Multifunctional genetically modified immune cell, and preparation method and use therefor

Abstract

Provided are a multifunctional genetically modified immune cell, and a preparation method and use therefor. The multifunctional gene modification vector 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 a NKp30 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 multifunctional genetically modified immune cell prepared by using the multifunctional gene modification vector can improve the specific killing ability, survival ability, proliferation ability and tumor infiltration ability of the immune cell, so that the clinical curative effect of the immune cell is further improved.

Description

Multifunctional Genetically Modified Immune Cells, Their Preparation Methods and Applications Technical Field

[0001] This invention relates to the field of biopharmaceuticals, specifically to multifunctional gene-modified immune cells, their preparation methods and applications, and more specifically, to an isolated nucleic acid, expression vector, transgenic immune cells, reagent kit, pharmaceutical composition and its uses. Background Technology

[0002] Natural killer (NK) cells are a major component of the innate immune system, exerting immune surveillance and immunomodulation functions through various pathways. In clinical practice, NK cells are abundant, can be reinfused from other cells, and have few side effects and high safety, thus NK cell therapy has enormous application and development potential. However, NK cell therapy still faces many technical challenges that need to be overcome or resolved, such as low cell viability after cryopreservation and thawing, weak proliferation and persistence in vivo, poor tumor infiltration, and susceptibility to immunosuppression, which severely limit the effectiveness and market value of NK cell therapy.

[0003] Most tumors possess a physical barrier of fibrous tissue, within which exists a tumor microenvironment characterized by hypoxia, low pH, nutritional deficiencies, and high osmotic pressure, and a lack of mature blood vessels. Therefore, the tumor microenvironment is highly unfavorable for the localization, infiltration, survival, and proliferation of immune cells within the tumor, and also easily leads to immunosuppression and exhaustion. Research and development of core technologies in areas such as enhancing the tumor-killing activity and specificity of immune cells, strengthening their infiltration, survival, and proliferation capabilities within tumors, and enhancing their resistance to immunosuppression and exhaustion hold promise for overcoming the bottlenecks in immunocellular therapy for tumor treatment.

[0004] Gene-modified cell therapy is a novel treatment method that modifies the genome of cells in a patient's body to treat diseases. Its principle involves using genetic engineering techniques to introduce exogenous genes or regulatory factors into cells, giving these cells new functions or enhanced therapeutic potential. Gene-modified cell technology is not only applied to T cells but is also extensively studied 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 and has good specific killing activity, cell proliferation, resistance to immune exhaustion and ability to 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 a certain extent.

[0008] This invention is based on the following discoveries of the inventors:

[0009] The inventors unexpectedly discovered that NK cells are prone to immune exhaustion or immunosuppression within tumors, have weak in vivo expansion and persistence, and poor tumor infiltration ability. To address these issues and further enhance the killing activity and specificity of NK cells, the inventors constructed a multifunctional vector expressing a chimeric antigen receptor (NKp30-NKR) with NKp30 as an extracellular recognizer, a cell membrane-expressed IL15 / IL15Rα fusion protein, and a chemokine receptor CXCR2. This vector can be used to genetically modify immune cells such as NK cells, T cells, and macrophages, thereby enhancing the recognition and killing activity of immune cells, increasing their in vivo survival time and cell proliferation, and improving their ability to infiltrate tumors. These capabilities can synergistically enhance the anti-tumor activity of immune cells.

[0010] Therefore, in a first aspect, the present invention provides an isolated nucleic acid. According to embodiments 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, the first, second, and third nucleic acid fragments being linked together; wherein the first nucleic acid fragment encodes an antigen-chimeric receptor targeting the NKp30 ligand; the second nucleic acid fragment encodes a fusion protein comprising IL-15 and IL-15Rα, the IL-15 and IL-15Rα being linked together; and the third nucleic acid fragment encodes a CXCR2 receptor. Through extensive inventive experiments, the inventors have discovered that when immune cells carrying the isolated nucleic acid express the antigen-chimeric receptor (first nucleic acid fragment), the immune cells can bind to the NKp30 ligand, thereby effectively targeting and killing various hematologic malignancies and solid tumor cells expressing the NKp30 ligand; furthermore, immune cells carrying the isolated nucleic acid can also express the IL15 / IL15Rα fusion protein and the CXCR2 receptor, greatly improving their ability to survive and proliferate in vivo, and enhancing their infiltration into tumor tissue, thereby better exerting their anti-tumor effect.

[0011] In a second aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the isolated nucleic acid described in the first aspect. Therefore, the transgenic immune cells prepared using the expression vector of the present invention possess higher tumor recognition and killing capabilities, and can improve their survival time, amplification capacity, and tumor infiltration capacity in vitro and in vivo.

[0012] In a third aspect, the present invention provides a transgenic immune cell. According to embodiments of the present invention, the transgenic immune cell expresses an antigen-chimeric receptor targeting the NKp30 ligand, a fusion protein, and a CXCR2 receptor; wherein the fusion protein includes IL-15Rα and IL-15, and the IL-15 and IL-15Rα are linked. The transgenic immune cell of the present invention possesses high tumor recognition, killing, and tumor infiltration capabilities, can survive long-term in vivo, and exhibits strong expansion, proliferation, and chemotaxis abilities.

[0013] In a 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 cells described in the third aspect. The pharmaceutical composition of the present invention exhibits high tumor-killing efficiency and strong anti-tumor activity, and can be used for the prevention or treatment of various tumor diseases.

[0014] In a fifth aspect, the present invention provides a kit. According to embodiments 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. Using the kit of the present invention, transgenic immune cells can be prepared, resulting in transgenic immune cells with high tumor recognition, killing, and tumor infiltration capabilities, the ability to survive long-term in vivo, and strong amplification, proliferation, and chemotaxis abilities.

