Multifunctional gene-modified immune cells and preparation method and application thereof

Abstract

The present invention discloses multifunctional gene-modified immune cells and their preparation methods and applications. The multifunctional gene-modified vector carries an isolated nucleic acid, and the isolated nucleic acid 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 NKp30 ligand; the second nucleic acid fragment is used to encode a fusion protein, wherein the fusion protein includes IL-15 and IL-15Rα, wherein the IL-15 and IL-15Rα are connected; and the third nucleic acid fragment is used to encode a CXCR2 receptor. Thus, the multifunctional gene-modified immune cells prepared using the multifunctional gene-modified vector can improve their specific killing ability, survival ability, proliferation ability and tumor infiltration ability, thereby further improving the clinical efficacy of immune cells.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular, to multifunctional gene-modified immune cells and preparation methods and applications thereof, and in particular, 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 innate immunity, which 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, so NK cell therapy has great 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 resuscitation of NK cells, weak proliferation and persistence in the body, poor infiltration in tumors and susceptibility to immunosuppression, which seriously limit the effectiveness and market value of NK cell therapy.

[0003] Most tumors have a physical barrier of fibrous tissue, and within the barrier there is a tumor microenvironment with low oxygen, low pH, nutritional deficiencies, high osmotic pressure, and lack of mature blood vessels. Therefore, the microenvironment within the tumor is very unfavorable for the localization and infiltration, survival and proliferation of immune cells within the tumor, and is also prone to immunosuppression and exhaustion. How to enhance the killing activity and specificity of immune cells against tumors, strengthen their ability to infiltrate, survive and proliferate within tumors, and enhance their ability to resist immunosuppression and exhaustion, etc., the research and development of core key technologies in these areas is expected to break through the bottleneck of immune cell therapy for tumor treatment.

[0004] Gene-modified cell therapy is a new type of therapy that treats diseases by modifying the genome of cells in the patient's body. The principle is to use genetic engineering technology to introduce exogenous genes or regulatory factors into cells, so that these cells acquire new functions or have stronger therapeutic potential. Gene-modified cell technology is not only applied to T cells, but has also been widely 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 have good specific killing activity, cell proliferation, resistance to immune exhaustion and improved ability to infiltrate tumors. Summary of the invention

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

[0007] The present invention is accomplished based on the following findings of the inventors:

[0008] The inventors unexpectedly discovered that NK cells are prone to immunoexhaustion or immunosuppression in tumors, have weak proliferation and persistence in vivo, and poor infiltration ability in tumors. In order to solve such problems and further improve 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, IL15 / IL15Rα fusion protein expressed on the cell membrane, and chemokine receptor CXCR2. The 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 the survival cycle and cell proliferation in vivo, and improving the ability of immune cells to infiltrate into tumors. These abilities can synergistically improve the anti-tumor activity of immune cells.

[0009] Therefore, in the first aspect of the present invention, the present invention proposes an isolated nucleic acid. According to an embodiment of the present invention, the isolated nucleic acid 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 NKp30 ligand; the second nucleic acid fragment is used to encode a fusion protein, wherein the fusion protein includes IL-15 and IL-15Rα, wherein the IL-15 and IL-15Rα are connected; the third nucleic acid fragment is used to encode a CXCR2 receptor. The inventor has found through a large number of creative experiments that when the immune cells carrying the isolated nucleic acid express the antigen chimeric receptor (the first nucleic acid fragment), the immune cells can bind to the NKp30 ligand, thereby effectively targeting and killing a variety of hematological tumors and solid tumor cells expressing NKp30 ligands; in addition, the immune cells carrying the above-mentioned isolated nucleic acid can also express IL15 / IL15Rα fusion protein and CXCR2 receptor, greatly improving their ability to survive and proliferate in vivo, and enhancing their infiltration inside the tumor tissue, thereby better exerting an anti-tumor effect.

[0010] In the second aspect of the present invention, 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. Thus, the use of the expression vector of the present invention to prepare transgenic immune cells can enable the prepared transgenic immune cells to have higher tumor recognition and killing capabilities, and can improve their survival time in vivo and in vitro, amplification ability and infiltration ability in tumors.

[0011] In the third aspect of the present invention, the present invention proposes a transgenic immune cell. According to an embodiment of the present invention, the transgenic immune cell expresses an antigen chimeric receptor targeting 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 connected. The transgenic immune cell of the present invention has high tumor recognition, killing ability and tumor infiltration ability, can survive in vivo for a long time, and has strong amplification ability, proliferation and chemotaxis ability.

