Preparation of a genetically engineered cell and its application in in vitro expansion of NK cells

Abstract

The present application discloses a genetically engineered cell, in which an exogenous gene 4-1BBL, IL-15 or CD155 is integrated. The present application also discloses a method for preparing the above-mentioned genetically engineered cell, comprising the following steps: transfecting a host cell with an expression vector carrying the genes of 4-1BBL, IL-15, CD155α and / or CD155δ to obtain the genetically engineered cell. The present application also discloses the use of the above-mentioned genetically engineered cell in in vitro expansion of NK cells, increasing the anti-tumor activity of NK cells, increasing the purity or survival time of NK cells. The present application also discloses a method for expanding NK cells, comprising co-culturing the above-mentioned genetically engineered cell with NK cells. The present application also discloses the use of NK cells prepared by the above method in the preparation of anti-cancer drugs.

Description

Technical Field

[0001] This specification relates to the field of disease immunotherapy, and particularly to the preparation of genetically engineered cells and their application in the in vitro expansion of NK cells. Background Art

[0002] Human natural killer (NK) cells are one of the important members of the innate immune system of the body and play an important role in immune surveillance of the body. The main advantages of NK cell immunotherapy are that NK cells do not need to be pre-sensitized to recognize target cells, can directly attack virus-infected cells and tumor cells, and the recognition and killing of tumor cells by NK cells are not restricted by MHC-I. Moreover, adoptive immunotherapy with NK cells does not cause graft-versus-host disease (GvHD), and there are no adverse reactions after reinfusion. Therefore, tumor immunotherapy based on NK cells has its unique advantages and good application prospects.

[0003] Although adoptive immunotherapy with NK cells has good anti-tumor effects and application prospects, NK cell immunotherapy is still difficult to be routinely applied clinically. An important obstacle is that human NK cells only account for 10-15% of peripheral blood mononuclear cells, and the number of NK cells is difficult to meet the huge clinical needs. Summary of the Invention

[0004] In the existing culture method of NK cells without feeder cells, the amplification multiple of cells is mostly within 500 times. Without sorting, the purity of NK cells is low, and there are certain defects both in terms of the purity of the obtained cells and the number of cells.

[0005] To solve the above problems, the present application provides a genetically engineered cell as a feeder cell for expanding NK cells.

[0006] The present application provides a genetically engineered cell, in which the exogenous genes 4-1BBL, IL-15 or CD155 are integrated.

[0007] The present application also provides a method for preparing the above-mentioned genetically engineered cell, including the following steps: transfecting a host cell with an expression vector carrying the genes 4-1BBL, IL-15, CD155α and / or CD155δ to obtain the genetically engineered cell. The present application also discloses the application of the above-mentioned genetically engineered cell in the in vitro expansion of NK cells, increasing the anti-tumor activity of NK cells, increasing the purity or survival time of NK cells.

[0008] The present application also provides a method for expanding NK cells, including co-culturing the above-mentioned genetically engineered cell with NK cells.

[0009] The present application also provides the use of NK cells prepared by the above method in the preparation of anti-cancer drugs.

[0010] The beneficial effects brought by the examples in the present application include but are not limited to: (1) The genetically engineered cells can be used as feeder cells for expanding NK cells, enabling the NK cell proliferation multiple to exceed 2 million times. (2) Using such feeder cells to expand NK cells can obtain highly pure gene-edited NK cells, increase the survival time of NK cells, and significantly improve the anti-tumor activity of NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The present application will be further described by way of exemplary embodiments, which will be described in detail through the accompanying drawings. These embodiments are not restrictive, where:

[0012] Figure 1 FIG. is a flow cytometry analysis diagram of the expression levels of 4-1BBL molecules on the surface of four 4-1BBL-K562 cells according to some embodiments of the present application;

[0013] Figure 2 FIG. is a flow cytometry analysis diagram of the expression levels of CD155 protein on the surface of three 4-1BBL-IL15-CD155-K562 cells according to some embodiments of the present application;

[0014] Figure 3 FIG. is a flow cytometry analysis diagram of the expression levels of 4-1BBL and CD155 on the surface of monoclonal 4-1BBL-IL15-CD155α-K562 cells according to some embodiments of the present application;

[0015] Figure 4 FIG. is a flow cytometry analysis diagram of the expression levels of 4-1BBL and CD155 on the surface of monoclonal 4-1BBL-IL15-CD155δ-K562 cells according to some embodiments of the present application;

[0016] Figure 5 FIG. is a flow cytometry analysis diagram of the expression levels of 4-1BBL and CD155 on the surface of monoclonal 4-1BBL-IL15-CD155α / δ-K562 cells according to some embodiments of the present application;

[0017] Figure 6 FIG. is a growth curve diagram of NK cells after co-culture with 4-1BBL-IL15-CD155-K562 cells according to some embodiments of the present application;

[0018] Figure 7Flow cytometry analysis chart of the influence of 4-1BBL-IL15-CD155-K562 cells and 4-1BBL-IL15-K562-#3 cells on the purity of TIGIT gene-edited NK cells as shown in some embodiments of the present application;

[0019] Figure 8 Flow cytometry analysis chart of the expression level of the activating receptor CD226 of TIGIT gene-edited NK cells after co-culture as shown in some embodiments of the present application;

[0020] Figure 9 Analysis chart of the killing activity of TIGIT gene-edited NK cells after co-culture as shown in some embodiments of the present application. Detailed implementation manners

[0021] To more clearly illustrate the technical solutions of the embodiments of this specification, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some examples or embodiments of this specification. For those of ordinary skill in the art, without creative efforts, this specification can also be applied to other similar scenarios based on these drawings. Unless obvious from the language context or otherwise stated, the same reference numerals in the figures represent the same structure or operation.

[0022] As shown in this specification and the claims, unless the context clearly indicates an exceptional situation, words such as "a", "an", "one" and / or "the" are not specifically singular and may also include plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of the clearly identified steps and elements, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.

[0023] Flowcharts are used in this specification to illustrate the operations performed by the systems according to the embodiments of this specification. It should be understood that the operations before or after do not necessarily need to be executed precisely in sequence. On the contrary, they can be executed in reverse order or simultaneously. At the same time, other operations can also be added to these processes, or one or several steps can be removed from these processes.