[0015] In a 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 includes: introducing the expression vector described in the second aspect into immune cells; and culturing the immune cells infused with the expression vector. Using the method described in this invention, the killing efficiency of immune cells can be enhanced, the antitumor activity of immune cells can be enhanced, and the long-term survival and expansion capacity of immune cells in vitro and in vivo can be improved, as well as their proliferation and chemotaxis abilities. In particular, it can prepare immune cells with strong killing, activation, proliferation, and chemotaxis in vitro for constructing desired immune cell models.

[0016] In a seventh aspect of the invention, the invention provides for the use of the isolated nucleic acid described in the first aspect, the expression vector described in the second aspect, the transgenic immune cells described in the third aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of a medicament for the treatment or prevention of tumors.

[0017] In an eighth aspect of the invention, the invention provides for 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 prevention and / or treatment of tumors.

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

[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 is a schematic diagram of the gene element structure of the multifunctional vector of Embodiment 1 of the present invention;

[0022] Figure 2 is a flow cytometry graph showing the expression of each element of the multifunctional carrier in Embodiment 2 of the present invention; wherein:

[0023] A shows the flow cytometry detection of mbIL15RF and CXCR2 element expression on uninfected NK cells;

[0024] B is a flow cytometry diagram showing the expression of mbIL15RF and CXCR2 elements on NK cells infected with a multifunctional vector lentivirus.

[0025] C shows the MFI results for detecting NKp30 expression on NK cells in the uninfected and infected groups;

[0026] Figure 3 is a diagram showing the effect of multifunctional NK cells on tumor cell killing activity in Example 3 of the present invention.

[0027] Figure 4 is a graph showing the survival rate of NK cells and pluripotent NK cells under different IL-2 culture concentrations in Example 3 of the present invention.

[0028] Figure 5 shows the results of detecting the chemotactic ability of NK cells and pluripotent NK cells at different CXCL8 concentrations in Example 3 of the present invention.

[0029] Figure 6 shows the tumor-suppressing effect of peripheral blood-derived multifunctional primary NK cells on a mouse model bearing human colorectal cancer NCI-H716 cells in Example 4 of the present invention.

[0030] Figure 7 shows the killing efficiency of NK cells and pluripotent NK cells against human chronic myeloid leukemia K562 cells in Example 5 of the present invention.

[0031] Figure 8 shows the chemotactic capacity of various transgenic immune cells with different chemokine receptors in Example 6 of the present invention. Detailed Implementation

[0032] 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.

[0033] 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 technical features indicated, nor are they in any particular order. 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.

[0034] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is 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.

[0035] 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.

[0036] In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0037] In this invention, the terms “optionally,” “optionally,” or “optionally” generally refer to events or conditions described subsequently 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.

[0038] In this document, the term "vector" or "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, which transfers the inserted nucleic acid molecule to host cells and / or between host cells. The 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 vector also includes vectors having a variety of the functions described above. The 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 vector, the vector can produce the desired expression product.

[0039] In this document, the term "pharmaceutical composition" generally refers to a unit dosage 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 sufficiently combining the active compound with a liquid carrier, a solid carrier, or both.

[0040] In this document, the term "treatment" refers to the use of drugs to achieve desired pharmacological and / or physiological effects. These effects may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in susceptible individuals who have not yet been diagnosed with the disease; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of drugs or transgenic immune cells to an individual to treat, cure, alleviate, improve, reduce, or suppress the individual's disease.

[0041] The term "immune cell" generally refers to cells capable of generating an immune response (e.g., an antigen-specific immune response). For example, the immune cell may contain, or already contains, the isolated nucleic acids and / or vectors described in this application, or individual cells, cell lines, or cell cultures capable of expressing the antigen chimeric receptor, fusion protein, and CXCR2 receptor described in this application. In this application, the 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.

[0042] In this article, "carbon terminus" and "C-terminus" are synonymous; "nitrogen terminus" and "N-terminus" are synonymous.

[0043] The inventors have proposed a multifunctional vector expressing a chimeric antigen recognition receptor targeting the NKp30 ligand, enhanced cytokine and chemokine receptors expressed on the cell membrane, which can be used to modify NK cells with multifunctional genes and enhance their anti-tumor activity. In experiments, the inventors found that the vector gene transduction into NK cells has the following beneficial effects: 1) The expressed NKp30-NKR can enhance the killing efficiency of NK cells against tumors with high NKp30 ligand expression, thus enhancing the anti-tumor activity of NK cells; 2) The expression of the IL15 / IL15Rα fusion protein on the cell membrane can further improve the long-term survival and expansion capacity of NK cells in vitro and in vivo; 3) The expression of the CXCR2 receptor can further enhance the tumor infiltration capacity of NK cells.

[0044] Specifically, this invention proposes an isolated nucleic acid, an expression vector, transgenic immune cells, a pharmaceutical composition, a reagent kit, a method for enhancing the killing, activation, proliferation, and chemotaxis of immune cells, and their uses, which will be described in detail below.

[0045] isolated nucleic acids

[0046] This invention proposes an isolated nucleic acid comprising: 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 together; wherein the first nucleic acid fragment encodes an antigen-chimeric receptor targeting the NKp30 ligand; the second nucleic acid fragment encodes a fusion protein comprising IL-15 and IL-15Rα, wherein IL-15 and IL-15Rα are linked together; and the third nucleic acid fragment encodes a CXCR2 receptor.

[0047] Through extensive and creative experiments, the inventors discovered that when immune cells carrying the isolated nucleic acid express antigen-chimeric receptors, they can bind to NKp30 ligands, thereby effectively targeting and killing various hematologic malignancies and solid tumor cells expressing NKp30 ligands. In addition, immune cells carrying the isolated nucleic acid can also express IL15 / IL15Rα fusion protein and CXCR2 receptors, greatly improving their ability to survive and proliferate in vivo and enhancing their infiltration into tumor tissues, thus better exerting their anti-tumor effects.

