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 for knocking out KIR2DL5 and targeting HER2, a preparation method and application thereof, and belongs to the technical field of genetic engineering. Background Art

[0002] In recent years, cell immunotherapy has become an effective means of treating 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 based on the design ideas of CAR-T and combined with the characteristics of NK cells, has also emerged. At present, many 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 found that KIR2DL5, a new immune checkpoint in the KIRs family, can bind to the poliovirus receptor (PVR) protein overexpressed in cancer; it was also found that KIR2DL5-positive immune cells exist in a variety of PVR+ cancers, such as bladder cancer, kidney cancer, breast cancer and other solid tumor tissues, indicating that the interaction between KIR2DL5 and PVR may affect the function of immune cells in the tumor microenvironment to achieve immune escape and inhibit the function of NK cells. Therefore, blocking the KIR2DL5-PVR pathway helps to restore NK cell activity 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 in breast cancer. The overexpression of this gene is also an important clinical treatment monitoring and prognostic indicator, and is an important target for the selection of tumor targeted therapy drugs. At present, there are many HER2 targeted drugs, mainly including monoclonal antibody drugs (trastuzumab and pertuzumab), antibody-drug conjugates developed on monoclonal antibodies (enmetuzumab), small molecule tyrosine kinase inhibitors (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 explains that blocking the immunosuppressive KIR2DL5 / PVR pathway can induce effective human NK cell-mediated antitumor immunity; the literature reports that KIR2DL5 is mainly expressed on NK cells, studies the mechanism of KIR2DL5-PVR interaction, and blocks the KIR2DL5-PVR interaction by preparing KIR2DL5 monoclonal antibodies, thereby enhancing NK tumor cytotoxicity (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 KIR2DL5 monoclonal antibody (F8B30), thereby enhancing the cytotoxicity of NK cells against tumors. However, under the condition of E:T=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 the patent highly express HER2 antibodies and can be used to treat HER2-related tumors. Moreover, immune cells overexpress sGP130, blocking the binding of IL-6 to its receptors, improving their safety in vivo, but the killing rate of sGP130-HER2-CAR-NK92 cells prepared in the patent on breast cancer cells JIMT-1 is only about 60%.

[0008] Patent CN117683113A discloses the preparation and application of HER2-targeted CAR-NK cells expressing mIL-15. The patent provides a membrane-bound IL-15, which 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 IL-15 receptor downstream signaling pathway, promoting the self-proliferation of CAR-NK cells, and avoiding the cytokine toxicity caused by secretory IL-15. However, the CAR-NK cells prepared by this patent have low killing efficiency against target cells SKBR3 and NCI-N87, which are 55% and 65% respectively.

[0009] Patent CN117801121A discloses a HER2, MAPK4-specific chimeric antigen receptor, dual CAR-T cells and their applications for the treatment of breast cancer. The HER2, MAPK4-specific chimeric antigen receptor in this patent contains an extracellular antigen binding domain, a transmembrane domain and an intracellular signal transduction domain composed of a HER2 antigen binding domain and a MAPK4 antigen binding domain. It has a significant killing effect on HER2-positive target cells and MAPK4-positive target cells, and can improve the effect of T cells in killing tumors. Compared with single-target CAR-, it can better eliminate tumor cells, reduce the antigen escape phenomenon caused by tumor heterogeneity, and further enhance the ability of CAR-T cells to kill tumors. However, the killing rate of the dual-target CAR-T cells on the target cell SKBR3 is up to 80%.

[0010] At present, there are no reports on the application of KIR2DL5 in CAR, nor are there any reports on the combination of KIR2DL5 and HER2 targets to construct CAR-NK cells for tumor treatment. Summary of the invention

[0011] In view of the shortcomings of the prior art, the present invention provides a CAR-NK cell with KIR2DL5 knockout and HER2 targeting, a preparation method and application thereof, to achieve the following invention objectives: to improve the cytokine secretion ability of CAR-NK cells, to improve the killing rate of human breast adenocarcinoma cells, and to improve the inhibitory effect on tumors in vivo.

[0012] In order to solve the above technical problems, the present invention adopts the following technical solutions: A CAR-NK cell with KIR2DL5 knocked out and targeting HER2 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 artificial nucleic acid sequence of the HER2 single-chain antibody is 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, and CD3ζ signal transduction region.

[0014] 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 SEQID NO.7; and the artificial nucleic acid sequence of the CD3ζ signaling region is shown in SEQ ID NO.8.

