Transgenic NK cells and their construction method and application

Abstract

The present application relates to the field of immune cell technology, and in particular to a transgenic NK cell and its construction method and application. The transgenic NK cell expresses IL-15 fusion peptide and chimeric antigen receptor by fusion, and a self-cleaving peptide is connected between the IL-15 fusion peptide and the chimeric antigen receptor; the IL-15 fusion peptide includes a signal peptide and the 49aa-162aa region of IL-15. The transgenic NK cells constructed in this application have effectively improved killing activity.

Description

Technical Field

[0001] The present application relates to the field of immune cell technology, and in particular to a transgenic NK cell and a construction method and application thereof. Background Art

[0002] In recent years, tumor immunotherapy has become a hot topic in the field of anti-tumor research. As one of the adoptive immunotherapy technologies for tumors, NK cells and CAR-NK cells have shown strong cytotoxicity, good safety, can be used as universal "off-the-shelf" products, and are easy to prepare on a large scale. They have shown good efficacy in clinical applications of solid tumors, and are expected to become the key active ingredients of the next generation of tumor immunotherapy.

[0003] Interleukin-15 (IL-15) and interleukin-2 (IL-2) are both key cytokines that regulate the development, maturation, survival, proliferation and effector function of NK cells. In comparison, IL-15's stimulation of regulatory T cells (Treg) is significantly lower than that of IL-2, and it can inhibit IL-2-mediated activation-induced cell death (AICD). Therefore, IL-15 is more in line with the requirements of adoptive immunotherapy than IL-2, and NK cell clinical trials often use IL-15 instead of IL-2.

[0004] However, the killing activity of traditional CAR-NK cells expressing interleukin-15 needs to be further improved.

[0005] In view of this, this application is hereby filed. Summary of the invention

[0006] Based on this, the present application provides one or more embodiments. The technical solution includes:

[0007] One or more embodiments of the present application provide a transgenic NK cell, wherein the transgenic NK cell expresses an IL-15 fusion peptide and a chimeric antigen receptor, wherein a self-cleaving peptide is connected between the IL-15 fusion peptide and the chimeric antigen receptor;

[0008] The IL-15 fusion peptide includes a signal peptide and the 49aa-162aa region of IL-15.

[0009] In some embodiments of the present application, the IL-15 fusion peptide is linked to the N-terminus of the chimeric antigen receptor.

[0010] In some embodiments of the present application, the IL-15 fusion peptide is secretory IL-15, and the amino acid sequence is shown in SEQ ID NO.2.

[0011] In some embodiments of the present application, the IL-15 fusion peptide is a membrane-bound IL-15. Compared with the secretory IL-15, the C-terminus of the membrane-bound IL-15 is sequentially connected to the hinge region and the transmembrane region.

[0012] In some embodiments of the present application, the membrane-bound IL-15 and the transmembrane region in the chimeric antigen receptor are both derived from CD8α.

[0013] In some embodiments of the present application, the intracellular co-stimulatory domain in the chimeric antigen receptor is a 4-1BB co-stimulatory domain.

[0014] In some embodiments of the present application, the amino acid sequence of the intracellular signal transduction domain in the chimeric antigen receptor is shown in SEQ ID NO.10.

[0015] In some embodiments of the present application, the transgenic NK cells include NK92 cells.

[0016] In some embodiments of the present application, the hinge regions in the membrane-bound IL-15 and the chimeric antigen receptor are each independently selected from the hinge region of at least one of the following proteins: CD8, IgG4, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154.

[0017] In some embodiments of the present application, the signal peptides in the IL-15 fusion peptide and the chimeric antigen receptor are each independently selected from the signal peptides of at least one of the following proteins: CD8α, GM-CSF, CD4, CD28, CD137 and IgG.

[0018] In some embodiments of the present application, the target-specific binding domain in the chimeric antigen receptor heterosexually binds to one or more of the following targets: Her2, AFP, ANGPT2, AXL, B7H3, BAFFR, BCMA, CAIX, CD1a, CD4, CD5, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD39, CD44, CD46, CD47, CD56, CD64, CD70, CD73, CD79, CD117, CD123, CD126, CD133, CD138, CD147, CD171, CD205, CDH17, CEA, Claudin6, Claudin 18.2, CLL1, CS1, CSF-1R, CSPG4, CTLA-4, CXCR4, DLL3, EGFR, EGFRvIII, EpCAM, EphA2, FAP, FBP, FGFR, FR, FSHR, GD2, glycolipid F77, GPC2, GPC3, GPRC5D, GPR20, GPR78, GSPG4, GUCY2C, Her3, Her4, ICAM-1, IGF1R, IL-13R, integrin, L1-CAM, Lewis Y, LILRB4, LMP1, LMP2, MAGEA3, mesothelin, Met, MUC-1, MUC-16, Nectin-4, NY-ESO-1, ROR1, PD-L1, PSCA, PSMA, TAG-72, TACI, Trop-2, VEGFR1, VEGFR2, VEGFR3 and WT-1.

[0019] In some embodiments of the present application, the self-cleaving peptide is selected from one or more of T2A, P2A, F2A and E2A.

[0020] In some embodiments of the present application, the transgenic NK cells express the polypeptide shown in SEQ ID NO.6 or SEQ ID NO.7.

[0021] One or more embodiments of the present application further provide a method for constructing the transgenic NK cells, the method comprising:

[0022] Provide NK cells to be genetically modified to express the IL-15 fusion peptide and chimeric antigen receptor.

[0023] One or more embodiments of the present application further provide a drug, which includes the transgenic NK cells.

[0024] One or more embodiments of the present application further provide a method for producing the drug, which comprises the step of culturing the transgenic NK cells.

[0025] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0027] Figure 1 The technical roadmap for this application;

[0028] Figure 2a is the lentiviral plasmid map (pCDH-GFP);

[0029] Figure 2b is the lentiviral plasmid map (pCDH-sIL15);

[0030] Figure 2c is the lentiviral plasmid map (pCDH-mbIL15);

[0031] Figure 2d is the lentiviral plasmid map (pCDH-HER2);

[0032] Figure 2e is the lentiviral plasmid map (pCDH-HER2-T2A-sIL15);

[0033] Figure 2f is the lentiviral plasmid map (pCDH-HER2-T2A-mbIL15);

[0034] Figure 3 The cell status was observed under a microscope (40×);

[0035] Figure 4 is the HER2-CAR positive rate on the 7th, 14th, and 25th days after lentiviral infection;

[0036] Figure 5 The positive rate of HER2-CAR after 14 days of resuscitation;

[0037] Figure 6 To determine the IL-15 content in cell culture supernatant and cell lysate;

[0038] Figure 7 After 2 h of EdU labeling, flow cytometry was performed. 488-channel fluorescence;

[0039] Figure 8 Flow cytometry was used to detect the expression of HER2 in three cell lines;

[0040] Fig. 9 For the determination of cytotoxicity;

[0041] Fig.10 For the determination of effector cell CD107a;

[0042] Fig.11 This is a statistical analysis chart of TNF-α;

[0043] Fig.12 This is a statistical analysis chart of IFN-γ;

[0044] Fig.13 This is a statistical analysis chart of GM-CSF;

[0045] Fig.14 The expression of HER2-CAR was determined by flow cytometry. DETAILED DESCRIPTION

[0046] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0048] the term

[0049] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0050] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0051] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0052] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0053] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0054] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0055] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0056] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0057] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0058] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0059] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0060] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0061] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0062] All documents mentioned in this application are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features may also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description in this application. In a first aspect of an embodiment of the present application, a transgenic NK cell is provided, wherein the transgenic NK cell expresses an IL-15 fusion peptide and a chimeric antigen receptor, and a self-cleaving peptide is connected between the IL-15 fusion peptide and the chimeric antigen receptor;

[0063] The IL-15 fusion peptide includes a signal peptide and the 49aa-162aa region of IL-15.

