CAR-NK cell for knocking out KIR2DL5 targeting HER2, preparation method and application thereof

Abstract

The invention provides a CAR-NK cell for knocking out KIR2DL5 targeting HER2 as well as a preparation method and application of the CAR-NK cell, and belongs to the technical field of genetic engineering.The CAR-NK cell is obtained by adopting a chimeric antigen receptor with HER2 as a target spot to infect an NK cell and then knocking out a KIR2DL5 gene; the chimeric antigen receptor taking the HER2 as the target spot comprises an HER2 single-chain antibody; the nucleic acid artificial sequence of the HER2 single-chain antibody is as shown in SEQ ID NO. 2. According to the application disclosed by the invention, the interaction of KIR2DL5-PVR is blocked by knocking out NK cell immune checkpoints, so that tumor immune escape is avoided, and the activity of killing tumor cells by CAR-NK cells is remarkably enhanced; according to the invention, by optimizing the nucleic acid artificial sequence of HER2, the secretion ability of CAR-NK cell factors, the tumor killing efficiency and the in-vivo inhibition effect on tumors can be improved.

Description

Technical Field

[0001] The present invention relates to a CAR-NK cell that knocks out KIR2DL5 and targets HER2, a preparation method thereof, and an application thereof, belonging to the technical field of genetic engineering. Background Art

[0002] In recent years, cell immunotherapy has become an effective means for the treatment of malignant tumors. Adoptive cell therapy represented by chimeric antigen receptor-engineered T cells (CAR-T) has made great progress in the field of tumor treatment. With the rise of CAR-T therapy, CAR-NK (Chimeric Antigen Receptor-Nature Killer Cell) therapy, which is constructed based on the design idea of CAR-T and combines the characteristics of NK cells, has also stood out. At present, a number of studies have confirmed the feasibility of CAR-NK cells in the treatment of solid tumors, mainly including ovarian cancer, breast cancer, pancreatic cancer, colon cancer, glioblastoma, hepatocellular carcinoma, etc.

[0003] The latest research has found that the novel immune checkpoint KIR2DL5 of the KIRs family can bind to the poliovirus receptor (PVR) protein overexpressed in cancer; it has also been found that there are KIR2DL5-positive immune cells in various PVR+ cancers, such as solid tumor tissues of bladder cancer, renal cancer, breast cancer, etc., indicating that the interaction between KIR2DL5 and PVR may achieve immune escape by affecting the function of immune cells in the tumor microenvironment and inhibit the function of NK cells. Therefore, blocking the KIR2DL5-PVR pathway helps to restore the activity of NK cells and significantly enhance the ability of NK cells to kill tumor cells.

[0004] The proto-oncogene human epidermal growth factor receptor 2 (Her-2), also known as HER2, is expressed in many tumors, and HER2 overexpression is one of the most common phenomena in malignant tumors, especially very common in breast cancer. The overexpression of this gene is also an important clinical treatment monitoring and prognosis index, and is an important target for the selection of tumor-targeted therapeutic drugs. At present, there are many HER2-targeted drugs, mainly including monoclonal antibody drugs (trastuzumab and pertuzumab), antibody-drug conjugates developed on monoclonal antibodies (ado-trastuzumab emtansine), small molecule tyrosine kinase inhibitor drugs (lapatinib and neratinib), etc. Although these drugs have significantly improved the prognosis of patients with HER2-positive diseases, they will produce acquired drug resistance, and the tumor will relapse and further deteriorate.

[0005] Existing literature has explained that blocking the immunosuppressive KIR2DL5 / PVR pathway can elicit effective human NK cell-mediated antitumor immunity; this literature reported that KIR2DL5 is mainly expressed on NK cells, studied the mechanism of KIR2DL5-PVR interaction, and blocked the KIR2DL5-PVR interaction by preparing a monoclonal antibody against KIR2DL5, thereby enhancing the cytotoxicity of NK tumors (Xiaoxin Ren, Mou Peng, Peng Xing, et al. Blockade of the immunosuppressive KIR2DL5 / PVR pathway elicits potent human NK cell–mediated antitumor immunity, Journal of Clinical Investigation (2022)).

