Preparation and application of TGF-beta activated CAR-NK (Chimeric Antigen Receptor-Natural Killer) cell

Abstract

The invention belongs to the technical field of tumor cell immunotherapy, and particularly relates to preparation and application of a TGF-beta activated CAR-NK cell. The preparation method disclosed by the invention comprises the following steps: constructing lentivirus expression plasmids, transforming, screening, packaging viruses, preparing virus particles, infecting cells, amplifying the cells and the like. The CD276CAR.TGFBR2-KO-T4z-NK92 cell constructed by the invention can be effectively combined with a receptor protein of TGF-beta and a CD276 antigen, the NK cell is activated by TGF-beta1 to avoid tumor escape of the CAR-NK cell caused by antigen deletion or down-regulation, and the immunosuppression effect of the TGF-beta1 is thoroughly avoided, so that a certain technical foundation is laid for a prevention and treatment technology for improving esophageal squamous cell carcinoma. The infiltration condition of the NK-92 cells in tumor tissues is evaluated through flow cytometry, and the tumor infiltration rate of the CD276CAR.TGFBR2-KO-T4z-NK92 cells is obviously higher than that of other control cells. In general, the data shows that the CD276CAR.TGFBR2-KO-T4z-NK92 cells have excellent anti-tumor activity in vivo and in vitro, and the durability of the CD276CAR.TGFBR2-KO-T4z-NK92 cells in tumors is enhanced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tumor cell immunotherapy, and particularly relates to a preparation method and application of TGF-beta activated CD276 targeted chimeric antigen receptor (CAR) NK cells. BACKGROUND

[0002] In recent years, tumor prevention and treatment methods based on immune cells have made great progress, especially the efficacy of CAR-T cell therapy in hematological malignancies has made people full of expectations for tumor immune cell therapy. Therefore, researchers have also actively followed the CAR engineering technology for the engineering modification of other types of immune cells, thereby deriving a series of new cell therapies centered on CAR technology, such as CAR-NK cells, CAR-NKT cells, CAR-macrophages, CAR-Treg cells. However, a considerable part of experimental results show that CAR-T and CAR-NK cell therapy methods have limited effect in the prevention and treatment of solid tumors. One possible reason for this is that the tumor microenvironment has a strong inhibitory effect on the relevant modified immune cells.

[0003] Transforming growth factor-beta (TGF-beta) as a cytokine can not only inhibit the activity of immune cells in the tumor microenvironment, thereby reducing the attack ability of immune cells on tumors, but also promote the growth, metastasis and invasion of tumor cells. Therefore, blocking TGF-beta signaling by targeting TGF-beta is a feasible method for tumor prevention and treatment. In order to overcome tumor immune evasion, NK cells can be designed to recognize specific tumor targets (such as CD276). CD276 is a member of the B7 family, and there are two isozymes in humans (2Ig-B7H3 and 4Ig-B7H3). It is highly expressed in esophageal cancer samples and not detected in normal esophageal cancer tissues, so it is an ideal target for immunotherapy. Studies have shown that TGF-beta not only inhibits the activity of NK cells, but also has an inhibitory effect on various CAR-NK cells, and because a single antigen is prone to mutation or deletion, leading to the inability of CAR-NK cells to recognize target cells and kill them. Some studies have introduced dominant negative TGFBR2 into CAR-NK cells or knocked out TGFBR2 in CAR-NK cells, but its defect is that it cannot activate NK cells at the same time, and it cannot resist the risk of deletion of a single antigen.

[0004] Therefore, a potential method for solving the low cure rate of esophageal squamous cell carcinoma (ESCC) is to design a CAR-NK cell that can specifically target tumor cells, resist the immunosuppressive molecule TGF-β1 in the tumor microenvironment, and be activated by TGF-β1. Even if the ESCC lacks the CD276 antigen, it can still exert an anti-tumor effect, and the crispr-cas9 knockout of the endogenous TGFBR2 in the NK cell completely avoids the negative regulation of the NK cell by TGF-β1 in the microenvironment. This design method can "kill three birds with one stone": specifically target tumor cells, reduce the probability of tumor cell immune escape, and resist the immunosuppressive effect of TGF-β1, thereby improving the effectiveness of CAR-NK cell therapy and significantly improving the treatment effect of ESCC. SUMMARY

[0005] The purpose of the present application is to overcome the deficiencies in the prior art, provide a preparation method of TGF-β activated CD276 targeted chimeric antigen receptor NK cell, and construct CD276 CAR.TGFBR2-KO-T4z-NK92 cell, which can effectively combine the receptor protein of TGF-β and the CD276 antigen, and avoid tumor escape caused by antigen deletion or down-regulation of CAR-NK cell through TGF-β1 activated NK cell, and completely avoid the immunosuppressive effect of TGF-β1, thereby laying a certain technical foundation for improving the prevention and treatment technology of ESCC.