[0012] In the fourth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the 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 various tumor diseases.

[0013] In a fifth aspect of the present invention, the present invention proposes 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 described in the present invention can be used to prepare transgenic immune cells, so that the prepared transgenic immune cells have high tumor recognition, killing ability and tumor infiltration ability, can survive in vivo for a long time, and have strong amplification ability, proliferation and chemotaxis ability.

[0014] In the sixth aspect of the present invention, the present invention proposes 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 introduced with 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, improve the long-term survival and expansion ability of immune cells in vivo and in vitro, and the proliferation and chemotaxis ability, especially in vitro preparation of immune cells with strong killing, activation, proliferation and chemotaxis, for constructing the required immune cell model.

[0015] 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.

[0016] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0018] Figure 1 This is a schematic diagram of the gene element structure of the multifunctional vector of Example 1 of the present invention;

[0019] Figure 2 This is a flow cytometry diagram of the expression of each element of the multifunctional vector of Example 2 of the present invention; wherein:

[0020] A is the flow cytometry analysis of the expression of mbIL15RF and CXCR2 elements on NK cells in the uninfected group;

[0021] B is a flow cytometry image of the expression of mbIL15RF and CXCR2 elements on NK cells in the multifunctional vector lentivirus infection group;

[0022] C is the MFI result of detecting NKp30 expression on NK cells in the uninfected group and the infected group;

[0023] Figure 3 This is a graph showing the investigation of the killing activity of multifunctional NK-92 cells on tumor cells according to Example 3 of the present invention;

[0024] Figure 4 It is a curve diagram showing the change in survival rate of NK-92 cells and multifunctional NK-92 cells in Example 3 of the present invention under different IL-2 culture concentrations;

[0025] Figure 5 This is a result diagram of detecting the chemotactic ability of NK-92 cells and multifunctional NK-92 cells at different CXCL8 concentrations in Example 3 of the present invention;

[0026] Figure 6 This is a diagram showing the tumor inhibition effect of the peripheral blood-derived multifunctional primary NK cells of Example 4 of the present invention on the human colorectal cancer NCI-H716 cell tumor-bearing mouse model. DETAILED DESCRIPTION

[0027] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0028] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated, nor is there any order of precedence. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0029] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0030] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in the present invention, all other technical and scientific terms used in the present invention have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0031] In the present invention, the terms "comprise" or "include" are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0032] In the present invention, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or situation can but does not necessarily occur, and the description includes cases where the event or situation occurs and cases where it does not occur.

[0033] In this article, the term "vector" or "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and / or translation of DNA or RNA. The vector also includes vectors with multiple of the above functions. The vector can 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.

[0034] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a solid carrier, or both.

[0035] As used herein, the term "treatment" refers to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be 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, including: (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 blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or transgenic immune cell to an individual to treat, cure, alleviate, improve, mitigate or inhibit the individual's disease.

[0036] The term "immune cell" generally refers to a cell capable of producing an immune response (e.g., an antigen-specific immune response). For example, the immune cell may or may contain an isolated nucleic acid and / or a vector, or may express an individual cell, cell line or cell culture of an antigen chimeric receptor, a fusion protein and a CXCR2 receptor as described herein. In the present application, the immune cell may include a T cell, a B cell, a natural killer cell (NK cell), a macrophage, a NKT cell, a monocyte, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte and / or a peripheral blood mononuclear cell.

[0037] In this document, "carbon terminus" and "C-terminus" are synonymous; "nitrogen terminus" and "N-terminus" are synonymous.

[0038] The inventors proposed a multifunctional vector that expresses a chimeric antigen recognition receptor targeting NKp30 ligand, and an enhanced cytokine and chemokine receptor expressed on the cell membrane, which can be used for multifunctional gene modification of NK cells to enhance the anti-tumor activity of NK cells. The inventors found in experiments that after the vector gene transduction of NK cells proposed by the present invention, it has the following beneficial effects: 1) The expressed NKp30-NKR can enhance the killing efficiency of NK cells against tumors with high expression of NKp30 ligands, and enhance the anti-tumor activity of NK cells; 2) The cell membrane expresses IL15 / IL15Rα fusion protein, which can further improve the long-term survival and expansion ability of NK cells in vivo and in vitro; 3) The expression of CXCR2 receptor can further improve the tumor infiltration ability of NK cells.