[0015] The nucleotide sequence of the sgRNA of the KIR2DL5 gene is shown in 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, which is packaged by a lentivirus and then infected with NK cells 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 electroporate HER2-CAR-NK cells to prepare HER2-targeting CAR-NK cells with KIR2DL5 knocked out.

[0017] The use of the CAR-NK cells in the preparation of medicines for treating breast cancer.

[0018] Compared with the prior art, the present invention achieves the following beneficial effects: The present invention is the first to combine KIR2DL5 with HER2 targets to construct CAR-T cells for tumor treatment.

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

[0020] The KIR2DL5 knockout CAR-NK cells targeting HER2 prepared by the present invention have an in vitro killing rate of 97.5% on breast cancer cells SKBR3, and have a significant inhibitory effect on in vivo tumors in a breast cancer mouse model. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Example 1 Construction of expression vector 1. Construction of HER2-CAR expression vector The modules of HER2-CAR and their artificial nucleic acid sequences are as follows: (1) CD8 signal peptide, the artificial nucleic acid sequence of which is shown in SEQ ID NO.1 in the sequence listing; (2) HER2 single-chain antibody, whose artificial nucleic acid sequence is shown in SEQ ID NO.2 in the sequence listing; (3) CD8 Hinge region, the artificial nucleic acid sequence of which is shown in SEQ ID NO.4 in the sequence listing; (4) CD8 transmembrane region, whose artificial nucleic acid sequence is shown in SEQ ID NO.5 in the sequence list; (5) CD28 co-stimulatory region, whose artificial nucleic acid sequence is shown in SEQ ID NO.6 in the sequence list; (6) 4-1BB co-stimulatory region, whose artificial nucleic acid sequence is shown in SEQ ID NO.7 in the sequence list; (7) CD3ζ signal transduction region, whose artificial nucleic acid sequence is shown in SEQ ID NO.8 in the sequence list; The artificial nucleic acid 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 were connected in sequence, and the entire expression frame was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. and inserted into the standard vector pUC57 to obtain pUC-HER2-CAR, which was transformed into E. coli (DH5α) and the plasmid DNA was extracted. After correct sequencing, the plasmid was extracted using the plasmid extraction kit of OMEGA and named HER2-CAR. The concentration of the plasmid was 1.0 μg / μL.

[0023] 2. Construction of CRISPR / CAS9 Knockout KIR2DL5 Expression Vector KIR2DL5 sgRNA was designed according to the website https: / / www.benchling.com / crispr, and sgRNA with high On-Target and Off-Target scores was selected from the KIR2DL5 sequence (its nucleic acid artificial sequence is shown in SEQ ID NO.9 in the sequence list) (see Table 1 below); the nucleic acid artificial sequence of KIR2DL5 sgRNA obtained by screening was commissioned to Sangon Biotech (Shanghai) Co., Ltd. for synthesis and ligation into the CRISPR / CAS9 expression vector pX330A to obtain three pX330A-KIR2DL5 vectors. After correct sequencing, plasmid pX330A-KIR2DL5 was extracted and named pX330A-KIR2DL5-1, pX330A-KIR2DL5-2, and pX330A-KIR2DL5-3, respectively. The concentration of the plasmids was 1.0 μg / μL.

[0024] Table 1 sgRNA sequences of KIR2DL5 gene

[0025] Example 2 Lentivirus packaging and titer detection The lentiviral packaging cell line 293T was inoculated into a culture dish containing DMEM medium containing 10 vol% FBS and maintained at 37°C and 5% CO 2 The cells were cultured under the same conditions and ready for transfection when the adhesion rate reached 70%.

[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 (lentiviral packaging auxiliary plasmid mixture): 10 μL (10 μg); HG Transgene TM Reagent (high-efficiency transfection reagent): 60 μL.

[0027] After mixing, place at room temperature for 20 min, then drop the mixture in the EP tube evenly into the culture dish containing 293T cells and place in CO 2Culture in an incubator. 24 hours after transfection, carefully remove the cell culture medium and discard it in a waste liquid cup containing disinfectant, then add 15mL of fresh DMEM medium containing 10vol% serum to continue culturing. After 48 hours of changing the medium, aspirate the cell supernatant into a 50mL centrifuge tube, centrifuge at 4°C, 500g for 5 minutes, filter the supernatant with a 0.45μm filter and transfer it to a new centrifuge tube. After harvesting the virus, concentrate it to obtain concentrated HER2-CAR recombinant lentivirus, with a detection titer of 6.2×10 7 pfu / mL, and stored in a -80℃ refrigerator for later use.