[0024] The present application provides a genetically engineered cell, and an exogenous gene 4-1BBL, IL-15 or CD155 is integrated in the genetically engineered cell. Therefore, the genetically engineered cell membrane surface in the present application stably expresses 4-1BBL and CD155 molecules, and stably secretes IL-15.

[0025] As used herein, "genetically engineered cell" refers to a genetically modified cell or its descendants. In some embodiments, genetically engineered cells can be produced by using a coupling reagent to link an exogenous polypeptide to the cell surface.

[0026] As used herein, an "exogenous gene" refers to a gene that is not native to a cell but has been introduced into the cell or a cell progenitor. An exogenous gene may include regions or open reading frames that are homologous or identical to endogenous genes native to the cell. In some embodiments, the exogenous gene may be RNA. In some embodiments, the exogenous gene may be DNA. In some embodiments, the exogenous gene may be integrated into the genome of the cell.

[0027] In some embodiments, the nucleotide sequence of the 4-1BBL may be as shown in SEQ ID NO.1. In some embodiments, the nucleotide sequence of the 4-1BBL may have a sequence identity of more than 80% with the nucleotide sequence shown in SEQ ID NO:1 and possess the function or activity of 4-1BBL. 4-1BBL is a ligand of 4-1BB. Activation of 4-1BB by the ligand 4-1BBL can stimulate the proliferation of NK cells and the secretion of cytokines, thereby enhancing the anti-tumor immune response level of the body.

[0028] As used herein, "sequence" generally should be understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding said amino acid sequence, unless a more restrictive interpretation is required herein.

[0029] "Sequence identity" between two nucleotide sequences indicates the percentage of identical nucleotides between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, nucleotide sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity.

[0030] As used herein, "cytokine" refers to a small soluble protein substance secreted by cells that has various effects on other cells. Cytokines mediate many important physiological functions, including growth, development, wound healing, and immune responses. They act by binding to their cell-specific receptors located in the cell membrane, which allows a unique signal transduction cascade to initiate in the cell, ultimately leading to biochemical and phenotypic changes in the target cell. Cytokines can act locally and at sites distant from the release site. They include type I cytokines, which include many interleukins and several hematopoietic growth factors; type II cytokines, including interferons and interleukin 10; tumor necrosis factor ("TNF")-related molecules, including TNF-α and lymphotoxin; members of the immunoglobulin superfamily, including interleukin 1 ("IL-1"); and chemokines, a family of molecules that play a key role in a vast variety of immune and inflammatory functions. Depending on the cell state, the same cytokine can have different effects on cells. Cytokines often regulate the expression of other cytokines and trigger their cascades.

[0031] In some embodiments, the nucleotide sequence of the IL-15 may be as shown in SEQ ID NO.2. In some embodiments, the nucleotide sequence of the IL-15 may have a sequence identity of more than 80% with the nucleotide sequence shown in SEQ ID NO:2 and have the function or activity of IL-15.

[0032] IL-15 plays an important role in stimulating the proliferation and activation of NK cells and is often used for in vitro expansion of immune cells. On NK cells, IL-15 can stimulate their proliferation, promote the production of granzymes and perforins in NK cells, enhance the killing activity of NK cells, and maintain the survival of NK cells. And IL-15 can increase the survival time of NK cells.

[0033] Activation refers to the process (e.g., a signal transduction event) that causes or leads to one or more cellular responses of NK cells, and the cellular responses may be selected from: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers.

[0034] In some embodiments, the CD155 includes a CD155α subunit and a CD155δ subunit. In some embodiments, the nucleotide sequence of the CD155α subunit may be as shown in SEQ ID NO.3. In some embodiments, the nucleotide sequence of the CD155α subunit may have a sequence identity of more than 80% with the nucleotide sequence shown in SEQ ID NO:3 and have the function or activity of CD155α. In some embodiments, the nucleotide sequence of the CD155δ subunit may be as shown in SEQ IDNO.4. In some embodiments, the nucleotide sequence of the CD155δ subunit may have a sequence identity of more than 80% with the nucleotide sequence shown in SEQ ID NO:4 and have the function or activity of CD155δ.

[0035] In some embodiments, the host cell for constructing the genetically engineered cell may be a mammalian tumor cell. In some embodiments, preferably, the host cell for constructing the genetically engineered cell may be a human erythroleukemia cell, and most preferably a K562 cell.

[0036] This application also provides a method for preparing the above-mentioned genetically engineered cell, which includes the following steps: transfecting a host cell with an expression vector carrying the 4-1BBL, IL-15, CD155α, and / or CD155δ genes to obtain the genetically engineered cell.

[0037] In some embodiments, the expression vector may be an expression vector separately integrated with genes of 4-1BBL, IL-15, CD155α, and / or CD155δ. In some embodiments, the expression vector may be any one of a lentiviral vector, a retroviral vector, an electrotransduction vector, or a transposon vector.

[0038] In some embodiments, the expression vector may be a transposon vector. In some embodiments, the transposon vector may be a Sleeping Beauty transposon vector. Compared with other commonly used gene delivery vectors, the Sleeping Beauty transposon vector has incomparable advantages: 1. It can permanently integrate and insert foreign genes into the host chromosome genome; 2. It has low immunogenicity and high in vivo safety; 3. It has a large foreign gene packaging capacity and can even package BACs of more than 100 kb.

[0039] In some embodiments, the Sleeping Beauty transposon vector may be co-transfected with a transposase into genetically engineered cells. In some embodiments, the transposase may be a Sleeping Beauty transposase.

[0040] As used herein, a "vector" refers to a nucleic acid molecule that can transport another nucleic acid linked thereto. In some embodiments, a vector can effect extrachromosomal replication and / or expression of the nucleic acid linked thereto in a host cell such as a eukaryotic and / or prokaryotic cell. A vector capable of directing the expression of an operably linked gene is referred to herein as an "expression vector".

[0041] As used herein, a "transposon" refers to a mobile segment of DNA that can move from one locus to another.