[0006] The above-mentioned literature blocked the KIR2DL5-PVR interaction by preparing a monoclonal antibody against KIR2DL5 (F8B30), thereby enhancing the cytotoxicity of NK cells against tumors. However, at an E:T ratio of 5:1, the killing rate of F8B30 against tumor cell lines (A427, K562) was about 80%.

[0007] Patent CN115820697A discloses an immune cell and its preparation method and application. The sGP130-HER2-CAR-NK92 cells obtained in this patent highly express HER2 antibodies and can be used for the treatment of HER2-related tumors. Moreover, the immune cells overexpress sGP130, block the binding of IL-6 to its receptor, and improve their in vivo safety. However, the killing rate of the sGP130-HER2-CAR-NK92 cells prepared in this patent against breast cancer cell line JIMT-1 is only about 60%.

[0008] Patent CN117683113A discloses the preparation and application of HER2-targeted CAR-NK cells expressing mIL-15. This patent provides a membrane-bound IL-15, and the membrane-bound IL-15 contains an IL-15 core sequence, a hinge region, and a transmembrane region. The membrane-bound IL-15 can better bind to the cell's own IL-15 receptor, thereby activating the downstream signaling pathway of the IL-15 receptor and promoting the self-proliferation of CAR-NK cells, while avoiding the cytokine toxicity caused by secreted IL-15. However, the killing efficiency of the CAR-NK cells prepared in this patent against target cells SKBR3 and NCI-N87 is relatively low, 55% and 65% respectively.

[0009] Patent CN117801121A discloses a HER2, MAPK4-specific chimeric antigen receptor, dual CAR-T cell for treating breast cancer and its application. The HER2, MAPK4-specific chimeric antigen receptor in this patent comprises an extracellular antigen-binding domain composed of a HER2 antigen-binding domain and a MAPK4 antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. It has a significant killing effect on HER2-positive target cells and MAPK4-positive target cells, can enhance the tumor-killing effect of T cells, and can better eliminate tumor cells and reduce the antigen escape phenomenon caused by tumor heterogeneity compared with single-target CAR-, further strengthening the tumor-killing ability of CAR-T cells. However, the killing rate of this dual-target CAR-T cell against the target cell SKBR3 is at most 80%.

[0010] Currently, there are no relevant reports on the application of KIR2DL5 to CAR, nor are there reports on constructing CAR-NK cells by combining KIR2DL5 with the HER2 target for tumor treatment. Summary of the Invention

[0011] Aiming at the deficiencies of the existing technology, the present invention provides a KIR2DL5-knocked-out HER2-targeted CAR-NK cell, its preparation method and application, and realizes the following invention purposes: improving the cytokine secretion ability of CAR-NK cells, increasing the killing rate of human breast adenocarcinoma cells, and enhancing the inhibitory effect on in-vivo tumors.

[0012] To solve the above technical problems, the present invention adopts the following technical solutions: A KIR2DL5-knocked-out HER2-targeted CAR-NK cell is obtained by infecting NK cells with a chimeric antigen receptor targeting HER2 and then knocking out the KIR2DL5 gene; the chimeric antigen receptor targeting HER2 includes a HER2 single-chain antibody; the nucleic acid artificial sequence of the HER2 single-chain antibody is as shown in SEQ ID NO.2.

[0013] The chimeric antigen receptor targeting HER2 includes the following modules: CD8 signal peptide, HER2 single-chain antibody, CD8 Hinge region, CD8 transmembrane region, CD28 co-stimulatory region, 4-1BB co-stimulatory region, CD3ζ signaling region.

[0014] The artificial nucleic acid sequence of the CD8 signal peptide is shown as SEQ ID NO.1; the artificial nucleic acid sequence of the CD8 Hinge region is shown as SEQ ID NO.4; the artificial nucleic acid sequence of the CD8 transmembrane region is shown as SEQ ID NO.5; the artificial nucleic acid sequence of the CD28 co-stimulatory region is shown as SEQ ID NO.6; the artificial nucleic acid sequence of the 4-1BB co-stimulatory region is shown as SEQ ID NO.7; the artificial nucleic acid sequence of the CD3ζ signaling region is shown as SEQ ID NO.8.