[0064] The chimeric antigen receptor is not particularly limited in the present embodiment. For example, it has the following structure: signal peptide-target specific binding domain-hinge region-transmembrane region-intracellular co-stimulatory domain-intracellular signal transduction domain, where "-" represents a peptide bond.

[0065] The IL-15 in the IL-15 fusion peptide of the embodiment of the present application can be selected from the extracellular 49aa-162aa region of IL-15, and can also be mutated or modified on this basis, for example, it can be a conservative mutant thereof.

[0066] "Conservative mutants" refer to mutants modified on the basis of wild-type IL-15, and the activity or function (including affinity with receptors) of the resulting mutants are consistent with wild-type IL-15. The modified NK cells with "conservative mutants" have the same biological properties as the modified NK cells with wild-type IL-15.

[0067] For example, the conservative mutants of IL-15 have at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids replaced by amino acids with similar or similar properties compared to wild-type interleukin-15. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A below.

[0068] Table A

[0069]

[0070] For example, a conservative mutant of IL-15 is prepared by a method of replacing, deleting or adding at least one amino acid in wild-type IL-15 without changing the function of IL-15, or a conservative mutant of IL-15 is prepared by a method of replacing, deleting or adding at least one amino acid in wild-type IL-15 without changing the function of IL-15, and these conservative mutants have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with their corresponding wild-type.

[0071] Traditionally, conservative mutants may have some conservative amino acid substitutions relative to wild-type subunits, and such conservative amino acid substitutions have no effect or weak effect on the overall protein charge, i.e., polarity or hydrophobicity. For conservative amino acid substitutions, reference may be made to Table B below.

[0072] Table B

[0073]

[0074]

[0075] For each amino acid, additional conservative substitutions include "homologues" of the amino acid. Specifically, "homologues" refer to amino acids obtained by inserting a methylene group (CH2) into the β-side chain of the amino acid side chain. Examples of "homologues" may include homophenylalanine, homoarginine, homoserine, etc., but are not limited thereto.

[0076] The present application does not specifically limit the species source of IL-15, and it can be mammals (humans, mice, rats, guinea pigs, rabbits, macaques, goats, sheep, horses, cattle, pigs, dogs, etc.), or poultry such as chickens, geese, and ducks. In some embodiments, IL-15 is derived from mammals. Preferably, IL-15 is derived from humans, and IL-15 from other species can replace human IL-15 to achieve similar functions.

[0077] The examples of the present application do not specifically limit the types of NK cells, such as haploid NK cells, cord blood NK cells, stem cell-derived NK cells, NK cell lines, adaptive NK cells, cytokine-induced memory-like NK cells, and CARNK cells, etc. In one embodiment, the modified NK cells are NK92 cells.

[0078] The embodiments of the present application do not particularly limit the positional relationship between the IL-15 fusion peptide and the chimeric antigen receptor. In one example, the IL-15 fusion peptide is connected to the N-terminus of the chimeric antigen receptor.

[0079] In one embodiment, the IL-15 fusion peptide is secretory IL-15, and the amino acid sequence is shown in SEQ ID NO.2.

[0080] In one embodiment, the IL-15 fusion peptide is a membrane-bound IL-15, and relative to the secretory IL-15, the C-terminus of the membrane-bound IL-15 is sequentially connected to a hinge region and a transmembrane region.

[0081] In one embodiment, the transmembrane regions in the membrane-bound IL-15 and the chimeric antigen receptor are both derived from CD8α, or have at least 70% (71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) homology with the transmembrane region derived from CD8α, or are the entire region or a partial region of the transmembrane region derived from CD8α.

[0082] In one embodiment, the intracellular co-stimulatory domain in the chimeric antigen receptor is a 4-1BB co-stimulatory domain, or has at least 70% (71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) homology with the 4-1BB co-stimulatory domain, or is the entire region of the 4-1BB co-stimulatory domain, or it can be a partial region.

[0083] In one embodiment, the amino acid sequence of the intracellular signal transduction domain in the chimeric antigen receptor is as shown in SEQ ID NO.10, or has at least 70% (71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) homology to the sequence.

[0084] In one embodiment, the hinge regions in the membrane-bound IL-15 and the chimeric antigen receptor are independently selected from the hinge region of at least one of the following proteins: CD8, IgG4, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154. The hinge region can be selected from one or more. For the hinge region derived from a certain protein, the entire region or a partial region can be selected, the wild type or a mutant can be selected, for example, the selected mutant has at least 70% (71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) homology with the wild-type sequence.

[0085] In one embodiment, the signal peptides in the IL-15 fusion peptide and the chimeric antigen receptor are independently selected from the signal peptides of at least one of the following proteins: CD8α, GM-CSF, CD4, CD28, CD137 and IgG. The signal peptides can be selected from one or more. For a signal peptide derived from a certain protein, its entire region can be selected, or a partial region can be selected, the wild type can be selected, or its mutant can be selected, for example, the selected mutant has at least 70% (71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) homology with the wild-type sequence.

[0086] In one embodiment, the target-specific binding domain in the chimeric antigen receptor heterosexually binds to one or more of the following targets: Her2, AFP, ANGPT2, AXL, B7H3, BAFFR, BCMA, CAIX, CD1a, CD4, CD5, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD39, CD44, CD46, CD47, CD56, CD64, CD70, CD73, CD79, CD117, CD123, CD126, CD133, CD138, CD147, CD171 、CD205、CDH17、CEA、Claudin6、Claudin18.2、CLL1、CS1、CSF-1R、CSPG4、CTLA-4、CXCR4、DLL3、EGFR、EGFRvIII、EpCAM、EphA2、FAP、FBP、FGFR、FR、FSHR、GD2、glycolipid F77、GPC2、GPC3、GPRC5D、GPR20、GPR78、GSPG4、GUCY2C、Her3、Her4、ICAM-1、IGF1R、IL-13R、integrin、L1-CAM、Lewis Y, LILRB4, LMP1, LMP2, MAGEA3, mesothelin, Met, MUC-1, MUC-16, Nectin-4, NY-ESO-1, ROR1, PD-L1, PSCA, PSMA, TAG-72, TACI, Trop-2, VEGFR1, VEGFR2, VEGFR3 and WT-1. In one embodiment, the number of target-specific binding domains in the chimeric antigen receptor is 1. In one embodiment, the target-specific binding domain in the chimeric antigen receptor targets Her2. In one embodiment, the sequence of the target-specific binding domain in the chimeric antigen receptor is as shown in 22aa-263aa of SEQ ID NO.4, or has at least 70% (71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) homology to the sequence shown in 22aa-263aa of SEQ ID NO.4.

[0087] The present application embodiment does not specifically limit the type of self-cleaving peptide, which can be selected from but not limited to: T2A, P2A, F2A and E2A. In one embodiment, the self-cleaving peptide is T2A, and the amino acid sequence is as shown in SEQ ID NO.5, or has at least 70% (71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%) homology with the sequence.

[0088] In one embodiment, the transgenic NK cells express the polypeptide shown in SEQ ID NO.6, and in another embodiment, the transgenic NK cells express the polypeptide shown in SEQ ID NO.7.

[0089] In a second aspect of the embodiment of the present application, a method for constructing the transgenic NK cells is provided, the method comprising:

[0090] Provide NK cells to be genetically modified to express the IL-15 fusion peptide and chimeric antigen receptor.