[0042] As used herein, "transfection" refers to the process by which exogenous nucleic acid is transferred or introduced into a cell. Methods for introducing genes into cells and expressing genes in cells are known in the art. A vector can be easily introduced into a cell by any method in the art, such as mammalian, bacterial, yeast, or insect cells. For example, an expression vector can be transferred into a host cell by physical, chemical, or biological means. Physical methods for introducing polynucleotides into cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing polynucleotides of interest into cells include the use of DNA and RNA vectors. Chemical means for introducing polynucleotides into cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes.

[0043] This application also provides the use of the above-mentioned genetically engineered cells in in vitro expansion of NK cells, increasing the anti-tumor activity of NK cells, increasing the purity or survival time of NK cells.

[0044] As used herein, "amplification" refers to the process by which NK cells undergo a series of cell divisions to increase the number of cells.

[0045] This application also provides a method for amplifying NK cells, which includes co-culturing the above-mentioned genetically engineered cells with NK cells. In some embodiments, the method may include the following steps: 1) chemically treating or irradiating the genetically engineered cells; 2) co-culturing the treated genetically engineered cells with NK cells in an NK cell amplification culture medium to obtain NK cells.

[0046] In some embodiments, the chemical treatment may be mitomycin treatment. In some embodiments, the irradiation treatment may be γ-ray irradiation. The genetically engineered cells after chemical treatment or irradiation treatment can no longer divide and proliferate but still maintain metabolic activity.

[0047] In some embodiments, the NK cell amplification culture medium may be RPMI-1640 complete culture medium containing 10% FBS. In some embodiments, the co-culturing conditions of the genetically engineered cells and NK cells may be 37°C and 5% CO2. In some embodiments, IL-2 may be added to the NK cell amplification culture medium. In some embodiments, the final concentration of IL-2 in the NK cell amplification culture medium may be 800 IU / mL - 1300 IU. In some embodiments, the final concentration of IL-2 in the NK cell amplification culture medium may be 1000 IU / mL.

[0048] In some embodiments, in step 2), the treated genetically engineered cells may be added to the NK cell amplification culture medium once or multiple times. In some embodiments, preferably, the treated genetically engineered cells may be added each time at a ratio of 0.5:1 to 2:1 of genetically engineered cells to NK cells. In some embodiments, the treated genetically engineered cells are added each time at a ratio of 1:1 of genetically engineered cells to NK cells.

[0049] In some embodiments, preferably, the interval time for adding the treated genetically engineered cells multiple times may be 3 - 9 days. In some embodiments, the interval time for adding the treated genetically engineered cells multiple times may be 7 days.

[0050] In some embodiments, the co-culturing time may be 5 days to 23 days. In some embodiments, preferably, the co-culturing time may be 7 days to 21 days.

[0051] In some embodiments, the NK cells may knockout the TIGIT receptor.

[0052] As used herein, "knockout" refers to a technique that inactivates or deletes a specific gene of NK cells through certain means.

[0053] The binding of CD155 on the surface of tumor cells to the TIGIT receptor on the surface of NK cells can significantly inhibit the anti-tumor immune response of NK cells. Knocking out the TIGIT receptor on the surface of NK cells using gene editing technology can make NK cells immune to the inhibition of CD155 on the surface of cancer cells. In order to obtain a large number of highly pure TIGIT receptor-knockout NK cells, in this application, 4-1BBL-CD155-IL-15-K562 cells were constructed, and the CD155 molecule on the surface of this feeder cell was used to inhibit the proliferation of impurity cells in gene-edited NK cells, specifically enabling the large proliferation and expansion of TIGIT receptor-knockout NK cells, thereby enhancing the tolerance of NK cells. At the same time, IL-15 was used to increase the survival time of NK cells.

[0054] This application also provides the use of the NK cells prepared by the above method in the preparation of anti-cancer drugs.

[0055] In some embodiments, the drug can be an antibody drug, a nucleic acid drug, a small molecule drug, an oncolytic virus drug or a cell drug. In some embodiments, the application can be the application in the combined treatment of NK cells with antibody drugs, nucleic acid drugs, small molecule drugs, oncolytic virus drugs and cell drugs.

[0056] "Cancer" herein refers to a disease in which abnormal cells divide uncontrollably. In some embodiments, cancer can invade other tissues. In some embodiments, the cancer can be solid cancer and non-solid cancer. In some embodiments, the cancer can include cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, cutaneous squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, thyroid cancer, urothelial carcinoma of the bladder, brain tumor, gastric cancer, peritoneal cancer, head and neck cancer, endometrial cancer, renal cancer, female genital tract cancer, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hematopoietic system tumors, multiple myeloma, non-Hodgkin lymphoma, acute myeloid leukemia, B-cell lymphoma, T-cell lymphoma.

[0057] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all purchased from regular biochemical reagent companies unless otherwise specified. In the following examples, quantitative tests are all set with three repeated experiments, and the results are averaged.

[0058] Example 1: Preparation of 4-1BBL-K562 cells

[0059] 1) Search the NCBI (The National Center for Biotechnology Information) database to obtain the 4-1BBL coding sequence SEQ ID NO.1 (Table 1), numbered NM_003811.4.

[0060] 2) Design PCR amplification primers SEQ ID NO.5 and SEQ ID NO.6 (Table 2) based on the 4-1BBL sequence, and amplify the 4-1BBL coding fragment from PBMC.

[0061] 3) Insert the 4-1BBL coding fragment into the Sleeping Beauty transposon vector through cloning construction to construct the 4-1BBL transposon.

[0062] 4) Co-transfect the 4-1BBL transposon and the transposase into K-562 cells. During the transfection process, the number of K-562 cells and the 4-1BBL coding plasmid are combined according to Table 3 to obtain K-562 cells with stable expression of 4-1BBL (4-1BBL-K562).

[0063] Table 1 4-1BBL coding sequence

[0064]

[0065] Table 2 4-1BBL amplification primer sequences

[0066]

[0067] Table 3 4-1BBL expression vector transfection conditions

[0068]

[0069] Example 2: Detection of 4-1BBL expression level

[0070] 1) Collect wild-type K562, 4-1BBL-K562-#1, 4-1BBL-K562-#2, 4-1BBL-K562-#3, 4-1BBL-K562-#4 cells, and wash them twice with 1×PBS containing 1% fetal bovine serum (FBS).