[0048] It should be noted that the three nucleic acid fragments mentioned above in this invention are intended for independent expression. Therefore, the connection order of the three nucleic acid fragments in the gene mentioned above in this invention can be selected according to actual needs and does not require special limitation.

[0049] In some embodiments, the isolated nucleic acid may further include at least one of the following additional technical features:

[0050] In some embodiments, the antigen-chimeric receptor includes an extracellular region and a transmembrane region of an NKp30 receptor, and an intracellular region, wherein the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region.

[0051] In some embodiments, the extracellular region and transmembrane region have an amino acid sequence as shown in SEQ ID NO:11.

[0052] In some embodiments, the intracellular region includes a co-stimulatory domain and an intracellular signal transduction domain.

[0053] In some embodiments, the co-stimulatory domain is selected from the intracellular segment of the CD28 molecule.

[0054] In some embodiments, the intracellular segment of the CD28 molecule has an amino acid sequence as shown in SEQ ID NO:12.

[0055] In some embodiments, the intracellular signal transduction domain is selected from the intracellular segment of the CD3ζ molecule.

[0056] In some embodiments, the intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO:13.

[0057] In some embodiments, the C-terminus of the CD28 molecule is connected to the N-terminus of the CD3ζ molecule.

[0058] In some embodiments, the antigen chimeric receptor has an amino acid sequence as shown in SEQ ID NO:1.

[0059] In some embodiments, the first nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:2.

[0060] In some embodiments, 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.

[0061] In some embodiments, the IL-15 has an amino acid sequence as shown in SEQ ID NO:6.

[0062] In some embodiments, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO:5.

[0063] In some embodiments, the fusion protein further includes a linker peptide, wherein the C-terminus of the IL-15 is linked to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is linked to the N-terminus of IL-15Rα; or the N-terminus of the IL-15 is linked to the C-terminus of the linker peptide, and the N-terminus of the linker peptide is linked to the C-terminus of IL-15Rα.

[0064] In some embodiments, the linker peptide is selected from either a flexible linker or a rigid linker.

[0065] In some embodiments, the linker peptide has an amino acid sequence as shown in SEQ ID NO:14.

[0066] In some embodiments, the fusion protein has an amino acid sequence as shown in SEQ ID NO:3.

[0067] In some embodiments, the second nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:4.

[0068] In some embodiments, the CXCR2 receptor has an amino acid sequence as shown in SEQ ID NO:9.

[0069] In some embodiments, the third nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:10.

[0070] In some embodiments, the isolated nucleic acid further comprises two fourth nucleic acid fragments, wherein each pair of nucleic acid fragments in the first, second, and third nucleic acid fragments is connected by one of the fourth nucleic acid fragments, wherein each of the fourth nucleic acid fragments independently encodes P2A or a fragment thereof.

[0071] In some embodiments, the P2A or a fragment thereof includes at least one of P2A, T2A, E2A, and F2A or a fragment thereof.

[0072] In some embodiments, the fourth nucleic acid fragment encodes P2A or a fragment thereof.

[0073] In some embodiments, the P2A or a fragment thereof has an amino acid sequence as shown in SEQ ID NO:7.

[0074] In some embodiments, the fourth nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:8.

[0075] In some embodiments, the isolated nucleic acid further includes a promoter connected to the 5' end of a nucleic acid fragment composed of the first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment.

[0076] In some embodiments, the promoter is selected from EF1α, SFFV, CAG, or CMV.

[0077] In some embodiments, the promoter is selected from EF1α.

[0078] In some embodiments, the EF1α has a nucleotide sequence as shown in SEQ ID NO:15.

[0079] In some embodiments, the isolated nucleic acid further includes a fifth nucleic acid fragment encoding a signal peptide, the C-terminus of which is linked to the N-terminus of IL-15, the C-terminus of which is linked to the N-terminus of a linker peptide, and the C-terminus of which is linked to the N-terminus of IL-15Rα; or the N-terminus of which is linked to the C-terminus of which is linked to the N-terminus of which is linked to the C-terminus of IL-15Rα, and the N-terminus of which is linked to the C-terminus of the signal peptide.

[0080] In some embodiments, the signal peptide has an amino acid sequence as shown in SEQ ID NO:16.

[0081] In some embodiments, the fifth nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:17.

[0082] It should be noted that the "fusion protein" mentioned in this article may or may not contain the "signal peptide". When constructing the multifunctional vector shown in Figure 1, the "fusion protein" contains the "signal peptide". However, when the multifunctional vector is introduced into immune cells to obtain transgenic immune cells, the "signal peptide" will be cleaved off. In this case, the "fusion protein" does not contain the "signal peptide". Therefore, whether the "fusion protein" contains the "signal peptide" should be considered on a case-by-case basis. Both "fusion proteins" that contain or do not contain the "signal peptide" are within the protection scope of this invention.

[0083] In some embodiments, the isolated nucleic acid, from the 5' end to the 3' end, is sequentially 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.

[0084] expression carrier

[0085] This invention provides an expression vector carrying the isolated nucleic acid described above. Therefore, transgenic immune cells prepared using the expression vector of this invention exhibit higher tumor recognition and killing capabilities, and can improve their survival time, amplification capacity, and tumor infiltration capacity both in vivo and in vitro.

[0086] When linking the aforementioned nucleic acid molecules to an expression vector, the nucleic acid molecules can be directly or indirectly connected to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. These control elements can be directly derived from the expression vector itself or be exogenous, i.e., not originating from the vector itself. Of course, the connection between the nucleic acid molecules and the control elements must be operably established.

