VHH antibody, CAR-NK cell and application

Abstract

The present application relates to the field of biotechnology and cell therapy, in particular to a VHH antibody, CAR-NK cell and application.Under the condition of gradient progression of effector-target ratio, based on the luciferase method detection, it is found that the cytotoxicity decreases with the decrease of effector-target ratio, the BCMA-CAR-NK cell corresponding to the four VHH antibodies can effectively kill MM.1S, and the killing ability is significantly stronger than that of the control group.The above proves that hBCMAnb-CAR-NK has the tumor response reaction specific to BCMA.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and cell therapy, specifically to a VHH antibody, CAR-NK cells, and their applications. Background Technology

[0002] VHH antibody is a nanobody derived from heavy chain IgG antibody. It has the advantages of small size and strong antigen affinity, and can overcome the limitations of traditional antibodies. It has broad application prospects in disease diagnosis, treatment, food safety, and environmental protection.

[0003] The optimization and screening of VHH antibodies is currently a hot topic and a major challenge in the industry. Each VHH antibody that is screened and shows significant efficacy against a specific target has enormous value and makes a significant contribution to further conquering and treating various solid tumors in humans. Summary of the Invention

[0004] The purpose of this invention is to provide a VHH antibody, CAR-NK cells, and their applications to solve the problems mentioned in the background art.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0006] A VHH antibody, wherein the VHH antibody is any one of 1F, 7B, 11F, and 12A.

[0007] One, a fragment or variant thereof, wherein:

[0008] The base sequence of 1F is shown in SEQ ID NO.1;

[0009] The base sequence of 7B is shown in SEQ ID NO.2;

[0010] The base sequence of 11F is shown in SEQ ID NO.3;

[0011] The base sequence of 12A is shown in SEQ ID NO.4.

[0012] Preferably, the corresponding amino acid sequence is as follows:

[0013] The amino acid sequence of 1F is shown in SEQ ID NO.6;

[0014] The amino acid sequence of 7B is shown in SEQ ID NO.7;

[0015] The amino acid sequence of 11F is shown in SEQ ID NO.8;

[0016] The amino acid sequence of 12A is shown in SEQ ID NO.9.

[0017] A CAR-NK cell expressing a VHH antibody as described in any of the preceding descriptions.

[0018] Preferably, the CAR-NK cells are autologous, allogeneic, or xenogeneic.

[0019] A pharmaceutical composition comprising a VHH antibody as described in any one of the preceding claims or a CAR-NK cell as described in any one of the preceding claims, and at least one pharmaceutically acceptable carrier.

[0020] Use of any of the preceding VHH antibodies, any of the preceding CAR-NK cells, or the aforementioned pharmaceutical compositions in the preparation of kits for the diagnosis / treatment of malignant tumors or autoimmune diseases.

[0021] The use of any of the preceding VHH antibodies, any of the preceding CAR-NK cells, or the aforementioned pharmaceutical compositions in the preparation of medicaments for the diagnosis / treatment of malignant tumors or autoimmune diseases.

[0022] Preferably, the malignant tumor is multiple myeloma.

[0023] Compared with existing technologies, the beneficial effects achieved by this invention are as follows: Under gradient effector-to-target ratio conditions, luciferase assay revealed that cytotoxicity decreased as the effector-to-target ratio decreased; BCMA-CAR-NK cells corresponding to the four VHH antibodies could effectively kill MM.1S cells, and their killing ability was significantly stronger than that of the control group. This demonstrates that hBCMAnb-CAR-NK cells possess a BCMA-specific tumor response. Attached Figure Description

[0024] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0025] Figure 1 Image of a culture dish used for the second round of clone count detection in the Alpaca VHH phage display library;

[0026] Figure 2 Figure showing the results of flow cytometry analysis and screening of hBCMAnb highly bound to MM.1S in cells expressing BCMA.

[0027] Figure 3 Figure 1 shows the results of flow cytometry analysis of the binding ability of four types of hBCMAnb to tumor cells MM.1S.

