Coding gene, protein, NKG2DL-FAP double-target CAR-NK cell for expressing IL-15 and construction method and application of NKG2DL-FAP double-target CAR-NK cell

Abstract

The invention discloses a coding gene, a protein, an NKG2DL-FAP double-target CAR-NK cell for expressing IL-15 as well as a construction method and application of the NKG2DL-FAP double-target CAR-NK cell. The nucleotide sequence of the coding gene is as shown in SEQ NO1. On one hand, compared with a single target spot, tumor immune escape can be prevented, and specific recognition and killing of tumor cells are enhanced; on the other hand, the function stimulating factor IL-15 is secreted, so that enough killing activity can be kept in the target cell clearing process, and the lasting function effect is achieved. The cell therapy is expected to promote a huge breakthrough in the field of tumor cell therapy.

Description

Technical field:

[0001] The present invention belongs to the field of biomedicine, and specifically relates to NKG2DL-FAP dual-target CAR-NK cells encoding genes, proteins, and expressing IL-15, as well as construction methods and uses thereof. Background technology:

[0002] In recent years, chimeric antigen receptor T-cell immunotherapy (CAR-T) has become the most promising tumor cell immunotherapy. The CAR structure generally consists of four parts: antigen binding domain, hinge region, transmembrane region, and intracellular signal transduction domain. Through genetic engineering technology, the four parts are fused in series and expressed on the T cell membrane in sequence, thereby giving T cells targeted killing ability. Although CAR-T cells have achieved remarkable results in hematological tumors, there are many problems that cannot be ignored, including: cytokine storm, graft-versus-host disease, high preparation cost, long preparation cycle, etc. More importantly, CAR-T cells have poor efficacy for solid tumors, and the immunosuppression of the tumor microenvironment makes it difficult for T cells to exert the ideal killing effect. It is very necessary to find a safe and effective cell replacement therapy for the treatment of solid tumors.

[0003] Natural killer (NK) cells are an important component of innate immunity, and they mainly exert cell killing effects by releasing perforin, granzymes, and secreting cytokines such as IFN-γ and TNF-α. NK cells can exert cell killing effects without antigen sensitization. Due to the toxic side effects and high costs of CAR-T cells, CAR-NK cell therapy has gradually emerged. CAR-NK expresses CAR molecules on the surface of NK cells to recognize target antigens and then kill target cells. Compared with CAR-T therapy, CAR-NK cells have unique advantages, including: ① CAR-NK cells do not secrete inflammatory factors such as IL-1 and IL-6 that cause cytokine storms, reducing toxic side effects such as cytokine storms and neurotoxicity; ② CAR-NK cells have multiple killing mechanisms that can eliminate tumor cells with low or no expression of target antigens, and reduce the recurrence of tumors with target antigen-negative cells; ③ Allogeneic transplanted CAR-NK cells will not cause graft-versus-host disease; ④ NK cells have multiple sources to meet the needs of patients whose own cells are affected by multiple chemotherapy; ⑤ CAR-NK cells can provide timely "spot" treatment.

[0004] Natural killer cell family 2 member D (NKG2D) is one of the most important activating receptors of NK cells. It transmits activation signals by recognizing NKG2D ligands (NKG2DL) on the surface of target cells, and activates NK cells to kill target cells. NKG2DL is located at different positions on chromosome 6, and there are 8 members, including: MICA, MICB, ULBP1-6. NKG2DL is not expressed or expressed in very low amounts in normal cells, but in cases of tumors, infections, and autoimmune diseases, the expression of ligands increases significantly. In recent years, NKG2D / NKG2DL has become a new target for tumor immunotherapy. The drugs under development are mainly CAR-T cells, concentrated in the early clinical stage, and the indications are mainly hematological tumors. In solid tumors, there are problems such as poor infiltration and activation of tumor sites, and the efficacy is poor.