[0006] To achieve the above-mentioned purpose, the technical scheme created by the present application is as follows: A preparation method of gene edited NK cell, comprising the following steps: (1) Construction of lentiviral expression plasmid An online tool Benchling is used to design sgRNA of TGFBR2, and a CRISPR-Cas9 vector is used to construct a recombinant plasmid crisper-cas9-TGFBR2; according to the transmembrane and extracellular sequences of TGFBR2 gene and the intracellular sequences of 4-1BB and CD3ζ, a pCDH-eGFP vector is used to construct a recombinant plasmid pCDH-T4z; (2) Transformation and selection The recombinant plasmid in step (1) is transformed into competent cells of Escherichia coli, and resistance (ampicillin resistance) screening is performed, and the recombinant plasmid is extracted and saved for later use through bacterial liquid PCR screening and sequencing verification; (3) Virus packaging and virus particle preparation 293T cells are cultured, and trypsin digestion is performed, and the digested cells are transferred and inoculated into a cell culture plate, and the plate is cultured for a certain period of time to obtain the cultured host cells; Then the lentivirus expression plasmid, packaging plasmid and buffer are mixed, added to the cell culture plate, and then transfected with transfection reagent for a certain time, and then cultured in DMEM complete medium at 35-37℃ for 24-72h, and the supernatant (virus supernatant) is collected, which is the packaged virus particles; (4) Infection of cells After the CAR-NK cells are collected and resuspended with the culture medium, they are placed in a cell culture plate. The virus supernatant prepared in step (3) is added to the transfection enhancer and added to the CAR-NK cells for infection, and the gene-edited CAR-NK cells are obtained. (5) Cell expansion The gene-edited CAR-NK cells are added to the NK92 cell culture medium containing nutritional components, mixed uniformly, and then transferred to an incubator at 35-37℃ for 24-72h. During this period, the expression of CAR and TGFBR2 is detected to evaluate the transfection efficiency.

[0007] Specifically, in step (1), the construction steps of the recombinant plasmid pCDH-T4z (pCDH-EF1A-TGFBR2-4-1BB-CD3ζ-P2A-eGFP) are as follows: a. TGFBR2 fragment amplification Polymerase chain reaction (PCR) was used to amplify the TGFBR2 truncated fragment and the intracellular 4-1BB fragment and CD3ζ fragment from the esophageal cancer cell line KYSE70 cell cDNA and pCDH-EF1-CD19-4-1BB-CD3ζ-eGFP plasmid, respectively. b. Ligation The pCDH-eGFP vector was obtained by using restriction endonuclease Nhe I and Sal I to digest the pCDH-EF1-CD19-4-1BB-CD3ζ-eGFP plasmid, and the amplified fragments in step a were ligated to obtain the recombinant plasmid pCDH-T4z (pCDH-EF1A-TGFBR2-4-1BB-CD3ζ-P2A-eGFP).

[0008] Specifically, the primers used in step a amplification are as follows: TGFBR2-forward: 5'-GGCGCCTACTCTAGAGCTAG-3'; TGFBR2BBz-reverse: 5'-TGCCCCGTTTGTAGAAGATG-3'; TGFBR2BBz-forward: 5'-CATCTTCTACAAACGGGGCA-3'; GFP-reverse: 5'-ATCCAGAGGTTGATTGTCGA-3'.

[0009] Specifically, in step (1), the nucleotide sequence of the obtained pCDH-T4z plasmid is shown in SEQ ID No. 1.

[0010] Specifically, in step (1), the construction steps of the recombinant plasmid crisper-cas9-TGFBR2 are as follows: 1) Design sgRNA (single guide RNA): determine the target editing region (exon region) of the TGFBR2 gene, and use the online tool Benchling to design sgRNA; 2) Construct CRISPR-Cas9 vector: design and construct a vector (plasmid) lentiCRISPRv2 (Addgene, #52961) with Cas9 sequence and sgRNA expression frame, and cut the vector into linearity with restriction enzyme BsmbI; 3) Primer annealing: the primers used are: F: 5'-CACCGAACGTGCGGTGGGATCGTGC-3' R: 5'-AaacGCACGATCCCACCGCACGTTC-3' 4) Ligation: ligate the product of step 2) with the product of step 3) to obtain the recombinant plasmid crisper-cas9-TGFBR2.

[0011] Specifically, in step (1), the nucleotide sequence of the obtained crisper-cas9-TGFBR2 recombinant plasmid is shown in SEQ ID No. 2.

[0012] Specifically, the sgRNA sequence is: 5'-CACCGAACGTGCGGTGGGATCGTGC-3'.

[0013] Specifically, during primer annealing, the reaction program is: 37℃ for 30min, 95℃ for 5min, and then cooling to 25℃ (5℃ / min).

[0014] Specifically, the lentivirus expression plasmid is the recombinant plasmid crisper-cas9-TGFBR2 or the recombinant plasmid pCDH-T4z.

[0015] Specifically, in step (3), the culture reaches about 90% confluence before trypsin digestion.

[0016] Specifically, in step (3), during trypsin digestion, add DMEM medium to terminate digestion after digestion at 35-37℃ for 1-3min.

[0017] Specifically, in step (3), during plating culture, 1x10 6 cells were placed in each of 6-well plates.