[0091] The present application embodiment does not specifically limit the construction method, for example, a fusion expression vector can be constructed and then introduced into the NK cells to be genetically modified. For example, a fusion expression vector including a nucleic acid molecule shown in SEQ ID NO.8 or SEQ ID NO.9 can be constructed and then introduced into the NK cells to be genetically modified.

[0092] The third aspect of the embodiment of the present application provides a drug, which includes the transgenic NK cells. The present application does not specifically limit the dosage form of the drug, and it can be any dosage form required by clinical needs. Of course, the excipients contained in the corresponding drug are different depending on the dosage form.

[0093] The fourth aspect of the present application provides a method for producing the drug, which comprises the step of culturing the transgenic NK cells.

[0094] It is understood that the culture is carried out under appropriate culture conditions, for example, the culture medium used includes MEMα culture medium, 0.08mM-0.12mMβ-mercaptoethanol, 0.01mM-0.03mM folic acid, 10%-15% (v / v) horse serum and 10%-15% (v / v) fetal bovine serum.

[0095] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0096] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0097] Overexpression of HER2 is closely related to a variety of malignant tumors, such as breast cancer, gastric cancer, ovarian cancer, prostate cancer, and lung cancer, and has a high degree of malignancy and a poor prognosis. Currently, HER2 has been widely used as a target for tumor treatment and can effectively inhibit the growth of tumor cells. Therefore, using HER2 as a target for cell therapy has a very broad prospect in the field of solid tumor treatment.

[0098] The applicant previously constructed a second-generation CAR lentiviral vector targeting HER2, and packaged the lentivirus to infect mbIL15-NK92 cells and sIL15-NK92 cells. The results showed that the positive rate of HER2-CAR was only 24.24% and 23.76%. In order to further improve the positive rate of CAR-NK cells and facilitate functional research, we connected HER2-CAR and IL-15 through the T2A element, constructed a lentiviral vector and packaged the lentivirus, and removed IL-2 from the culture medium after infecting NK92 cells, and indirectly increased the positive rate of HER2-CAR by promoting the expression of IL-15. The results showed that the positive rate of HER2-CAR gradually increased during the culture process, and the positive rate of HER2-T2A-mbIL15-NK92 cells increased faster than that of HER2-T2A-sIL15-NK92 cells. After stabilization, the positive rate of the former was 94.5%, slightly higher than that of the latter (88.2%), indicating that this experiment successfully constructed NK92 cells that stably and highly expressed HER2-CAR and could autosecrete IL-15. Proliferation experiments showed that HER2-T2A-IL15-NK92 cells had similar proliferation activity to IL15-NK92 cells.

[0099] The in vitro anti-tumor ability of the CAR-NK cells we constructed was verified by in vitro killing experiments and detection of related factor expression. The results showed that compared with IL15-NK92 cells, HER2-IL15-NK92 cells can kill HER2-positive tumor cells more effectively and have targeting specificity. The release of effector factors also depends on the presence of the target HER2. In summary, HER2-CAR can guide NK92 cells to specifically target HER2-positive tumor cells and enhance the killing ability of NK92 cells, which provides a research basis for the preparation of enhanced CAR-IL15-NK92 cells.

[0100] Technical route see Figure 1 shown.

[0101] Example 1

[0102] 1. Sequence determination and lentiviral plasmid construction

[0103] 1.1 Sequence determination

[0104] The amino acid sequence of human IL-15 (NP_000576.1) was found on the NCBI website as shown in SEQ ID NO.1:

[0105] Using UniProt (https: / / www.uniprot.org) website for analysis, amino acids 1-29 were signal peptides, 30-48 were propeptides, 49-162 were extracellular regions, and there was a glycosylation site at position 127. Secreted and membrane-bound IL-15 were constructed, respectively.

[0106] The CD8α signal peptide is linked to the N-terminus of amino acids 49-162 of natural IL-15 to obtain the amino acid sequence of secretory IL-15 (sIL-15) as shown in SEQ ID NO.2:

[0107] MALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS. The structure of sIL-15 is: CD8α signal peptide-IL-15 fragment.

[0108] In order to localize IL-15 on the cell membrane, this embodiment adds a hinge region and a transmembrane region on the basis of sIL-15, wherein the hinge region connects the extracellular region and the transmembrane region, which can provide flexibility to overcome spatial barriers, facilitate the binding of the extracellular region to the receptor, and the transmembrane region can anchor the protein on the cell membrane. The hinge region and transmembrane region derived from CD8α are selected, and sIL-15, CD8α hinge region, and CD8α transmembrane region are connected in sequence to obtain the amino acid sequence of membrane-bound IL-15 (mbIL-15) as shown in SEQ ID NO.3:

[0109] MALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC. The structure of mbIL-15 is: CD8α signal peptide-IL-15 fragment-CD8α hinge region-CD8α transmembrane region.

[0110] Construct HER2-CAR: First, the scFv fragment of the extracellular antigen recognition domain. This example selects the HER2 antigen as the target. The amino acid sequence of trastuzumab is searched through the NCBI website. The antibody structure is analyzed through the NovoPro website (https: / / www.novopro.cn / tools / ) to find the sequence of the heavy chain variable region (VH) and light chain variable region (VL) that specifically binds the antibody to the antigen. The sequence of ScFv is connected by a short peptide in the middle. The signal peptide is the second. The CD8α signal peptide is selected. The scFv fragment is connected in series with the CD8α hinge region, CD8α transmembrane region, 4-1BB costimulatory domain, and CD3ζ signal transduction domain found on NCBI to obtain the sequence of the chimeric antigen receptor. The target fragment is codon-optimized according to the preference of human cell expression and inserted between the NotI and SalI restriction sites of the lentiviral vector pCDH-GFP. Beijing Liuhe Huada Gene Technology Co., Ltd. is commissioned to synthesize it.

[0111] HER2-CAR amino acid sequence (SEQ ID NO.4):

[0112] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGG DGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQ QHYTTPPTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. The structure of HER2-CAR is: CD8α signal peptide-HER2scFv fragment-CD8α hinge region-CD8α transmembrane region-4-1BB costimulatory domain-CD3ζ signal transduction domain.

[0113] The HER2-CAR sequence and the IL-15 sequence were connected through the T2A element by homologous recombination.

[0114] GSGEGRGSLLTCGDVEENPGP (T2A, SEQ ID NO. 5).

[0115] Secretory HER2-T2A-sIL15 (SEQ ID NO.6):

[0116] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS。

[0117] Membrane-bound HER2-T2A-mbIL15 (SEQ ID NO.7):

[0118] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC。

[0119] Nucleic acid molecule encoding secreted HER2-T2A-sIL15 (SEQ ID NO.8):

[0120]

[0121] Nucleic acid molecule encoding membrane-bound HER2-T2A-mbIL15 (SEQ ID NO.9):

[0122]

[0123] SEQ ID NO.10:

[0124] LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0125] 1.2 Lentiviral plasmid construction

[0126] In this example, the lentiviral transduction method was used, the second-generation lentiviral vector pCDH-GFP was selected, the target fragment was inserted between the NotI and SalI restriction sites, codon optimization was performed according to the preference of human cell expression (SEQ ID NO.8, SEQ ID NO.9), and the DNA sequences of the recombinant lentiviral vector plasmids of pCDH-sIL15, pCDH-mbIL15, pCDH-HER2, pCDH-HER2-T2A-sIL15 and pCDH-HER2-T2A-mbIL15 were designed and constructed, and Beijing Liuhe BGI Gene Technology Co., Ltd. was commissioned to synthesize them.

