to the C-terminus of the second fragment.

[0059] In some embodiments, the third fragment comprises the sequence of P2A or a fragment thereof.

[0060] In some embodiments, the P2A or a fragment thereof has an amino acid sequence as shown in SEQ ID NO:14.

[0061] In some embodiments, the antigen chimeric receptor has the amino acid sequence shown in SEQ ID NO:3.

[0062] In some embodiments, the first nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:4.

[0063] In some embodiments, the C-terminus of IL-15 is linked to the N-terminus of IL-15Rα, or the N-terminus of IL-15 is linked to the C-terminus of IL-15Rα.

[0064] In some embodiments, the IL-15 has the amino acid sequence shown in SEQ ID NO:5.

[0065] In some embodiments, the IL-15Rα has the amino acid sequence shown in SEQ ID NO:6.

[0066] In some embodiments, the fusion protein further includes a connecting peptide, wherein the C-terminus of the IL-15 is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of IL-15Rα, or the N-terminus of the IL-15 is connected to the C-terminus of the connecting peptide, and the N-terminus of the connecting peptide is connected to the C-terminus of IL-15Rα.

[0067] In some embodiments, the connecting peptide is selected from any one of a flexible linker and a rigid linker.

[0068] In some embodiments, the connecting peptide has an amino acid sequence as shown in SEQ ID NO:7.

[0069] In some embodiments, the fusion protein has the amino acid sequence shown in SEQ ID NO:8.

[0070] In some embodiments, the second nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:9.

[0071] In some embodiments, the CXCR2 receptor has the amino acid sequence shown in SEQ ID NO:10.

[0072] In some embodiments, the third nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:11.

[0073] In some embodiments, the isolated nucleic acid further comprises two fourth nucleic acid fragments, wherein each two nucleic acid fragments of the first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment are connected by one fourth nucleic acid fragment, wherein each fourth nucleic acid fragment independently encodes P2A or a fragment thereof.

[0074] In some embodiments, the P2A or a fragment thereof includes at least one of P2A, T2A, E2A and F2A or a fragment thereof.

[0075] In some embodiments, the fourth nucleic acid segment encodes P2A or a fragment thereof.

[0076] In some embodiments, the P2A or a fragment thereof has an amino acid sequence as shown in SEQ ID NO:14.

[0077] In some embodiments, the fourth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO:15.

[0078] In some embodiments, the isolated nucleic acid further comprises a promoter linked to the 5' end of the nucleic acid fragment consisting of the first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment.

[0079] In some embodiments, the promoter is selected from EF1α, SFFV, CAG, or CMV.

[0080] In some embodiments, the promoter is selected from EF1α.

[0081] In some embodiments, the EF1α has the nucleotide sequence shown in SEQ ID NO:16.

[0082] In some embodiments, the isolated nucleic acid further includes a fifth nucleic acid fragment, which encodes a signal peptide, the C-terminus of the signal peptide is connected to the N-terminus of the IL-15, the C-terminus of the IL-15 is connected to the N-terminus of the connecting peptide, the C-terminus of the connecting peptide is connected to the N-terminus of IL-15Rα, or the N-terminus of the IL-15 is connected to the C-terminus of the connecting peptide, the N-terminus of the connecting peptide is connected to the C-terminus of IL-15Rα, and the N-terminus of IL-15Rα is connected to the C-terminus of the signal peptide.

[0083] In some embodiments, the signal peptide has the amino acid sequence shown in SEQ ID NO:17.

[0084] In some embodiments, the fifth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO:18.

[0085] It should be noted that the "fusion protein" described herein may or may not contain the "signal peptide". When the multifunctional vector shown in Figure 1 is constructed, the "fusion protein" contains the "signal peptide". When the multifunctional vector is introduced into immune cells to obtain transgenic immune cells, the "signal peptide" will be cut off. At this time, the "fusion protein" does not contain the "signal peptide". Therefore, whether the "fusion protein" contains the "signal peptide" should be considered according to the specific circumstances, and "fusion proteins" containing or not containing the "signal peptide" are all within the scope of protection of the present invention.

[0086] In some embodiments, the isolated nucleic acid comprises the promoter, the first nucleic acid fragment, one of the fourth nucleic acid fragments, the fifth nucleic acid fragment, the second nucleic acid fragment, another fourth nucleic acid fragment, and the third nucleic acid fragment from the 5' end to the 3' end.

[0087] expression vector

[0088] The present invention provides an expression vector carrying the isolated nucleic acid described above. Thus, using the expression vector of the present invention to prepare transgenic immune cells can enhance tumor recognition and killing capabilities, and can improve their survival time, proliferation capacity, and tumor infiltration ability in vivo and in vitro.