[0015] The nucleotide sequence of the sgRNA of the KIR2DL5 gene is shown as SEQ ID NO.10.

[0016] The preparation method of the CAR-NK cells is as follows: a chimeric antigen receptor targeting HER2 is constructed to obtain a HER2-CAR expression vector. After being packaged by lentivirus, NK cells are infected to obtain HER2-CAR-NK cells; the sgRNA of the KIR2DL5 gene is constructed to obtain a pX330A-KIR2DL5 vector, and the pX330A-KIR2DL5 vector is used to electrotransform HER2-CAR-NK cells to prepare CAR-NK cells targeting HER2 with KIR2DL5 knocked out.

[0017] The application of the described CAR-NK cells in the preparation of drugs for treating breast cancer.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention first combines KIR2DL5 and the HER2 target to construct CAR-T cells for tumor treatment.

[0019] The present invention knocks out the NK cell immune checkpoint (KIR2DL5), blocks the interaction between KIR2DL5 and PVR, avoids tumor immune escape, and significantly enhances the activity of CAR-NK cells in killing tumor cells; by optimizing the artificial nucleic acid sequence of HER2, the present invention can improve the cytokine secretion ability, tumor killing efficiency and in vivo inhibitory effect on tumors of CAR-NK cells; the NK cells with KIR2DL5 knocked out target and bind to the HER2 antigen, having a synergistic effect, jointly improving the cytokine secretion ability and tumor cell killing efficiency of CAR-NK cells, and making them have stronger anti-tumor activity.

[0020] The CAR-NK cells targeting HER2 with KIR2DL5 knocked out prepared by the present invention have an in vitro killing rate of 97.5% against breast cancer cells SKBR3; they have an obvious inhibitory effect on in vivo tumors in a breast cancer mouse model. Description of the Drawings

[0021] Figure 1 It is a flow cytometry graph of the expression rate of CD3-CD16+CD56+ in NK cells; Figure 2 It is a flow cytometry graph of the HER2 expression rate in HER2-CAR-NK cells; Figure 3 It is a bar graph of the cytokine secretion amount of the CAR-NK cells prepared by the present invention; Figure 4 It is a bar graph of the killing rate of the CAR-NK cells prepared by the present invention against target cells; Figure 5 It is a tumor growth curve graph after the CAR-NK cells prepared by the present invention are used in a mouse tumor model. Detailed implementation manners

[0022] Example 1 Construction of expression vector 1. Construction of HER2-CAR expression vector The modules of HER2-CAR and their nucleic acid artificial sequences are as follows: (1) CD8 signal peptide, whose nucleic acid artificial sequence is shown as SEQ ID NO.1 in the sequence listing; (2) HER2 single-chain antibody, whose nucleic acid artificial sequence is shown as SEQ ID NO.2 in the sequence listing; (3) CD8 Hinge region, whose nucleic acid artificial sequence is shown as SEQ ID NO.4 in the sequence listing; (4) CD8 transmembrane region, whose nucleic acid artificial sequence is shown as SEQ ID NO.5 in the sequence listing; (5) CD28 co-stimulatory region, whose nucleic acid artificial sequence is shown as SEQ ID NO.6 in the sequence listing; (6) 4-1BB co-stimulatory region, whose nucleic acid artificial sequence is shown as SEQ ID NO.7 in the sequence listing; (7) CD3ζ signal transduction region, whose nucleic acid artificial sequence is shown as SEQ ID NO.8 in the sequence listing; Sequentially connect the nucleic acid artificial sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, entrust Sangon Biotech (Shanghai) Co., Ltd. to synthesize its entire expression cassette and insert it onto the standard vector pUC57 to obtain pUC-HER2-CAR. Transform it into E. coli (DH5α), extract plasmid DNA, and after correct sequencing, use the plasmid extraction kit of OMEGA company to extract the plasmid, named HER2-CAR, and the concentration of the plasmid is 1.0 μg / μL.