[0127] Figure 2a to Figure 2f This is a map of lentiviral plasmids, corresponding to pCDH-GFP, pCDH-sIL15, pCDH-mbIL15, pCDH-HER2, pCDH-HER2-T2A-sIL15, and pCDH-HER2-T2A-mbIL15, respectively.

[0128] The steps of transformation, identification and extraction of plasmids involved in this section are as follows:

[0129] (1) After receiving the plasmid, centrifuge at 5000 rpm for 1 min to collect the dry powder, add 20 μL of sterile water to dissolve it, and let it stand at room temperature for 1 min;

[0130] (2) Thaw the DH5α competent cells on ice and label them;

[0131] (3) Add 2 μL of plasmid to 100 μL of competent medium and incubate on ice for 30 min;

[0132] (4) Heat shock at 42°C for 90 seconds, followed by an ice bath for 2 minutes;

[0133] (5) Add 900 μL of LB liquid medium without antibiotics and culture at 37°C, 180 rpm for 45 min;

[0134] (6) Centrifuge at 6000 rpm for 5 min, remove 900 μL of the supernatant and discard it, then use the remaining supernatant to mix the cells.

[0135] (7) Add the mixed bacterial solution to an ampicillin-resistant LB plate and spread the liquid evenly;

[0136] (8) Incubate the plate in the normal direction for 1 hour, then invert and incubate for 12 to 16 hours;

[0137] (9) Pick a single colony on the plate, expand the culture, and sequence and identify it.

[0138] Use the Novozyme Endotoxin-Free Plasmid Miniprep Kit to extract the pCDH-sIL15 and pCDH-mbIL15 plasmids. The specific steps are as follows:

[0139] (1) E. coli carrying pCDH-sIL15 and pCDH-mbIL15 plasmids were inoculated into 10 mL of ampicillin-resistant LB liquid culture medium, respectively, and cultured at 37°C and 180 rpm for 12 h-16 h;

[0140] (2) Centrifuge the bacterial solution at 10,000 g for 5 min at room temperature;

[0141] (3) Add RNase A to Solution I in advance, discard the culture medium, add 500 μL Solution I, and vortex;

[0142] (4) Add 500 μL of Solution II, gently invert and mix, and leave at room temperature for 2 min-3 min;

[0143] (5) Add 250 μL of pre-cooled N3 Buffer and invert until a white flocculent precipitate appears;

[0144] (6) Centrifuge at 13000g for 10 min at room temperature and transfer the supernatant to a centrifuge tube;

[0145] (7) Add 1 / 10 volume of ETR Solution to the supernatant, invert to mix, and place on ice for 10 min;

[0146] (8) Incubate at 42°C for 5 min, and the solution becomes turbid again;

[0147] (9) Centrifuge at 12,000 g for 3 min at 25°C. The solution will separate into two layers. Transfer the clear upper layer to a centrifuge tube. Add 1 / 2 volume of anhydrous ethanol to the supernatant. Mix by inversion and let stand at room temperature for 1 min to 2 min.

[0148] (10) The DNA binding column was mounted on a collection tube, and the mixture was transferred (up to 700 μL at a time) to the column, centrifuged at 10,000 g for 1 min at room temperature, and the filtrate was discarded;

[0149] (11) Repeat step 10 until all the mixed solution is transferred;

[0150] (12) Add 500 μL HBC Buffer to the column, centrifuge at 10,000 g for 1 min at room temperature, and discard the filtrate;

[0151] (13) Add 700 μL DNA Wash Buffer to the column, centrifuge at 10,000 g for 1 min at room temperature, discard the filtrate, and repeat the wash process;

[0152] (14) Centrifuge the empty column at 13000 g for 2 min and leave it at room temperature for 10 min until the anhydrous ethanol evaporates completely;

[0153] (15) Place the DNA binding column in a clean centrifuge tube, add 30 μL of ddH2O to the column, let stand for 1 min, and centrifuge at 10,000 g for 1 min at room temperature;

[0154] (16) Discard the DNA binding column and store the DNA product at -20°C until use.

[0155] 2. Lentiviral Packaging and Construction of Transgenic NK92 Cells

[0156] 2.1 Lentiviral packaging

[0157] Lentivirus packaging uses a three-plasmid packaging system, which is modified from HIV-1 (human immunodeficiency virus type 1), removing genes that are not necessary for packaging and retaining only important regulatory genes, reducing the possibility and pathogenicity of replicable virus production. The components required for virus production are divided into three plasmids, pCDH-sIL15, pCDH-mbIL15, pCDH-HER2, pCDH-HER2-T2A-sIL15, and pCDH-HER2-T2A-mbIL15 are transfer plasmids containing target genes. In addition, there is a packaging plasmid psPAX2, which encodes the main structural proteins, specific enzymes and regulatory factors of the virus, and an envelope plasmid pMD2.G encodes the envelope protein of vesicular stomatitis virus (VSV-G). The above three plasmids are co-transfected into the packaging cell 293T through a transfection reagent, and the transcribed target gene RNA and the protein translated by the auxiliary plasmid can be assembled into a lentivirus. Through the above process, five lentiviruses, sIL15, mbIL15, HER2, HER2-T2A-sIL15 and HER2-T2A-mbIL15, were packaged and concentrated, and their titers were detected.

[0158] Lentivirus packaging process:

[0159] 2.1.1 Recovery and culture of 293T cells

[0160] The recovery, culture and passage steps of 293T cells are as follows:

[0161] (1) Take the cryovial out of the liquid nitrogen tank and quickly place it in a 37°C water bath to shake it continuously to dissolve;

[0162] (2) Before the cells are almost completely thawed (about 1 minute), spray the outer surface of the cryotube with 75% ethanol and wipe it clean;

[0163] (3) Open the cryopreserved tube in a biosafety cabinet and transfer the cells to a 15 mL centrifuge tube containing 9 mL of DMEM complete medium;

[0164] (4) Centrifuge at 1000 rpm for 5 min at room temperature, remove the supernatant, add 15 mL of fresh DMEM complete medium to the centrifuge tube to resuspend the cells, transfer the cell suspension to the culture flask, and mix gently;

[0165] (5) Place in an incubator and culture at 37°C, 5% CO2;

[0166] (6) When the cells grow close to saturation density, they need to be subcultured, usually every 48 hours, and the cell confluence should not exceed 90%;

[0167] (7) When subculturing, discard the culture medium and rinse the cells with 5 mL of PBS;

[0168] (8) Discard PBS, add 1 mL of trypsin, and place the cells in a 37°C incubator for 2-3 min until the cells become round, the intercellular spaces are obvious, and some cells begin to fall off the flask wall;

[0169] (9) Add 4 mL of DMEM complete medium to terminate digestion and carefully blow off the cells;

[0170] (10) Pipette 50 μL of cell suspension into a centrifuge tube, add 50 μL of 0.2% trypan blue and mix well;

[0171] (11) Add the cell suspension mixed with trypan blue into a cell counting plate, place it in a cell counter, and measure the cell density and viability;

[0172] (12) According to the cell subculture density (2.0×10 5 The required volume of cell suspension was calculated based on the volume of 1% DMEM cells / mL, the required cell suspension was pipetted into a culture bottle, the required volume of DMEM complete medium was added, and the bottle was placed in an incubator at 37°C and 5% CO2 for culture.