[0087] In this article, "operable ligation" refers to ligating a foreign gene into an expression vector so that the control elements within the vector, such as transcriptional and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include plasmids and bacteriophages.

[0088] In some embodiments, the expression vector may further include at least one of the following additional technical features:

[0089] In some embodiments, the expression vector is selected from viruses, prokaryotic expression vectors, or eukaryotic expression vectors.

[0090] In some embodiments, the expression vector is selected from viruses.

[0091] Transgenic immune cells

[0092] This invention proposes a transgenic immune cell that expresses an antigen-chimeric receptor targeting the NKp30 ligand, a fusion protein, and a CXCR2 receptor; wherein the fusion protein includes IL-15Rα and IL-15, and the IL-15 and IL-15Rα are linked. The transgenic immune cell of this invention exhibits high tumor recognition and killing capabilities, and can improve its survival time, proliferation capacity, and tumor infiltration capacity both in vivo and in vitro.

[0093] In some embodiments, the above-described transgenic immune cells may further include at least one of the following additional technical features:

[0094] In some embodiments, the antigen-chimeric receptor includes an extracellular region and a transmembrane region of an NKp30 receptor, and an intracellular region, wherein the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region.

[0095] In some embodiments, the extracellular region and transmembrane region have an amino acid sequence as shown in SEQ ID NO:11.

[0096] In some embodiments, the intracellular region includes a co-stimulatory domain and an intracellular signal transduction domain.

[0097] In some embodiments, the co-stimulatory domain is selected from the intracellular segment of the CD28 molecule.

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

[0099] In some embodiments, the C-terminus of the CD28 molecule is connected to the N-terminus of the CD3ζ molecule.

[0100] In some embodiments, the intracellular segment of the CD28 molecule has an amino acid sequence as shown in SEQ ID NO:12.

[0101] In some embodiments, the intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO:13.

[0102] In some embodiments, the antigen chimeric receptor has an amino acid sequence as shown in SEQ ID NO:1.

[0103] In some embodiments, 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.

[0104] In some embodiments, the IL-15 has an amino acid sequence as shown in SEQ ID NO:6.

[0105] In some embodiments, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO:5.

[0106] In some embodiments, the fusion protein further includes a linker peptide, wherein the C-terminus of the IL-15 is linked to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is linked to the N-terminus of IL-15Rα; or the N-terminus of the IL-15 is linked to the C-terminus of the linker peptide, and the N-terminus of the linker peptide is linked to the C-terminus of IL-15Rα.

[0107] In some embodiments, the linker peptide is selected from either a flexible linker or a rigid linker.

[0108] In some embodiments, the linker peptide has an amino acid sequence as shown in SEQ ID NO:14.

[0109] In some embodiments, the fusion protein has an amino acid sequence as shown in SEQ ID NO:3.

[0110] In some embodiments, the CXCR2 receptor has an amino acid sequence as shown in SEQ ID NO:9.

[0111] In some embodiments, the transgenic immune cells are obtained by introducing the above-described expression vector into immune cells.

[0112] In some embodiments, the transgenic immune cells are derived from at least one of T cells, NKT cells, NK cells, and macrophages.

[0113] In some specific embodiments, the transgenic immune cells are derived from NK cells.

[0114] 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.

[0115] In some embodiments, the T cells include CD4+ T cells, CD8+ T cells, and γδ T cells.

[0116] In some embodiments, the T cells include CD4+ T cells, CD8+ T cells, Treg cells, and γδ T cells.

[0117] Pharmaceutical Composition

[0118] This invention provides a pharmaceutical composition comprising: the isolated nucleic acid described above, the expression vector described above, or the transgenic immune cells described above. The pharmaceutical composition of this invention exhibits high tumor-killing efficiency and strong anti-tumor activity, and can be used for the prevention or treatment of various tumor diseases.

[0119] Reagent test kit

[0120] This invention provides a kit comprising: the isolated nucleic acid or the expression vector described above. Using the kit of this invention, transgenic immune cells can be prepared, resulting in transgenic immune cells with high tumor recognition, killing, and tumor infiltration capabilities, long-term survival in vivo, and strong amplification, proliferation, and chemotaxis abilities.

[0121] A method to enhance the killing, activation, proliferation and chemotaxis of immune cells

[0122] This invention proposes a method to enhance the killing, activation, proliferation, and chemotaxis of immune cells. The method includes: introducing the aforementioned expression vector into immune cells; and culturing the immune cells infused with the expression vector. Using the method described in this invention, the killing efficiency of immune cells can be enhanced, the anti-tumor activity of immune cells can be increased, and the long-term survival and expansion capabilities of immune cells in vitro and in vivo can be improved, as well as their proliferation and chemotaxis abilities. In particular, it can prepare immune cells with strong killing, activation, proliferation, and chemotaxis in vitro for constructing desired immune cell models.

[0123] In some embodiments, the above-described methods for enhancing the killing, activation, proliferation, and chemotaxis of immune cells may further include at least one of the following additional technical features:

[0124] In some embodiments, the introduction of the expression vector into immune cells is performed by electroporation, transfection, or infection.

[0125] In some embodiments, the immune cells are at least one of T cells, NKT cells, NK cells, and macrophages.

[0126] In some specific embodiments, the immune cells are NK cells.

[0127] 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.

[0128] In some embodiments, the T cells include CD4+ T cells, CD8+ T cells, and γδ T cells.

[0129] In some embodiments, the T cells include CD4+ T cells, CD8+ T cells, Treg cells, and γδ T cells.

[0130] use

[0131] The use of the isolated nucleic acid, the expression vector, the transgenic immune cell, or the pharmaceutical composition described above in the preparation of a drug for the treatment or prevention of tumors.

[0132] The use of the isolated nucleic acid, the expression vector, the transgenic immune cell, or the pharmaceutical composition described above in the prevention and / or treatment of tumors.