[0028] Figure 4 Schematic diagram of BCMA CAR;

[0029] Figure 5 Figure 1. Experimental results of high-efficiency expression of hBCMAnb-CAR in NK92MI cells;

[0030] Figure 6 Figure showing the results of in vitro experiments verifying the killing function of CAR-NK;

[0031] Figure 7 A graph showing the transduction efficiency of CAR in NK cells;

[0032] Figure 8 Figure 1. Flow cytometry results of the binding ability of two CD19-expressing CAR-NK proteins to CD19-Fc protein;

[0033] Figure 9 Figure showing the results of flow cytometry analysis of the binding ability of CD19 / BCMA CAR-NK to BCMA-Fc protein;

[0034] Figure 10 Figure 1. Flow cytometry results of the binding ability of three BCMA-expressing CAR-NK proteins to BCMA-Fc protein.

[0035] Figure 11 Figure 1 shows the results of an in vitro experiment verifying the killing function of two CAR-NK cells in Jeko-1 tumor cells.

[0036] Figure 12 Figure 1 shows the results of an in vitro experiment verifying the killing function of two CAR-NK cells in MM.1S tumor cells.

[0037] Figure 13 Figure 1 shows the results of in vitro experiments verifying the killing function of three BCMA-expressing CAR-NK cells in MM.1S tumor cells.

[0038] Figure 14 Figure showing the experimental results of flow cytometry detection of the degranulation ability of two CAR-NK cells in Jeko-1 tumor cells;

[0039] Figure 15 Figure showing the experimental results of flow cytometry detection of the degranulation ability of two CAR-NK cells in MM.1S tumor cells;

[0040] Figure 16 Figure 1 shows the experimental results of flow cytometry detection of the degranulation ability of three BCMA-expressing CAR-NK cells in MM.1S tumor cells;

[0041] Figure 17 Fluorescence imaging of CD19 / BCMA CAR-NK in the MM.1S animal model;

[0042] Figure 18Statistical graph of fluorescence imaging data of CD19 / BCMA CAR-NK in MM.1S animal model;

[0043] Figure 19 Weight statistics of the MM.1S animal model after treatment;

[0044] Figure 20 Fluorescence imaging of CD19 / BCMA CAR-NK in the Jeko-1 animal model;

[0045] Figure 21 Statistical graph of fluorescence imaging data of CD19 / BCMA CAR-NK in Jeko-1 animal model;

[0046] Figure 22 Weight statistics of the Jeko-1 animal model after treatment. Detailed Implementation

[0047] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] I. Phage Library Screening for Alpaca Nanobody Library

[0049] 1) Recombinant human BCMA protein (Kaikai Biotechnology, catalog number BCM-HM217) was selected as the antigen for immune activation of VHH antibody;

[0050] 2) Select one healthy alpaca for immunization. Use a 1:1 mixture of complete Freund's adjuvant and antigen, emulsify, and inject subcutaneously at multiple points. The dosage is 0.2 mg per alpaca, and a total of 5 immunizations are performed, with an interval of 2 weeks.

[0051] 3) After the 5th immunization, 200 mL of peripheral blood was collected from alpacas, and mononuclear cells were obtained by density centrifugation. RNA was extracted using the TRIZOL lysis method and reverse transcribed into cDNA, which was then amplified by nested PCR to obtain the VHH gene fragment.

[0052] 4) The VHH gene fragment was ligated into the pCANTAB-5E phage display plasmid and then electroporated into TG1 competent cells to establish a phage library;

[0053] 5) Coat the immunotubes with BCMA antigen and block with 5% skim milk powder. Add 1 mL of phage library and incubate at room temperature for 1 hour by rotation. Wash the immunotubes with 2 mL of PBST (1×PBS with 0.1% Tween 20), then neutralize with 500 μL of Gly-HCl neutralization buffer. Transfer the solution from the immunotubes to a new 1.5 mL centrifuge tube; this is the first round of phage elution buffer. 6) Infect TG1 strain with the first round of phage elution buffer and centrifuge in a 1.5 mL tube for 10 minutes. 5 and 10 6 Two gradient dilutions ( Figure 1 Plate counting was performed. The number of single colonies at different dilutions can be clearly distinguished on the plate. The screening efficiency was calculated by clonal counting.

[0054] II. Phage ELISA

[0055] 1) Select single clones from the second round of screening and culture. When the OD reaches 0.5, add M13KO7 helper phage (brand: NEB, catalog number: N0315S), incubate overnight at 25°C, and then centrifuge to collect the supernatant.