[0023] 2. Construction of CRISPR / CAS9 Knockout KIR2DL5 Expression Vector Design sgRNA of KIR2DL5 according to the website https: / / www.benchling.com / crispr, and select the sgRNA with relatively high On-Target targeting score and Off-Target off-target score from the KIR2DL5 sequence (its nucleic acid artificial sequence is shown as SEQ ID NO.9 in the sequence listing) (see Table 1 below); entrust the nucleic acid artificial sequence of the screened KIR2DL5 sgRNA to Sangon Biotech (Shanghai) Co., Ltd. for synthesis and ligate it to the CRISPR / CAS9 expression vector pX330A to obtain 3 pX330A-KIR2DL5 vectors. After correct sequencing, extract the plasmid pX330A-KIR2DL5, and name them pX330A-KIR2DL5-1, pX330A-KIR2DL5-2, and pX330A-KIR2DL5-3 respectively. The concentration of the plasmids is all 1.0 μg / μL.

[0024] Table 1 sgRNA Sequences of KIR2DL5 Gene

[0025] Example 2 Lentivirus Packaging and Titer Detection Inoculate the lentivirus packaging cell line 293T into a culture dish containing DMEM medium with 10 vol% FBS, and culture it under the conditions of 37 °C and 5% CO2. When the adherent rate reaches 70%, prepare for transfection.

[0026] Take a sterile 5 mL EP tube and prepare the reaction system according to the following components: Serum-free DMEM: 3 mL; HER2-CAR plasmid: 10 μg; GM easyTM Lentiviral Mix (lentivirus packaging auxiliary plasmid mixture): 10 μL (10 μg); HG Transgene TM Reagent (high-efficiency transfection reagent): 60 μL.

[0027] After mixing, place it at room temperature for 20 min, then evenly drop the mixture in the EP tube into the culture dish containing 293T cells, and place it in a CO2 incubator for culture. After 24 h of transfection, carefully suck out the cell culture medium and discard it into the waste liquid cup containing disinfectant, and then add 15 mL of fresh DMEM medium containing 10 vol% serum and continue to culture. After changing the medium for 48 h, aspirate the cell supernatant into a 50 mL centrifuge tube, centrifuge at 4 °C and 500 g for 5 min, filter the supernatant through a 0.45 μm filter and transfer it to a new centrifuge tube. After harvesting the virus, concentrate it to obtain concentrated HER2-CAR recombinant lentivirus, and the detected titer is 6.2×107 pfu / mL, and store it for future use in a -80°C low-temperature refrigerator.

[0028] Example 3 Preparation of HER2-CAR-NK cells (1) Preparation of NK cells Take 50 mL of the patient's autologous peripheral blood, use TBD sample density separation solution (purchased from Tianjin Haoyang Huake Biotechnology), separate to obtain PBMC (peripheral blood mononuclear cells), culture to obtain NK cells, and detect the expression rate of CD3-CD16+CD56+ in NK cells by flow cytometry (CD3-FITC, CD16 / CD56-PE antibodies are purchased from BECKMAN company, A07735). The expression rate of CD3-CD16+CD56+ in NK cells is 81.4% (as Figure 1 ).

[0029] (2) Lentivirus infection of NK cells Take out the above-prepared HER2-CAR recombinant lentivirus from -80°C, thaw it and add it to NK medium, dilute the virus titer to 5×10 7 pfu / mL, resuspend 1×10 6 NK cells with the diluted recombinant lentivirus solution to obtain a cell suspension. Add the cell suspension to a 6-well plate, 1 mL per well, so that the ratio of the number of virus particles to the number of NK cells is 50:1. After culturing in a 37°C, 5% CO2 incubator for 6 h, add 1 mL of NK medium to each well and continue culturing. In one week, the infected NK cells are obtained and named HER2-CAR-NK cells.

[0030] Use flow cytometry to detect the expression of HER2 in HER2-CAR-NK cells. In the present invention, the expression rate of HER2 in HER2-CAR-NK cells is 85.1% ( Figure 2 ).

[0031] Example 4 Preparation of CAR-NK cells with KIR2DL5 gene knockout Prepare CAR-NK cells with KIR2DL5 knockout by electrotransfecting HER2-CAR-NK cells with the pX330A-KIR2DL5 vector. The specific steps are as follows: (1) Wash the electroporation cuvette, soak it in 75% alcohol for 2 h, and then irradiate it under ultraviolet light for 15 min.