[0173] 2.1.2 Lentivirus packaging

[0174] Use of Lipofectamine TM 3000 transfection reagent was used to co-transfect 293T cells with the plasmid carrying the target gene and the auxiliary plasmids psPAX2 and pMD2.G to package lentivirus. The specific steps are as follows:

[0175] (1) Digest 293T cells within ten generations and adjust the volume to 5×10 5 cells / mL, take 10mL and spread evenly in a 10cm culture dish;

[0176] (2) Place the cells in a 37°C incubator for about 24 hours to allow the cells to recover. When the cell confluence reaches about 90%, lentivirus packaging is performed.

[0177] (3) Take a 2 mL centrifuge tube and add 725 μL Opti-MEM medium and 29 μL Lipofectamine TM 3000 reagent, mix thoroughly;

[0178] (4) Take another 2 mL centrifuge tube, add 725 μL Opti-MEM medium, the target plasmid and the auxiliary plasmid (the plasmid needs to be filtered with a 0.22 μm filter membrane in advance), and then add 29 μL P3000 reagent and mix well;

[0179] (5) Add the liposome dilution obtained in step 3 to the DNA mixture obtained in step 4 and incubate at room temperature for 15 min;

[0180] (6) Add the above DNA-liposome mixture dropwise into the cells, cross-mix, and place in an incubator;

[0181] (7) After 6-8 hours, replace the cell supernatant with 10 mL of fresh DMEM complete medium;

[0182] (8) 48 h after transfection, the viral supernatant was collected and replaced with 10 mL of fresh DMEM complete medium. The supernatant was temporarily stored at 4°C.

[0183] (9) 72 h after transfection, the viral supernatant was collected again and mixed with that collected 48 h after transfection, and centrifuged at 1500 rpm for 5 min to remove impurities.

[0184] 2.1.3 Concentration of lentivirus

[0185] (2) Lenti-X TM Concentrator Reagent Concentrator

[0186] (1) Transfer the lentiviral supernatant collected in 2.3.5 into a sterile container and mix with Lenti-XTM Concentrator reagents were mixed in a 3:1 volume ratio and gently inverted to mix;

[0187] (2) Incubate the mixture at 4°C overnight;

[0188] (3) Centrifuge at 1500 g for 45 min at 4°C. A white precipitate can be seen at the bottom of the centrifuge tube.

[0189] (4) Discard the supernatant and resuspend the cells in an appropriate amount of culture medium according to the cells to be infected;

[0190] (5) The concentrated virus solution was packaged and stored at -80°C until use.

[0191] 2.1.4 Determination of lentiviral titer

[0192] Method 1 HIV-1p24 detection kit (enzyme-linked immunosorbent assay)

[0193] (1) Allow each component to equilibrate at room temperature for 30 min, take out the strips required for the experiment and mark them;

[0194] (2) Dilute the standard to 15 ng / mL with diluent, and then prepare a series of standard dilutions using a 3-fold serial dilution method;

[0195] (3) Add 10 μL of lysis buffer to each reaction well;

[0196] (4) Add the diluted standard and the virus sample obtained in 2.3.6 to the ELISA plate, 90 μL / well, shake for 30s-60s to mix, and incubate at 37°C for 30min;

[0197] (5) After incubation, discard the liquid in the wells, add washing solution (1×), shake gently, and pat dry on clean filter paper. Repeat three times;

[0198] (6) Add 100 μL of enzyme-labeled p24 antibody to each well and incubate at 37°C for 30 min;

[0199] (7) Wash the plate three times, add 100 μL / well of the pre-prepared colorimetric solution, and incubate at 37°C in the dark for 15 min;

[0200] (8) Add 100 μL / well of stop solution and read the result after uniform color development (usually within 20 minutes after adding the stop solution).

[0201] (9) Set the ELISA reader to dual wavelengths of 450 / 630 nm to read the absorbance value.

[0202] Methods Detection of HER2-CAR positive rate by flow cytometry

[0203] (1) Take 6×10 6 HER2-sIL15-NK92 cells and HER2-mbIL15-NK92 cells were placed in a centrifuge tube and centrifuged at 1800 rpm for 8 min at 4°C;

[0204] (2) Discard the supernatant, add 1 mL of FACS Buffer to wash once, centrifuge at 1800 rpm for 8 min at 4°C, discard the supernatant, and add 100 μL of FACS Buffer to resuspend;

[0205] (3) Add 10 μL of FITC-Labeled Human HER2 working solution and incubate at 4°C in the dark for 15 min-30 min;

[0206] (4) Add FACS buffer to the top, centrifuge at 1800 rpm at 4°C for 8 min, and discard the supernatant;

[0207] (5) Add 100 μL FACS Buffer to resuspend the sample and test on the instrument.

[0208] 2.2 Construction of transgenic NK92 cells

[0209] Four lentiviruses, sIL15, mbIL15, HER2-T2A-sIL15 and HER2-T2A-mbIL15, were used to infect NK92 cells in the logarithmic growth phase at an MOI of 50. At this time, MEMα medium supplemented with 0.1mM β-mercaptoethanol, 0.02mM folic acid, 12.5% ​​horse serum and 12.5% ​​fetal bovine serum contained 100U / mL IL-2 to maintain the growth of NK92 cells. 48h-72h after infection, the genes carried by the virus began to be expressed. At 72h, the IL-2 in the culture medium was removed and replaced with a culture medium without IL-2 for continued culture.

[0210] The results showed that after 72 hours of culture without IL-2, the NK92 cells in the control group began to apoptosis in large numbers, while the cells in the experimental group remained in a growth state. This phenomenon preliminarily judged that both IL-15 and HER2-CAR were expressed. The cells in the experimental group could be cultured continuously for more than 30 days and passaged for more than 15 times. Four types of cells were obtained and named sIL15-NK92 cells, mbIL15-NK92 cells, HER2-T2A-sIL15-NK92 cells, and HER2-T2A-mbIL15-NK92 cells. Figure 3The cell status was observed under a microscope (40×); the left picture is the control group, which is NK92 cells that have not undergone any modification; the right picture is any one of the NK92 cells infected with lentiviruses including sIL15, mbIL15, HER2-T2A-sIL15, and HER2-T2A-mbIL15 in the experimental group, showing clumping growth, and the pictures are not much different.

[0211] Compared with the construction process of HER2-T2A-mbIL15-NK92 cells, in the construction process of HER2-mbIL15-NK92 cells, two lentiviruses HER2 and mbIL15 were used to infect NK92 cells in the logarithmic growth phase, that is, T2A was not connected between HER2 and mbIL15. Similarly, in the construction process of HER2-sIL15-NK92 cells, HER2 and sIL15 were also independent, and T2A was not connected between the two.

[0212] 2.2.1.Resuscitation and culture of NK-92 cells

[0213] The recovery, culture and passage steps of NK-92 cells are as follows:

[0214] (1) Take the cryotube out of the liquid nitrogen tank and quickly place it in a 37°C water bath and shake it continuously to dissolve;

[0215] (2) Before the cells are almost completely thawed (about 1 minute), spray the outer surface of the cryotube with 75% ethanol and wipe it clean;

[0216] (3) Open the cryovial in a biosafety cabinet and transfer the cells to a 15 mL centrifuge tube containing 9 mL of NK-92 complete medium;

[0217] (4) Centrifuge at 500 rpm for 5 min at room temperature, remove the supernatant, add 15 mL of fresh NK-92 complete medium to the centrifuge tube to resuspend the cells, transfer the cell suspension to the culture flask, and mix gently;

[0218] (5) Place in an incubator and culture at 37°C, 5% CO2;

[0219] (6) When the cells grow close to saturation density, they need to be subcultured, usually every 48 hours, and the cell confluence should not exceed 90%;

[0220] (7) Gently shake the culture flask to suspend all the cells in the flask, and use a pipette to transfer the cell suspension into a centrifuge tube;

[0221] (8) Centrifuge at 500 rpm for 5 min at room temperature;

[0222] (9) Discard the culture supernatant in a biosafety cabinet and resuspend the cells in 5 mL of fresh NK-92 complete medium;

[0223] (10) Pipette 50 μL of cell suspension into a centrifuge tube, add 50 μL of 0.2% trypan blue and mix well;

[0224] (11) Add the cell suspension mixed with trypan blue into a cell counting plate, place it in a cell counter, and measure the cell density and viability;

[0225] (12) According to the cell subculture density (1.5×10 5 The required volume of cell suspension was calculated based on the cell volume per mL, the required cell suspension was pipetted into a culture bottle, the required volume of NK-92 complete medium was added, and the bottle was placed in an incubator at 37°C and 5% CO2 for culture.