[0039] Specifically, the present invention proposes an isolated nucleic acid, an expression vector, a transgenic immune cell, a pharmaceutical composition, a kit, a method for enhancing the killing, activation, proliferation and chemotaxis of immune cells and uses thereof, which will be described in detail below.

[0040] Isolated nucleic acids

[0041] 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 NKp30 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.

[0042] After a large number of creative experiments, the inventors found that when the immune cells carrying the isolated nucleic acid express the antigen chimeric receptor, the immune cells can bind to the NKp30 ligand, thereby effectively targeting and killing a variety of blood tumors and solid tumor cells expressing the NKp30 ligand; in addition, the immune cells carrying the above-mentioned isolated nucleic acid can also express IL15 / IL15Rα fusion protein and CXCR2 receptor, greatly improving their ability to survive and proliferate in vivo, and enhancing their infiltration into tumor tissues, thereby better exerting anti-tumor effects.

[0043] 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 without special limitation.

[0044] In some embodiments, the isolated nucleic acid may further include at least one of the following additional technical features:

[0045] In some embodiments, the antigen chimeric receptor comprises: an extracellular region and a transmembrane region of the NKp30 receptor, and an intracellular region, wherein the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region.

[0046] In some embodiments, the extracellular region and the transmembrane region have an amino acid sequence as shown in SEQ ID NO:11.

[0047] In some embodiments, the intracellular region comprises a co-stimulatory domain and an intracellular signaling domain.

[0048] In some embodiments, the costimulatory domain is selected from the intracellular segment of the CD28 molecule.

[0049] In some embodiments, the intracellular segment of the CD28 molecule has an amino acid sequence as shown in SEQ ID NO:12.

[0050] In some embodiments, the intracellular signaling domain is selected from the intracellular segment of the CD3zeta molecule.

[0051] In some embodiments, the intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO:13.

[0052] In some embodiments, the C-terminus of the CD28 molecule is linked to the N-terminus of the CD3ζ molecule.

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

[0054] In some embodiments, the first nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:2.

[0055] 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.

[0056] In some embodiments, the IL-15 has the amino acid sequence shown in SEQ ID NO:6.

[0057] In some embodiments, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO:5.

[0058] 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.

[0059] In some embodiments, the connecting peptide is selected from any one of a flexible linker and a rigid linker.

[0060] In some embodiments, the connecting peptide has an amino acid sequence as shown in SEQ ID NO:14.

[0061] In some embodiments, the fusion protein has an amino acid sequence as shown in SEQ ID NO:3.

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

[0063] In some embodiments, the CXCR2 receptor has an amino acid sequence as shown in SEQ ID NO:9.

[0064] In some embodiments, the third nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:10.

[0065] In some embodiments, the isolated nucleic acid further comprises two fourth nucleic acid fragments, and each two nucleic acid fragments of the first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are respectively connected by one fourth nucleic acid fragment, wherein each fourth nucleic acid fragment independently encodes P2A or a fragment thereof.

[0066] In some embodiments, the P2A or a fragment thereof includes at least one of P2A, T2A, E2A and F2A or a fragment thereof.

[0067] In some embodiments, the fourth nucleic acid segment encodes P2A or a fragment thereof.

[0068] In some embodiments, the P2A or a fragment thereof has an amino acid sequence as shown in SEQ ID NO:7.

[0069] In some embodiments, the fourth nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:8.

[0070] 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.

[0071] In some embodiments, the promoter is selected from EF1α, SFFV, CAG or CMV.

[0072] In some embodiments, the promoter is selected from EF1α.

[0073] In some embodiments, the EF1α has a nucleotide sequence as shown in SEQ ID NO:15.

[0074] 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.

[0075] In some embodiments, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 16

[0076] In some embodiments, the fifth nucleic acid fragment has a nucleotide sequence as shown in SEQ ID NO:17.

[0077] It should be noted that the "fusion protein" described herein may or may not contain the "signal peptide". Figure 1 When the multifunctional vector is introduced into immune cells to obtain transgenic immune cells, the "fusion protein" contains the "signal peptide", and when the multifunctional vector is introduced into immune cells to obtain transgenic immune cells, the "signal peptide" will be cut off, and the "fusion protein" does not contain the "signal peptide" at this time. Therefore, whether the "fusion protein" contains the "signal peptide" should be considered according to the specific circumstances, and the "fusion protein" containing or not containing the "signal peptide" is within the protection scope of the present invention.

[0078] 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.