[0133] In some embodiments, the above uses may further include at least one of the following additional technical features:

[0134] In some embodiments, the tumor includes solid tumors and hematomas.

[0135] In some embodiments, 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, glioma, kidney cancer, breast cancer, thyroid cancer, nasopharyngeal carcinoma, oral cancer, sarcoma, prostate cancer, melanoma, and squamous cell carcinoma of the skin.

[0136] In some embodiments, the hematologic malignancy includes at least one selected from acute myeloid leukemia, acute lymphoblastic 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 neoplasm.

[0137] In some embodiments, 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, glioma, kidney cancer, breast cancer, thyroid cancer, osteosarcoma, prostate cancer, and melanoma.

[0138] In some embodiments, the hematologic malignancy includes at least one selected from acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma.

[0139] This invention provides a method for treating or preventing tumors. According to embodiments of the invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned transgenic immune cells or the aforementioned pharmaceutical composition.

[0140] The effective dosage described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective dosage 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.

[0141] 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, such as liquid, semi-solid, 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, intramuscular or subcutaneous injection, intraperitoneal injection, intrapleural injection, hepatic artery infusion, bladder infusion, or intrathecal administration.

[0142] According to embodiments of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0143] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting 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 art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0144] The sequence descriptions involved in this invention are detailed in Table 1.

[0145] Table 1: Amino Acid / Nucleotide Sequence Description

[0146] Example 1: Preparation of NK-92 cells modified with a multifunctional vector

[0147] 1. Construction of a multifunctional carrier

[0148] The nucleotide sequence shown in SEQ ID NO:21 was synthesized through whole-genome synthesis. After synthesis, it was cloned into the lentiviral vector pLVX-EF1-IRES-Puro via the EcoRI and MluI restriction sites. After sequencing verification, the pLVX-EF1-multi-functional-vector plasmid was obtained, which is the multifunctional vector plasmid involved in this invention. A schematic diagram of the gene element structure of the multifunctional vector in this embodiment is shown in Figure 1.

[0149] 2. Packaging of lentiviruses

[0150] Take 5 × 10⁵ 293T cells in the logarithmic growth phase 6 Inoculate cells into 10cm cell culture dishes, add 10mL of DMEM medium, and incubate overnight at 37°C with 5% CO2. When the cell density reaches 80-90%, replace with 10mL of fresh DMEM medium and keep the cell culture dishes in the incubator for later use. Prepare the lentiviral packaging system by adding 6μg of psPAX2 and 3μg of pMD2.G lentiviral packaging helper plasmids, and 6μg of lentiviral vector plasmid to 250μL of serum-free DMEM medium and mixing thoroughly. Add 15μL of... Add to 235 μL of serum-free DMEM medium and mix well. Add the mixture to the plasmid mixture in one go, mix well, and incubate at room temperature for 15 min. Add the mixture to 293T cell culture dishes. After 24 h, change the medium and return the culture dishes to a 37℃, 5% CO2 incubator. After 48 h, collect the cell supernatant, centrifuge at 400×g for 5 min to remove cell debris, and filter the supernatant through a 0.45 μm filter into a 50 mL centrifuge tube. Add 5×PEG8000 solution to concentrate the virus solution, mix well by inverting the centrifuge tube, and incubate overnight at 4℃. Centrifuge at 4℃, 4000×g for 20 min, discard the supernatant, resuspend the virus pellet in an appropriate amount of serum-free DMEM, transfer to an EP tube, and store at -80℃.

[0151] 3. Lentiviral infection of human NK cells

[0152] Collect NK cells in the logarithmic growth phase, centrifuge at 100×g for 5 min to harvest the cells, resuspend the cells in an appropriate amount of α-MEM medium, and adjust the cell density to 5×10⁶ cells / year. 5 Cells / mL. 5 × 10⁵ cells / mL were added to each well of a 24-well plate. 5 One NK cell, 0.2 mL of virus concentrate, 0.8 mL of α-MEM medium, and protamine (final concentration 8 μg / mL) were mixed thoroughly. The mixture was incubated at 37℃ in a 5% CO2 incubator. After 24 h, the cell status was observed, the medium was changed, and the infected cells were transferred to EP tubes. The cells were centrifuged at 100×g for 5 min, and a small amount of fresh α-MEM medium was added to resuspend the cells. The cells were then transferred to a cell culture flask, and 10 mL of fresh α-MEM medium and IL-2 (final concentration 200 IU / mL) were added. The cells were cultured for another 48 h. The cells were then transferred to fresh α-MEM medium, and IL-2 was completely removed. The cells were then subjected to pressure selection for 2 weeks to obtain multifunctional vector gene-modified NK cells (multifunctional NK cells), which were used for subsequent functional experiments.

[0153] Example 2: Expression Verification of Each Component of the Multifunctional Carrier

[0154] Peripheral blood mononuclear cells (PBMCs) were isolated and cultured in pre-coated culture flasks, following CN 202310035787.4, and induced with cytokines such as IL-2. Lentiviral infection was performed on day 7 of culture. After medium replacement on day 9, culture continued, and flow cytometry was performed on day 11 to detect the expression of various elements on NK cells. The flow cytometry method is as follows: 1×10⁶ PBMCs were cultured in pre-coated culture flasks. 6Cells were added to flow cytometry tubes for staining. Different staining protocols were followed by antibody administration, and the cells were incubated at room temperature for 30 min. After washing twice with 1×PBS, the cell pellet was resuspended and analyzed by flow cytometry. Protocol 1 used PerCP / Cyanine 5.5-labeled anti-human CD3 antibody (purchased from Biolegend), Brilliant Violet 785™-labeled anti-human CD56 antibody (purchased from Biolegend), APC-labeled anti-human NKp30 antibody (purchased from Biolegend), and PE-labeled anti-human CXCR2 antibody (purchased from Biolegend). Protocol 2 used the same antibodies.