[0056] 2) Coat the ELISA plate with BCMA antigen (KaiKa Bio, catalog number BCM-HM217) (1 ng / μL, 100 μL / well), block and wash, add 100 μL of phage culture supernatant to each well, incubate and wash, add horseradish peroxidase-labeled anti-M13 antibody (diluted 10,000 times with PBS), incubate and wash, add TMB chromogenic solution for color development, and finally add stop solution to terminate the reaction. Measure the optical density at 450 nm.

[0057] 3) After removing redundant sequences through sequencing, 20 unique antibody sequences were obtained.

[0058] The first round of ELISA colorimetric readings are shown in Table 1:

[0059] Table 1

[0060]

[0061] The second round of ELISA colorimetric readings are shown in Table 2:

[0062] Table 2

[0063]

[0064] III. Expression of hBCMAnb protein

[0065] 1) According to the product instructions of Array Mini (product number: profac_ary0601000), a cell-free protein expression product of Kangma (Shanghai) Biotechnology Co., Ltd., the screened hBCMAnb antibody sequence was constructed into a Fusion PCR fragment;

[0066] 2) Add the Fusion PCR template to the water-dissolved ProteinFactory Rxn reaction system at a volume ratio of 1:45, mix well and adjust the total reaction volume to 10 mL. Place the above reaction solution in a disposable shaker flask, mix well, seal with a breathable membrane or cover with a lid (do not completely seal), and react overnight on a shaker at 30℃ and 220 rpm.

[0067] 3) Magnetic bead purification: Take 1.5 mL of ProteinFactory reaction solution, centrifuge at 4000 rpm for 3 min at 4℃, and collect the supernatant; take His-Monster beads, wash twice with 5 mL of Binding Buffer, magnetically absorb the beads, and set aside; add the washed His-Monster beads to the supernatant, shake thoroughly, and rotate to mix at 4℃ for 1 h; after incubation, magnetically absorb the beads, and discard the supernatant; add washing buffer, shake thoroughly, magnetically absorb the beads, and discard the supernatant; add Elution buffer to the beads, pipette tip to mix thoroughly, let stand, magnetically absorb the beads, and collect the Elution supernatant, which is the target protein. Repeat 5-8 times.

[0068] The binding buffer is a mixed solution of 20 mM Tris-HCl and 500 mM NaCl, pH 8.0; the washing buffer is a mixed solution of 20 mM Tris-HCl, 500 mM NaCl, and 20 mM Imidazole, pH 8.0; and the elution buffer is a mixed solution of 20 mM Tris-HCl, 500 mM NaCl, and 250 mM Imidazole, pH 8.0. IV. Flow Cytometry Detection of BCMA-Expressing Cells with Highly Binding hBCMAnb (Human BCMA Nanobody) to MM.1S

[0069] 1) Obtain MM.1S cells (brand: Cell Bank of the Chinese Academy of Sciences / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; catalog number: SCSP-5017), count, resuspend, and adjust the density to 1×10⁻⁶. 4 / 50μL;

[0070] 2) Different concentrations of hBCMAnb protein (10 ng / mL, 40 ng / mL, 160 ng / mL) were added to the cell suspension and incubated at 4°C for 30 min. Myc-Tag (9B11) was added and diluted 400-fold with Mouse mAb, and incubated at 4°C for 30 min. 3) 2 mL of PBS was added and centrifuged. The cells were centrifuged at 400 g for 5 min. The cells were resuspended in 100 μL of PBS and subjected to flow cytometry. Four hBCMAnb strains were screened, namely 1F, 7B, 11F, and 12A, with the absence of hBCMAnb as a negative control. The flow cytometry screening results are as follows: Figure 2 As shown.

[0071] V. Flow cytometry detection of the binding ability of four hBCMAnb to tumor cell MM.1S.

[0072] 1) Count MM.1S cells, resuspend them, and adjust their density to 2 × 10⁻⁶. 4 / 50μL;

[0073] 2) Add different concentrations of four hBCMAnb proteins (10 ng / mL, 40 ng / mL, 160 ng / mL) to 50 μL of cell suspension and incubate at 4℃ for 30 min.