[0071] 2) Resuspend the cells with PBS containing 1% FBS and count them, and adjust the cell concentration to 3×10^6 cells / mL.

[0072] 3) Take 50 μL of the above cell suspension and add it to a new 1.5 mL centrifuge tube, add 1 μL of PE-anti human 4-1BBL antibody, and incubate at 4°C in the dark for 30 min.

[0073] 4) Wash twice with 1% FBS 1×PBS.

[0074] 5) Resuspend the cells with 300 μL of 1% FBS 1×PBS and perform flow cytometry analysis.

[0075] The results of flow cytometry analysis are as Figure 1 shown in Table 4. Figure 1 The results showed that K-562 cells were transfected after being mixed with 4-1BBL expression plasmid at different ratios. The fluorescence signal intensity of 4-1BBL on the surface of K-562 cells was detected by flow cytometry. It was found that the peak values of 4-1BBL fluorescence signals generated by the four parameter combinations shifted to the right relative to the untransfected wild-type K-562, indicating that the four parameter combinations could all express 4-1BBL molecules on the surface of K-562 cells. Among them, the peak value of the 4-1BBL fluorescence signal of the 4-1BBL-K562-#4 parameter combination shifted the most to the right, indicating that this parameter combination could achieve the highest expression level of 4-1BBL molecules on the surface of K-562 cells. According to the results in Table 4, the K-562 cells obtained with the 4-1BBL-K562-#4 parameter combination had the highest average fluorescence signal intensity of 4-1BBL, indicating that this parameter combination could achieve the highest average expression level of 4-1BBL molecules on the surface of K-562 cells. Therefore, the K-562 cells transfected with the 4-1BBL-K562-#4 parameter combination were selected for subsequent cell construction.

[0076] Table 4 Detection results of 4-1BBL expression intensity

[0077] Group Mean fluorescence signal intensity of 4-1BBL Wild-type K562 1587 4-1BBL-K562-#1 6106 4-1BBL-K562-#2 15333 4-1BBL-K562-#3 17686 4-1BBL-K562-#4 22058

[0078] Example 3: Preparation of 4-1BBL-IL15-K562 cells

[0079] 1) Search the NCBI (The National Center for Biotechnology Information) database to obtain the IL-15 coding sequence SEQ ID NO.2 (Table 5), with the accession number: NM_000585.5.

[0080] 2) Design PCR amplification primers SEQ ID NO.7 and SEQ ID NO.8 (Table 6) according to the IL-15 sequence, and amplify the IL-15 coding fragment from PBMC.

[0081] 3) Insert the IL-15 coding fragment into the Sleeping Beauty transposon vector by cloning to construct the IL-15 transposon.

[0082] 4) Co-transfect the IL-15 transposon and transposase into 4-1BBL-K562-#4 cells. During the transfection process, the number of 4-1BBL-K562-#4 cells and the 4-1BBL coding plasmid were combined according to Table 7, and K562 cells stably expressing 4-1BBL-IL15 (4-1BBL-IL15-K562) were obtained by ELISA screening.

[0083] Table 5 IL-15 coding sequence

[0084]

[0085] Table 6 IL-15 amplification primer sequences

[0086]

[0087] Table 7 IL-15 expression vector transfection conditions

[0088]

[0089]

[0090] Screening of 4-1BBL-IL15-K562 cells:

[0091] 1) Collect 4-1BBL-IL15-K562-#1, 4-1BBL-IL15-K562-#2, 4-1BBL-IL15-K562-#3, and 4-1BBL-IL15-K562-#4 cells, and wash them twice with 1×PBS.

[0092] 2) Resuspend the cells with RPMI-1640 medium containing 10% FBS and count them, and adjust the cell concentration to 5×10^4 cells / mL.

[0093] 3) Take 200 μL of the above cell suspension and add it to a 96-well plate, and culture it at 37°C for 24 hours.

[0094] 4) After culturing for 24 hours, aspirate 50 μL of the culture supernatant from each well.

[0095] 5) Detect the concentration of IL-15 in the culture supernatant using a human IL-15 ELISA detection kit (product number: abs510018, Absin).

[0096] The ELISA detection results are shown in Table 8. All four combinations of the number of 4-1BBL-K562-#4 cells and the IL-15 coding plasmid can enable 4-1BBL-K562-#4 cells to secrete IL-15. Among them, the 4-1BBL-IL15-K562-#3 parameter combination can enable

[0097] 4-1BBL-K562-#4 cells secrete the most IL-15. Therefore, 4-1BBL-IL15-K562-#3 with the highest IL-15 secretion concentration was selected for subsequent cell construction.

[0098] Table 8 Detection of IL-15 secretion level

[0099] Group IL-15 concentration (ng / mL) 4-1BBL-IL15-K562-#1 2.35 4-1BBL-IL15-K562-#2 5.18 4-1BBL-IL15-K562-#3 8.65 4-1BBL-IL15-K562-#4 8.53

[0100] Example 4: Preparation of 4-1BBL-IL15-CD155-K562 cells

[0101] 1) Search the NCBI (The National Center for Biotechnology Information) database to obtain the coding sequence SEQ ID NO.3 of the CD155 α subunit, numbered: NM_006505.5; the coding sequence SEQ ID NO.4 of the CD155 δ subunit, numbered: NM_001135770.4 (Table 9).

[0102] 2) Design PCR amplification primers SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12 according to the CD155 sequence (Table 10). The primer combination of SEQ ID NO.9 and SEQ ID NO.10 was used to amplify the CD155 α coding fragment from the lung cancer cell line H1299; the primer combination of SEQ ID NO.11 and SEQ ID NO.12 was used to amplify the CD155 δ coding fragment from the lung cancer cell line H1299. The two coding fragments were inserted into the Sleeping Beauty transposon vector respectively to construct two transposons of CD155 α and δ.