[0155] Analysis of CD3 and CD56 using their respective gate systems. - CD56 + The cells exhibiting the desired phenotype were NK cells. Next, the positivity rates of mbIL15RF (double-positive for IL-15 and IL-15Rα) or CXCR2 expression on NK cells were analyzed, along with the mean fluorescence intensity (MFI) of NKp30 expression. The results are shown in Figures 2A and 2B. Uninfected NK cells did not express CXCR2 or mbIL15RF molecules. After infection with the multifunctional vector lentivirus, the expression of CXCR2 and mbIL15RF molecules on NK cells significantly increased. As shown in Figure 2C, the mean fluorescence intensity of NKp30 expression on infected NK cells was also significantly higher than that on uninfected NK cells. These results indicate that all three functional elements designed in the multifunctional vector can be expressed efficiently.

[0156] Example 3: In vitro functional detection of NK cells modified by multifunctional vector

[0157] In this embodiment, after obtaining the multifunctional vector gene-modified NK cells (multifunctional NK cells) of the present invention through Example 1, the inventors examined the killing activity, survival and chemotaxis of the multifunctional NK cells in vitro.

[0158] 1. Multifunctional vector gene modification promotes the killing activity of NK cells.

[0159] The inventors tested the cytotoxic activity of NK cells modified with a multifunctional vector gene. The specific method is as follows: Colorectal cancer NCI-H716 cells were fluorescently labeled using CFSE at a ratio of 2×10⁻⁶. 4Cells were inoculated into 96-well plates, and NK cells or pluripotent NK cells were then inoculated into the plates and incubated for 4 hours. Cells were collected in flow cytometry tubes, and PI staining was added to distinguish between live and dead cells. The killing efficiency was detected by flow cytometry. The ratio of effector cells to target cells was 2:1.

[0160] The experimental results are shown in Figure 3. The multifunctional NK cells modified with the multifunctional vector gene of this invention have a significantly higher killing efficiency against colorectal cancer NCI-H716 cells than the unmodified NK cells.

[0161] 2. Multifunctional vector gene modification promotes NK cell survival.

[0162] The inventors further verified the effect of multifunctional vector gene modification on NK cell survival promotion. The specific method is as follows: NK cells and multifunctional NK cells of equal numbers were inoculated into 24-well plates, and different IL-2 concentrations (0, 20, and 200 IU / mL) were set. Flow cytometry was performed every 24 hours to detect apoptosis rate. The flow cytometry detection of apoptosis rate was performed according to the kit instructions (Linke Biotechnology, catalog number AP101), briefly as follows: cells were collected in EP tubes, washed once by centrifugation with 1×PBS solution, and resuspended. 5 μL of Annexin V-FITC and 10 μL of PI were added to each tube. After gentle vortexing, the cells were incubated at room temperature in the dark for 5 min, and then resuspended for flow cytometry detection. Cell viability was determined by the proportion of cells that were negative for both Annexin V-FITC and PI staining.

[0163] Figure 4 shows the results of NK cell viability assessment after 96 hours of culture: In the absence of IL-2 (0 IU / mL), the viability of unmodified NK cells decreased significantly from 24 hours, with most cells undergoing apoptosis by 72 hours. In contrast, the multifunctional NK cell group maintained high viability even under complete IL-2 removal conditions, with very few cells undergoing apoptosis. These results suggest that multifunctional vector gene modification can play an important role in promoting NK cell survival.

[0164] 3. Multifunctional vector genes promote the chemotaxis of NK cells.

[0165] The inventors further investigated the chemotactic ability of pluripotent NK cells using a transwell assay. 1×10⁻⁶ cells were introduced into the upper chamber of the transwell. 6NK cells or pluripotent NK cells were cultured in a lower chamber with 600 μL of serum-free α-MEM medium, and chemokine CXCL8 at concentrations of 1, 10, and 100 ng / mL were added, respectively, for chemotaxis. The cells were then returned to the cell culture incubator, and after 48 h, cells in the lower chamber were collected for cell counting. The results are shown in Figure 5. Compared with unmodified NK cells, pluripotent NK cells showed a significantly higher number of cells migrating to the lower chamber. These results indicate that pluripotent vector gene modification can significantly promote the chemotactic ability of NK cells.

[0166] Example 4: NK cells modified with multifunctional vector genes exhibit potent in vivo tumor-suppressing activity.

[0167] The inventors established a mouse ectopic xenograft model of human colorectal cancer using the human colorectal cancer cell line NCI-H716, and prepared peripheral blood-derived multifunctional primary NK cells by infecting human peripheral blood primary NK cells with a multifunctional vector lentivirus. The therapeutic effect of multifunctional primary NK cells on the colorectal cancer model was then observed.

[0168] The specific method is as follows: Six-week-old NCG mice were selected for subcutaneous tumor implantation in the axilla, with a tumor-bearing dose of 1×10⁻⁶. 7 NCI-H716 cells / device. On day 9 post-tumor implantation, tumors with a volume of 50 mm were selected. 3 Approximately 100 mice were used in the experiment and randomly divided into three groups based on tumor size: an untreated group, a non-genetically modified NK cell therapy group, and a multifunctional primary NK cell therapy group. Mice in the non-genetically modified NK cell therapy group were treated every two days for a total of three treatments, with each treatment involving a tail vein infusion dose of 8 × 10⁻⁶. 6 CD56 + NK cells, a total of 2.4 × 10⁻⁶ were infused. 7 CD56 + NK cells, and intraperitoneal injection of 5×10 every 2 days. 4 IU of IL-2 maintains NK cell activity in vivo; mice in the multifunctional primary NK cell therapy group received one tail vein infusion treatment at a dose of 2×10⁻⁶. 6 Multifunctional primary NK cells / cell, without adjuvant IL-2 injection. Tumor volume was observed 1-2 times weekly, and tumor growth curves were plotted. The results are shown in Figure 6. Compared with the unmodified NK cell therapy group, the multifunctional primary NK cell therapy group showed stronger tumor suppression effect at a lower cell therapy dose and without the need for additional IL-2 injection.