[0018] Specifically, in step (3), during plating culture, the culture medium was DMEM medium, and the plating culture was performed for 12-48h.

[0019] Specifically, in step (3), during preparation of virus particles, a "jetPRIME kit" was used; the packaging plasmids were pMD2.G and psPAX2, and the mass ratio of the lentiviral expression plasmid, pMD2.G, and psPAX2 was 1:(0.2-0.5):(0.6-1).

[0020] Specifically, in step (3), the transfection culture temperature was 35-37℃, and the transfection culture time was 48-72h.

[0021] Specifically, in step (4), during resuspension, the culture medium used was α-MEM (Gibco) medium.

[0022] Specifically, in step (4), the final concentration of the transfection enhancer added to the virus supernatant was 8-10 ng / ml, and polybrene (also known as hexadimethrine bromide) was preferably used.

[0023] Specifically, in step (4), 1x10 6 NK92 cells were added to 1-5ml of virus supernatant for infection.

[0024] Specifically, in step (4), during the infection process, the infection was performed by centrifugation at 30-35℃ and 600g-1000g for 1-2h.

[0025] Specifically, in step (5), the NK92 culture medium containing nutritional components was α-MEM (Gibco) medium, and preferably, the NK92 cell culture medium contained 12.5% heat-inactivated fetal bovine serum, 12.5% heat-inactivated horse serum, 0.2mM myo-inositol, 0.02mM folic acid, 0.1mM mercaptoethanol, 200IU / ml of IL-2.

[0026] Further, based on the overall inventive concept, the present application also provides a plurality of gene-edited NK cells prepared by the above method, specifically, CD276CAR-NK92 cells, CD276CAR.TGFBR2-KO-NK92 cells, or CD276CAR.TGFBR2-KO-T4z-NK92 cells.

[0027] Specifically, wherein the CD276CAR.TGFBR2-KO-T4z-NK92 cell is endogenous TGFBR2 knockout, can express a chimeric antigen receptor polypeptide, the chimeric antigen receptor polypeptide includes signal peptide, humanized single chain antibody (scFv), hinge region, transmembrane domain, cytoplasmic signaling domain 41BB or CD3 zeta chain;And still express another chimeric receptor (T4z), the chimeric receptor includes TGFBR2 extracellular and transmembrane domain, cytoplasmic signaling domain 41BB or CD3 zeta chain.

[0028] Further, based on a general inventive concept, the application also provides the use of the genetically edited NK cell in the preparation of an anti-tumor drug.

[0029] Specifically, the tumor includes solid tumors and blood tumors, and the solid tumor includes but is not limited to lung cancer, liver cancer, pancreatic cancer, breast cancer, gastric cancer, colorectal cancer, esophageal cancer, esophageal squamous cell carcinoma, etc.

[0030] Further, based on a general inventive concept, the application also provides the use of the genetically edited NK cell in the preparation of an anti-tumor drug.

[0031] Compared with the prior art, the application has the following advantages: The application evaluates the infiltration of NK-92 cells in tumor tissues by flow cytometry. The tumor infiltration rate of CD276CAR.TGFBR2-KO-T4z-NK92 cells is significantly higher than that of the remaining control cells. Overall, these data show that CD276CAR.TGFBR2-KO-T4z-NK92 cells have superior anti-tumor activity in vitro and in vivo, and the persistence of CD276CAR.TGFBR2-KO-T4z-NK92 cells in tumors is enhanced.

[0032] The application constructs CD276CAR.TGFBR2-KO-T4z-NK92 cells, which can effectively bind to TGF-β receptor protein and CD276 antigen, and activate NK cells by TGF-β1, avoiding tumor escape caused by antigen deletion or down-regulation of CAR-NK, and completely avoiding the immunosuppressive effect of TGF-β1, thereby laying a certain technical foundation for improving the prevention and treatment technology of ESCC. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The figure is a schematic diagram of the gene editing structure of the CD276CAR.TGFBR2-KO-T4z-NK92 cell of the application; Figure 2 The figure is a flow cytometry sorting efficiency diagram of CD276CAR.T4z expression; The figure is a flow cytometry sorting efficiency diagram of CD276CAR.T4z expression;Figure 3 Figure for flow cytometry detection of CD276 expression efficiency in esophageal squamous cell line; Figure 4 Results of TGF-β1 inhibiting the in vitro tumor cell killing ability of CD276 CAR-NK92 cells; Figure 5 Results of CD276 CAR.TGFBR2-KO-T4z-NK92 cells resisting activation of TGF-β1 downstream signaling pathway; Figure 6 Results of enhanced in vitro tumor cell killing ability of CD276 CAR.TGFBR2-KO-T4z-NK92 cells; Figure 7 Results of enhanced cytotoxic granule expression of CD276 CAR.TGFBR2-KO-T4z-NK92 cells; Figure 8 Results of enhanced anti-tumor ability of CD276 CAR.TGFBR2-KO-T4z-NK92 cells in vivo in nude mice. DETAILED DESCRIPTION

[0034] The present application will be further described in conjunction with the following examples, but the embodiments of the present application are not limited thereto. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the art. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as understood by those skilled in the art. In addition, any method and material similar or equivalent to those described can be used in the present application.