[0089] When the nucleic acid molecule is linked to an expression vector, the nucleic acid molecule can be directly or indirectly linked to the control elements on the expression vector, as long as these control elements are capable of controlling translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the expression vector itself or exogenous, i.e., not derived from the vector itself. Of course, it is sufficient that the nucleic acid molecule and the control elements are operably linked.

[0090] As used herein, "operably linked" means that the exogenous gene is linked to the expression vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can function as intended to regulate the transcription and translation of the exogenous gene. Commonly used vectors include plasmids, bacteriophages, and the like.

[0091] In some embodiments, the above expression vector may further include at least one of the following additional technical features:

[0092] In some embodiments, the expression vector is selected from a virus, a prokaryotic expression vector, or a eukaryotic expression vector.

[0093] In some embodiments, the expression vector is selected from a virus.

[0094] Genetically modified immune cells

[0095] The present invention proposes a transgenic immune cell that expresses an antigen-targeting chimeric receptor targeting an NKG2D ligand, a fusion protein, and the CXCR2 receptor. The fusion protein comprises IL-15Rα and IL-15, with the IL-15 and IL-15Rα linked. The transgenic immune cell has enhanced tumor recognition and killing capabilities, and can improve its survival, proliferation, and tumor infiltration capabilities both in vivo and in vitro.

[0096] In some embodiments, the antigen chimeric receptor comprises: a first fragment and a second fragment, the first fragment and the second fragment are connected; the first fragment comprises an extracellular region, a transmembrane region and an intracellular region, the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region, and the extracellular region and the transmembrane region are selected from the NKG2D receptor; the second fragment comprises the full-length sequence of DAP10.

[0097] In some embodiments, the NKG2D receptor has the amino acid sequence shown in SEQ ID NO: 1.

[0098] In some embodiments, the intracellular region is selected from the intracellular segment of the CD3ζ molecule.

[0099] In some embodiments, the intracellular segment of the CD3ζ molecule has the amino acid sequence shown in SEQ ID NO:2.

[0100] In some embodiments, the full-length DAP10 sequence has the amino acid sequence shown in SEQ ID NO: 12. Therefore, the antigen chimeric receptor can have stronger and broader NKG2D ligand binding activity, thereby enabling the transgenic immune cells to better bind to the NKG2D ligand and better play a role in killing tumors.

[0101] In some embodiments, the full-length sequence encoding DAP10 has a nucleotide sequence as shown in SEQ ID NO:13.

[0102] In some embodiments, the antigen chimeric receptor further includes a third fragment, the N-terminus of the third fragment is connected to the C-terminus of the first fragment, and the C-terminus of the third fragment is connected to the N-terminus of the second fragment, or the C-terminus of the third fragment is connected to the N-terminus of the first fragment, and the N-terminus of the third fragment is connected to the C-terminus of the second fragment.

[0103] In some embodiments, the third fragment comprises the sequence of P2A or a fragment thereof.

[0104] In some embodiments, the P2A or a fragment thereof has an amino acid sequence as shown in SEQ ID NO:14.

[0105] In some embodiments, the antigen chimeric receptor has the amino acid sequence shown in SEQ ID NO:3.

[0106] In some embodiments, the C-terminus of IL-15 is linked to the N-terminus of IL-15Rα, or the N-terminus of IL-15 is linked to the C-terminus of IL-15Rα.

[0107] In some embodiments, the IL-15 has the amino acid sequence shown in SEQ ID NO:5.

[0108] In some embodiments, the IL-15Rα has the amino acid sequence shown in SEQ ID NO:6.

[0109] In some embodiments, the fusion protein further includes a connecting peptide, wherein the C-terminus of the IL-15 is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of IL-15Rα, or the N-terminus of the IL-15 is connected to the C-terminus of the connecting peptide, and the N-terminus of the connecting peptide is connected to the C-terminus of IL-15Rα.

[0110] In some embodiments, the connecting peptide is selected from any one of a flexible linker and a rigid linker.

[0111] In some embodiments, the connecting peptide has an amino acid sequence as shown in SEQ ID NO:7.

[0112] In some embodiments, the fusion protein has the amino acid sequence shown in SEQ ID NO:8.

[0113] In some embodiments, the CXCR2 receptor has the amino acid sequence shown in SEQ ID NO:10.

[0114] In some embodiments, the DAP10 protein has the amino acid sequence shown in SEQ ID NO:12.

[0115] In some embodiments, the transgenic immune cells are obtained by introducing the above-mentioned expression vector into immune cells.

[0116] In some embodiments, the transgenic immune cells are derived from at least one of T cells, NKT cells, NK cells, and macrophages.

[0117] In some embodiments, the transgenic immune cells are derived from NK cells.

[0118] In some embodiments, the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells, and NK-92 cells.

[0119] In some embodiments, the T cells include CD4+ T cells, CD8+ T cells, and γδ T cells.

[0120] In some embodiments, the T cells include CD4+ T cells, CD8+ T cells, Treg cells, and γδ T cells.