[0103] 3) Co-transfect the two transposons of CD155 α and δ with the transposase into 4-1BBL-IL15-K562-#3 cells respectively. During the transfection process, the ratio of the number of 4-1BBL-IL15-K562-#3 cells to the CD155 α or CD155 δ plasmid was combined according to Table 11 to obtain three kinds of K562 cells with stable expression of 4-1BBL-IL15-CD155: 4-1BBL-IL15-CD155α-K562, 4-1BBL-IL15-CD155δ-K562, 4-1BBL-IL15-CD155α / δ-K562.

[0104] Table 9 CD155α and CD155δ coding sequences

[0105]

[0106]

[0107] Table 10 CD155α and CD155δ Amplification Primers

[0108]

[0109] Table 11 CD155 Expression Vector Transfection Conditions

[0110]

[0111] Example 5: Detection of CD155 Expression Level

[0112] 1) Collect 4-1BBL-IL15-K562-#3, 4-1BBL-IL15-CD155α-K562, 4-1BBL-IL15-CD155δ-K562, 4-1BBL-IL15-CD155α / δ-K562, and wash twice with 1×PBS containing 1% fetal bovine serum (FBS).

[0113] 2) Resuspend the cells in PBS containing 1% FBS and count, then adjust the cell concentration to 3×10^6 cells / mL.

[0114] 3) Take 50 μL of the above cell suspension and add it to a new 1.5 mL centrifuge tube, then add 1 μL of APC-anti human CD155 antibody, and incubate at 4°C in the dark for 30 min.

[0115] 4) Wash twice with 1×PBS containing 1% FBS.

[0116] 5) Resuspend the cells in 300 μL of 1×PBS containing 1% FBS and perform flow cytometry analysis.

[0117] The flow cytometry results are as Figure 3 shown in Table 12. Figure 2The results showed that after transfection by mixing 4-1BBL-IL15-K562#3 cells with CD155α and / or CD155δ expression plasmids, the peak fluorescence signals of CD155 on the surface of 4-1BBL-IL15-K562#3 cells shifted to the right relative to the untransfected 4-1BBL-IL15-K562#3 cells, indicating that all three parameter combinations could express CD155 molecules on the surface of K-562 cells. Among them, the peak fluorescence signal of CD155 in the 4-4-1BBL-IL15-CD155δ-K562 parameter combination shifted the most to the right, indicating that this parameter combination could achieve the highest expression level of CD155 molecules expressed on the surface of K-562 cells. According to the results in Table 12, the K-562 cells obtained with the 4-1BBL-IL15-CD155δ-K562 parameter combination had the highest average fluorescence signal intensity of CD155, indicating that this parameter combination could achieve the highest average expression level of CD155 molecules expressed on the surface of K-562 cells. Therefore, the K-562 cells transfected with the 4-1BBL-IL15-CD155δ-K562 parameter combination were selected for subsequent cell construction.

[0118] Table 12 Detection Results of CD155 Expression Level

[0119] Group Mean fluorescence signal intensity of CD155 4-1BBL-IL15-K562-#3 587 4-1BBL-IL15-CD155α-K562 10668 4-1BBL-IL15-CD155δ-K562 10696 4-1BBL-IL15-CD155α / δ-K562 5300

[0120] Example 6: Screening of 4-1BBL-IL15-CD155-K562 Monoclonal Cells

[0121] 1) Take the cells of 4-1BBL-IL15-CD155α-K562, 4-1BBL-IL15-CD155δ-K562, and 4-1BBL-IL15-CD155α / δ-K562 in Example 5, inoculate them into a 96-well plate by the limiting dilution method, and use an inverted microscope to examine and record the monoclonal cell wells.

[0122] 2) After 14 weeks, monoclonal amplification formed obvious precipitates and filled the chambers of the 96-well plate. For each type of cell, 10 clones were selected, and each clone was transferred to a 6-well plate and cultured for another 14 days.

[0123] The numbers are as follows:

[0124] 4-1BBL-IL15-CD155α-K562: A1, A2, A3, A4, A5, A6, A7, A8, A9, A10

[0125] 4-1BBL-IL15-CD155δ-K562: D1, D2, D3, D4, D5, D6, D7, D8, D9, D10

[0126] 4-1BBL-IL15-CD155α / δ-K562: AD1, AD2, AD3, AD4, AD5, AD6, AD7, AD8, AD9, AD10

[0127] 3) After 14 days, collect the cloned cells in the 6-well plate and wash them twice with 1×PBS containing 1% fetal bovine serum (FBS).

[0128] 4) Resuspend the cells with PBS containing 1% FBS and count them, then adjust the cell concentration to 3×10^6 cells / mL.

[0129] 5) Take 50 μL of the above cell suspension and add it to a new 1.5 mL centrifuge tube. Add 1 μL each of PE-anti human 4-1BBL antibody and APC-anti human CD155 antibody, and incubate in the dark at 4°C for 30 min.

[0130] 6) Wash twice with 1% FBS 1×PBS.

[0131] 7) Resuspend the cells with 300 μL of 1% FBS 1×PBS and perform flow cytometry analysis.

[0132] The flow cytometry results are as Figures 3 to 5 and shown in Table 13. After stable expansion of monoclonal cells derived from 4-1BBL-IL15-CD155α-K562, 4-1BBL-IL15-CD155δ-K562, and 4-1BBL-IL15-CD155α / δ-K562, cells with clone numbers A9, D8, and AD6 had relatively high levels of 4-1BBL and CD155 expression. Therefore, clones A9, D8, and AD6 were selected for subsequent comparison of the NK cell expansion fold.

[0133] Table 13 Flow cytometry results of 4-1BBL and CD155 expression levels

[0134]

[0135]

[0136] Example 7: Preparation of TIGIT gene-edited NK cells

[0137] 1. Expansion and culture of primary NK cells

[0138] 1) Take out the cryopreserved human peripheral blood mononuclear cells (PBMC) from liquid nitrogen and quickly thaw them in a 37°C water bath.

[0139] 2) Add 4 mL of RPMI-1640 complete culture medium containing 10% FBS to a new 15 mL centrifuge tube, and transfer 1 mL of the PBMC suspension to the 15 mL centrifuge tube.

[0140] 3) Centrifuge at 250×g for 5 min at room temperature.

[0141] 4) Discard the supernatant, and resuspend the cells with 1 mL of RPMI-1640 culture medium.