[0079] Expression vector

[0080] The present invention provides an expression vector, which carries the above-mentioned isolated nucleic acid. Thus, the transgenic immune cells prepared by using the expression vector of the present invention can have higher tumor recognition and killing capabilities, and can improve their survival time in vivo and in vitro, proliferation ability and infiltration ability in tumors.

[0081] When the above nucleic acid molecule is connected to an expression vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the expression vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule and the control element can be operably connected.

[0082] Herein, "operably linked" means connecting the foreign gene to the expression vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors may be, for example, plasmids, bacteriophages, etc.

[0083] In some embodiments, the above expression vector may further include at least one of the following additional technical features:

[0084] In some embodiments, the expression vector is selected from a virus, a prokaryotic expression vector, or a eukaryotic expression vector.

[0085] In some embodiments, the expression vector is selected from a virus.

[0086] Genetically modified immune cells

[0087] The present invention proposes a transgenic immune cell, which expresses an antigen chimeric receptor targeting 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 connected. The transgenic immune cell of the present invention has a high tumor recognition and killing ability, and can improve its survival time, expansion ability and infiltration ability in tumors in vitro and in vivo.

[0088] In some embodiments, the above-mentioned transgenic immune cells may further include at least one of the following additional technical features:

[0089] In some embodiments, the antigen chimeric receptor comprises: an extracellular region and a transmembrane region of the NKp30 receptor, and an intracellular region, wherein the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region.

[0090] In some embodiments, the extracellular region and the transmembrane region have an amino acid sequence as shown in SEQ ID NO:11.

[0091] In some embodiments, the intracellular region comprises a co-stimulatory domain and an intracellular signaling domain.

[0092] In some embodiments, the costimulatory domain is selected from the intracellular segment of the CD28 molecule.

[0093] In some embodiments, the intracellular signaling domain is selected from the intracellular segment of the CD3zeta molecule.

[0094] In some embodiments, the C-terminus of the CD28 molecule is linked to the N-terminus of the CD3ζ molecule.

[0095] In some embodiments, the intracellular segment of the CD28 molecule has an amino acid sequence as shown in SEQ ID NO:12.

[0096] In some embodiments, the intracellular segment of the CD3ζ molecule has an amino acid sequence as shown in SEQ ID NO:13.

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

[0098] 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.

[0099] In some embodiments, the IL-15 has the amino acid sequence shown in SEQ ID NO:6.

[0100] In some embodiments, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO:5.

[0101] 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.

[0102] In some embodiments, the connecting peptide is selected from any one of a flexible linker and a rigid linker.

[0103] In some embodiments, the connecting peptide has an amino acid sequence as shown in SEQ ID NO:14.

[0104] In some embodiments, the fusion protein has an amino acid sequence as shown in SEQ ID NO:3.

[0105] In some embodiments, the CXCR2 receptor has an amino acid sequence as shown in SEQ ID NO:9.

[0106] In some embodiments, the transgenic immune cells are obtained by introducing the above-mentioned expression vector into immune cells.

[0107] In some embodiments, the transgenic immune cells are derived from at least one of T cells, NKT cells, NK cells, and macrophages.

[0108] In some specific embodiments, the transgenic immune cells are derived from NK cells.

[0109] 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.

[0110] In some embodiments, the T cells include CD4+ T cells, CD8+ T cells, and γδ T cells.

[0111] Pharmaceutical composition

[0112] The present invention provides a pharmaceutical composition, which comprises: the above-mentioned isolated nucleic acid, the above-mentioned expression vector or the above-mentioned transgenic immune cell. 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 various tumor diseases.

[0113] Reagent test kit

[0114] The present invention provides a kit, which includes: the above-mentioned isolated nucleic acid or the above-mentioned expression vector. The kit of the present invention can be used to prepare transgenic immune cells, so that the prepared transgenic immune cells have high tumor recognition, killing ability and tumor infiltration ability, can survive in the body for a long time, and have strong amplification ability, proliferation and chemotaxis ability.

[0115] A method for enhancing immune cell killing, activation, proliferation and chemotaxis

[0116] The present invention proposes a method for enhancing the killing, activation, proliferation and chemotaxis of immune cells, the method comprising: introducing the above-mentioned expression vector into immune cells; culturing the immune cells introduced with 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, improve the long-term survival and expansion ability of immune cells in vivo and in vitro, and the proliferation and chemotaxis ability, especially can prepare immune cells with strong killing, activation, proliferation and chemotaxis in vitro, and use them to construct the required immune cell model.