[0121] Pharmaceutical composition

[0122] The present invention provides a pharmaceutical composition comprising the aforementioned isolated nucleic acid, the aforementioned expression vector, or the aforementioned transgenic immune cell. The pharmaceutical composition of the present invention has high tumor-killing efficiency and strong anti-tumor activity, and can be used to prevent or treat a variety of tumor diseases.

[0123] Reagent test kit

[0124] The present invention provides a kit comprising the isolated nucleic acid or expression vector described above. The kit can be used to prepare transgenic immune cells. The prepared transgenic immune cells have high tumor recognition and killing abilities, as well as tumor infiltration abilities, are capable of long-term survival in vivo, and exhibit strong expansion, proliferation, and chemotaxis abilities.

[0125] Methods for enhancing immune cell killing, activation, proliferation and chemotaxis

[0126] The present invention provides a method for enhancing the killing, activation, proliferation, and chemotaxis of immune cells. The method comprises: introducing the aforementioned expression vector into immune cells; and culturing the immune cells containing the expression vector. The method can enhance the killing efficiency of immune cells, strengthen their anti-tumor activity, and improve their long-term survival and expansion, proliferation, and chemotaxis in vitro and in vivo. In particular, immune cells with enhanced killing, activation, proliferation, and chemotaxis can be prepared in vitro for use in constructing desired immune cell models.

[0127] In some embodiments, the above method for enhancing immune cell killing, activation, proliferation and chemotaxis may further include at least one of the following additional technical features:

[0128] In some embodiments, the introduction of the expression vector into immune cells is performed by electroporation, transfection or infection.

[0129] In some embodiments, the immune cell is at least one of a T cell, an NKT cell, a NK cell, and a macrophage.

[0130] In some embodiments, the immune cells are NK cells.

[0131] In some embodiments, the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC)-derived NK cells, and NK-92 cells.

[0132] In some embodiments, the T cells include CD4+ T cells, CD8+ T cells, and γδ T cells.

[0133] In some embodiments, the T cells include CD4+ T cells, CD8+ T cells, Treg cells, and γδ T cells.

[0134] use

[0135] The present invention provides the use of the isolated nucleic acid described in the first aspect, the expression vector described in the second aspect, the transgenic immune cell described in the third aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of a drug for treating or preventing tumors.

[0136] The present invention proposes the use of the isolated nucleic acid described in the first aspect, the expression vector described in the second aspect, the transgenic immune cell described in the third aspect, or the pharmaceutical composition described in the fourth aspect in preventing and / or treating tumors.

[0137] In some embodiments, the above-mentioned use may further include at least one of the following additional technical features:

[0138] In some embodiments, the tumor includes solid tumors and hematological tumors.

[0139] In some embodiments, the solid tumor comprises at least one selected from pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, thyroid cancer, nasopharyngeal cancer, oral cancer, sarcoma, prostate cancer, melanoma and squamous cell carcinoma of the skin.

[0140] In some embodiments, the hematological tumor comprises at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, and myeloproliferative neoplasms.

[0141] In some embodiments, the solid tumor comprises at least one selected from pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, kidney cancer, breast cancer, thyroid cancer, osteosarcoma, prostate cancer and melanoma.

[0142] In some embodiments, the hematological tumor comprises at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma.

[0143] The present invention provides a method for treating or preventing tumors. According to an embodiment of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the transgenic immune cell of the third aspect or the pharmaceutical composition of the fourth aspect.

[0144] The effective amount of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, and the like. For example, depending on the exigencies of the treatment, several divided doses may be administered daily, or the dose may be proportionally reduced.

[0145] The transgenic immune cells or pharmaceutical compositions of the present invention can be incorporated into drugs suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. For example, liquid, semisolid and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical drugs are in the form of injection solutions or infusion solutions. The aforementioned transgenic immune cells or pharmaceutical compositions can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection or intraperitoneal injection or intrathoracic injection or hepatic artery perfusion or bladder perfusion or intrathecal administration, etc.

[0146] According to an embodiment of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0147] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0148] The sequences involved in the present invention are detailed in Table 1.

[0149] Table 1: Amino acid / nucleotide sequence description

[0150] Example 1

[0151] 1. Construction of Multifunctional Vectors

[0152] The nucleotide sequence of SEQ ID NO:22 was synthesized by whole gene synthesis and cloned into the lentiviral vector pLVX-EF1-IRES-Puro via the restriction enzyme sites EcoRI and MluI. After verification by sequencing, the pLVX-EF1-multi-functional-vector plasmid was obtained, which is the multifunctional vector plasmid of the present invention. A schematic diagram of the genetic element structure of the multifunctional vector of this example is shown in Figure 1.