[0035] Biological materials, experimental reagents, culture medium 1. Biological materials: Escherichia coli competent cells Stabl3 (TransGen Biotech, CD521); human kidney epithelial cell line 293T cells, esophageal cancer cell lines KYSE150, KYSE70, TE7 cells are from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0036] 2. Vector plasmid: CRISPR-Cas9 (Addgene, #52961), pCDH-EF1α-CD276 (the method in Chinese patent CN118620844A is referred to for obtaining).

[0037] 3. Culture medium: NK-92 cells were cultured in a-MEM (Gibco) supplemented with 12.5% horse serum (Gibco), 12.5% bovine serum (Gibco), 200 U / mL IL-2 (Beijing Sihuan Biopharmaceutical Co., Ltd.), 0.02 mM folic acid (Sigma-Aldrich), 0.2 mM myo-inositol (Sigma-Aldrich), and 0.1 mM mercaptoethanol (Sigma-Aldrich).

[0038] DMEM medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 units / mL penicillin, and 100 μg / mL streptomycin.

[0039] Example 1 Example 1 uses CRISPR-Cas9 technology to edit the TGFBR2 gene, providing a method for preparing CD276CAR.TGFBR2-KO-NK92 cells, and the specific steps are as follows: 1) Design sgRNA (single guide RNA) Determine the target editing region (exon region) of the TGFBR2 gene (Gene ID: 7048), and use the online tool Benchling to design sgRNA (the specific sequence is: 5'-CACCGAACGTGCGGTGGGATCGTGC-3').

[0040] 2) Construct CRISPR-Cas9 vector Design and construct a vector (plasmid) lentiCRISPRv2 (Addgene, #52961) with a Cas9 sequence and sgRNA expression frame, and cut the vector into a linear form with restriction enzyme BsmbI; 3) Primer annealing (the specific reaction program is: 37°C for 30 min, 95°C for 5 min, and then cool to 25°C (5°C / min)); The primers used are: F: 5'-CACCGAACGT RGCGGTGGGATCGTGC-3' R: 5'-AaacGCACGATCCCACCGCACGTTC-3' 4) Ligation Ligate the product of step 2) with the product of step 3) (the ligation conditions are: 25°C, 60 min) to obtain the crisper-cas9-TGFBR2 recombinant plasmid; The nucleotide sequence of the obtained crisper-cas9-TGFBR2 recombinant plasmid is shown in SEQ ID No. 2.

[0041] 5) Transformation, screening The ligation product in step 4) is transformed into Stabl3 competent cells, and resistance (ampicillin resistance (100 μg / ml)) screening, and screening and sequencing verification by bacterial liquid PCR are carried out to ensure that the plasmid recombination construction is correct, and the recombinant plasmid (crisper-cas9TGFBR2) with correct recombination is extracted and saved for future use.

[0042] 6) Virus packaging and virus particle preparation Taking human embryonic kidney cell line 293T cells as an example, the specific virus packaging and virus particle preparation process is as follows: First, the 293T cells are pre-cultured to about 90% confluence, then washed twice with PBS, then trypsin digestion (37°C for 3 min) is used, then DMEM medium is added to terminate the reaction, and then the cells are plated and cultured in DMEM medium for 24 hours; when plating, taking a 6-well plate as an example, 1×10 6 cells per well are plated; Then, according to the instructions of the "jetPRIME kit", 200 μl of buffer, 5 μl of jetPRIME transfection solution, crisper-cas9-TGFBR2 recombinant plasmid (1 μg) and packaging plasmid (pMD2.G (0.4 μg) (Addgene, #12259) and psPAX2 (0.8 μg) (Addgene, #12260) are added to the well plate (with the above 6-well plate as the reference ratio), gently shake to mix evenly, and then incubate in the incubator (37°C, 5% CO2) for 6 hours, replace the culture medium with DMEM complete medium (2 ml per well); continue to culture for 48 hours, and take the supernatant (virus supernatant), which is the packaged virus particles.

[0043] 7) Infection of cells The CD276 CAR-NK92 cells (obtained by referring to the method in Chinese patent CN118620844A) are collected and resuspended in NK92 culture medium α-MEM (5×10 5 / ml), and then added to a 6-well plate (2 ml per well); at the same time, the virus supernatant prepared in step 6) is added with polybrene (polybrene, also known as hexadimethrine bromide) to a final concentration of 10 ng / ml; 1×10 6 CD276 CAR-NK92 cells are added to 2 ml of virus supernatant, and the adjusted virus supernatant is added to the 6-well plate with CD276 CAR-NK92 cells, mixed evenly, and then centrifuged at 32°C and 800g for 2h to infect, obtaining infected cells.