[0169] The above experimental results show that the multifunctional vector gene-modified NK cells based on the present invention have significantly enhanced anti-tumor activity against solid tumors such as colorectal cancer, and the dosage is lower when applied and no additional injection of IL-2 is required (avoiding the side effects of systemic IL-2 application), which is expected to break through the bottleneck of poor efficacy of immunotherapy for tumor treatment.

[0170] Example 5

[0171] In this embodiment, after obtaining the multifunctional vector gene-modified NK cell (multifunctional NK cell) of the present invention through Example 1, the inventors investigated the killing activity of the multifunctional NK cell against human chronic myeloid leukemia K562 cells.

[0172] The specific method is as follows: K562 cells were fluorescently labeled using CFSE at a ratio of 2×10⁻⁶. 4 Cells were inoculated into 96-well plates, and NK or multifunctional NK-92 cells were then inoculated into the plates and incubated for 4 hours. Cells were collected in flow cytometry tubes, and PI staining was added to distinguish between live and dead cells. The killing efficiency was then detected by flow cytometry.

[0173] The experimental results are shown in Figure 7. The results show that the multifunctional NK cells modified by the multifunctional vector gene of this invention have a significantly higher killing efficiency against human chronic myeloid leukemia K562 cells than the unmodified NK cells.

[0174] Example 6

[0175] In this embodiment, the method described in step 1 of Example 1 was used to combine NKp30-NKR expression with the aforementioned fusion protein and different chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4) to obtain various transgenic immune cells expressing different chemokine receptors. The chemotactic ability of these various transgenic immune cells expressing different chemokine receptors was detected using the method described in Example 3.

[0176] The experimental results are shown in Figure 8. The results indicate that although other chemokine receptors have similar effects to CXCR2, compared to other chemokine receptors (CXCR1, CXCR3, CXCR4), the transgenic immune cells expressing NKp30-NKR combined with the aforementioned fusion protein and CXCR2 exhibited the strongest chemotactic activity towards tumor sites; while transgenic immune cells prepared with other chemokine receptors showed weaker tumor chemotactic activity. Furthermore, the transgenic immune cells expressing NKp30-NKR combined with the aforementioned fusion protein and CXCR2 exhibited stronger antitumor activity than combinations of other chemokine receptors.

[0177] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is 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.

[0178] 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. An isolated nucleic acid, comprising, include: The first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment are linked together; among them... The first nucleic acid fragment is used to encode an antigen-chimeric receptor that targets the NKp30 ligand; The second nucleic acid fragment is used to encode a fusion protein, the fusion protein comprising IL-15 and IL-15Rα linked together; The third nucleic acid fragment is used to encode the CXCR2 receptor.

2. The isolated nucleic acid of claim 1, wherein, The antigen-chimeric receptor includes: The extracellular and transmembrane regions of the NKp30 receptor, and The intracellular region has its N-terminus connected to the C-terminus of the transmembrane region.

3. The isolated nucleic acid of claim 2, wherein, The extracellular and transmembrane regions have the amino acid sequences shown in SEQ ID NO:11; The intracellular region includes a co-stimulatory domain and an intracellular signal transduction domain; The co-stimulatory domain is selected from the intracellular segment of the CD28 molecule; The intracellular signal transduction domain is selected from the intracellular segment of the CD3ζ molecule; The C-terminus of the CD28 molecule is connected to the N-terminus of the CD3ζ molecule.

4. The isolated nucleic acid of claim 3, wherein, The intracellular segment of the CD28 molecule has the amino acid sequence shown in SEQ ID NO:12; The intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO:

13.

5. The isolated nucleic acid of claim 4, wherein, The antigen-chimeric receptor has the amino acid sequence shown in SEQ ID NO:

1.

6. The isolated nucleic acid of claim 5, wherein, The first nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:

2.

7. The isolated nucleic acid of claim 1, wherein 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.

8. The isolated nucleic acid of claim 7, wherein, The IL-15 has the amino acid sequence shown in SEQ ID NO:6; The IL-15Rα has the amino acid sequence shown in SEQ ID NO:

5.

9. The isolated nucleic acid of claim 8, wherein, The fusion protein further includes a linker peptide, wherein the C-terminus of the IL-15 is linked to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is linked to the N-terminus of IL-15Rα, or... The N-terminus of IL-15 is connected to the C-terminus of the linker peptide, and the N-terminus of the linker peptide is connected to the C-terminus of IL-15Rα.

10. The isolated nucleic acid of claim 9, wherein, The linker peptide is selected from either a flexible linker or a rigid linker.

11. The isolated nucleic acid of claim 10, wherein, The linker peptide has an amino acid sequence as shown in SEQ ID NO:

14.

12. The isolated nucleic acid of claim 11, wherein, The fusion protein has the amino acid sequence shown in SEQ ID NO:

3.

13. The isolated nucleic acid of claim 12, wherein, The second nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:

4.

14. The isolated nucleic acid of claim 1, wherein The CXCR2 receptor has the amino acid sequence shown in SEQ ID NO:

9.

15. The isolated nucleic acid of claim 1, wherein The third nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:

10.

16. The isolated nucleic acid of claim 1, wherein It further includes two fourth nucleic acid fragments, wherein each pair of nucleic acid fragments in the first, second, and third nucleic acid fragments is connected by one of the fourth nucleic acid fragments, wherein each of the fourth nucleic acid fragments independently encodes P2A or a fragment thereof.