[0153] 2. Lentivirus Packaging

[0154] 5×10 293T cells in the logarithmic growth phase were taken 6 Inoculate in a 10cm cell culture dish, add 10mL DMEM medium, and culture overnight in a 37℃, 5% CO2 incubator. When the cell density in the cell culture dish reaches 80-90%, replace with 10mL fresh DMEM medium, and continue to place the cell culture dish in the incubator for standby use. Prepare the lentiviral packaging system, add 6μg psPAX2 and 3μg pMD2.G, 6μg lentiviral vector plasmid to 250μL serum-free DMEM medium to prepare a plasmid mixture, and mix well. Add 15μL Add to 235 μL serum-free DMEM medium and mix well. Add the mixed solution to the above plasmid mixture at one time, mix well, and incubate at room temperature for 15 minutes. Add the mixed solution to the 293T cell culture dish. Change the medium after 24 hours, put the culture dish back into the 37°C, 5% CO2 incubator, collect the cell supernatant after 48 hours, centrifuge at 400×g for 5 minutes, remove cell debris, and filter the supernatant with a 0.45μm filter into a 50mL centrifuge tube. Add 5×PEG8000 solution to concentrate the virus solution, mix evenly by inverting the centrifuge tube, and place it in a 4°C refrigerator overnight. Centrifuge at 4000×g for 20 minutes at 4°C, discard the supernatant, add an appropriate amount of serum-free DMEM to resuspend the virus pellet, transfer it to an EP tube, and store it in a -80°C refrigerator.

[0155] 3. Lentiviral infection of human NK cells

[0156] NK cells in the logarithmic growth phase were aspirated and centrifuged at 100 × g for 5 min to harvest the cells. Appropriate amount of α-MEM medium was added to resuspend the cells and the cell density was adjusted to 5 × 10 5 5×10 5NK cells, 0.2mL virus concentrate, 0.8mL α-MEM medium and protamine (final concentration 8μg / mL) are mixed evenly. Place in a 37°C, 5% CO2 incubator for culture. After 24 hours, observe the cell status, change the medium, transfer the infected cells into an EP tube, centrifuge at 100×g for 5 minutes, add a small amount of fresh α-MEM medium to resuspend the cells, transfer the cells into a cell culture flask, add 10mL fresh α-MEM medium and IL-2 (final concentration of 200IU / mL) and continue to culture for 48 hours. Transfer the cells into new α-MEM medium and completely remove IL-2 for pressure screening for 2 weeks to obtain multifunctional vector gene-modified NK cells (multifunctional NK cells) for subsequent functional experiments.

[0157] Example 2: Verification of expression of various elements of the multifunctional vector

[0158] Peripheral blood mononuclear cells (PBMCs) were isolated and inoculated into pre-coated culture flasks for culture. IL-2 and other cytokines were used for induction culture according to CN 202310035787.4. Lentivirus infection was performed on the 7th day of culture. After changing the medium on the 9th day, culture was continued. On the 11th day, flow cytometry was performed to detect the expression of various elements on NK cells. The flow cytometry method was as follows: 1×10 6 Each cell was added to the flow cytometry tube for staining. According to different staining schemes, antibodies were added for staining and incubated at room temperature for 30 minutes. After washing twice with 1× PBS solution, the cell pellet was resuspended and then subjected to flow cytometry detection. Scheme 1: Add PerCP / Cyanine5.5 labeled anti-human CD3 antibody (purchased from Biolegend), Brilliant Violet 785 TM Anti-human CD56 antibody (purchased from Biolegend), APC-labeled anti-human NKG2D antibody (purchased from Biolegend) and PE-labeled anti-human CXCR2 antibody (purchased from Biolegend). TM Labeled anti-human CD56 antibody (purchased from Biolegend), APC-labeled anti-human IL-15Rα antibody (purchased from Biolegend) and PE-labeled anti-human IL-15 antibody (purchased from Invitrogen).

[0159] CD3 analysis by gating on CD3 and CD56 - CD56 +Phenotype of cells, namely NK cells. Next, the positive rate of mbIL15RF (double positive for IL-15 and IL-15Rα) or CXCR2 expression on NK cells and the mean fluorescence intensity (MFI) of NKG2D expression were analyzed. The experimental results are shown in A and B in Figure 2. CXCR2 and mbIL15RF molecules are not expressed on the NK cells of the uninfected group. After infection with the multifunctional vector lentivirus, the expression of CXCR2 and mbIL15RF molecules on NK cells was significantly increased. As shown in Figure 2C, the mean fluorescence intensity of NKG2D expression on NK cells in the infected group was also significantly higher than that of NK cells in the uninfected group. These results indicate that the three functional elements designed in the multifunctional vector can be efficiently expressed.

[0160] Example 3: In vitro functional detection of NK cells modified with multifunctional vector genes

[0161] In this example, after the inventors obtained the multifunctional vector gene-modified NK cells (multifunctional NK cells) of the present invention through Example 1, they investigated the killing activity, survival and chemotaxis of the multifunctional NK cells in vitro.