[0032] (2) Take 1×10 7 HER2-CAR-NK cells, add 500 μL of electroporation buffer to resuspend the cells, and pipette up and down evenly to obtain a HER2-CAR-NK cell suspension.

[0033] (3) Add the pX330A-KIR2DL5-1 plasmid (10 μg) to the cell suspension and pipette up and down to mix evenly.

[0034] (4) Transfer the HER2-CAR-NK cell suspension containing the pX330A-KIR2DL5-1 plasmid into an electroporation cuvette of the corresponding specification, and set the parameters according to the predetermined conditions: 300 V, 10 ms, and perform 2 electroporation operations.

[0035] (5) After the electroporation is completed, place the electroporation cuvette on ice and incubate for 10 min to allow the nucleic acid to fully enter the cells.

[0036] (6) Take out the electroporation cup from the ice, transfer the cells out of the electroporation cup, filter and count, and inoculate them in fresh DMEM medium at a certain cell density (3×10 6 / mL), and place it in an incubator at 37 °C and 5% CO2 for culture.

[0037] (7) After culturing for 48 h, the electroporation efficiency of the cells can be detected. The formula for calculating the electroporation efficiency is: ((gene expression rate before electroporation - gene expression rate after electroporation) / gene expression rate before electroporation) × 100%.

[0038] The expression rate of KIR2DL5 in HER2-CAR-NK cells before electroporation detected by flow cytometry was 50%. After electroporation with pX330A-KIR2DL5-1, the expression rate of KIR2DL5 was 5%. The electroporation efficiency = ((50% - 5%) / 50%) × 100% = 90%. That is, the efficiency of knocking out the KIR2DL5 gene by the pX330A-KIR2DL5-1 vector constructed in the present invention is 90%.

[0039] Electroporate HER2-CAR-NK cells with pX330A-KIR2DL5-2 and pX330A-KIR2DL5-3 respectively according to the above method, and calculate the electroporation efficiency (see Table 2).

[0040] As can be seen from Table 2, the electroporation efficiency of pX330A-KIR2DL5-1 is the highest. Therefore, the HER2-CAR-NK cells electroporated with pX330A-KIR2DL5-1 (named HER2-CAR-NK cells with KIR2DL5 knocked out) are used for subsequent experiments.

[0041] Table 2 Electroporation efficiency of pX330A-KIR2DL5 plasmid

[0042] Example 6 Detection of cytokine (IFN-γ, TNF-α) secretion Preparation of HER2 pre-optimization - CAR - NK cells with KIR2DL5 knocked out: Construct a HER2 pre-optimization - CAR expression vector according to the method of Example 1 of the present invention, with the following changes: the HER2 single-chain antibody uses the pre-optimized nucleic acid artificial sequence as shown in SEQ ID NO.3 in the sequence listing; prepare HER2 pre-optimization - CAR - NK cells according to the methods of Example 2 and Example 3; continue to electrotransfer HER2 pre-optimization - CAR - NK cells with pX330A - KIR2DL5 - 1 according to the method of Example 4, and HER2 pre-optimization - CAR - NK cells with KIR2DL5 knocked out can be obtained.

[0043] Use human breast adenocarcinoma cell line SKBR3 as the target cell, and use HER2 - CAR - NK cells with KIR2DL5 knocked out, HER2 - CAR - NK cells, HER2 pre-optimization - CAR - NK cells with KIR2DL5 knocked out, and NK cells as effector cells. Respectively, according to E:T = 5:1, seed the effector cells (5×10 5 cells / well) and the target cells (1×10 5 cells / well) into 96-well plates, 100 μL / well, and place them in an incubator at 37°C and 5% CO2 for co-culture. Collect the culture supernatant at 48 h of culture, and use the ELISA method to detect the secretion levels of cytokines (IFN-γ, TNF-α) respectively.

[0044] The specific grouping is as follows: Experimental group A: Co-culture HER2 - CAR - NK cells with KIR2DL5 knocked out and SKBR3 cells; Experimental group B: Co-culture HER2 - CAR - NK cells and SKBR3 cells; Experimental group C: Co-culture HER2 pre-optimization - CAR - NK cells with KIR2DL5 knocked out and SKBR3 cells; Experimental group D: Co-culture HER2 pre-optimization - CAR - NK cells and SKBR3 cells; Control group: Co-culture NK cells and SKBR3 cells.