[0028] Example 3 Preparation of HER2-CAR-NK cells (1) Preparation of NK cells 50 mL of the patient's autologous peripheral blood was collected and separated with TBD sample density separation fluid (purchased from Tianjin Haoyang Huake Biological) to obtain PBMC (peripheral blood mononuclear cells), and NK cells were cultured. Flow cytometry was used to detect the expression rate of CD3-CD16+CD56+ in NK cells (CD3-FITC, CD16 / CD56-PE antibodies were purchased from BECKMAN, A07735). The expression rate of CD3-CD16+CD56+ in NK cells was 81.4% (such as Figure 1 ).

[0029] (2) Lentivirus infection of NK cells Take out the HER2-CAR recombinant lentivirus prepared above from -80℃, thaw it, add NK culture medium, and dilute the virus titer to 5×10 7 pfu / mL, and resuspended with diluted recombinant lentivirus solution to 1×10 6 NK cells were added to obtain a cell suspension. The cell suspension was added 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 was 50:1, and the culture was carried out at 37°C and 5% CO 2 After culturing in the incubator for 6 hours, 1 mL of NK culture medium was added to each well and culture was continued. Infected NK cells were obtained in one week and named HER2-CAR-NK cells.

[0030] The expression of HER2 in HER2-CAR-NK cells was detected by flow cytometry. The expression rate of HER2 in HER2-CAR-NK cells in the present invention was 85.1% ( Figure 2 ).

[0031] Example 4 Preparation of CAR-NK cells with KIR2DL5 gene knockout HER2-CAR-NK cells were electroporated with pX330A-KIR2DL5 vector to prepare KIR2DL5 knockout CAR-NK cells. The specific steps are as follows: (1) Clean the electrophoresis cup, soak it in 75% alcohol for 2 hours, and then irradiate it under ultraviolet light for 15 minutes.

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

[0033] (3) Add 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 electric transfer cup of corresponding specifications, set the parameters according to the predetermined conditions: 300V, 10ms, and perform 2 electric shock operations.

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

[0036] (6) Take the electroporation cup out of the ice, transfer the cells out of the electroporation cup, filter and count them, and count them according to a certain cell density (3×10 6 / mL) were inoculated in fresh DMEM medium and placed at 37°C and 5% CO 2 Culture in incubator.

[0037] (7) After 48 hours of culture, the cell electroporation efficiency can be tested. The electroporation efficiency is calculated as follows: ((gene expression rate before electroporation - gene expression rate after electroporation) / gene expression rate before electroporation) × 100%.

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

[0039] According to the above method, pX330A-KIR2DL5-2 and pX330A-KIR2DL5-3 were electroporated into HER2-CAR-NK cells, and the electroporation efficiency was calculated (see Table 2).

[0040] As shown in Table 2, pX330A-KIR2DL5-1 had the highest electroporation efficiency, so HER2-CAR-NK cells electroporated with pX330A-KIR2DL5-1 (named as HER2-CAR-NK cells with KIR2DL5 knockout) were selected for subsequent experiments.

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

[0042] Example 6 Cytokine (IFN-γ, TNF-α) secretion detection Preparation of HER2-optimized pre-CAR-NK cells with KIR2DL5 knockout: According to the method of Example 1 of the present invention, a HER2 pre-optimization-CAR expression vector is constructed, with the following changes: the HER2 single-chain antibody adopts an artificial nucleic acid sequence before optimization, as shown in SEQ ID NO.3 in the sequence table; HER2 pre-optimization-CAR-NK cells are prepared according to the methods of Examples 2 and 3; and HER2 pre-optimization-CAR-NK cells are electroporated with pX330A-KIR2DL5-1 according to the method of Example 4 to obtain HER2 pre-optimization-CAR-NK cells with KIR2DL5 knocked out.

[0043] Human breast cancer cells SKBR3 were used as target cells, and HER2-CAR-NK cells with KIR2DL5 knockout, HER2-CAR-NK cells, HER2-optimized pre-CAR-NK cells with KIR2DL5 knockout, and NK cells were used as effector cells. The effector cells (5×10 5 cells / well) and target cells (1×10 5 100 μL / well) were inoculated into a 96-well plate and placed in a 37°C, 5% CO 2 The cells were co-cultured in an incubator, and the culture supernatant was collected at 48 h of culture. The secretion of cytokines (IFN-γ, TNF-α) was detected by ELSA method.

[0044] The specific groups are as follows: Experimental group A: HER2-CAR-NK cells with KIR2DL5 knockout were co-cultured with SKBR3 cells; Experimental group B: HER2-CAR-NK cells were co-cultured with SKBR3 cells; Experimental group C: HER2-optimized pre-CAR-NK cells with KIR2DL5 knockout were co-cultured with SKBR3 cells; Experimental group D: HER2-optimized pre-CAR-NK cells co-cultured with SKBR3 cells; Control group: NK cells were co-cultured with SKBR3 cells.