[0226] 2.2.2. Lentivirus infection of NK-92 cells

[0227] (1) Collect NK-92 cells by centrifugation and adjust the cell density to 1×10 6 cells / mL, 100 μL per well was evenly plated in a 24-well plate;

[0228] (2) Remove the lentivirus and melt it on ice to infect NK-92 cells;

[0229] (3) Supplement NK-92 complete medium to 1 / 2 of the culture volume and add polybrene at a final concentration of 8 μg / mL;

[0230] (4) Place the 24-well plate in a centrifuge and centrifuge at 1500 rpm for 1 h at room temperature;

[0231] (5) Place in a 37°C incubator and continue infection for 1 h;

[0232] (6) Add 1 / 2 volume of NK-92 complete medium and return to the incubator for continued culture;

[0233] (7) 24 h after infection, the virus-containing medium was replaced with fresh NK-92 complete medium and the culture was continued;

[0234] (8) After 72 h, the cells were collected, the supernatant was removed by centrifugation, and the cells were washed three times with PBS. The cells were then resuspended in NK-92 complete medium (without IL-2) and subcultured.

[0235] 3. Flow cytometry detection of HER2-CAR positive rate in transgenic NK92 cells

[0236] On days 7, 14, and 25 after lentiviral infection, FITC-labeled HER2 antigen was used to detect the percentage of HER2-CAR positive cells. The flow cytometry results are shown in Figure 2. Figure 4 As shown, the HER2-CAR positive rate was 62.1% (HER2-T2A-mbIL15-NK92 cells) and 49.1% (HER2-T2A-sIL15-NK92 cells) on the seventh day after infection, and the proportion of positive cells gradually increased over time, reaching 83.4% and 95.2% (HER2-T2A-mbIL15-NK92 cells) and 66.4% and 89.8% (HER2-T2A-sIL15-NK92 cells) on the 14th and 25th days after viral transduction, respectively. The reason may be that the two target genes share a promoter, and their transcription is continuous. Only in the translation stage do the ribosomes skip the T2A element to separate the two polypeptides. NK92 cells will upregulate the expression of endogenous IL-15 under the culture conditions without exogenous IL-2, and thus increase the expression of HER2-CAR. At the same time, the present application found that the positive rate of HER2-T2A-mbIL15-NK92 cells increased faster, and after stabilization, it was slightly higher than that of HER2-T2A-sIL15-NK92 cells. This shows that screening using mbIL-15 is better than sIL-15. The advantage of this construction method is that only one infection is required to express HER2-CAR and IL-15 at the same time, reducing the amount of lentivirus used, and can significantly increase the positive rate of HER2-CAR, without the need for flow sorting, which reduces costs and simplifies operations.

[0237] Figure 4 The positive rates of HER2-CAR on days 7, 14, and 25 after lentiviral infection. Figure A: The positive rate of HER2-T2A-mbIL15-NK92 cells; Figure B: The positive rate of HER2-T2A-sIL15-NK92 cells.

[0238] The cells were frozen and revived using MEMα medium supplemented with 0.1 mM β-mercaptoethanol, 0.02 mM folic acid, 12.5% ​​horse serum and 12.5% ​​fetal bovine serum without IL-2. The cells were subcultured for 14 days and the positive rate of HER2-CAR was detected. Figure 5 The positive rates of HER2-T2A-mbIL15-NK92 cells were 94.5%, and the positive rates of HER2-T2A-sIL15-NK92 cells were 88.2%, which were not much different from those before freezing, indicating that HER2-CAR can not only be stably expressed on HER2-T2A-mbIL15-NK92 cells and HER2-T2A-sIL15-NK92 cells, but also not affected by freezing and thawing. Figure 5The positive rate of HER2-CAR after 14 days of recovery. Figure A: The positive rate of HER2-T2A-mbIL15-NK92 cells; Figure B: The positive rate of HER2-T2A-sIL15-NK92 cells.

[0239] 4.ELISA detection of IL-15 expression

[0240] Equal amounts of sIL15-NK92 cells, mbIL15-NK92 cells, HER2-T2A-sIL15-NK92 cells, and HER2-T2A-mbIL15-NK92 cells were cultured in the same culture volume for 48 h, and the supernatant and cell pellet were separated after centrifugation. The cells were washed and ultrasonically disrupted, and resuspended in PBS equal to the culture volume. The Human IL-15 Precoated ELISA kit was used to detect the IL-15 content in the cell culture supernatant and cell disrupted fluid, and sIL15-NK92 cells and mbIL15-NK92 cells were used as controls.

[0241] The results showed that even when co-expressed with HER2-CAR, the levels of secreted and membrane-bound IL-15 did not change significantly. In addition, since we constructed the IL-15 sequence after the transmembrane region of HER2-CAR, its successful expression also demonstrated that the T2A element effectively cut the two proteins and did not affect the expression level of downstream proteins.

[0242] Figure 6 Determination of IL-15 content in cell culture supernatant and cell lysis fluid. Figure A: IL-15 content in the culture supernatant of mbIL15-NK92 cells, HER2-T2A-mbIL15-NK92 cells, sIL15-NK92 cells, and HER2-T2A-sIL15-NK92 cells; Figure B: IL-15 content in mbIL15-NK92 cells, HER2-T2A-mbIL15-NK92 cells, sIL15-NK92 cells, and HER2-T2A-sIL15-NK92 cell lysis fluid.

[0243] For secretory IL-15, take the cell culture supernatant. For membrane-bound IL-15, the mechanical vibration of ultrasonic cell disruptor is needed to destroy the cell membrane and release the protein inside the cell. Human IL-15 Precoated ELISA kit is used to detect the content of IL-15 in the cell disrupted fluid. The specific steps are as follows:

[0244] (1) Take out all components 20 minutes in advance and equilibrate them to room temperature. Take out the strips required for the experiment and mark them.

[0245] (2) Dissolve the lyophilized powder of the standard in diluent, let stand for 5 min-10 min, and then grade dilute the standard to concentrations of 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.3 pg / mL, and 15.6 pg / mL;

[0246] (3) Add the serially diluted standards and cell culture supernatant samples to the pre-coated ELISA plate in sequence, 100 μL / well, and incubate at room temperature for 2 h;

[0247] (4) Discard the liquid in the wells, add 1× Washing buffer, shake gently, pat dry on clean filter paper, and repeat 3 times;

[0248] (5) Add 100 μL / well of Biotinylated antibody working solution and incubate at room temperature for 30 min.

[0249] (6) Repeat step 4, add 100 μL / well of Streptavidin-HRP working solution, and incubate at room temperature for 30 min;

[0250] (7) Repeat step 4, add 100 μL / well of TMB working solution, and incubate at room temperature in the dark for 10 min-15 min;

[0251] (8) 100 μL / well Stop solution was quickly added to terminate the reaction;

[0252] (9) Read the absorbance at a wavelength of 450 nm within 10 minutes after termination.