[0117] 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:

[0118] In some embodiments, the introduction of the expression vector into immune cells is performed by electroporation, transfection or infection.

[0119] In some embodiments, the immune cell is at least one of a T cell, a NKT cell, a NK cell, and a macrophage.

[0120] In some specific embodiments, the immune cells are NK cells.

[0121] 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.

[0122] In some embodiments, the T cells include CD4+ T cells, CD8+ T cells, and γδ T cells.

[0123] use

[0124] Use of the above-mentioned isolated nucleic acid, the above-mentioned expression vector, the above-mentioned transgenic immune cell or the above-mentioned pharmaceutical composition in the preparation of a drug, wherein the drug is used for treating or preventing tumors.

[0125] In some embodiments, the above-mentioned use may further include at least one of the following additional technical features:

[0126] In some embodiments, the tumor includes solid tumors and hematological tumors.

[0127] 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.

[0128] In some embodiments, the blood tumor includes 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.

[0129] 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. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product instructions are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0130] The sequence descriptions involved in the present invention are shown in Table 1.

[0131] Table 1: Amino acid / nucleotide sequence description

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142] Example 1: Preparation of multifunctional vector gene-modified NK-92 cells

[0143] 1. Construction of multifunctional vector

[0144] The nucleotide sequence shown in SEQ ID NO: 21 was synthesized by whole gene synthesis, and then cloned into the lentiviral vector pLVX-EF1-IRES-Puro through restriction sites EcoRI and MluI. After sequencing and verification, the pLVX-EF1-multi-functional-vector plasmid was obtained, i.e., the multifunctional vector plasmid involved in the present invention. The schematic diagram of the gene element structure of the multifunctional vector of this embodiment is shown in Figure 1 .

[0145] 2. Lentivirus packaging

[0146] Take 5×10 293T cells in the logarithmic growth phase 6 The cells were inoculated into a 10 cm cell culture dish and 10 mL of DMEM medium was added to the dish and incubated at 37°C with 5% CO 2 Culture overnight in an incubator. When the cell density in the cell culture dish reaches 80-90%, replace with 10 mL of fresh DMEM medium and continue to place the cell culture dish in the incubator for later use. Prepare a lentiviral packaging system. Add 6 μg psPAX2 and 3 μg pMD2.G, a lentiviral packaging auxiliary plasmid, and 6 μg lentiviral vector plasmid to 250 μL serum-free DMEM medium to prepare a plasmid mixture and mix well. Add 15 μL Add to 235 μL serum-free DMEM medium and mix well. Add the mixture to the above plasmid mixture at once, mix well, and incubate at room temperature for 15 minutes. Add the mixture to the 293T cell culture dish. After 24 hours, change the medium and place the culture dish back to 37°C and 5% CO 2 Incubator, collect cell supernatant after 48h, centrifuge at 400×g for 5min, 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 the centrifuge tube upside down, and place it in a 4℃ refrigerator overnight. Centrifuge at 4000×g for 20min at 4℃, discard the supernatant, add an appropriate amount of serum-free DMEM to resuspend the virus precipitate, transfer it into an EP tube, and store it in a -80℃ refrigerator.

[0147] 3. Lentivirus infection of human NK-92 cells

[0148] NK-92 cells in the logarithmic growth phase were aspirated and centrifuged at 100 × g for 5 min to harvest the cells. An 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-92 cells, 0.2 mL of virus concentrate, 0.8 mL of α-MEM medium and protamine (final concentration 8 μg / mL), mixed evenly. Place at 37°C, 5% CO 2 Culture in an incubator. 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 bottle, add 10mL of 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-92 cells (multifunctional NK-92 cells) for subsequent functional experiments.

[0149] Example 2: Verification of expression of each element of the multifunctional vector

[0150] Peripheral blood mononuclear cells (PBMC) were isolated and inoculated in pre-coated culture flasks for culture. Referring to CN202310035787.4, IL-2 and other cytokines were used for induction culture. 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 is 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 added PerCP / Cyanine5.5 labeled anti-human CD3 antibody (purchased from Biolegend), BrilliantViolet 785 TM 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). 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).

[0151] Analysis of CD3 by gating on CD3 and CD56 - CD56 +Then, we analyzed the positive rate of mbIL15RF (IL-15 and IL-15Rα double positive) or CXCR2 expression on NK cells, and the mean fluorescence intensity (MFI) of NKp30 expression. Figure 2 As shown in A and B, CXCR2 and mbIL15RF molecules were not expressed on NK cells in the uninfected group. After infection with the multifunctional vector lentivirus, the expression of CXCR2 and mbIL15RF molecules on NK cells increased significantly. Figure 2 As shown in C, the average fluorescence intensity of NKp30 expression on NK cells in the infected group was also significantly higher than that in the uninfected group. These results indicate that the three functional elements designed in the multifunctional vector can be efficiently expressed.