[0044] 8) Cell expansion The infected cells were added to the α-MEM medium and resuspended with the α-MEM medium (5x10 5 / ml), and were added to a 6-well plate (2 ml per well); at the same time, the virus supernatant prepared in step (4) was added with polybrene (also known as hexadimethrine bromide) to a final concentration of 10 ng / ml; 1x10 6 CD276CAR.TGFBR2-KO-NK92 cells were added to 2 ml of the virus supernatant, and the virus supernatant after the above adjustment was added to the 6-well plate with the CD276CAR.TGFBR2-KO-NK92 cells, mixed uniformly, and then centrifuged at 32°C and 800g for 2 h to perform infection, thereby obtaining the infected cells.

[0045] Example 2 Example 2 provides a preparation method of TGF-β-activated CAR-NK cells, comprising the following steps: (I) Construction of lentiviral expression plasmid (1) Construction of T4z plasmid KYSE70 cells were collected, RNA was extracted and reverse transcribed into cDNA, and polymerase chain reaction (PCR) was used to amplify the TGFBR2 truncated fragment and the intracellular 4-1BB fragment and CD3 zeta fragment in the KYSE70 cell cDNA and pCDH-EF1-CD19-4-1BB-CD3 zeta-eGFP plasmid (obtained by referring to the method in Chinese patent CN114921497A), respectively. The primers used in amplification are as follows: TGFBR2-forward: 5'-GGCGCCTACTCTAGAGCTAG-3'; TGFBR2BBz-reverse: 5'-TGCCCCGTTTGTAGAAGATG-3'; TGFBR2BBz-forward: 5'-CATCTTCTACAAACGGGGCA-3'; GFP-reverse: 5'-ATCCAGAGGTTGATTGTCGA-3'; (2) Ligation The pCDH-eGFP vector was obtained by using restriction endonuclease Nhe I and Sal I to digest the pCDH-EF1-CD19-4-1BB-CD3zeta-eGFP plasmid, and then connecting the amplified fragment in step (1) (the connection condition is 50°C, 60 min) to obtain the recombinant plasmid: pCDH-TGFBR2-4-1BB-CD3zeta (the specific name is pCDH-EF1A-TGFBR2-4-1BB-CD3zeta-P2A-eGFP). The nucleotide sequence of the obtained pCDH-TGFBR2-4-1BB-CD3zeta plasmid is shown in SEQ ID No. 1.

[0046] (3) Transformation, screening The connection product in step (2) was transformed into Stabl3 competent cells, and resistance (ampicillin resistance (100 μg / ml)) screening, and liquid bacterial PCR screening and sequencing verification were performed to ensure that the plasmid recombination construction was correct, and the correctly recombined recombinant plasmid was extracted and saved for later use.

[0047] (4) Virus packaging and virus particle preparation Taking the human embryonic kidney cell line 293T cells as an example, the specific virus packaging and virus particle preparation process is as follows: First, the 293T cells were pre-cultured to about 90% confluence, then washed twice with PBS, then added trypsin for digestion (37°C for 3 min) and then added DMEM medium to terminate, and then plated and cultured in DMEM medium for 24 hours; when plating, taking a 6-well plate as an example, 1×10 6 cells were plated per well; Then, according to the instructions of the “jetPRIME kit”, 200 μl of buffer, 5 μl of jetPRIME transfection solution, T4z (pCDH-TGFBR2-4-1BB-CD3zeta) recombinant plasmid (1 μg) and packaging plasmid (pMD2.G (0.4 μg) (Addgene, #12259) and psPAX2 (0.8 μg)) (Addgene, #12260) were added to the well plate (taking the aforementioned 6-well plate as an example), gently shaken to mix evenly, and then cultured in an incubator (37°C, 5% CO2) for 6 hours, and the culture medium was replaced with DMEM complete medium (2 ml per well); continue to culture for 48 hours, and take the supernatant (virus supernatant), which is the packaged virus particles.

[0048] (II) Infection, cell expansion (5) Infection of cells The CD276CAR.TGFBR2-KO-NK92 cells were collected and resuspended with a-MEM medium (5×10 5 / ml) into 6-well plates (2 ml per well); at the same time, the virus supernatant prepared in step (4) was added with polybrene (also known as hexadimethrine bromide) to a final concentration of 10 ng / ml; 1x10 6 (6) Cell expansion The infected cells were added with α-MEM medium, mixed uniformly, and then transferred into an incubator (37°C, 5% CO2) for further culture; during the culture, fresh NK92 cell culture medium was replaced every 24 h; after 48 h of culture, flow cytometry was used to detect the expression of CAR and TGFBR2 to evaluate the transfection efficiency; then, flow cytometry sorting was used for further purification and continued culture.

[0049] Experimental results (I) Construction idea of lentiviral expression plasmid Figure 1 The CD276CAR and TGFBR2-4-1BB-CD3z structure are illustrated in the following figure, Figure 2 The flow cytometry detection of CD276CAR, CD276CAR-NK92 cell knockout of TGFBR2, and the efficiency of transfection of T4z based on the knockout of TGFBR2 are shown in the following figure. The results show that the positive rate of CD276CAR expression can reach more than 90%, and the TGFBR2 on the surface is successfully knocked out, with a TGFBR2 expression of only 0.82%, and the T4z plasmid is further successfully transfected, with a CD276CAR positive and T4z double positive rate of 81%, which can be used for the detection of the next step of the experiment.