[0162] 1. Multifunctional vector gene modification promotes the killing activity of NK cells

[0163] The inventors tested the killing activity of NK cells modified by the multifunctional vector gene. The specific method is as follows: CFSE was used to fluorescently label colorectal cancer NCI-H716 or HCT-116 cells, and 2×10 4 Cells were plated at 100 μg / well in a 96-well plate. NK cells or multifunctional NK cells were then plated and incubated for 4 hours. The cells were collected in flow cytometry tubes and stained with PI to distinguish live and dead cells. The killing efficiency was measured by flow cytometry. The effector cell to target cell ratio was 2:1.

[0164] The test results are shown in Figure 3A and B. The multifunctional NK cells modified with the multifunctional vector gene of the present invention have a significantly higher killing efficiency against colorectal cancer NCI-H716 or HCT-116 cells than the NK cells that have not been genetically modified.

[0165] 2. Multifunctional vector gene modification promotes NK cell survival

[0166] The inventors further verified the effect of multifunctional vector gene modification on NK cell survival. The specific method is as follows: NK cells and multifunctional NK cells with the same cell number were plated in 24-well plates, and different IL-2 concentrations (0, 20 and 200 IU / mL) were set, and the apoptosis rate was detected by flow cytometry every 24 hours. The flow cytometry detection of apoptosis rate was performed according to the steps of the kit instructions (purchased from Lianke Bio, product number AP101), which is briefly as follows: collect cells in EP tubes, add 1× PBS solution, centrifuge and wash once, and resuspend the cells. Add 5μL Annexin V-FITC and 10μL PI to each tube. After gently vortexing to mix, incubate at room temperature in the dark for 5 minutes, and resuspend the cells for flow cytometry. The cell viability is the ratio of double-negative Annexin V-FITC and PI staining.

[0167] Figure 4 shows the results of NK cell survival studies up to 96 hours of culture. In the absence of IL-2 (IL-2 0 IU / mL), cell survival in the unmodified NK cell group decreased significantly starting at 24 hours, with most cells undergoing apoptosis by 72 hours. In contrast, the multifunctional NK-92 cell group maintained a high cell viability even in the complete withdrawal of IL-2, with very few cells undergoing apoptosis. These results suggest that multifunctional vector gene modification can play a significant role in promoting NK cell survival.

[0168] 3. Multifunctional vector gene promotes NK cell chemotaxis

[0169] The inventors further investigated the chemotactic ability of multifunctional NK cells by transwell experiment. 6 NK cells or multifunctional NK-92 cells were added to the lower chamber, and 600 μL of serum-free α-MEM medium was added to the lower chamber, and the chemokine CXCL8 was added at concentrations of 1, 10, and 100 ng / mL for chemotaxis. The cells were returned to the cell culture incubator, and after 48 hours, the cells in the lower chamber were collected and counted. The experimental results are shown in Figure 5. Compared with unmodified NK cells, the number of multifunctional NK cells migrating to the lower chamber was significantly greater. The above experimental results show that gene modification with multifunctional vectors can significantly promote the chemotactic ability of NK cells.

[0170] Example 4: Multifunctional vector gene-modified NK cells have strong in vivo tumor suppression activity

[0171] The inventors used the human colorectal cancer cell line NCI-H716 cells to establish a human colorectal cancer mouse heterotopic transplant tumor model, infected human peripheral blood primary NK cells with a multifunctional vector lentivirus to prepare peripheral blood-derived multifunctional primary NK cells, and observed the therapeutic effect of multifunctional primary NK cells on the colorectal cancer model.

[0172] The specific method is as follows: 6-week-old NCG mice were selected for subcutaneous tumor bearing in the armpits, with a tumor-bearing dose of 1×10 7 NCI-H716 cells / mouse. On day 9 after tumor implantation, the screening tumor volume was 50 mm 3 About 100 mice were used in the experiment and randomly divided into an untreated group, an unmodified NK cell treatment group, and a multifunctional primary NK cell treatment group according to the size of the tumor. The mice in the unmodified NK cell treatment group were treated once every 2 days for a total of 3 times, with a tail vein infusion dose of 8×10 6 CD56 + NK cells, a total of 2.4×10 7 CD56 + NK cells were injected intraperitoneally every 2 days at a dose of 5 × 10 4 IU of IL-2 maintained the activity of NK cells in vivo; the mice in the multifunctional primary NK cell treatment group were reinfused into the tail vein once, with a dose of 2×10 6 Multifunctional primary NK cells were administered per group, without IL-2 injection as adjuvant therapy. Tumor volume was monitored 1-2 times weekly, and tumor growth curves were plotted. The results, shown in Figure 6, show that compared to the unmodified NK cell treatment group, the multifunctional primary NK cell treatment group demonstrated a stronger tumor suppression effect, even at a lower cell therapy dose and without the need for additional IL-2 injections.