[0253] 5: Proliferation activity of transgenic NK92 cells

[0254] To detect whether there is a difference in the proliferation of HER2-T2A-IL15-NK92 cells and IL15-NK92 cells, the EdU method was used to detect the proliferation activity of cells.

[0255] like Figure 7 The positive rate of EdU in HER2-T2A-IL15-NK92 cells was not much different from that in IL15-NK92 control cells. The cell proliferation curve was drawn using the CCK-8 method. Figure 7 , HER2-T2A-IL15-NK92 cells showed a proliferation trend similar to that of IL15-NK92 cells, which proves that the HER2-T2A-IL15-NK92 cells constructed in this example have the same proliferation activity as that of IL15-NK92 cells.

[0256] Figure 7 After 2 h of EdU labeling, flow cytometry was performed. 488 channel fluorescence. A: sIL15-NK92 cells; B: mbIL15-NK92 cells; C: HER2-T2A-sIL15-NK92 cells; D: HER2-T2A-mbIL15-NK92 cells.

[0257] EdU cell proliferation assay

[0258] (1) Take cells in the logarithmic growth phase and add 1×10 6 The cells were inoculated into six-well plates and cultured until the cells grew normally;

[0259] (2) Dilute the EdU solution with NK92 complete medium (without IL-2) to prepare 50 μM EdU medium;

[0260] (3) Centrifuge the cells to remove the culture medium, resuspend them in EdU culture medium, 1 mL per well, and incubate for 2 h;

[0261] (4) Collect cells into a centrifuge tube, centrifuge at 500 rpm for 5 min, and discard the supernatant;

[0262] (5) Resuspend the cells in 1 mL of PBS, centrifuge at 500 rpm for 5 min, and discard the supernatant;

[0263] (6) Add 4% paraformaldehyde, 1 mL per tube, fix for 15 min-30 min, centrifuge at 500 rpm for 10 min, and discard the supernatant;

[0264] (7) Add 2 mg / mL glycine solution, 2 mL per tube for neutralization for 5 min, centrifuge at 500 rpm for 10 min, discard the supernatant, and wash once with 2 mL PBS;

[0265] (8) Add 0.5% TritonX-100 permeabilization agent, 1 mL per tube, incubate at room temperature for 10 min, centrifuge at 500 rpm for 10 min, discard the supernatant, and wash once with 2 mL PBS;

[0266] (9) Join For the staining reaction solution, resuspend the cells in 500 μL per tube, incubate at room temperature in the dark for 10 min, centrifuge at 500 rpm for 10 min, and discard the supernatant;

[0267] (10) Add 0.5% TritonX-100 permeabilization agent, 3 mL per tube, centrifuge at 500 rpm for 10 min, and discard the supernatant;

[0268] (11) Immediately after staining, use flow cytometry to detect 488 channels of fluorescence.

[0269] 6. Study on the function of transgenic cells

[0270] 6.1 Expression of HER2 in tumor target cells

[0271] Flow cytometry analysis was performed to determine the expression of HER2 antigen on the surface of SK-BR-3, NCI-N87, and K562 cells. As shown in the figure, SK-BR-3 and NCI-N87 cells highly expressed HER2, while K562 cells did not express HER2.

[0272] Figure 8 The expression of HER2 in three cell lines was detected by flow cytometry, and the control group was a mixture of the three cells without adding flow cytometry antibodies.

[0273] In order to prove that HER2-CAR has targeting, can specifically recognize tumor surface antigens and activate NK92 cells, the basic information of tumor cells was queried from The Human Protein Atlas (https: / / www.proteinatlas.org / ) database, and the tumor cell lines with high expression and non-expression of HER2 were selected from the existing cell library in the laboratory, namely: SK-BR-3 cell line, which is a human breast adenocarcinoma cell, isolated from the pleural effusion of a 43-year-old female patient with breast cancer, overexpressing HER2 gene products; NCI-N87 cell line, which is a human gastric cancer cell, was isolated from the stomach of a male patient with gastric adenocarcinoma in 1976; K562 cell line, which is a human chronic myeloid leukemia cell, was originally isolated from the bone marrow of a patient with chronic myeloid leukemia, and is also a common target cell in in vitro NK cell cytotoxicity assays. The expression of HER2 on the cell surface was detected by flow cytometry. The specific steps are as follows:

[0274] (1) The tumor cells in the logarithmic growth phase were digested with trypsin, centrifuged at 1000 rpm for 5 min after digestion, and the supernatant was discarded;

[0275] (2) Resuspend the cells in FACS Buffer and adjust the cell density to 3×10 6 cells / mL, 100 μL of cell suspension was evenly spread in each well;

[0276] (3) Dilute the primary antibody (trastuzumab) to 200 nM with FACS Buffer, centrifuge the cells at 1000 rpm for 5 min, discard the supernatant, resuspend the cells with 100 μL / well of the primary antibody diluent, and incubate at 4°C for 60 min;

[0277] (4) After incubation, wash twice with 200 μL / well of PBS, centrifuge and discard the supernatant;

[0278] (5) Dilute the secondary antibody (Goat anti-Human IgG Sceondary Antibody) with FACS Buffer at a ratio of 1:500, add 100 μL / well of the secondary antibody dilution to resuspend the cells, and incubate at 4°C for 60 min;

[0279] (6) Wash the cells twice with PBS, resuspend them in 100 μL FACS Buffer, and analyze them using a flow cytometer.

[0280] 6.2 Detection of HER2-CAR-NK92 cell killing activity

[0281] The killing efficiency was calculated according to the formula and plotted using Graphpad prism 6 software. Fig. 9 The killing efficiency of effector cells for the three target cells increased with the increase of the effector-target ratio. For SK-BR-3 and NCI-N87 target cells with high HER2 expression, the killing rate of CAR-IL15-NK92 cells was higher than that of IL15-NK92 cells at the same effector-target ratio, and increased with the increase of HER2-CAR positivity. For K562 cells that do not express HER2, at the same effector-target ratio, there was no significant difference in their killing activity regardless of whether the effector cells expressed HER2-CAR. The above results show that HER2-CAR can guide NK92 cells to specifically target HER2-positive tumor cells and can enhance the killing ability of NK92 cells.

[0282]

[0283] Killing rate = 1-target cell survival rate

[0284] Fig. 9 For the determination of cytotoxicity. A: The killing efficiency of 6 effector cells on SK-BR-3 cells; B: The killing efficiency of 6 effector cells on NCI-N87 cells; C: The killing efficiency of 6 effector cells on K562 cells.

[0285] After incubating HER2-T2A-IL15-NK92, HER2-IL15-NK92, IL15-NK92 effector cells with SK-BR-3, NCI-N87, K562 target cells at effector-target ratios of 5:1, 1:1, 1:5, and 1:25 for 24 hours, the absorbance of each experimental well and control well was measured using CCK-8 reagent. The specific steps were as follows:

[0286] (1) Count target cells and effector cells respectively, and adjust the cell concentration according to the effector-target ratio of 5:1, 1:1, 1:5, and 1:25;

[0287] (2) As shown in Table 1, 50 μL of target cells and effector cells were added to a 96-well cell culture plate and incubated for 24 h, and negative controls such as blank control wells, effector cell spontaneous wells, and target cell spontaneous wells were set up;

[0288] Table 1. Settings of control group and experimental group

[0289] name content background 50μL target cell culture medium + 50μL effector cell culture medium Target cells spontaneously 50μL target cells + 50μL effector cell culture medium Effector cells spontaneously 50μL target cell culture medium + 50μL effector cells Experimental Group 50μL target cells + 50μL effector cells

[0290] (3) After incubation, 10 μL of CCK-8 solution was added to each well;

[0291] (4) Incubate the culture plate in a 37°C incubator for 2 h;

[0292] (5) Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0293] 6.3 Detection of CD107a secretion

[0294] During the killing process of NK cells, the toxic granules in the cells are transported to the vicinity of the cell membrane and released, and CD107a is transferred to the cell surface during this process. Therefore, the expression of CD107a represents NK cells with killing activity, which can further prove the targeting and activation effect of HER2-CAR on NK92 cells.