[0152] Example 3: In vitro functional detection of NK cells modified by multifunctional vector gene

[0153] In this example, after the inventors obtained the multifunctional vector gene-modified NK-92 cells (multifunctional NK-92 cells) of the present invention through Example 1, they investigated the killing activity, survival and chemotaxis of the multifunctional NK-92 cells in vitro.

[0154] 1. Multifunctional vector gene modification promotes the killing activity of NK cells

[0155] The inventors tested the killing activity of NK-92 cells modified with the multifunctional vector gene. The specific method is as follows: CFSE was used to fluorescently label the colorectal cancer NCI-H716 cells, and the number of cells was 2×10 4 Cells / well were inoculated into 96-well plates, and NK-92 or multifunctional NK-92 cells were inoculated into the plates and incubated for 4 hours. The cells were collected in flow cytometry tubes, and PI was added to distinguish between live and dead cells, and the killing efficiency was detected by flow cytometry. The ratio of effector cells to target cells was 2:1.

[0156] The test results are as follows Figure 3 As shown, the killing efficiency of the multifunctional NK-92 cells modified by the multifunctional vector gene of the present invention on colorectal cancer NCI-H716 cells is significantly higher than that of NK-92 cells.

[0157] 2. Multifunctional vector gene modification promotes NK cell survival

[0158] The inventors further verified the effect of multifunctional vector gene modification on the survival of NK-92 cells. The specific method is as follows: NK-92 cells and multifunctional NK-92 cells with the same number of cells were plated in 24-well plates, and different IL-2 concentrations (0, 20 and 200 IU / mL) were set, and flow cytometry was performed every 24 hours to detect the apoptosis rate. The flow cytometry detection of apoptosis rate was performed according to the steps in the kit instructions (Lianke Bio, catalog 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 gentle vortex mixing, incubate at room temperature in the dark for 5 minutes, and resuspend the cells for flow detection. The cell viability is the ratio of double negatives of Annexin V-FITC and PI staining.

[0159] The results of the NK cell survival rate test after 96 hours of culture are as follows Figure 4 As shown in the figure: In the absence of IL-2 (IL-2 0IU / mL), the cell survival rate of the NK-92 cell group decreased significantly from 24h, and most cells had undergone apoptosis at 72h; while the multifunctional NK-92 cell group was able to maintain a high cell survival rate even under the condition of complete withdrawal of IL-2, and few cells underwent apoptosis. These results suggest that multifunctional vector gene modification can play an important role in promoting the survival of NK cells.

[0160] 3. Multifunctional vector gene promotes chemotaxis of NK cells

[0161] The inventors further investigated the chemotactic ability of multifunctional NK-92 cells by transwell experiment. 1×10 6 NK-92 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 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 the cells in the lower chamber were collected after 48 hours for cell counting. The experimental results are shown in Figure 5 As shown, compared with NK-92 cells, the number of multifunctional NK-92 cells migrating to the lower chamber is significantly greater. The above test results show that multifunctional vector gene modification can significantly promote the chemotactic ability of NK cells.

[0162] Example 4: Multifunctional vector gene-modified NK cells have strong in vivo tumor-suppressing activity

[0163] The inventors established a human colorectal cancer mouse heterotopic transplant tumor model using human colorectal cancer cell line NCI-H716 cells, infected human peripheral blood primary NK cells with 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.

[0164] The specific method is as follows: 6-week-old NCG mice were selected for subcutaneous tumor bearing in the armpits, and the tumor bearing dose was 1×10 7 NCI-H716 cells / mouse. On day 9 after tumor loading, the screening tumor volume was 50mm 3 About 100 mice were used in the experiment and randomly divided into an untreated group, a non-genetically modified NK cell treatment group, and a multifunctional primary NK cell treatment group according to the size of the tumor. The mice in the non-genetically modified 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 + 5×10 NK cells were injected intraperitoneally every 2 days 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 / cell, no IL-2 injection adjuvant therapy. Tumor volume was observed 1-2 times a week, and tumor growth curve was drawn. Figure 6 As shown, compared with the non-genetically modified NK cell treatment group, the multifunctional primary NK cell treatment group still showed a stronger tumor inhibition effect at a lower cell treatment dose.