[0173] The above test results show that the NK cells modified by the multifunctional vector gene of the present invention have significantly enhanced anti-tumor activity against solid tumors such as colorectal cancer, and the dosage is lower when used, and no additional injection of IL-2 is required (avoiding the side effects of systemic application of IL-2). It is expected to break through the bottleneck of poor treatment effect of immune cell therapy on solid tumors.

[0174] Example 5

[0175] In this example, the inventors obtained the multifunctional vector gene-modified NK cells (multifunctional NK cells) of the present invention through Example 1 and then investigated the killing activity of the multifunctional NK cells against human chronic myeloid leukemia K562 cells.

[0176] The specific method is as follows: K562 cells were fluorescently labeled with CFSE and the cells were stained with 2×10 4 Cells were plated into 96-well plates at a concentration of 100 cells / well. NK cells or multifunctional NK cells were then plated and incubated for 4 hours. Cells were collected into flow cytometry tubes, stained with PI to distinguish live and dead cells, and cytotoxicity was assessed by flow cytometry. Effector cell:target cell ratios of 0.5:1, 1:1, and 2:1 were tested.

[0177] The experimental results are shown in FIG7 , which show that the multifunctional NK cells modified with the multifunctional vector gene of the present invention have a significantly higher killing efficiency against human chronic myeloid leukemia K562 cells than the NK cells that have not been genetically modified.

[0178] Example 6

[0179] In this example, the expression of NKG2D-NKR was combined with the above-mentioned fusion protein and different chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4) using the method described in step 1 of Example 1 to obtain a variety of transgenic immune cells expressing different chemokine receptors. The chemotactic ability of the various transgenic immune cells expressing different chemokine receptors was tested using the method described in Example 3.

[0180] The test results are shown in Figure 8. The results show that while other chemokine receptors have similar effects to CXCR2, compared to other chemokine receptors (CXCR1, CXCR3, and CXCR4), transgenic immune cells expressing NKG2D-NKR in combination with the aforementioned fusion protein and CXCR2 have the strongest chemotactic ability towards tumors; whereas transgenic immune cells prepared with other chemokine receptors have weaker chemotactic ability towards tumors. Furthermore, transgenic immune cells expressing NKG2D-NKR in combination with the aforementioned fusion protein and CXCR2 have stronger anti-tumor activity than other chemokine receptor combinations.

[0181] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0182] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An isolated nucleic acid, characterized in that include: A first nucleic acid fragment, a second nucleic acid fragment and a third nucleic acid fragment, wherein the first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are connected; wherein The first nucleic acid fragment is used to encode an antigen chimeric receptor targeting NKG2D ligand; The second nucleic acid fragment is used to encode a fusion protein, wherein the fusion protein includes IL-15 and IL-15Rα, and the IL-15 and IL-15Rα are connected; The third nucleic acid fragment is used to encode the CXCR2 receptor.

2. The isolated nucleic acid according to claim 1, characterized in that The antigen chimeric receptor comprises: a first segment and a second segment, wherein the first segment and the second segment are connected; The first fragment includes an extracellular region, a transmembrane region and an intracellular region, the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region, and the extracellular region and the transmembrane region are selected from NKG2D receptors; The second piece includes the full-length sequence of DAP10.

3. The isolated nucleic acid according to claim 1, characterized in that The NKG2D receptor has an amino acid sequence as shown in SEQ ID NO: 1; The intracellular region is selected from the intracellular segment of the CD3ζ molecule.

4. The isolated nucleic acid according to claim 3, characterized in that The intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO: 2; The full-length sequence of DAP10 has the amino acid sequence shown in SEQ ID NO:

12.

5. The isolated nucleic acid according to claim 4, characterized in that The full-length sequence encoding DAP10 has a nucleotide sequence as shown in SEQ ID NO:

13.

6. The isolated nucleic acid according to claim 5, characterized in that The antigen chimeric receptor further comprises a third fragment, the N-terminus of the third fragment is connected to the C-terminus of the first fragment, and the C-terminus of the third fragment is connected to the N-terminus of the second fragment, or The C-terminus of the third fragment is connected to the N-terminus of the first fragment, and the N-terminus of the third fragment is connected to the C-terminus of the second fragment; The third fragment includes the sequence of P2A or a fragment thereof.

7. The isolated nucleic acid according to claim 6, characterized in that The P2A or its fragment has the amino acid sequence shown in SEQ ID NO:

14.

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

3.

9. The isolated nucleic acid according to claim 8, characterized in that The first nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:

4.

10. The isolated nucleic acid according to claim 1, characterized in that The C-terminus of IL-15 is connected to the N-terminus of IL-15Rα, or the N-terminus of IL-15 is connected to the C-terminus of IL-15Rα; The IL-15 has an amino acid sequence as shown in SEQ ID NO: 5; The IL-15Rα has the amino acid sequence shown in SEQ ID NO:

6.