[0005] Recent studies have shown that tumor-associated fibroblasts and extracellular matrix form a physical barrier for T cells to enter the tumor microenvironment, preventing T cells from killing solid tumors. At the same time, these cells also inhibit the killing function of T cells. Fibroblast activation protein (FAP) is a surface antigen expressed by activated fibroblasts in the tumor stroma, which plays an important role in the occurrence, development and metastasis of tumors. Under physiological conditions, FAP is expressed at a low level in most adult tissues. In the tumor environment, FAP is elevated to varying degrees in a variety of tumor cells, with the highest median being pancreatic cancer and breast cancer. In most epithelial cancers, FAP is highly expressed in cancer-associated fibroblasts in the tumor stroma. It can be seen that FAP can be expressed on the surface of tumor cells and is also widely present in fibroblasts in the tumor microenvironment. When CAR cells target and kill these tumor cells and fibroblasts expressing FAP, it is expected to achieve infiltration into the microenvironment and enhance the ability to attack tumor cells, and have more effective tumor killing ability.

[0006] As a member of the chemokine family, IL-15 has been found in recent years to stimulate NK cell proliferation and activation and enhance the ability to clear target cells. Both soluble recombinant IL-15 and IL-15 agonists have shown significant anti-tumor effects in preclinical mouse models. This patent constructs CAR-NK cells that can secrete the functional stimulatory factor IL-15, enhancing its anti-tumor ability. At the same time, this patent also provides a new transmembrane structure NCR2TM and a new co-stimulatory molecule structure CD226 to further enhance the targeted killing performance of CAR-NK cells.

[0007] Constructing a CAR-NK cell with excellent safety and effectiveness, which can accurately target specific targets of tumor cells, enhance microenvironment infiltration and strengthen tumor killing activity, is crucial for conquering solid tumors. The present invention will develop dual-target CAR-NK cells that express IL-15 and target FAP and NKG2DL, and provide new structural sequences of transmembrane NCR2TM and co-stimulatory molecule CD226. On the one hand, compared with single targets, it helps to prevent tumor immune escape and enhance specific recognition and killing of tumor cells; on the other hand, by secreting the functional stimulating factor IL-15, the process of clearing target cells can maintain sufficient killing activity and exert lasting functional effects. This cell therapy is expected to promote a huge breakthrough in the field of tumor cell therapy. Summary of the invention:

[0008] The purpose of the present invention is to provide a NKG2DL-FAP dual-target CAR-NK cell encoding gene, protein, and expressing IL-15, as well as a construction method and use thereof.

[0009] To solve the above problems, the technical solution of the present invention is:

[0010] A coding gene, the nucleotide sequence of which is shown in SEQ NO 1.

[0011] A protein, the amino acid sequence of the protein is shown as SEQ NO 2.

[0012] A NKG2DL-FAP dual-target CAR-NK cell expressing IL-15, wherein the NKG2DL-FAP dual-target CAR-NK cell contains a nucleotide sequence encoding a gene as shown in SEQ NO1.

[0013] A method for constructing NKG2DL-FAP dual-target CAR-NK cells expressing IL-15, comprising the following steps:

[0014] Step 1, constructing a pKN0153 retroviral vector plasmid, wherein the pKN0153 retroviral vector plasmid contains the amino acid sequence shown in SEQ NO1;

[0015] Step 2, amplifying the pKN0153 retroviral vector plasmid;

[0016] Step 3: Introduce the pKN0153 retroviral vector plasmid into NK cells to obtain NKG2DL-FAP dual-target CAR-NK cells.

[0017] In a further improvement, the NK cells are human NK cells.

[0018] A use of NKG2DL-FAP dual-target CAR-NK cells expressing IL-15, wherein the NKG2DL-FAP dual-target CAR-NK cells expressing IL-15 are as described in claim 3 or 4; the NKG2DL-FAP dual-target CAR-NK cells expressing IL-15 are used as a drug for treating tumors.