[0050] The CD276CAR plasmid involved is prior art (the specific name is pCDH-EF1A-MYC-CD276-P2A-eGFP, which can be referred to in documents such as Chinese patent CN118620844A), and the specific nucleotide sequence is as follows: ​

[0051] (ii) Detection of expression of CD276 CAR, T4z and KOTGFBR2 The detection method is specifically as follows: Add 1 μL of protein L antibody (which can mark the CD276 CAR structure) and 1 μL of TGFBR2 antibody (BioLegend) to the cell suspension, respectively, and incubate at 4°C in the dark for 20 minutes. The expression levels of protein L and TGFBR2 are detected by flow cytometry.

[0052] Figure 3 Flow cytometry was used to detect the expression of CD276 in different esophageal squamous cell carcinoma cell lines. The results are shown in Figure 3 The expression levels of CD276 in KYSE150 cells and KYSE70 cells are relatively high, and they can be used as target cells. The expression level of CD276 in TE7 cells is relatively low, and it can be used as a control target cell.

[0053] (iii) Inhibition of the in vitro killing ability of CD276 CAR-NK92 cells by TGF-β1 Figure 4 It is shown that when different concentrations of TGF-β1 are added exogenously, the anti-tumor effect of CD276 CAR-NK92 cells on KYSE150 cells is inhibited.

[0054] (iv) Resistance of CD276CAR.TGFBR2-KO-T4z-NK92 cells to activation of TGF-β1 downstream signaling pathway Studies have shown that TGF-βDNR (TGF-β dominant negative receptor) can block TGF-β1-mediated signaling in CAR-NK cells (see literature Chaudhry K, Geiger A, Dowlati E, Lang H, Sohai DK, Hwang EI, et al. Co-transducing B7H3 CAR-NK cells with the DNR preserves their cytolytic function against GBM in the presence of exogenous TGF-beta. Mol Ther Methods Clin Dev. 2022;27:415-30.). Next, the present application evaluates whether the modification of TGFBR2-KO-T4z can block TGF-β1-mediated signaling in NK-92 cells. As shown in Figure 5As shown, CD276CAR-NK92 cells, CD276CAR.TGFBR2-KO-NK92 cells and CD276CAR.TGFBR2-KO-T4z-NK92 cells were treated with recombinant TGF-βΙ (10 ng / ml) (BioLegend) for 6 hours. Western blot analysis showed that there was strong phosphorylation of Smad2 in control CD276CAR-NK92 cells, while there was no phosphorylation of Smad2 in CD276CAR.TGFBR2-KO-NK92 cells and CD276CAR.TGFBR2-KO-T4z-NK92 cells after treatment with TGF-βΙ. These data demonstrate that either knocking out TGFBR2 in CD276CAR-NK92 cells or further expressing chimeric receptor T4z on the basis of knock-out blocks TGF-βΙ -induced intracellular inhibitory signals.

[0055] (V) Detection of tumor cell apoptosis After exogenous application and non-application of TGF-βΙ (10 ng / ml) to treat CD276CAR.TGFBR2-KO-T4z-NK92 cells and control cells (CD276CAR-NK92, CD276CAR.TGFBR2-KO-NK92) for 24 h, flow cytometry was used to analyze the cytotoxicity of CD276CAR.TGFBR2-KO-T4z-NK92 cells. Tumor cells were labeled with Cell Proliferation Dye eFluor 670, and then incubated with different types of NK92 cells (CD276CAR-NK92, CD276CAR.TGFBR2-KO-NK92, CD276CAR.TGFBR2-KO-T4z-NK92) as effector cells in 96-well low-adhesion plates at different effector-to-target ratios (5:1 and 1:1) at 37°C in a 5% carbon dioxide incubator for 6 hours. The cells were collected, the cell pellets were resuspended with PBS, 1 μΐ of Propidium (iodinated propyl, Sigma) was added to the cell suspension before being loaded onto the machine, and all samples were analyzed using a FACSCanto II flow cytometer, and the data were analyzed using FlowJo software. TM 670, and then incubated with different types of NK92 cells (CD276CAR-NK92, CD276CAR.TGFBR2-KO-NK92, CD276CAR.TGFBR2-KO-T4z-NK92) as effector cells in 96-well low-adhesion plates at different effector-to-target ratios (5:1 and 1:1) at 37°C in a 5% carbon dioxide incubator for 6 hours. The cells were collected, the cell pellets were resuspended with PBS, 1 μΐ of Propidium (iodinated propyl, Sigma) was added to the cell suspension before being loaded onto the machine, and all samples were analyzed using a FACSCanto II flow cytometer, and the data were analyzed using FlowJo software.