11. The isolated nucleic acid according to claim 10, characterized in that The fusion protein further comprises a connecting peptide, the C-terminus of the IL-15 is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of IL-15Rα, or The N-terminus of the IL-15 is connected to the C-terminus of the connecting peptide, and the N-terminus of the connecting peptide is connected to the C-terminus of IL-15Rα; The connecting peptide is selected from any one of a flexible linker and a rigid linker; The connecting peptide has an amino acid sequence as shown in SEQ ID NO:

7.

12. The isolated nucleic acid according to claim 11, characterized in that The fusion protein has an amino acid sequence as shown in SEQ ID NO:

8.

13. The isolated nucleic acid according to claim 12, characterized in that The second nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:

9.

14. The isolated nucleic acid according to claim 1, characterized in that The CXCR2 receptor has an amino acid sequence as shown in SEQ ID NO:

10.

15. The isolated nucleic acid according to claim 1, characterized in that The third nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:

11.

16. The isolated nucleic acid according to claim 1, characterized in that It further comprises two fourth nucleic acid fragments, wherein every two nucleic acid fragments among the first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are connected by one fourth nucleic acid fragment, respectively, wherein each fourth nucleic acid fragment independently encodes P2A or a fragment thereof.

17. The isolated nucleic acid according to claim 16, characterized in that The P2A or fragment thereof includes at least one of P2A, T2A, E2A and F2A or a fragment thereof.

18. The isolated nucleic acid according to claim 17, characterized in that The fourth nucleic acid fragment encodes P2A or a fragment thereof; The P2A or its fragment has the amino acid sequence shown in SEQ ID NO:

14.

19. The isolated nucleic acid according to claim 18, characterized in that The fourth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO:

15.

20. The isolated nucleic acid according to claim 1, characterized in that further comprising a promoter, the promoter being connected to the 5' end of the nucleic acid fragment consisting of the first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment; The promoter is selected from EF1α, SFFV, CAG or CMV.

21. The isolated nucleic acid according to claim 20, characterized in that The promoter is selected from EF1α; The EF1α has a nucleotide sequence as shown in SEQ ID NO:

16.

22. The isolated nucleic acid according to claim 1, characterized in that further comprising a fifth nucleic acid fragment, the fifth nucleic acid fragment encoding a signal peptide, the C-terminus of the signal peptide is connected to the N-terminus of the IL-15, the C-terminus of the IL-15 is connected to the N-terminus of the connecting peptide, the C-terminus of the connecting peptide is connected to the N-terminus of IL-15Rα, or The N-terminus of the IL-15 is connected to the C-terminus of the connecting peptide, the N-terminus of the connecting peptide is connected to the C-terminus of IL-15Rα, and the N-terminus of the IL-15Rα is connected to the C-terminus of the signal peptide; The signal peptide has an amino acid sequence as shown in SEQ ID NO:

17.

23. The isolated nucleic acid according to claim 22, characterized in that The fifth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO:

18.

24. The isolated nucleic acid according to any one of claims 1 to 23, characterized in that The isolated nucleic acid comprises the promoter, the first nucleic acid fragment, one of the fourth nucleic acid fragments, the fifth nucleic acid fragment, the second nucleic acid fragment, another of the fourth nucleic acid fragments and the third nucleic acid fragment from the 5' end to the 3' end.

25. An expression vector carrying the isolated nucleic acid according to any one of claims 1 to 24; The expression vector is selected from a virus, a prokaryotic expression vector or a eukaryotic expression vector.

26. The expression vector according to claim 25, characterized in that The expression vector is selected from viruses.

27. A transgenic immune cell, characterized in that: The transgenic immune cells express antigen chimeric receptors, fusion proteins and CXCR2 receptors targeting NKG2D ligands; The fusion protein includes IL-15Rα and IL-15, and the IL-15 and IL-15Rα are connected.

28. The transgenic immune cell according to claim 27, characterized in that The antigen chimeric receptor comprises: a first segment and a second segment, wherein the first segment and the second segment are connected; The first fragment includes an extracellular region, a transmembrane region and an intracellular region, the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region, and the extracellular region and the transmembrane region are selected from NKG2D receptors; The second fragment includes the full-length sequence of DAP10; The NKG2D receptor has an amino acid sequence as shown in SEQ ID NO: 1; The intracellular region is selected from the intracellular segment of the CD3ζ molecule.

29. The transgenic immune cell according to claim 28, characterized in that The intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO: 2; The full-length sequence of DAP10 has the amino acid sequence shown in SEQ ID NO:

12.

30. The transgenic immune cell according to claim 29, characterized in that The full-length sequence encoding DAP10 has a nucleotide sequence as shown in SEQ ID NO:

13.