[0019] In a further improvement, the tumor is a solid tumor.

[0020] In a further improvement, the solid tumor is a human breast cancer cell tumor.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] The present invention develops dual-target CAR-NK cells that express IL-15 and target FAP and NKG2DL, and provides a new structural sequence of transmembrane NCR2TM and co-stimulatory molecule CD226. On the one hand, compared with single targets, it helps prevent tumor immune escape and enhances specific recognition and killing of tumor cells; on the other hand, by secreting the functional stimulating factor IL-15, the process of clearing target cells can maintain sufficient killing activity and exert a lasting functional effect. This cell therapy is expected to promote a huge breakthrough in the field of tumor cell therapy. Description of the drawings:

[0023] Figure 1 A diagram of a chimeric antigen receptor targeting human FAP and NKG2DL;

[0024] Figure 2 The plasmid map of the bispecific CAR targeting hFAP and NKG2DL;

[0025] Figure 3 This is a diagram showing the expansion of NK cells.

[0026] Figure 4 This is the detection chart of CAR double expression positive rate.

[0027] Figure 5 This is the IL-15 expression detection diagram.

[0028] Figure 6 This is a graph showing the expression of NKG2DL in the MDA-MB-436 cell line.

[0029] Figure 7 This is a graph showing the expression of FAP in the MDA-MB-436 cell line.

[0030] Figure 8 This is a test chart of the performance of CAR-NK cells in killing target cells.

[0031] Fig. 9This is a diagram showing the therapeutic effect of CAR-NK cells on breast cancer in mice. Specific implementation method:

[0032] (1) Structural composition

[0033] Design of a chimeric antigen receptor targeting human FAP and NKG2DL, the structure of which is shown in Figure 1 , including an extracellular domain, a hinge region domain (hinge), a transmembrane domain (TM) and an intracellular signaling domain. The extracellular domain contains a single-chain antibody targeting FAP and a NKG2D protein. The amino acid sequence of the single-chain antibody of Anti-hFAP is derived from a PCT patent (PCT / US92 / 08038). The amino acid sequence of the NKG2D protein that binds to the NKG2D ligand is derived from the 91st to 216th amino acid sequence of the gene. The constructed retroviral vector plasmid containing the chimeric antigen receptor is numbered pKN0153. The invention designs a new transmembrane domain (NCR2TM) and a costimulatory molecule domain (CD226).

[0034] (2) Retrovirus preparation

[0035] The pKN0153 retroviral vector plasmid and the retroviral auxiliary packaging plasmid were co-transfected into 293T cells to prepare retrovirus. The plasmid map is shown in Figure 2 .

[0036] The experimental steps for retrovirus preparation are as follows:

[0037] ①Resuscitate and culture 293T cells. Add 12E+06 cells to a T75 cell flask. The culture medium is DMEM medium containing 10% FBS. Culture the flask in a CO2 cell culture incubator.

[0038] ②When the cell confluence reaches about 80%, replace with fresh cell culture medium (DMEM medium containing 10% FBS);

[0039] ③Take 500 μL of opti-DMEM medium, add 10 μg of pMSCV plasmid (pKN0153), 10 μg of pUMVC plasmid and 10 μg of pRD114A plasmid and mix well;

[0040] ④ Take another 500 μL of opti-DMEM medium, add 30 μg of polyetherimide (PEI) solution and mix well;

[0041] ⑤ Add the PEI solution to the plasmid solution in step ③ and mix well. After standing at room temperature for 20 minutes, add it to 293T cells. After incubation for 8 hours, remove the culture medium and add 15 mL of fresh cell culture medium.

[0042] ⑥ 72 hours after transfection, collect the culture medium supernatant, centrifuge at 1000g for 10 minutes, take the supernatant virus liquid and store it in a -80℃ refrigerator for NK cell transduction.

[0043] ⑦The titer of the packaged retrovirus is shown in Table 1.