[0045] The ELISA results (see Figure 3 and Table 3) show that: The contents of IFN-γ and TNF-α in the experimental groups are significantly higher than those in the control group, and there are significant differences. Among them, the contents of IFN-γ and TNF-α in experimental group A are the highest, 6955 pg / mL and 8522 pg / mL respectively, indicating that the CAR - NK cells constructed by the present invention have stronger cytokine secretion ability.

[0046] The IFN-γ and TNF-α contents in experimental group A were higher than those in experimental group B, indicating that knocking out KIR2DL5 could enhance the immune response of CAR-NK cells and enable them to release more cytokines; the IFN-γ and TNF-α contents in experimental group A were higher than those in experimental group C, indicating that after codon optimization of the artificial HER2 nucleic acid sequence, the cytokine secretion ability of CAR-NK cells could be improved.

[0047] Table 3 Cytokine secretion

[0048] Example 7 In vitro killing experiment of CAR-NK cells The effector cells (5×10 5 cells / well) and target cells SKBR3 (1×10 5 cells / well) were inoculated into 96-well plates, 100 μL / well, and divided into five groups. The specific grouping is as follows: Experimental group A: Co-culture of HER2-CAR-NK cells with knocked-out KIR2DL5 and SKBR3 cells; Experimental group B: Co-culture of HER2-CAR-NK cells and SKBR3 cells; Experimental group C: Co-culture of HER2-pre-optimized-CAR-NK cells with knocked-out KIR2DL5 and SKBR3 cells; Experimental group D: Co-culture of HER2-pre-optimized-CAR-NK cells and SKBR3 cells; Control group: Co-culture of NK cells and SKBR3 cells.

[0049] They were placed in a 5% CO2, 37 °C incubator for co-culture. After 24 h, 20 mL of CCK-8 was added to each well. After continued incubation for 2 h, the OD value was read at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell killing rate was calculated. The results are as Figure 4 shown in Table 4.

[0050] Table 4 Killing rate of CAR-NK cells against breast cancer cells

[0051] As Figure 4 shown in and Table 4, the killing rate of the experimental groups was significantly higher than that of the control group, showing a significant difference. Among them, the cell killing rate of experimental group A was the highest, indicating that the CAR-NK cells constructed in the present invention could produce a strong immune response, had specific cytotoxic activity against breast cancer cells, and had a strong tumor killing ability.

[0052] Compared with experimental group D, the killing rates of experimental groups B and C were significantly increased, indicating that the optimization of the HER2 nucleic acid sequence or the knockout of KIR2DL5 had a significant impact on the tumor killing effect of CAR-NK cells; Compared with experimental group D, the killing rate of experimental group A increased by 32 percentage points, indicating that the knockout of KIR2DL5 on the basis of the optimization of the HER2 nucleic acid sequence could play a synergistic role and jointly enhance the killing ability and anti-tumor activity of CAR-NK against tumor cells.

[0053] Example 8 Inhibitory effect of CAR-NK cells on tumors in vivo Male C57BL / 6J mice weighing 18 - 22 g (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were raised in an animal house (room temperature 23 ± 2 °C, humidity 50% ± 10%). Log-phase breast cancer cells SKBR3 cells were collected and diluted to 2×10 5 cells / mL with phosphate buffer (PBS) to obtain a SKBR3 cell suspension. Under sterile conditions, 0.2 mL of the SKBR3 cell suspension was inoculated into the left axilla of the mice. When a hard nodule the size of a soybean appeared under the axilla as the standard for successful modeling, it was counted as day 0, and the length and width of the tumor were measured with a vernier caliper to calculate the tumor volume (about 400 mm 3 ), tumor volume (mm3) = length × width 2 × 0.5.