[0074] 3) Add Myc-Tag(9B11)Mouse mAb (1:400) and incubate at 4℃ for 30 min;

[0075] 4) Add 2 mL of PBS and centrifuge at 400 g for 5 min;

[0076] 5) Resuspend cells in 100 μL PBS and perform flow cytometry. Use cells without hBCMAnb as a negative control.

[0077] The results are as follows Figure 3 As shown, all four nanobody proteins can bind to cell lines expressing BCMA antigen.

[0078] VI. Preparation of Lentivirals

[0079] The specific steps for preparing a lentiviral vector for infecting NK cells expressing CAR molecules are as follows:

[0080] 1) Transfect 293T cells when the confluence is 80-90%. Change the Opti-MEM medium (Thermo, catalog number 31985070) used for packaging the virus 2 hours before transfection.

[0081] 2) Lentiviral preparation using a four-plasmid packaging system: This system includes three helper plasmids, specifically pLP1, pLP2, and pVSVG, and a target plasmid (all purchased from Thermo Fisher, catalog number K497500). The target plasmid pLenti is a lentiviral expression plasmid with a backbone based on pLenti-CMV-V5-LUC Blast (purchased from Addgene, catalog number 21474). The original CMV promoter of the plasmid is replaced with the EF1α promoter. Different CARs (containing the 38A positive control sequence and the four selected hBCMAnb sequences) are inserted into the expression framework to obtain hBCMAnb-CAR or the positive control (the commercial product sequence from Legend Biotech) 38A-CAR (e.g., hBCMAnb-CAR). Figure 4 (As shown). The four plasmids were mixed in a certain proportion and then the transfection reagent Lipo2000 was added to prepare the transfection reagent mixture;

[0082] 3) After mixing, incubate at room temperature for 15-25 minutes, then add to 293T cells along the side wall;

[0083] 4) Collect viral supernatant: Collect viral supernatant after 48 and 72 hours respectively;

[0084] 5) After filtering cell debris through a 0.45 μm pore size filter membrane, the supernatant was concentrated using ultracentrifugation;

[0085] 6) Lentiviral titers were detected using NK-92MI cell line (Cell Bank of the Chinese Academy of Sciences / Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), and the functional titers were found to be between 0.5 and 1 × 10⁻⁶. 8 Within the TU / mL range, lentiviruses that can effectively transfect cells can be obtained for subsequent experiments.

[0086] VII. Preparation of CAR-NK cells

[0087] 1) Collect NK92MI cells, wash twice with PBS by centrifugation, add RPMI 1640 medium (brand: Yuanpei Biotechnology; catalog number: L210KJ) containing 10% PAN serum (brand: PAN; catalog number: ST30-3302), mix well, and count. Seed 0.3M cells per well in a 96-well plate. Add hBCMAnb-CAR lentivirus (MOI=5) (positive control and the four selected sequences) to NK92MI cells. Add 8μg / mL Polybrene (brand: Beyotime; catalog number: C0351) to each well, and bring the volume up to 200μL with medium. Centrifuge at 1200g for 1.5h at 32℃, incubate at 37℃ for 4h, discard the supernatant, and transfer to a 24-well plate. After 48h, detect CAR expression using flow cytometry with NGFR antibody (brand: Biolegend; catalog number: 345106).

[0088] The results showed that hBCMAnb-CAR was highly expressed in NK92MI cells, with a transduction efficiency of over 60%. Figure 5 8. In vitro experimental verification of CAR-NK killing function

[0089] A cell line MM.1S-CBR-Luc-GFP co-expressing luciferase CBR and green fluorescent protein GFP was constructed using multiple myeloma cells MM.1S. (CBR stands for click beetle red luciferase, which can catalyze the excitation of red spectrum by luciferin substrates; CBR and GFP were simultaneously introduced into MM.1S cells). This cell line was used as target cells for in vitro killing assays.

[0090] 1) Target cell plating: Collect MM.1S tumor cells in the logarithmic growth phase, centrifuge, count, and adjust the cell density to 1×10⁻⁶. 5 The sample was injected at a rate of 100 μL / well into a 96-well U-plate, i.e., 1 × 10⁻⁶ μL / well. 4 One target cell;

[0091] 2) Adjusting CAR%: Collect hBCMAnb-CAR-NK cells / positive control antibody 38A-CAR-NK cells / negative control NK cells, and detect the CAR% of each CAR-NK cell by flow cytometry. Resuspend the CAR-NK cells in preheated NK medium and adjust the CAR% of each CAR-NK cell to be consistent with the total number of NK cells.