[0044] Table 1. Retrovirus titer test results

[0045]

[0046] (3) Activation and culture of human NK cells

[0047] The present invention uses Miltenyi Biotec CD3 sorting magnetic MicroBeads (Miltenyi Biotec, human-lyophilized, 130-097-043) to remove T cells from human cord blood mononuclear cells (CBMC) and activate NK cells. The experimental steps are as follows:

[0048] ① Prepare fresh MACS buffer (1×DPBS solution containing 2mM EDTA and 0.5% FBS) and pre-cool at 4℃;

[0049] ② Thaw 25M human umbilical cord blood mononuclear cells (CBMC) in a water bath at 37°C;

[0050] ③Take 10μL CBMC from the biosafety cabinet, count with a cell counter, transfer CBMC to a 15ml centrifuge tube, add 6mL pre-cooled 1×DPBS, and centrifuge at 400g for 5 minutes. After centrifugation, discard the supernatant and add MACS buffer to resuspend CBMC cells (80μL / 1E+07 cells);

[0051] ④Add CD3 Microbeads (20 μL / 1E+07 cells), mix well and incubate in a 4°C refrigerator for 15 minutes;

[0052] ⑤ Rinse the LS column (Miltenyi Biotec, 130-042-401) with 3 mL of MACS buffer, then add the cell suspension to the LS column. Then add 3 mL of MACS buffer, and after the cell suspension has all flowed into the 15 mL centrifuge tube, add another 3 mL of MACS buffer and collect all the cells into the 15 mL centrifuge tube;

[0053] ⑥ The cell suspension was centrifuged at 400g for 10 minutes, and the cells were resuspended in Icosai NK medium containing 10% FBS. 10 μL of cells were counted and K562 feeder cells were added to the CBMC T cell Deletion cells for activation culture at a ratio of NK:K562 feeder = 1:1. On the 5th day of NK cell activation culture, the NK cell multiple was 2.6 and the NK cell ratio was 55.5%, as shown in Table 2 and Figure 3 .

[0054] Table 2. NK cell expansion

[0055]

[0056]

[0057] (4) Preparation of Anti-hFAP-NKG2DL CAR-NK Cells

[0058] On the 5th day of NK cell activation culture, pKN0153 retrovirus was added to prepare pKN0153Anti-hFAP-NKG2DL-FAP CAR-NK cells. The experimental steps are as follows:

[0059] ① Take 5E+05 activated NK cells into a 12-well cell culture plate and add 0.9 ml of pKN0153 retroviral supernatant;

[0060] ② Add 10 μg / mL Vectofusin-1 (Miltenyi Biotec, 130-111-163) as a transduction agent, and use the cell culture medium containing 5% FBS and 200 IU / mL IL-2 in the Ecosense NK cell culture medium (Ecosense, NE000-N012), with a cell density of 5E+05 cells / ml;

[0061] ③ The cells were centrifuged at 1000 g for 30 minutes and then cultured in a CO2 cell culture incubator to prepare Anti-hFAP-NKG2DL CAR-NK (pKN0153-CAR-NK) cells.

[0062] (5) Detection of Anti-hFAP-NKG2DL-FAP CAR positive rate and IL-15 expression

[0063] Anti-hFAP-NKG2DLCAR-NK cells were cultured using Icosai NK cell culture medium containing 5% FBS and 200 IU / mL IL-2, and the CAR positive rate and IL-15 expression level were detected on the 8th and 12th days of culture. The experimental steps are as follows:

[0064] ① Take 5E+05 cells of each of the untransduced NK cells (UNK) and the transduced CAR-NK cells (pKN0153) into a 96-well V-bottom plate and centrifuge at 400g for 5 minutes;

[0065] ②Discard the supernatant, add 200 μL 1×DPBS to each tube and wash twice by centrifugation (400 g for 5 minutes);