[0054] The C57BL / 6J breast cancer model mice were randomly divided into 4 groups, with 5 mice in each group. The specific grouping is as follows: Experimental group A: Tail vein injection of 2×10 6 HER2-CAR-NK cells with KIR2DL5 knockout per mouse; Experimental group B: Tail vein injection of 2×10 6 HER2-CAR-NK cells per mouse; Experimental group C: Tail vein injection of 2×10 6 HER2-CAR-NK cells with KIR2DL5 knockout before optimization per mouse; Experimental group D: Tail vein injection of 2×10 6 HER2-CAR-NK cells before optimization per mouse; Control group: Tail vein injection of 2×10 6 NK cells per mouse; Tail vein injections were performed on days 0 and 7 respectively, and the tumor volume was measured on days 0, 7, 14, 21, 28, and 35, and a tumor growth curve graph was drawn with the tumor volume.

[0055] The results are as Figure 5As shown in Table 5, compared with the rapid and continuous growth of tumor volume in the control group mice, the tumor growth in the experimental group mice was slow, indicating that the CAR-NK cells in the experimental group had inhibitory effects on tumors. Among them, the inhibitory effects of CAR-NK cells in experimental group B and experimental group C on tumors were better than those in experimental group D, which indicated that after optimization of the HER2 nucleic acid sequence (experimental group B) and knockout of KIR2DL5 (experimental group C), the anti-tumor activity of CAR-NK cells and the ability to inhibit tumor growth could be improved.

[0056] In addition, compared with experimental group B and experimental group C, the inhibitory effect of the knockout of KIR2DL5-HER2-CAR-NK cells in experimental group A on tumors was the best, and it could reduce the tumor volume, which indicated that on the basis of the optimization of the HER2 nucleic acid sequence, the knockout of KIR2DL5 could play a synergistic role and jointly enhance the inhibitory effect of CAR-NK on tumors.

[0057] In summary, the HER2-CAR-NK cells with knockout of KIR2DL5 in the present invention can inhibit tumor growth, and have the best inhibitory effect on tumors.

[0058] Table 5 Changes in tumor size of mice immunized with CAR-NK cells (mm 3 )

Claims

1. A CAR-NK cell with KIR2DL5 knockout and targeting HER2, characterized in that: The CAR-NK cells are obtained by infecting NK cells with a chimeric antigen receptor targeting HER2, and then knocking out the KIR2DL5 gene; the chimeric antigen receptor targeting HER2 includes a HER2 single-chain antibody; the artificial nucleic acid sequence of the HER2 single-chain antibody is shown in SEQ ID NO.

2.

2. The CAR-NK cell with KIR2DL5 knockout and targeting HER2 according to claim 1, characterized in that: The chimeric antigen receptor targeting HER2 includes the following modules: CD8 signal peptide, HER2 single-chain antibody, CD8 Hinge region, CD8 transmembrane region, CD28 co-stimulatory region, 4-1BB co-stimulatory region, and CD3ζ signal transduction region.

3. The CAR-NK cell with KIR2DL5 knockout and HER2 targeting according to claim 2, characterized in that: The artificial nucleic acid sequence of the CD8 signal peptide is shown in SEQ ID NO.1; the artificial nucleic acid sequence of the CD8 Hinge region is shown in SEQ ID NO.4; the artificial nucleic acid sequence of the CD8 transmembrane region is shown in SEQ ID NO.5; the artificial nucleic acid sequence of the CD28 co-stimulatory region is shown in SEQ ID NO.6; the artificial nucleic acid sequence of the 4-1BB co-stimulatory region is shown in SEQ ID NO.7; and the artificial nucleic acid sequence of the CD3ζ signaling region is shown in SEQ ID NO.

8.

4. The CAR-NK cell with KIR2DL5 knockout and HER2 targeting according to claim 1, characterized in that: The nucleotide sequence of the sgRNA of the KIR2DL5 gene is shown in SEQ ID NO.

10.

5. The method for preparing the CAR-NK cell according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: constructing a chimeric antigen receptor targeting HER2 to obtain a HER2-CAR expression vector, packaging the vector with a lentivirus, and then infecting NK cells to obtain HER2-CAR-NK cells; constructing a pX330A-KIR2DL5 vector with sgRNA of the KIR2DL5 gene, and electroporating HER2-CAR-NK cells with the pX330A-KIR2DL5 vector to prepare HER2-targeting CAR-NK cells with KIR2DL5 knocked out.

6. Use of the CAR-NK cells according to any one of claims 1 to 4 in the preparation of medicines for treating breast cancer.

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