[0056] Figure 6 For flow cytometry detection of the killing efficiency of CD276CAR-NK92 cells, CD276CAR.TGFBR2-KO-NK92 cells and CD276CAR.TGFBR2-KO-T4z-NK92 cells on target cells, the results were as shown in FIG. 6. Figure 6As shown, it can be seen that CD276CAR.TGFBR2-KO-T4z-NK92 cells have the strongest effect on killing KYSE150 cells and KYSE70 cells, and the difference is more obvious when the effector-to-target ratio is 5:1. Moreover, the killing effect is not inhibited after TGF-β1 treatment, although the anti-tumor effect of CD276CAR.TGFBR2-KO-NK92 cells is also not inhibited by TGF-β1, but the killing efficiency of CD276CAR.TGFBR2-KO-NK92 cells is lower than that of CD276CAR.TGFBR2-KO-T4z-NK92 cells in both CD276 high expression (KYSE150 cells and KYSE70 cells) and low expression (TE7 cells) ESCC cell lines. Assuming that CD276 mutates or de-targets on tumor cells, the inventors knocked out CD276 in KYSE150 and KYSE70 using crisper-cas9 technology, and also found that CD276CAR.TGFBR2-KO-T4z-NK92 cells had better tumor cell clearance ability. Moreover, CD276CAR.TGFBR2-KO-T4z-NK92 cells secrete more IFN-γ when co-cultured with KYSE150, KYSE70, and TE7 cells, which also proves their strong anti-tumor effect, indicating that TGFBR2-KO-T4z gene editing is important for the anti-tumor function of CD276CAR-NK cells.

[0057] (VI) CD276CAR.TGFBR2-KO-T4z-NK92 cell degranulation detection NK cells kill tumor cells mainly by releasing cytotoxic molecules and degranulating, such as CD107A, perforin, etc. Therefore, the inventors detected the expression of CD107A and perforin in CD276CAR.TGFBR2-KO-T4z-NK92 cells after TGF-β1 (10 ng / ml) treatment. The levels of CD107A and perforin in cells were detected by flow cytometry. As shown, Figure 7 whether treated with TGF-β1 (10 ng / ml) or not, CD276CAR.TGFBR2-KO-T4z-NK92 cells release more cytotoxic molecules and particles than CD276CAR-NK92 cells and CD276CAR.TGFBR2-KO-NK92 cells, and the results also show that CD276CAR.TGFBR2-KO-T4z-NK92 cells are resistant to TGF-β1-mediated inhibition of CD276CAR-NK92 cell degranulation, and have superior anti-tumor efficacy.

[0058] (VII) Enhanced anti-tumor ability of CD276CAR.TGFBR2-KO-T4z-NK92 cells in vivo To directly evaluate the anti-tumor effect of CD276CAR.TGFBR2-KO-T4z-NK92 cells in vivo, KYSE150 cells were subcutaneously inoculated into nude mice (4-5 weeks, female mice) (Beijing Sbiopharm Biotechnology Co., Ltd.) to establish a human ESCC xenograft model. The mice were randomly divided into five groups and intravenously injected with PBS, NK-92-vector cells, CD276CAR-NK92 cells, CD276CAR.TGFBR2-KO-NK92 cells, and CD276CAR.TGFBR2-KO-T4z-NK92 cells twice a week for two consecutive weeks. In mice that were back-transfused with PBS or control NK-92-vector cells, CD276CAR-NK92 cells, and CD276CAR.TGFBR2-KO-NK92 cells through the tail vein, tumor progression was rapid, but back-transfusion of CD276CAR.TGFBR2-KO-T4z-NK92 cells resulted in a significant delay in tumor progression. In addition, there was no significant difference in the body weight of the five groups of mice, suggesting that back-transfusion of NK-92 gene edited cells is safe. CD276CAR.TGFBR2-KO-T4z-NK92 cell therapy significantly reduced the tumor size of KYSE150 cell-bearing mice. Finally, the infiltration of NK-92 cells in tumor tissue was evaluated by flow cytometry. The tumor infiltration rate of CD276CAR.TGFBR2-KO-T4z-NK92 cells was significantly higher than that of the remaining control cells. Overall, these data indicate that CD276CAR.TGFBR2-KO-T4z-NK92 cells have superior anti-tumor activity in vivo, and the persistence of CD276CAR.TGFBR2-KO-T4z-NK92 cells in tumors is enhanced.

[0059] Based on the above experimental results, it can be seen that CD276CAR.TGFBR2-KO-T4z-NK92 cells have the strongest anti-tumor effect and are not inhibited by exogenous TGF-β1. The present application is optimizing the currently reported CD276CAR, and the optimized CD276CAR.TGFBR2-KO-T4z structure has been packaged with lentivirus and NK92 cells have been infected, and CD276CAR.TGFBR2-KO-T4z-NK92 cells constructed by crispr-cas9 technology can effectively kill and control tumors.