[0066] ③ Add 1:100 diluted APC-Anti-NKG2D Antibody (Biolegend, 320808) and PE-Anti-FAP Antibody (ARCO Biosystems, FAP-HP245), 100 μL / well, and incubate at room temperature in the dark for 15 minutes;

[0067] ④ After incubation, centrifuge at 400g for 5 minutes. Discard the supernatant and add 200μL 1×DPBS to each sample and wash once by centrifugation (400g for 5 minutes);

[0068] ⑤Discard the supernatant and add 100 μL 1×DPBS to each well to resuspend the cells. CAR positive cells in UNK and pKN0153 cells were detected and analyzed by flow cytometry. The results are shown in Table 3 and Figure 4 The results showed that in NK cells transfected with FAP-NKG2DL dual CAR, the positive rates of dual expression of FAP CAR and NKG2DL CAR were detected to be 70.2% (D8) and 54.6% (D12), indicating that dual CAR NK cells targeting FAP and NKG2DL were successfully constructed.

[0069] ⑥ Anti-hFAP-NKG2DL-CAR-NK cells were collected, and the supernatant was centrifuged for IL-15 detection. The samples were prepared according to the instructions of the human IL-15 ELISA kit (ACRO CRS-A009), and the absorbance OD value was measured at a wavelength of 450nm to calculate the IL-15 expression level. The experimental results are as follows Figure 5 As shown, the results showed that the secretion of IL-15 by Anti-hFAP-NKG2DL-CAR-NK cells was significantly increased compared with UNK cells.

[0070] Table 3. CAR dual expression positive rate detection

[0071]

[0072] (6) Detection of NKG2DL and FAP expression levels on target cell surface

[0073] The MDA-MB-436 human breast cancer cell line was purchased, and the expression levels of NKG2DL and FAP in MDA-MB-436 cells were detected by flow cytometry (Beckman, CytoFlex). The experimental steps are as follows:

[0074] ① The culture conditions of MDA-MB-436 are L15+15% FBS+10μg / mL Insulin+16μg / mL Glutathione+1% P / S medium;

[0075] ② Take 2E+06 cells into a 1.5 mL centrifuge tube and centrifuge at 400 g for 5 minutes;

[0076] ③Discard the supernatant, add 800 μL 1×DPBS (Cytiva, SH30028.02) and wash twice by centrifugation (400 g for 5 minutes);

[0077] ④ Resuspend the cells with 900 μL 1×DPBS and add 100 μL each into a 96-well V-bottom plate as the blank group, isotype control group and experimental group;

[0078] ⑤ In the isotype control group cells, add 1:100 diluted Mouse IgG2A PE-conjugated Antibody antibody (R&D, IC003P), Mouse IgG2B PE-conjugated Antibody antibody (R&D, IC0041P), and APC Mouse IgG2a, κIsotype Ctrl (Biolegend, 981906) and incubate at room temperature in the dark for 15 minutes;

[0079] ⑥ In the experimental group, add 1:50 dilution of APC-anti-MICA / B antibody (Biolegend, 320908), Human ULBP-1 PE-conjugated Antibody (R&D, FAB1380P), Human ULBP-2 / 5 / 6 PE-conjugated Antibody (R&D, FAB1298P), Human ULBP-3 PE-conjugated Antibody (R&D, FAB1517P), Human ULBP-4 PE-conjugated Antibody (R&D, FAB6285P) and Human FAP PE-conjugated Antibody (Thermo, BMS168) to the cells, and incubate at room temperature in the dark for 15 minutes;

[0080] ⑦ After incubation, centrifuge at 400g for 5 minutes, discard the supernatant, add 200μL 1×DPBS and wash once by centrifugation (400g for 5 minutes);

[0081] ⑧Discard the supernatant and add 100 μL 1×DPBS to each well to resuspend the cells;

[0082] ⑨Flow cytometry analysis showed that MDA-MB-436 expressed ULBP-4 and FAP. Figure 6 and Figure 7 .