[0092] 3) Effector cell plating: Calculate the number of CAR-NK cells required for plating, resuspend the cells at the CAR-NK cell concentration required for the most effective target ratio (calculate cell density at 100 μL per well), and perform serial dilutions for other effector-target ratios. A positive control (K) is also included. maxUsing Triton-100 and a negative control (K) min (using NK medium);

[0093] 4) Gently mix the cell mixture in each well and incubate in an incubator for 4-6 hours. Preheat the Luciferin substrate. After washing the cells twice with FACS Buffer by centrifugation, add 0.5 mM luciferase substrate and incubate at 37°C in the dark for 10 minutes. Use a microplate reader in chemiluminescence mode to read the data.

[0094] 5) Calculate the kill efficiency % = (K min -K) / (K min -K max )×100%.

[0095] The results are as follows Figure 6 As shown, under gradient effector-to-target ratio conditions, luciferase assay revealed that cytotoxicity decreased with decreasing effector-to-target ratio. All four BCMA-CAR-NK cell lines effectively killed MM.1S cells, and their killing ability was significantly stronger than the control group. This demonstrates that hBCMAnb-CAR-NK possesses a BCMA-specific tumor response.

[0096] IX. Preparation of CD19 / BCMACAR-NK cells

[0097] NK cells were collected, washed twice with PBS by centrifugation, and mixed with NK medium. Cells were then counted, and 0.3M cells were seeded into each well of a 96-well plate. hCD19 / hBCMAnb-CAR lentivirus or hCD19-CAR (MOI=5) was added to the NK cells, along with protamine sulfate (final concentration 8 pg / ml). The volume was brought to 100 μL with NK medium. The plates were centrifuged at 32°C, 1200g, for 1.5 h. After centrifugation, the plates were incubated at 37°C for 4 h. The supernatant was discarded, and the cells were transferred to 24-well plates. CAR expression was detected by flow cytometry using FMC63 antibody (brand: BioSwan; catalog number: 300402) after 48 h. Results showed that the transduction efficiency of hCD19h / BCMAnb-CAR on NK cells was over 30%, and the transduction rate of hCD19-CAR on NK cells was over 70%. Figure 7 10. Flow cytometry detection of the affinity of CD19 / BCMACAR-NK cells

[0098] 1) Flow cytometry was used to detect the proportion of CAR in CD19 CAR-NK and CD19 / BCMA CAR-NK, and the CAR proportion was adjusted to be consistent.

[0099] 2) Count the two types of CAR-NK cells after CAR adjustment, resuspend them, and adjust their density to 1×10⁻⁶. 5 / 50μL

[0100] 3) Add CD19-Fc protein or BCMA-Fc protein at different dilution ratios to 50 μL of cell suspension and incubate at 4°C for 20 min.

[0101] 4) Add 2 mL of PBS and centrifuge at 400 g for 5 min;

[0102] 5) Discard the supernatant, resuspend the cells in 100 μL PBS, and perform flow cytometry. Use cells without CD19-Fc protein or BCMA-Fc protein as negative controls.

[0103] The results are as follows Figure 8 , Figure 9 and Figure 10 As shown, CD19 CAR-NK cells exhibit superior binding affinity to CD19-Fc protein compared to CD19 / BCMACAR-NK cells. At an antigen concentration of 1:80, CD19 CAR-NK cells showed approximately 88% binding affinity to CD19-Fc protein, while CD19 / BCMACAR-NK cells showed less than 10%. Furthermore, CD19 / BCMACAR-NK cells can bind to BCMA-Fc protein in a concentration-dependent manner. Compared to BCMACAR-NK, CD19 / BCMA CAR-NK cells showed stronger binding affinity to BCMA-Fc protein, but this affinity was still weaker than that of the positive control CD19 / BCMACAR-NK. XI. In vitro validation of the cytotoxic function of CD19 / BCMACAR-NK cells.

[0104] Multiple myeloma cells MM.1S and Jeko-1 were used to construct cell lines co-expressing luciferase CBR and green fluorescent protein GFP (CBR stands for click beetle redluciferase, which can catalyze the excitation of red spectrum by luciferin substrates; CBR and GFP were simultaneously introduced into MM.1S cells). These cell lines were used as target cells for in vitro killing assays.