Claims

1. A method of making a gene edited NK cell, comprising, Comprising the following steps: (1) Construction of lentiviral expression plasmid The sgRNA of TGFBR2 is designed using the online tool Benchling, and the recombinant plasmid crisper-cas9-TGFBR2 is constructed using the CRISPR-Cas9 vector; according to the transmembrane and extracellular sequences of TGFBR2 gene and the intracellular sequences of 4-1BB and CD3 zeta, the recombinant plasmid pCDH-T4z is constructed using the pCDH-eGFP vector; (2) Transformation and selection The recombinant plasmid in step (1) is transformed into E. coli competent cells, and resistance screening is performed. The recombinant plasmid is extracted and preserved for standby use through bacterial liquid PCR screening and sequencing verification; (3) Virus packaging and virus particle preparation Cultivate 293T cells, and digest them with trypsin. Transfer and inoculate the digested cells into a cell culture plate, and plate culture for a certain period of time to obtain the cultured host cells; Then mix the lentiviral expression plasmid, packaging plasmid and buffer, and add them to the cell culture plate. Then add the transfection reagent to transfect and culture for a certain period of time. Then use DMEM complete culture medium to culture at 35-37°C for 24-72 hours. Take the virus supernatant, which is the packaged virus particle; (4) Infection of cells Collect the CAR-NK cells, resuspend them with culture medium, and place them in a cell culture plate. Add the virus supernatant prepared in step (3) to the transfection enhancer and add it to the CAR-NK cells for infection to obtain the gene edited CAR-NK cells; (5) Cell expansion Add the gene edited CAR-NK cells to the NK92 cell culture medium containing nutritional components, mix uniformly, and transfer to a culture box at 35-37°C for 24-72 hours. During this period, detect the expression of CAR and TGFBR2 to evaluate the transfection efficiency.

2. The production method according to claim 1, wherein In step (1), the construction steps of the recombinant plasmid pCDH-T4z are as follows: a. TGFBR2 fragment amplification Polymerase chain reaction is used to amplify the TGFBR2 truncated fragment and the intracellular 4-1BB fragment and CD3 zeta fragment from the esophageal cancer cell line cell cDNA and pCDH-EF1-CD19-4-1BB-CD3 zeta-eGFP plasmid, respectively; b. Ligation Use restriction endonuclease Nhe I and Sal I to digest the pCDH-EF1-CD19-4-1BB-CD3 zeta-eGFP plasmid to obtain the pCDH-eGFP vector. Connect the amplified fragments in step a to obtain the recombinant plasmid pCDH-T4z; The primers used in step a amplification are as follows: TGFBR2-forward: 5'-GGCGCCTACTCTAGAGCTAG-3'; TGFBR2BBz-reverse: 5'-TGCCCCGTTTGTAGAAGATG-3'; TGFBR2BBz-forward: 5'-CATCTTCTACAAACGGGGCA-3'; GFP-reverse: 5'-ATCCAGAGGTTGATTGTCGA-3'; The nucleotide sequence of the obtained pCDH-T4z plasmid is shown as SEQ ID No.

1.

3. The production method according to claim 1, wherein In step (1), the construction steps of the recombinant plasmid crisper-cas9-TGFBR2 are as follows: 1) Design sgRNA: determine the target editing region of the TGFBR2 gene, and use the online tool Benchling to design sgRNA; 2) Construct CRISPR-Cas9 vector: design and construct a vector lentiCRISPRv2 with a Cas9 sequence and an sgRNA expression frame, and linearize the vector with restriction endonuclease BsmbI; 3) Primer annealing: the primers used are: F: 5'-CACCGAACGTGCGGTGGGATCGTGC-3' R: 5'-AaacGCACGATCCCACCGCACGTTC-3' 4) Ligation: ligate the product of step 2) with the product of step 3) to obtain the recombinant plasmid crisper-cas9-TGFBR2; The sgRNA sequence is: 5'-CACCGAACGTGCGGTGGGATCGTGC-3'; The nucleotide sequence of the obtained crisper-cas9-TGFBR2 recombinant plasmid is shown as SEQ ID No.

2.

4. The production method according to claim 1, wherein In step (3), the cells are cultured to a confluence of about 90% before trypsin digestion; In step (3), when trypsin digestion is performed, DMEM medium is added to terminate digestion after 1-3 min of digestion at 35-37°C; In step (3), when plating and culturing, the culture medium is DMEM medium, and the plating and culturing time is 12-48 h.

5. The production method according to claim 1, wherein In step (3), when preparing virus particles, the packaging plasmids are pMD2.G and psPAX2; the mass ratio of the lentiviral expression plasmid, pMD2.G, and psPAX2 is 1:(0.2-0.5):(0.6-1).

6. The production method according to claim 1, wherein In step (3), the transfection culture temperature is 35-37°C, and the transfection culture time is 48-72 h.

7. The production method according to claim 1, wherein In step (4), the final concentration of the transfection enhancer added to the virus supernatant is 8-10 ng / ml; In step (4), 1 x 10 6 NK92 cells were added to 1-5 ml of viral supernatant for infection. In step (4), during the infection process, the infection is performed by centrifugation at 30-35°C and 600g-1000g for 1-2 h.

8. The gene edited NK cells prepared by the method of any one of claims 1-7, characterized in that, Specifically, the CD276CAR-NK92 cell, the CD276CAR.TGFBR2-KO-NK92 cell, or the CD276CAR.TGFBR2-KO-T4z-NK92 cell.

9. The use of the genetically edited NK cell of claim 8 in the preparation of an anti-tumor drug.

10. The use of the genetically edited NK cell of claim 8 in the preparation of a drug for inhibiting the growth of esophageal cancer human xenograft tumor models.

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