[0083] (7) In vitro killing performance of CAR-NK cells

[0084] Anti-hFAP-NKG2DL-CAR-NK cells were co-cultured with target cells MDA-MB-436 to detect the tumor killing function of Anti-hFAP-NKG2DL-CAR-NK cells. The experimental steps are as follows:

[0085] ① Count the target MDA-MB-436 cells, label them with CFSE (Sigma, 87444-5mg-F) at a final concentration of 5 μM, and add 20,000 cells / well into a 96-well U-bottom plate in a volume of 100 μL;

[0086] ② Count UNK and pKN0153 CAR-NK cells and add them to the cells according to the CAR positive rate effect-target ratio (E:T) of 10:1, 5:1, 2:1, 1:1 and 0.5:1, 100 μL / well;

[0087] ③ Place in a CO2 cell culture incubator and incubate for 4 hours. After incubation, centrifuge at 2000 rpm for 3 minutes, discard the supernatant, add 100 μL 7-AAD (Biolegend, 420404) reagent to each well, mix well by pipetting, stain in the dark for 10 minutes, and detect with a flow cytometer (Beckman, CytoFlex).

[0088] The test results showed that compared with UNK cells, pKN0153 CAR-NK cells could effectively kill target cells MDA-MB-436 at effector-target ratios of 10:1, 5:1, 2:1, 1:1 and 0.5:1, with significant statistical differences. Figure 8 .

[0089] (8) CAR-NK cell therapy for mouse breast cancer model

[0090] Female 10-week-old NSG mice were selected and 1×10 6 MDA-MB-436 cells were subcutaneously inoculated in the axilla to construct a human breast cancer model. When the tumor grew to 50-100 mm3 5×10 6 Anti-hFAP-NKG2DL-CAR-NK cells and UNK cells were used to measure the tumor size every 5 days after the first cell injection. It can be seen that the efficacy of CAR-NK cells is significantly better than that of UNK cells, with significant statistical differences. Fig. 9 .

[0091] The above embodiment is only a specific implementation of the present invention and is not intended to be a limitation of the present invention. Any simple improvements and replacements made thereto are within the protection scope of the present invention.

Claims

1. A coding gene, characterized in that The nucleotide sequence of the coding gene is shown in SEQ NO 1.

2. A protein, characterized in that The amino acid sequence of the protein is shown in SEQ NO2.

3. A NKG2DL-FAP dual-target CAR-NK cell expressing IL-15, characterized in that: The NKG2DL-FAP dual-target CAR-NK cells contain a nucleotide sequence encoding a gene expressed as shown in SEQ NO1.

4. A method for constructing NKG2DL-FAP dual-target CAR-NK cells expressing IL-15, characterized in that: The steps include: Step 1, constructing a pKN0153 retroviral vector plasmid, wherein the pKN0153 retroviral vector plasmid contains the amino acid sequence shown in SEQ NO1; Step 2, amplifying the pKN0153 retroviral vector plasmid; Step 3: Introduce the pKN0153 retroviral vector plasmid into NK cells to obtain NKG2DL-FAP dual-target CAR-NK cells.

5. The method for constructing IL-15-expressing NKG2DL-FAP dual-target CAR-NK cells according to claim 4, characterized in that: The NK cells are human NK cells.

6. Use of NKG2DL-FAP dual-target CAR-NK cells expressing IL-15, characterized in that: The NKG2DL-FAP dual-target CAR-NK cells expressing IL-15 are as described in claim 3 or 4; the NKG2DL-FAP dual-target CAR-NK cells expressing IL-15 are used as a drug for treating tumors.

7. The use of IL-15-expressing NKG2DL-FAP dual-target CAR-NK cells according to claim 6, characterized in that: The tumor is a solid tumor.

8. The use of NKG2DL-FAP dual-target CAR-NK cells expressing IL-15 according to claim 7, characterized in that: The solid tumor is a human breast cancer cell tumor.

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