[0105] 1) Target cell plating: Collect MM.1S / Jeko-1 tumor cells in logarithmic growth phase, centrifuge, count, and adjust cell density to 2×10⁻⁶. 5 The sample was injected at a rate of 50 μL / well into a 96-well U-type substrate, i.e., 1 × 10⁻⁶ μL / well. 4 One target cell;

[0106] 2) Adjusting the CAR ratio: Collect CD19 CAR-NK cells, CD19 / BCMA CAR-NK cells, BCMA CAR-NK cells, CD19 / BCMA CAR-NK (positive control) cells and negative control NK cells. Detect the percentage of CAR in each CAR-NK cell by flow cytometry. Resuspend the CAR-NK cells in preheated NK medium and adjust the CAR% of each CAR-NK cell to be consistent with the total number of NK cells.

[0107] 3) Effector cell plating: Calculate the number of CAR-NK cells required for plating, resuspend the cells at the CAR-NK cell concentration required for the most effective target ratio (calculate cell density at 50 μL per well), and perform serial dilutions for other effector-target ratios. A positive control (K) is also included. max Using Triton-100 and a negative control (K) min (using NK medium);

[0108] 4) Gently mix the cell mixture in each well, incubate in an incubator for 4 hours, preheat the Luciferin substrate, wash the cells twice with FACS Buffer by centrifugation, add 0.5mM luciferase substrate, and incubate at 37°C in the dark for 10 minutes; read the data K using a microplate reader in chemiluminescence mode.

[0109] 5) Calculate the kill efficiency % = (K min -K) / (K min -K max )×100%.

[0110] The results are as follows Figure 11 and Figure 12 As shown, under gradient effector-to-target ratio conditions, cytotoxicity decreased with decreasing effector-to-target ratio, as detected by luciferase assay. In BCMA-negative cells (Jeko-1), there was no significant difference in cytotoxic activity between CD19 CAR-NK and CD19 / BCMA CAR-NK. In BCMA-positive cells (MM.1S), CD19 / BCMA CAR-NK exhibited the strongest cytotoxic activity, demonstrating the specificity of BCMA targeting.

[0111] like Figure 13 As shown, there was no significant difference in cytotoxicity between BCMA-positive cells (MM.1S), BCMACAR-NK, CD19 / BCMACAR-NK, and CD19 / BCMACAR-NK (positive control).

[0112] 12. In vitro verification of the degranulation ability of CD19 / BCMACAR-NK cells

[0113] 1) Target cell plating: Collect MM.1S / Jeko-1 tumor cells in logarithmic growth phase, centrifuge, count, and adjust cell density to 2×10⁻⁶. 6 The sample was injected at a rate of 50 μL / well into a 96-well U-type substrate, i.e., 1 × 10⁻⁶ μL / well. 5 One target cell;

[0114] 2) Adjusting CAR%: Collect CD19 CAR-NK cells, CD19 / BCMA CAR-NK cells, BCMA CAR-NK cells, CD19 / BCMA CAR-NK (positive control) cells and negative control NK cells. Detect the CAR% of each CAR-NK cell by flow cytometry. Resuspend the CAR-NK cells in preheated NK medium and adjust the CAR% of each CAR-NK cell to be consistent with the total number of NK cells.

[0115] 3) Effector cell plating: Calculate the number of CAR-NK cells required for plating, and resuspend the cells at the required CAR-NK cell concentration for an effector-target ratio of E:T = 1:1 (calculate cell density at 50 μL per well);

[0116] 4) Gently mix the cell mixture in each well, and add the protease inhibitor and CD107a fluid antibody at the same time, and incubate in an incubator for 4 hours;

[0117] 5) Collect the cells into a flow cytometry tube, add 2 ml of PBS, and centrifuge at 400 g for 5 min;

[0118] 6) Discard the supernatant, add FMC63 / NGFR for flow cytometry, and incubate at 4°C for 20 min;

[0119] 7) Add 2ml of PBS, centrifuge at 400g for 5 minutes;

[0120] 8) Discard the supernatant, add 100 μl PBS, and perform flow cytometry analysis.