[0142] 5) Add 19 mL of RPMI-1640 culture medium to a new 75 mL cell culture flask, and transfer the above cell suspension to the culture flask.

[0143] 6) Add human recombinant IL-2 protein to the culture flask at a final concentration of 200 U / mL.

[0144] 7) Place the culture flask in a 37°C, 5% CO2 incubator. After overnight culture, perform cell counting on the PBMC in the culture flask.

[0145] 8) Take out the cryopreserved irradiated 4-1BBL-IL-15-K562 cells from liquid nitrogen and quickly thaw them in a 37°C water bath.

[0146] 9) Add 4 mL of RPMI-1640 complete culture medium to a new 15 mL centrifuge tube, and transfer the 4-1BBL-IL-15-K562 cell suspension to the 15 mL centrifuge tube.

[0147] 10) Centrifuge at 250×g for 5 min at room temperature.

[0148] 11) Discard the supernatant, resuspend the cells with 1 mL of RPMI-1640 culture medium and perform cell counting.

[0149] 12) Add EK562 cells to the culture flask at an effector-to-target ratio of PBMC:4-1BBL-IL-15-K562 = 1:1.

[0150] 13) Culture the cells in a 37°C, 5% CO2 incubator. Change the medium every two days and perform cell counting. Control the cell density within the range of 0.5 to 1×10 6 cells / mL. Add IL-2 according to the volume of the culture medium at a final concentration of 100 U / mL.

[0151] 14) On the 7th day of culture, perform cell counting on the cells in the culture flask, and add 4-1BBL-IL-15-K562 cells to the culture flask again at a ratio of 1:1.

[0152] 15) The cells were cultured in an incubator at 37 °C with 5% CO2. The medium was changed every two days and cell counting was performed. The cell density was controlled within the range of 0.5 to 1×10 6 cells / mL. IL-2 was added according to the volume of the culture medium, with a final concentration of 100 U / mL.

[0153] 16) After culturing for 14 days, the purity of NK cells in the culture flask could reach over 95%.

[0154] 2. Preparation of sgRNA

[0155] By analyzing the CDS region of the TIGIT gene, the present invention selected target sequences that might mutate into stop codons. The corresponding sgRNA can be obtained by in vitro transcription or synthesized by a company. Both can achieve editing. Compared with in vitro transcription, the synthesized sgRNA can modify the ends of the sgRNA, which helps to improve the editing efficiency. Each synthesized sgRNA sequence contains 3 thiol groups and 3 methoxy groups at the 3' end and 5' end respectively.

[0156] The following will briefly describe the steps of in vitro transcription:

[0157] 1) Annealing: After adding the upstream sequence of the TAGG endonuclease sticky end and the downstream sequence with the endonuclease sticky end to each sgRNA, they were annealed together through a program (95 °C, 5 min; 95 °C - 85 °C (cooling at a rate of 2 °C / s); 85 °C - 25 °C (cooling at a rate of -0.1 °C / s); maintained at 4 °C) to obtain the annealed product, i.e., the oligo fragment.

[0158] 2) Linearization: The vector pUC57-sgRNA expression vector (Addgene#51132) was linearized by BsaI (NEB: R0539L). Linearization system: 2 μg of pUC57-sgRNA expression vector; 5 μL of buffer (NEB: R0539L); 1 μL of BsaI; made up to 50 μL with ddH2O.

[0159] 3) Ligation: The annealed product was ligated to the pUC57-sgRNA expression vector linearized by BsaI. Ligation system: 1 μL of T4 ligation buffer (NEB: M0202L), 20 ng of linearized vector, 5 μL of annealed oligo fragment (10 μM), 0.5 μL of T4 ligase (NEB: M0202L), made up to 10 μL with ddH2O, and ligated at 16 °C for 1 hour. The ligated vector was transformed, bacteria were picked, and identified. The positive clones were cultured in a shaker to extract plasmids (Axygene: AP-MN-P-250G) and the concentration was measured.

[0160] 4) PCR amplification: Use the primer sgRNA-F to amplify the sgRNA sequence containing the T7 promoter by PCR. After identifying the band by gel electrophoresis, treat it with RNA-SECURE (Thermo, #AM7005) at 65 °C for 15 minutes, and then recover the amplification product (Axygene: AP-PCR-250G) and measure its concentration.

[0161] 5) In vitro transcription: Use the T7 transcription kit (Thermo, #AM1354) to transcribe the DNA of sgRNA into RNA in vitro. After identifying by gel electrophoresis, recover the sgRNA with the RNA recovery kit (Thermo, #AM1908) and measure its concentration.

[0162] 3. Electroporation transfection of NK cells

[0163] 1) RNP mixing: Mix the sgRNA prepared in step 2 and the BE protein prepared in step 3 according to the ratio at a mass ratio of 1:4, and gently pipette and mix with a pipette tip.

[0164] 2) Prepare NK cells. Wash the NK cells prepared in step 1 with PBS once and centrifuge.

[0165] 3) Electroporation transfection: Perform electroporation transfection using an electroporator. After resuspending and mixing the NK cells and RNP with the electroporation solution, add them to the electroporation cuvette, and select an appropriate electroporation program for electroporation. After electroporation, aspirate the liquid with a pipette tip and place it in a pre-warmed culture medium for culture.

[0166] Example 8: Amplification of TIGIT gene-edited NK cells

[0167] 1) Take the TIGIT gene-edited NK cells 1 to 2 days after electroporation.

[0168] 2) Add 4 mL of RPMI-1640 complete culture medium containing 10% FBS to a new 15 mL centrifuge tube, and transfer 1 mL of the TIGIT gene-edited NK cell suspension to the 15 mL centrifuge tube.

[0169] 3) Centrifuge at 250 × g for 5 min at room temperature.

[0170] 4) Discard the supernatant, and resuspend the cells with 1 mL of RPMI-1640 culture medium.

[0171] 5) Add 19 mL of RPMI-1640 culture medium to a new 75 mL cell culture flask, and transfer the above cell suspension to the culture flask.

[0172] 6) Add human recombinant IL-2 protein to the culture flask at a final concentration of 200 U / mL.