[0045] ELISA results (see Figure 3 and Table 3) show: The IFN-γ and TNF-α contents in the experimental group were significantly higher than those in the control group, and there were significant differences. Among them, the IFN-γ and TNF-α contents in the experimental group A were 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 levels 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 release more cytokines; the IFN-γ and TNF-α levels in experimental group A were higher than those in experimental group C, indicating that the artificial nucleic acid sequence of HER2 could improve the factor secretion capacity of CAR-NK cells after codon optimization.

[0047] Table 3 Cytokine secretion

[0048] Example 7 CAR-NK cell killing experiment in vitro Effector cells (5 × 10 5 / well) and target cells SKBR3 (1×10 5 Each well) was inoculated in a 96-well plate, 100 μL / well, and divided into five groups. The specific groups are as follows: Experimental group A: HER2-CAR-NK cells with KIR2DL5 knockout were co-cultured with SKBR3 cells; Experimental group B: HER2-CAR-NK cells were co-cultured with SKBR3 cells; Experimental group C: HER2-optimized pre-CAR-NK cells with KIR2DL5 knockout were co-cultured with SKBR3 cells; Experimental group D: HER2-optimized pre-CAR-NK cells co-cultured with SKBR3 cells; Control group: NK cells were co-cultured with SKBR3 cells.

[0049] Place in 5% CO 2 , co-cultured in a 37°C incubator. After 24 hours, 20 mL of CCK-8 was added to each well. After incubation for another 2 hours, the ELISA reader was used to detect the wavelength at 450 nm, the OD value was read, and the cell killing rate was calculated. The results are as follows. Figure 4 And as shown in Table 4.

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

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

[0052] Compared with experimental group D, the killing rates of experimental groups B and C were significantly improved, indicating that the optimization of HER2 nucleic acid sequence or the knockout of KIR2DL5 had a significant effect 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, which indicates that knocking out KIR2DL5 based on the optimization of HER2 nucleic acid sequence can 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 Weitong Lihua Experimental Animal Technology Co., Ltd.) were housed in an animal room (room temperature 23±2°C, humidity 50%±10%). SKBR3 breast cancer cells in the logarithmic phase were collected and diluted to 2×10 in phosphate buffered saline (PBS). 5 SKBR3 cell suspension was obtained. Under sterile conditions, 0.2 mL of SKBR3 cell suspension was inoculated in the left armpit of the mouse. When a hard nodule the size of a soybean grain appeared in the armpit, it was counted as day 0 as the standard for successful modeling. 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, 5 mice in each group, and the specific groups were as follows: Experimental group A: 2×10 6 HER2-CAR-NK cells with KIR2DL5 knockout / cell; Experimental group B: 2×10 6 HER2-CAR-NK cells / cell; Experimental group C: 2×10 6 HER2 optimized pre-CAR-NK cells with KIR2DL5 knockout / cell; Experimental group D: 2×10 6 HER2 pre-optimization-CAR-NK cells / cell; Control group: 2×10 6 NK cells / cell; The cells were injected into the tail vein on days 0 and 7, and the tumor volumes were measured on days 0, 7, 14, 21, 28, and 35. The tumor growth curve was plotted using the tumor volume.

[0055] The results are as follows Figure 5 As shown in Table 5, compared with the rapid and continuous growth of tumor volume in the control group mice, the tumors in the experimental group mice grew slowly, indicating that the CAR-NK cells in the experimental groups had an inhibitory effect on tumors. Among them, the inhibitory effects of CAR-NK cells in experimental groups B and C on tumors were better than those in experimental group D, indicating that the optimization of HER2 nucleic acid sequence (experimental group B) and the knockout of KIR2DL5 (experimental group C) can improve the anti-tumor activity of CAR-NK cells and the ability to inhibit tumor growth.

[0056] In addition, compared with experimental groups B and C, the KIR2DL5-HER2-CAR-NK cells in experimental group A had the best inhibitory effect on tumors and could reduce tumor volume, which indicates that knocking out KIR2DL5 based on the optimization of HER2 nucleic acid sequence can play a synergistic role and jointly enhance the inhibitory effect of CAR-NK on tumors.

[0057] In summary, the HER2-CAR-NK cells with KIR2DL5 knockout in the present invention can inhibit tumor growth, and their tumor inhibition effect is the best.

[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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