[0121] The results are as follows Figure 14 and Figure 15 As shown, for CD19-positive Jeko-1 cells, CD19 CAR-NK cells exhibited superior degranulation ability compared to CD19 / BCMACAR-NK cells, while in BCMA-positive MM.1S cells, CD19 / BCMACAR-NK cells showed superior degranulation ability compared to CD19 CAR-NK cells. Figure 16 As shown, in BCMA-positive MM.1S cells, CD19 / BCMA CAR-NK showed the strongest degranulation ability, which was superior to BCMA CAR-NK and the positive control CD19 / BCMA CAR-NK.

[0122] 13. In vivo verification of the anti-tumor ability of CD19 / BCMACAR-NK

[0123] To further investigate the specific antitumor effects of dual-target CAR-NK (CD19 / BCMACAR-NK) in vivo, xenograft models of multiple myeloma and lymphoma were established. A peritoneal model was established to investigate the antitumor effects targeting BMCA, and MM.1S (CD19) was injected intraperitoneally. - BCMA + In NSG mice, CD19 / BCMACAR-NK cells were injected intraperitoneally, and tumor growth was monitored after infusion. An intravenous model was established to assess the anti-tumor effect of CD19-targeted therapy, using Jeko-1 (CD19) intravenously. + BCMA - CAR-NK cells were introduced into NSG mice, and tumor growth was monitored after intravenous infusion of CD19 / BCMACAR-NK (E:T = 10:1). Results showed that in a multiple myeloma animal model, BCMACAR-NK exhibited superior antitumor activity compared to CD19 / BCMACAR-NK in mice. Within 24 days, there were no significant changes in mouse body weight in any group, indicating that CAR-NK cells had good safety profiles. Figure 17 , Figure 18 and Figure 19 In a lymphoma animal model, CD19 CAR-NK demonstrated superior antitumor activity compared to CD19 / BCMACAT-NK in mice. Within 23 days, there was no significant change in body weight among the mouse groups, indicating that CAR-NK cells showed good safety profiles. Figure 20 , Figure 21 and Figure 22 ).

[0124] The base sequences corresponding to VHH gene fragments 1F, 7B, 11F, and 12A are as follows:

[0125] BCMA-12A (SEQ ID NO.1)

[0126] atggcagaagttcagctgcaggcaagcggtggtggttttgttcagcctggtggtagcctgcgtctgagctgtgcagccagcggtaccttttgggcatacatcatgggctggtttcgccaggcaccgggtaaagaacgtgaatttgttagcgcaatcagctacgatgatggtgttggttcttattatgccgatagcgtgaaaggtcgctttaccattagccgtgataatagcaaaaataccgtttacctgcagatgaatagtctgcgtgcagaagataccgcaacctattattgtgcattttggtcttggatgtttcatgcagtttattggggtcagggcacccaggttaccgttagcagcggcccgggaggccaa

[0127] >BCMA-11F(SEQ ID NO.2)

[0128] atggcagaagttcagctgcaggcaagcggtggtggttttgttcagcctggtggtagcctgcgtctgagctgtgcagccagcggttttggttctaaatggtacgaaatgggctggtttcgccaggcaccgggtaaagaacgtgaatttgttagcgcaatcagctggtacgatgatatctctgaatattatgccgatagcgtgaaaggtcgctttaccattagccgtgataatagcaaaaataccgtttacctgcagatgaatagtctgcgtgcagaagataccgcaacctattattgtgcaatctggtcttgggttaaaatgtacgcatattggggtcagggcacccaggttaccgttagcagcggcccgggaggccaa

[0129] >BCMA-7B(SEQ ID NO.3)

[0130] atggcagaagttcagctgcaggcaagcggtggtggttttgttcagcctggtggtagcctgcgtctgagctgtgcagccagcggtcgttactcttctaacgataacatgggctggtttcgccaggcaccgggtaaagaacgtgaatttgttagcgcaatcagctttctgccaaattttccagattattatgccgatagcgtgaaaggtcgctttaccattagccgtgataatagcaaaaataccgtttacctgcagatgaatagtctgcgtgcagaagataccgcaacctattattgtgcaaaatggatctgggatctgcaggttgcatattggggtcagggcacccaggttaccgttagcagcggcccgggaggccaa

[0131] >BCMA-1F(SEQ ID NO.4)