[0173] 7) Place the culture flask in a 37 °C, 5% CO2 incubator. After overnight culture, perform cell counting on the TIGIT gene-edited NK cells in the culture flask.

[0174] 8) Thaw the irradiated 4-1BBL-IL15-CD155(α, δ or α / δ)-K562 cells rapidly in a 37 °C water bath.

[0175] 9) Add 4 mL of complete RPMI-1640 culture medium to a new 15 mL centrifuge tube, and transfer the 4-1BBL-IL15-CD155(α, δ or α / δ)-K562 cell suspension to the 15 mL centrifuge tube.

[0176] 10) Centrifuge at 250×g for 5 min at room temperature.

[0177] 11) Discard the supernatant, resuspend the cells with 1 mL of RPMI-1640 culture medium and perform cell counting.

[0178] 12) Add 4-1BBL-IL15-CD155(α, δ or α / δ)-K562 cells to the TIGIT gene-edited NK cells at a ratio of 1:1.

[0179] 13) Culture the cells in a 37 °C, 5% CO2 incubator, and add IL-2 according to the volume of the culture medium to a final concentration of 100 U / mL.

[0180] 14) On the 14th day of culture, perform cell counting on the cells in the culture flask, and add 4-1BBL-IL15-CD155(α, δ or α / δ)-K562 cells to the TIGIT gene-edited NK cells at a ratio of 1:1.

[0181] 15) After the 14th day and on the 21st day of culture, take samples from the culture flask for cell counting and calculate the amplification fold.

[0182] The calculation results of the amplification fold are as Figure 6 shown in Table 14. According to the results, the 4-1BBL-IL15-CD155δ-K562 cells with clone number D8 had the highest NK cell amplification fold on the 7th, 14th, and 21st days after amplification.

[0183] Table 14 NK cell amplification fold

[0184]

[0185] Example 9: Detection of the expression levels of TIGIT and CD226 receptors in gene-edited NK cells

[0186] 1) Collect NK cells expanded for 21 days by 4-1BBL-IL15-K562-#3, 4-1BBL-IL15-CD155α-K562, 4-1BBL-IL15-CD155δ-K562, and 4-1BBL-IL15-CD155α / δ-K562 cells, and wash twice with 1×PBS containing 1% fetal bovine serum (FBS).

[0187] 2) Resuspend the cells with PBS containing 1% FBS and count them, and adjust the cell concentration to 3×10^6 cells / mL.

[0188] 3) Take 50 μL of the above cell suspension and add it to a new 1.5 mL centrifuge tube, then add 1 μL of BV421-anti human TIGIT antibody and 1 μL of FITC-anti human CD226 antibody, and incubate at 4°C in the dark for 30 min.

[0189] 4) Wash twice with 1×PBS containing 1% FBS.

[0190] 5) Resuspend the cells with 300 μL of 1×PBS containing 1% FBS and perform flow cytometry analysis.

[0191] The expression level of TIGIT in gene-edited NK cells is as Figure 7 shown in Table 15. Wild-type unedited NK cells or gene-edited NK cells were expanded by 4-1BBL-IL15-K562-#3 cells, and the positive ratios of TIGIT in NK cells were 81.1% and 31.9% respectively. It can be seen that gene editing can significantly reduce the expression level of TIGIT receptor on the surface of NK cells. CD155 stably expressing 4-1BBL-IL15-K562 cells A9, D8, and AD6 constructed by genetic engineering can further reduce the expression level of TIGIT receptor on the surface of NK cells. Among them, the gene-engineered 4-1BBL-IL15-CD155δ-K562 with clone number D8 has the strongest inhibitory effect on the expression of TIGIT receptor in NK cells, which can reduce the proportion of TIGIT-positive NK cells to 5.94%.

[0192] The expression level of CD226 in gene-edited NK cells is as Figure 8As shown in Table 15. The 4-1BBL-IL15-K562-#3 cells were used to expand wild-type unedited NK cells or gene-edited NK cells. The percentages of CD226-positive NK cells obtained were 88.3% and 88.6% respectively, indicating that gene editing does not affect the expression level of CD226 on NK cells. The CD155 cells stably expressing 4-1BBL-IL15-K562, namely A9, D8, and AD6, constructed by genetic engineering, could all increase the expression level of CD226 receptor on the surface of NK cells. Among them, the gene-engineered 4-1BBL-IL15-CD155δ-K562 with clone number D8 had the strongest promoting effect on the expression of CD226 receptor on NK cells, which could increase the percentage of CD226-positive NK cells to 99.5%.

[0193] Table 15 Expression levels of TIGIT and CD226 receptors on gene-edited NK cells

[0194]

[0195] Example 10: Investigation of the killing activity of in vitro-expanded NK cells

[0196] 1) Collect H1299 lung cancer cells for counting and adjust the cell suspension concentration to 2×10 5 cells / mL.

[0197] 2) Respectively collect unedited NK cells, NK cells expanded by 4-1BBL-IL15-K562, and NK cells expanded by feeder cells with clone numbers A9, D8, and AD6 for counting, and adjust the cell suspension concentration to 2×10 5 cells / mL.

[0198] 3) Mix NK cells and H1299 tumor cells at a ratio of 1:1.

[0199] 4) Add 50 μL of single NK cells, 50 μL of single lung cancer cells, or 100 μL of the above cell suspension to a 96-well plate.

[0200] 5) Add 50 μL of cell culture medium to the wells with single NK cells or single lung cancer cells to make up the volume to 100 μL.

[0201] 6) After culturing for 24 h, add 100 μL of CellTiter-Glo (purchased from Promega, catalog number G7571) bioluminescence detection reagent to each well.

[0202] 7) Shake at room temperature for 1 min, then let stand for 10 min, and then detect the bioluminescence intensity with an enzyme-linked immunosorbent assay (ELISA) reader.

[0203] 8) Calculate the killing activity of NK cells according to the following formula:

[0204]

[0205] Mean Mix : The average luminescence intensity of the co - culture group of NK cells and tumor cells;

[0206] Mean NK : The average luminescence intensity of the group with NK cells cultured alone;

[0207] Mean Tumor : The average luminescence intensity of the group with tumor cells cultured alone.