[0165] The above test results indicate 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 can be used at lower dosages, which is expected to break through the bottleneck of poor tumor treatment effects of immune cell therapy.

[0166] Example 5

[0167] In this example, after the inventors obtained the multifunctional vector gene-modified NK-92 cells (multifunctional NK-92 cells) of the present invention through Example 1, they investigated the killing activity of the multifunctional NK-92 cells against human chronic myeloid leukemia K562 cells.

[0168] The specific method is as follows: K562 cells were fluorescently labeled with CFSE at 2×10 4Cells / well were inoculated into 96-well plates, and NK-92 or multifunctional NK-92 cells were inoculated into the plates and incubated for 4 hours. The cells were collected in flow cytometry tubes, PI was added to distinguish between live and dead cells, and the killing efficiency was detected by flow cytometry.

[0169] The test results show that the multifunctional NK-92 cells modified by the multifunctional vector gene of the present invention have a significantly higher killing efficiency against human chronic myeloid leukemia K562 cells than NK-92 cells.

[0170] Example 6

[0171] In this example, the expression of NKp30-NKR was combined with the above fusion protein and different chemokine receptors by the method 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 detected by the method in Example 3.

[0172] The test results show that although other chemokine receptors have similar effects to CXCR2, compared with other chemokine receptors, the transgenic immune cells expressing NKp30-NKR combined with the above-mentioned expression fusion protein and CXCR2 have the strongest chemotaxis to the tumor site; while the transgenic immune cells prepared by changing the chemokine receptor have weaker chemotaxis to the tumor. Moreover, the transgenic immune cells expressing NKp30-NKR combined with the above-mentioned expression fusion protein and CXCR2 have stronger anti-tumor activity than other chemokine receptor combinations.

[0173] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0174] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An isolated nucleic acid, It is characterized in that include: A first nucleic acid fragment, a second nucleic acid fragment, a third nucleic acid fragment and two fourth nucleic acid fragments, wherein the first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are sequentially connected, and each two nucleic acid fragments in the first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are respectively connected through one fourth nucleic acid fragment; wherein The first nucleic acid fragment is used to encode an antigen chimeric receptor targeting NKp30 ligand, and the amino acid sequence of the antigen chimeric receptor is shown in SEQ ID NO: 1; The second nucleic acid fragment is used to encode a fusion protein, wherein the fusion protein is IL-15 and IL-15Rα, wherein the IL-15 and IL-15Rα are linked, wherein the amino acid sequence of the IL-15 is shown in SEQ ID NO:6, and the amino acid sequence of the IL-15Rα is shown in SEQ ID NO:5; and the amino acid sequence of the fusion protein is shown in SEQ ID NO:3; The third nucleic acid fragment is used to encode a CXCR2 receptor, and the amino acid sequence of the CXCR2 receptor is shown in SEQ ID NO: 9; Each of the fourth nucleic acid fragments independently encodes P2A.

2. The isolated nucleic acid according to claim 1, It is characterized in that The antigen chimeric receptor is composed of the extracellular region, the transmembrane region, and the intracellular region of the NKp30 receptor, and the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region; The amino acid sequences of the extracellular region and transmembrane region are shown in SEQ ID NO: 11; The intracellular region is composed of a co-stimulatory domain and an intracellular signaling domain; The co-stimulatory domain is the intracellular segment of the CD28 molecule; The intracellular signal transduction domain is the intracellular segment of the CD3ζ molecule; The C-terminus of the CD28 molecule is connected to the N-terminus of the CD3ζ molecule.

3. The isolated nucleic acid according to claim 2, It is characterized in that The amino acid sequence of the intracellular segment of the CD28 molecule is shown in SEQ ID NO: 12; The amino acid sequence of the intracellular segment of the CD3ζ molecule is shown in SEQ ID NO:

13.

4. The isolated nucleic acid according to claim 1, It is characterized in that The nucleotide sequence of the first nucleic acid fragment is shown in SEQ ID NO:

2.

5. The isolated nucleic acid according to claim 1, It is characterized in that The nucleotide sequence of the second nucleic acid fragment is shown in SEQ ID NO:

4.

6. The isolated nucleic acid according to claim 1, It is characterized in that The nucleotide sequence of the third nucleic acid fragment is shown in SEQ ID NO:

10.