17. The isolated nucleic acid of claim 16, wherein The P2A or a fragment thereof includes at least one of P2A, T2A, E2A and F2A or a fragment thereof.

18. The isolated nucleic acid of claim 17, wherein, The fourth nucleic acid fragment encodes P2A or a fragment thereof; The P2A or its fragments have an amino acid sequence as shown in SEQ ID NO:

7.

19. The isolated nucleic acid of claim 18, wherein, The fourth nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:

8.

20. The isolated nucleic acid of claim 1, wherein It further includes a promoter connected to the 5' end of a nucleic acid fragment composed 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 of claim 20, wherein, The promoter is selected from EF1α; The EF1α has a nucleotide sequence as shown in SEQ ID NO:

15.

22. The isolated nucleic acid of claim 1, wherein It further includes a fifth nucleic acid fragment encoding a signal peptide, the C-terminus of which is linked to the N-terminus of IL-15, the C-terminus of IL-15 being linked to the N-terminus of a linker peptide, and the C-terminus of the linker peptide being linked to the N-terminus of IL-15Rα, or... The N-terminus of IL-15 is connected to the C-terminus of the linker peptide, the N-terminus of the linker 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. The signal peptide has the amino acid sequence shown in SEQ ID NO:

16.

23. The isolated nucleic acid of claim 22, wherein The fifth nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:

17.

24. The isolated nucleic acid of any one of claims 1-23, wherein, The isolated nucleic acid, from the 5' end to the 3' end, consists of 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.

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

26. The expression vector of claim 25, wherein, The expression vector is selected from viruses.

27. A transgenic immune cell, wherein, The transgenic immune cells express an antigen-chimeric receptor, a fusion protein, and a CXCR2 receptor that target the NKp30 ligand. The fusion protein includes IL-15Rα and IL-15, with IL-15 and IL-15Rα linked together.

28. The genetically modified immune cell of claim 27, wherein, The antigen-chimeric receptor includes: The extracellular and transmembrane regions of the NKp30 receptor, and The intracellular region, wherein the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region; The extracellular and transmembrane regions have the amino acid sequences shown in SEQ ID NO:11; The intracellular region includes a co-stimulatory domain and an intracellular signal transduction domain; The co-stimulatory domain is selected from the intracellular segment of the CD28 molecule; The intracellular signal transduction domain is selected from the intracellular segment of the CD3ζ molecule; The C-terminus of the CD28 molecule is connected to the N-terminus of the CD3ζ molecule.

29. The genetically modified immune cell of claim 28, wherein, The intracellular segment of the CD28 molecule has the amino acid sequence shown in SEQ ID NO:12; The intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO:

13.

30. The genetically modified immune cell of claim 29, wherein, The antigen-chimeric receptor has the amino acid sequence shown in SEQ ID NO:

1.

31. The genetically modified immune cell of claim 30, wherein, 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 the amino acid sequence shown in SEQ ID NO:6; The IL-15Rα has the amino acid sequence shown in SEQ ID NO:

5.

32. The genetically modified immune cell of claim 31, wherein, The fusion protein further includes a linker peptide, wherein the C-terminus of the IL-15 is linked to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is linked to the N-terminus of IL-15Rα, or... The N-terminus of IL-15 is connected to the C-terminus of the linker peptide, and the N-terminus of the linker peptide is connected to the C-terminus of IL-15Rα. The linker peptide is selected from either a flexible linker or a rigid linker. The linker peptide has an amino acid sequence as shown in SEQ ID NO:

14.

33. The genetically modified immune cell of claim 32, wherein the immune cell is a T cell. The fusion protein has the amino acid sequence shown in SEQ ID NO:

3.

34. The genetically modified immune cell of claim 27, wherein the immune cell is a T cell. The CXCR2 receptor has the amino acid sequence shown in SEQ ID NO:

9.

35. The genetically modified immune cell of claim 27, wherein the immune cell is a T cell. The transgenic immune cells are obtained by introducing the expression vector according to any one of claims 25-26 into immune cells; 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 of 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 γδ T cells.

36. A pharmaceutical composition comprising, 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 35.

37. A kit comprising, 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.

38. A method of enhancing killing, activation, proliferation and chemotaxis of immune cells, comprising, include: The expression vector according to any one of claims 25-26 is introduced into immune cells; The immune cells introduced with the expression vector were cultured.

39. The method of claim 38, wherein, The expression vector is introduced into immune cells via electroporation, transfection, or infection. The immune cells are at least one of T cells, NKT cells, NK cells, and macrophages.

40. The method of claim 39, wherein, 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.

41. The method of claim 39, wherein, The T cells include CD4+ T cells, CD8+ T cells, Treg cells, and γδ T 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 35, or the pharmaceutical composition according to claim 36 in the preparation of a medicament, wherein the medicament is used to treat or prevent tumors; The tumors include solid tumors and hematologic tumors; The solid tumors include 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 carcinoma, oral cancer, sarcoma, prostate cancer, melanoma, and squamous cell carcinoma of the skin. The hematologic malignancy includes at least one of the following: acute myeloid leukemia, acute lymphoblastic 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 neoplasm.

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 35, or the pharmaceutical composition according to claim 36 in the prevention and / or treatment of tumors; The tumors include solid tumors and hematologic tumors; The solid tumors include 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 carcinoma, oral cancer, sarcoma, prostate cancer, melanoma, and squamous cell carcinoma of the skin. The hematologic malignancy includes at least one of the following: acute myeloid leukemia, acute lymphoblastic 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 neoplasm.

44. A method of treating or preventing a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-43. include: Administer to a subject a pharmaceutically acceptable amount of the transgenic immune cells of any one of claims 27 to 35 or the pharmaceutical composition of claim 36.

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