[0208] The detection results are as Figure 9 shown in Table 16. Wild - type unedited NK cells or gene - edited NK cells were expanded using 4 - 1BBL - IL15 - K562 - #3 cells. Gene - edited NK cells had stronger anti - tumor activity compared to unedited NK cells. The CD155 - stably - expressing 4 - 1BBL - IL15 - K562 cells A9, D8, and AD6 constructed by genetic engineering could further enhance the anti - tumor activity of gene - edited NK cells. Among them, the gene - engineered 4 - 1BBL - IL15 - CD155δ - K562 with clone number D8 had the strongest enhancing effect on the anti - tumor activity of NK cells.

[0209] Table 16 Detection of NK cell killing activity

[0210]

[0211]

[0212] In summary, NK cells expanded from 4 - 1BBL - L15 - CD155δ - K562 gene - engineered cells had a higher amplification multiple. At the same time, using 4 - 1BBL - L15 - CD155δ - K562 as feeder cells to expand TIGIT gene - edited NK cells could obtain TIGIT - negative NK cells with higher purity, and the activating receptor CD226 of NK cells was more expressed. This highly pure TIGIT gene - edited NK cell had stronger anti - tumor immune activity. The above results indicate that 4 - 1BBL - L15 - CD155δ - K562 gene - engineered cells have obvious practicality and innovation in expanding TIGIT gene - edited NK cells.

[0213] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only an example and does not constitute a limitation to this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification, so such modifications, improvements, and corrections still fall within the spirit and scope of the exemplary embodiments of this specification.

[0214] Meanwhile, this specification uses specific terms to describe the embodiments of this specification. Such as "one embodiment", "an embodiment", and / or "some embodiments" mean a certain feature, structure, or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that the "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more at different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0215] In some embodiments, numbers are used to describe the components and the quantity of attributes. It should be understood that such numbers used for the description of embodiments are, in some examples, modified by the modifiers "about", "approximately", or "substantially". Unless otherwise stated, "about", "approximately", or "substantially" indicate that the said numbers allow a variation of ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, and such approximate values can change according to the characteristics required by individual embodiments. In some embodiments, the numerical parameters should consider the specified significant digits and adopt the method of retaining the general number of digits. Although the numerical ranges and parameters used in some embodiments of this specification to confirm the breadth of their scope are approximate values, in specific embodiments, such numerical settings are made as precise as possible within the feasible range.

[0216] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, by way of example and not limitation, alternative configurations of the embodiments of this specification can be considered to be in accordance with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. A genetically engineered cell, characterized in that, The genetically engineered cells integrate exogenous genes 4-1BBL, IL-15, and CD155. The genetically engineered cells are K562 cells, and the CD155 is the CD155α subunit and / or the CD155δ subunit.

2. The genetically engineered cell according to claim 1, characterized in that, The nucleotide sequence of the 4-1BBL is as shown in SEQ ID NO. 1, or has a sequence identity of more than 80% with the nucleotide sequence shown in SEQ ID NO: 1, and has the function or activity of 4-1BBL; the nucleotide sequence of the IL-15 is as shown in SEQ ID NO.2, or has a sequence identity of more than 80% with the nucleotide sequence shown in SEQ ID NO: 2, and has the function or activity of IL-15.

3. The genetically engineered cell according to claim 2, characterized in that, The nucleotide sequence of the CD155α subunit is as shown in SEQ ID NO.3, or has a sequence identity of more than 80% with the nucleotide sequence shown in SEQ ID NO: 3, and has the function or activity of CD155α; the nucleotide sequence of the CD155δ subunit is as shown in SEQ ID NO.4, or has a sequence identity of more than 80% with the nucleotide sequence shown in SEQ ID NO: 4, and has the function or activity of CD155δ.

4. A method for preparing the genetically engineered cell according to any one of claims 1-3, characterized in that, It includes the following steps: Transfecting a host cell with an expression vector carrying the genes of 4-1BBL, IL-15, CD155α, and / or CD155δ to obtain the genetically engineered cells.

5. The method according to claim 4, wherein The expression vector is an expression vector that separately integrates the genes of 4-1BBL, IL-15, CD155α, and / or CD155δ.

6. The method according to claim 4, wherein The expression vector is a transposon vector.

7. The method according to claim 6, wherein The transposon vector is a sleeping beauty transposon vector.

8. The method according to claim 7, wherein The sleeping beauty transposon vector and a transposase are co-transfected into the genetically engineered cells.

9. Use of the genetically engineered cells according to any one of claims 1-3 in in vitro expansion of NK cells, increasing the anti-tumor activity of NK cells, increasing the purity or survival time of NK cells.

10. A method for NK cell expansion, characterized in that, It includes co-culturing the genetically engineered cells according to any one of claims 1-3 with NK cells.

11. The method according to claim 10, characterized in that, It includes the following steps: 1) Chemically treating or irradiating the genetically engineered cells with rays; 2) Co-culturing the treated genetically engineered cells with NK cells in an NK cell expansion culture medium to obtain NK cells.

12. The method according to claim 11, wherein The chemical treatment is mitomycin treatment; and / or, the ray treatment is γ-ray irradiation.

13. The method according to claim 11, wherein In step 2), the treated genetically engineered cells are added to the NK cell expansion culture medium once or multiple times.

14. The method according to claim 13, wherein In step 2), the treated genetically engineered cells are added each time at a ratio of 0.5:1 to 2:1 of the genetically engineered cells to NK cells.

15. The method according to claim 13, wherein In step 2), the interval time for adding the treated genetically engineered cells multiple times is 3-9 days.

16. The method according to claim 11, wherein IL-2 is added to the NK cell expansion culture medium; and / or, the co-culturing time is 5 days to 23 days; and / or, the TIGIT receptor of the NK cells is knocked out.

17. The method according to claim 14, wherein The treated genetically engineered cells are added each time at a ratio of 1:1 of the genetically engineered cells to NK cells.

18. The method according to claim 15, wherein The interval time for adding the treated genetically engineered cells multiple times is 7 days.

19. The method according to claim 11, characterized in that, The time of co-cultivation is 7 days to 21 days.

20. Use of NK cells prepared by the method according to any one of claims 10-19 in the preparation of anti-cancer drugs.

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