[0295] The results are as follows Fig.10 The NK92 cell population can be separated from the SK-BR-3 cell population using CD56 flow cytometry antibody. The CD107a positivity rate of the HER2-T2A-mbIL15-NK92 cell group (CAR positivity rate 94.5%) was 39.34%, which was significantly higher than that of the HER2-mbIL15-NK92 group (CAR positivity rate 24.24%) (9.38%) and the mbIL15-NK92 group (without CAR modification) (2.70%). The release of CD107a was also positively correlated with the dose of HER2-CAR expression, which demonstrated the targeting effect of HER2-CAR on HER2-positive tumor cells and the activation effect on NK92 effector cells.

[0296] Fig.10 For the determination of effector cell CD107a.

[0297] Detection of CD107a secretion can also be used to evaluate the killing activity of effector cells. After the effector cells mbIL15-NK92, HER2-mbIL15-NK92, HER2-T2A-mbIL15-NK92 and target cells SK-BR-3 were co-cultured at a 1:1 effector-target ratio for 4 hours, PE anti-human CD56 Antibody and APC anti-human CD107a Antibody were used for flow cytometry staining. The specific steps are as follows:

[0298] (1) Count the target cells and effector cells and adjust the cell density so that the effector-target ratio is 1:1;

[0299] (2) Add 100 μL of the two cell suspensions and 10 μL of APC anti-humanCD107a Antibody to each well of a 96-well cell culture plate and incubate in a 37°C, 5% CO2 constant temperature incubator for 4 h;

[0300] (3) After incubation, the mixture in the cell culture plate was transferred to a flow tube and the cells were washed twice with PBS;

[0301] (4) Prepare CD56 antibody dilution solution. Take 10 μL of PE anti-human CD56 Antibody and add it to 90 μL FACS Buffer in each flow cytometry tube.

[0302] (5) Resuspend the centrifuged cells with 100 μL of CD56 antibody diluent and incubate at 4°C in the dark for 20 min;

[0303] (6) After incubation, wash the cells once with PBS, resuspend them in 100 μL FACS Buffer, and analyze them using a flow cytometer.

[0304] 6.4 Determination of effector secretion levels

[0305] Activated NK cells can secrete a variety of effector factors to play a direct killing and immunomodulatory role. For example, TNF-α can induce apoptosis of tumor cells and effectively inhibit the formation of tumor blood vessels, thereby limiting the growth and spread of tumors. The secretion of TNF-α is one of the ways for NK cells to exert their cytotoxic effects; IFN-γ can further enhance the immune response by activating other immune cells (such as macrophages and T cells), which plays an important role in controlling and reducing the proliferation and spread of tumor cells; granulocyte-macrophage colony stimulating factor (GM-CSF) has the function of promoting DC cell maturation and macrophage generation.

[0306] The ELISA test results of the above three effectors are as follows Fig.11 , Fig.12 , Fig.13 The secretion trends of the three factors were similar. Compared with the IL15-NK92 control group, the stimulation of HER2-positive tumor cells could significantly increase the secretion of TNF-α, IFN-γ and GM-CSF by CAR-IL15-NK92 cells, and the secretion increased significantly with the increase of HER2-CAR positivity, which was consistent with the trend of the in vitro killing experiment, proving the targeting specificity of CAR-IL15-NK92 cells.

[0307] Fig.11 Figure 1 is a statistical analysis of TNF-α. Figure A: The secretion of TNF-α when the six effector cells were co-cultured with SK-BR-3 target cells; Figure B: The secretion of TNF-α when the six effector cells were co-cultured with NCI-N87 target cells.

[0308] Fig.12 Figure 1 shows the statistical analysis of IFN-γ. Figure A shows the secretion of IFN-γ when the six effector cells were co-cultured with SK-BR-3 target cells; Figure B shows the secretion of IFN-γ when the six effector cells were co-cultured with NCI-N87 target cells.

[0309] Fig.13 Figure 1 shows the statistical analysis of GM-CSF. Figure A shows the secretion of GM-CSF when the six effector cells were co-cultured with SK-BR-3 target cells; Figure B shows the secretion of GM-CSF when the six effector cells were co-cultured with NCI-N87 target cells.

[0310] Lack of nutrients, elevated levels of metabolic waste, and cellular dependence on factors and activation signals in the tumor microenvironment (TME) are also one of the reasons for dysfunction and shortened lifespan after adoptive transfer to patients. Studies have shown that after reinfusion of CD19 CAR-NK cells carrying human IL-15, CAR-NK cells in the patient's peripheral blood persist for at least 1 year. Other studies have found that NK cells quickly lose their anti-tumor effector function 24 hours after entering the TME, showing a unique tissue-resident phenotype and no longer actively promoting anti-tumor responses. IL-15 can reawaken the fighting spirit of NK cells and restore their anti-tumor efficacy. In addition, NK cells can exert immunomodulatory functions by secreting cytokines and chemokines, such as attracting dendritic cells to aggregate to solid tumors, leading to an inflammatory active microenvironment, commonly referred to as a "hot tumor", which is characterized by high T cell infiltration, elevated IFN-γ signaling pathways, and PD-L1 expression. Therefore, it has been proposed that CAR-NK cells can be used in combination with T cell therapy for solid tumors to improve the infiltration and function of subsequently infused CAR-T cells in mouse models. The above studies show that the CAR-IL15-NK92 cells constructed in the examples of the present application have great potential for persistence and killing activity in future in vivo experiments.

[0311] The specific steps for determining the secretion level of effector factors are as follows: After incubating HER2-T2A-IL15-NK92, HER2-IL15-NK92, and IL15-NK92 effector cells with SK-BR-3 and NCI-N87 target cells at a 1:1 effector-target ratio for 24 hours, the cell culture supernatant was collected and the concentrations of effector factors TNF-α, IFN-γ, and GM-CSF were detected using ELISA kits.

[0312] Comparative Example 1

[0313] HER2-CAR lentivirus was used to infect NK92 cells at an MOI of 50 to construct HER2-NK92 cells. The cells were collected 7 days after lentiviral infection and co-incubated with FITC-labeled HER2 antigen. The percentage of HER2-CAR positive cells was detected by flow cytometry. The positive rate of HER2-NK92 cells was 23.76%. Since it was not possible to screen by removing IL-2, the positive rate could not be increased any further. Fig.14 The expression of HER2-CAR was determined by flow cytometry.

[0314] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0315] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A genetically modified NK cell, characterized in that: The transgenic NK cells express the polypeptide shown in SEQ ID NO.

7.

2. The transgenic NK cell according to claim 1, characterized in that The transgenic NK cells include NK92 cells.

3. The method for constructing a transgenic NK cell according to any one of claims 1 to 2, characterized in that: The construction method comprises: Provide NK cells to be genetically modified so as to express the polypeptide.

4. A drug, characterized in that The drug comprises the transgenic NK cell according to any one of claims 1 to 2.

5. The method for producing the drug according to claim 4, characterized in that: The production method comprises the step of culturing the transgenic NK cell according to any one of claims 1 to 2.

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