[0132] atggcagaagttcagctgcaggcaagcggtggtggttttgttcagcctggtggtagcctgcgtctgagctgtgcagccagcggtcgttactttgcagatgataacatgggctggtttcgccaggcaccgggtaaagaacgtgaatttgttagcgcaatcagcgaaaattggaatgttgcatcttattatgccgatagcgtgaaaggtcgctttaccattagccgtgataatagcaaaaataccgtttacctgcagatgaatagtctgcgtgcagaagataccgcaacctattattgtgcatactggaaatggatgcataacatcgtttattggggtcagggcacccaggttaccgttagcagcggcccgggaggccaa

[0133] >Positive control 38A(SEQ ID NO.5)

[0134] caagtgaaactggaagagagcggcggcggcctggtccaggccggcaggtccctgagactgagctgtgccgccagcgagcacaccttcagcagccacgtgatgggctggttccggcaggcccctggcaaggaacgcgaatctgtggccgtgatcggctggcgggacatctccaccagctacgccgatagcgtgaagggcagattcacaatcagcagagataatgccaaaaagaccctgtacctgcagatgaacagcctgaagcccgaggacaccgccgtgtactactgcgccgctagacggatcgacgccgccgacttcgacagctggggacagggcacacaggtcaccgtgtccagc

[0135] The amino acid sequences corresponding to 1F, 7B, 11F, 12A, and 38A are as follows:

[0136] >BCMA-12A (SEQ ID NO.6)

[0137] MAEVQLQASGGGFVQPGGSLRLSCAASGTFWAYIMGWFRQAPGKEREFVSAISYDDGVGSYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAFWSWMFHAVYWGQGTQVTVSSGPGGQ

[0138] >BCMA-11F (SEQ ID NO.7)

[0139] MAEVQLQASGGGFVQPGGSLRLSCAASGFGSKWYEMGWFRQAPGKEREFVSAISWYDDISEYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAIWSWVKMYAYWGQGTQVTVSSGPGGQ

[0140] >BCMA-7B (SEQ ID NO.8)

[0141] MAEVQLQASGGGFVQPGGSLRLSCAASGRYSSNDNMGWFRQAPGKEREFVSAISFLPNFPDYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAKWIWDLQVAYWGQGTQVTVSSGPGGQ

[0142] >BCMA-1F (SEQ ID NO.9)

[0143] MAEVQLQASGGGFVQPGGSLRLSCAASGRYFADDNMGWFRQAPGKEREFVSAISENWNVASYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAYWKWMHNIVYWGQGTQVTVSSGPGGQ

[0144] Positive control 38A (SEQ ID NO.10)

[0145] QVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVAVIGWRDISTS YADSVKGRFTISRDNAKKTLYLQMNSLKPEDTAVYYCAARRIDAADFDSWGQGTQVTVSS

[0146] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A VHH antibody, characterized in that, The VHH antibody is any one of 1F, 7B, 11F, and 12A, wherein: The base sequence of 1F is shown in SEQ ID NO.1; The base sequence of 7B is shown in SEQ ID NO.2; The base sequence of 11F is shown in SEQ ID NO.3; The base sequence of 12A is shown in SEQ ID NO.

4.

2. The VHH antibody as described in claim 1, characterized in that, Its corresponding amino acid sequence is as follows: The amino acid sequence of 1F is shown in SEQ ID NO.6; The amino acid sequence of 7B is shown in SEQ ID NO.7; The amino acid sequence of 11F is shown in SEQ ID NO. 8; The amino acid sequence of 12A is shown in SEQ ID NO.

9.

3. A CAR-NK cell, characterized in that, Expressing a VHH antibody as described in any one of claims 1 to 2.

4. A CAR-NK cell as described in claim 3, characterized in that, The CAR-NK cells mentioned are autologous, allogeneic, or xenogeneic.

5. A pharmaceutical composition, characterized in that: It includes a VHH antibody as described in any one of claims 1 to 2 or a CAR-NK cell as described in any one of claims 3 to 4, and at least one pharmaceutically acceptable carrier.

6. The use of a VHH antibody according to any one of claims 1 to 2, or a CAR-NK cell according to any one of claims 3 to 4, or the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating malignant tumors; The malignant tumor mentioned is multiple myeloma.

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