31. The genetically modified immune cell according to claim 30, characterized in that: The antigen chimeric receptor further comprises a third fragment, the N-terminus of the third fragment is connected to the C-terminus of the first fragment, and the C-terminus of the third fragment is connected to the N-terminus of the second fragment, or The C-terminus of the third fragment is connected to the N-terminus of the first fragment, and the N-terminus of the third fragment is connected to the C-terminus of the second fragment; The third fragment includes the sequence of P2A or a fragment thereof; The P2A or its fragment has the amino acid sequence shown in SEQ ID NO:

14.

32. The transgenic immune cell according to claim 31, characterized in that The antigen chimeric receptor has an amino acid sequence as shown in SEQ ID NO:

3.

33. The genetically modified immune cell according to claim 32, characterized in that: The C-terminus of IL-15 is connected to the N-terminus of IL-15Rα, or the N-terminus of IL-15 is connected to the C-terminus of IL-15Rα; The IL-15 has an amino acid sequence as shown in SEQ ID NO: 5; The IL-15Rα has the amino acid sequence shown in SEQ ID NO:

6.

34. The transgenic immune cell according to claim 33, characterized in that The fusion protein further comprises a connecting peptide, the C-terminus of the IL-15 is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of IL-15Rα, or The N-terminus of the IL-15 is connected to the C-terminus of the connecting peptide, and the N-terminus of the connecting peptide is connected to the C-terminus of IL-15Rα; The connecting peptide is selected from any one of a flexible linker and a rigid linker; The connecting peptide has an amino acid sequence as shown in SEQ ID NO:

7.

35. The transgenic immune cell according to claim 34, characterized in that The fusion protein has an amino acid sequence as shown in SEQ ID NO:8; The CXCR2 receptor has an amino acid sequence as shown in SEQ ID NO: 10; The DAP10 protein has an amino acid sequence as shown in SEQ ID NO:

12.

36. The transgenic immune cell according to claim 35, characterized in that The transgenic immune cell is obtained by introducing the expression vector according to any one of claims 25 to 26 into an immune cell; The transgenic immune cells are derived from at least one of T cells, NKT cells, NK cells and macrophages, preferably NK cells; The NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell-derived NK cells and NK-92 cells; The T cells include CD4+T cells, CD8+T cells, Treg cells and γδT cells.

37. A pharmaceutical composition, characterized in that include: The isolated nucleic acid according to any one of claims 1 to 24, the expression vector according to any one of claims 25 to 26, or the transgenic immune cell according to any one of claims 27 to 36.

38. A kit, characterized in that include: The isolated nucleic acid according to any one of claims 1 to 24 or the expression vector according to any one of claims 25 to 26.

39. A method for enhancing immune cell killing, activation, proliferation and chemotaxis, characterized in that: include: Introducing the expression vector according to any one of claims 25 to 26 into immune cells; The immune cells introduced with the expression vector are cultured.

40. The method according to claim 39, characterized in that The introduction of the expression vector into the immune cells is carried out by electroporation, transfection or infection; The immune cell is at least one of a T cell, a NKT cell, a NK cell and a macrophage.

41. The method according to claim 40, characterized in that The immune cells are NK cells; The NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell-derived NK cells and NK-92 cells; The T cells include CD4+T cells, CD8+T cells, Treg cells and γδT cells.

42. Use of the isolated nucleic acid according to any one of claims 1 to 24, the expression vector according to any one of claims 25 to 26, the transgenic immune cell according to any one of claims 27 to 36, or the pharmaceutical composition according to claim 37 in the preparation of a drug for treating or preventing a tumor; The tumors include solid tumors and hematological tumors; The solid tumor includes at least one selected from pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, brain glioma, kidney cancer, breast cancer, thyroid cancer, nasopharyngeal cancer, oral cancer, sarcoma, prostate cancer, melanoma and skin squamous cell carcinoma; The blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma and multiple myeloma, myelodysplastic syndrome and myeloproliferative tumors.

43. Use of the isolated nucleic acid according to any one of claims 1 to 24, the expression vector according to any one of claims 25 to 26, the transgenic immune cell according to any one of claims 27 to 36, or the pharmaceutical composition according to claim 37 for preventing and / or treating tumors; The tumors include solid tumors and hematological tumors; The solid tumor includes at least one selected from pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, bile duct cancer, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, brain glioma, kidney cancer, breast cancer, thyroid cancer, nasopharyngeal cancer, oral cancer, sarcoma, prostate cancer, melanoma and skin squamous cell carcinoma; The blood tumor includes at least one selected from acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma and multiple myeloma, myelodysplastic syndrome and myeloproliferative tumors.

44. A method for treating or preventing tumors, characterized in that: include: A pharmaceutically acceptable amount of the transgenic immune cell according to any one of claims 27 to 36 or the pharmaceutical composition according to claim 37 is administered to a subject.

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