7. The isolated nucleic acid according to claim 1, It is characterized in that The amino acid sequence of P2A is shown in SEQ ID NO:7; the nucleotide sequence of the fourth nucleic acid fragment is shown in SEQ ID NO:

8.

8. The isolated nucleic acid according to claim 1, It is 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, the third nucleic acid fragment and the fourth nucleic acid fragment; The promoter is selected from EF1α, SFFV, CAG or CMV.

9. The isolated nucleic acid according to claim 8, It is characterized in that The promoter is selected from EF1α; The nucleotide sequence of EF1α is shown in SEQ ID NO:

15.

10. The isolated nucleic acid according to claim 1, It is characterized in that Further comprising a fifth nucleic acid fragment, the fifth nucleic acid fragment encoding a signal peptide.

11. The isolated nucleic acid according to claim 10, It is characterized in that The amino acid sequence of the signal peptide is shown in SEQ ID NO: 16; The nucleotide sequence of the fifth nucleic acid fragment is shown in SEQ ID NO:

17.

12. The isolated nucleic acid according to any one of claims 1 to 11, It is characterized in that The isolated nucleic acid comprises, from the 5' end to the 3' end, the promoter of claim 8 or 9, the first nucleic acid fragment, one of the fourth nucleic acid fragments, the fifth nucleic acid fragment of claim 10 or 11, the second nucleic acid fragment, another of the fourth nucleic acid fragments and the third nucleic acid fragment.

13. An expression vector carrying the isolated nucleic acid according to any one of claims 1 to 12; The expression vector is selected from a virus, a prokaryotic expression vector or a eukaryotic expression vector.

14. The expression vector according to claim 13, It is characterized in that The expression vector is selected from viruses.

15. A genetically modified immune cell, It is characterized in that The transgenic immune cells express antigen chimeric receptors, fusion proteins and CXCR2 receptors targeting NKp30 ligands; Wherein, the fusion protein includes IL-15Rα and IL-15, and the IL-15 and IL-15Rα are connected; The amino acid sequence of the antigen chimeric receptor is shown in SEQ ID NO: 1; The amino acid sequence of the fusion protein is shown in SEQ ID NO: 3; The amino acid sequence of the CXCR2 receptor is shown in SEQ ID NO:

9.

16. The transgenic immune cell according to claim 15, It is characterized in that The antigen chimeric receptor is composed of the extracellular region, the transmembrane region, and the intracellular region of the NKp30 receptor, and the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region; The amino acid sequences of the extracellular region and transmembrane region are shown in SEQ ID NO: 11; The intracellular region is composed of a co-stimulatory domain and an intracellular signaling domain; The co-stimulatory domain is the intracellular segment of the CD28 molecule; The intracellular signal transduction domain is the intracellular segment of the CD3ζ molecule; The C-terminus of the CD28 molecule is connected to the N-terminus of the CD3ζ molecule.

17. The transgenic immune cell according to claim 16, It is characterized in that The amino acid sequence of the intracellular segment of the CD28 molecule is shown in SEQ ID NO: 12; The amino acid sequence of the intracellular segment of the CD3ζ molecule is shown in SEQ ID NO:

13.

18. The transgenic immune cell according to claim 15, It is characterized in that The amino acid sequence of IL-15 is shown in SEQ ID NO: 6; The amino acid sequence of IL-15Rα is shown in SEQ ID NO:

5.

19. The transgenic immune cell according to claim 15, It is characterized in that The transgenic immune cell is obtained by introducing the expression vector according to claim 13 or 14 into an immune cell; The transgenic immune cells are derived from at least one of T cells, NK cells and macrophages; 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 and γδT cells.

20. The transgenic immune cell according to claim 19, It is characterized in that The transgenic immune cells are derived from NK cells.

21. A pharmaceutical composition, It is characterized in that include: The isolated nucleic acid according to any one of claims 1 to 12, the expression vector according to claim 13 or 14, or the transgenic immune cell according to any one of claims 15 to 20.

22. A kit, It is characterized in that include: The isolated nucleic acid according to any one of claims 1 to 12 or the expression vector according to claim 13 or 14.

23. Use of the isolated nucleic acid according to any one of claims 1 to 12, the expression vector according to claim 13 or 14, the transgenic immune cell according to any one of claims 15 to 20, or the pharmaceutical composition according to claim 21 in the preparation of a drug for treating or preventing a tumor; The tumors include solid tumors and hematological tumors.

24. The use according to claim 23, It is characterized in that 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, osteosarcoma, prostate cancer and melanoma; The blood tumor includes 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.

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