Antibodies against human mica / b alpha3 region and uses thereof

Abstract

This invention relates to the field of biomedicine and discloses an antibody against the human MICA / B α3 region and its applications. The antibody comprises a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region includes SEQ ID NO:2 or an amino acid sequence with similar or near-identical biological activity; the amino acid sequence of the light chain variable region includes SEQ ID NO:4 or an amino acid sequence with similar or near-identical biological activity. This antibody can reduce MICA / B shedding from the cell membrane surface, enhance NK cell killing ability, inhibit tumor growth and metastasis in vivo, and, when used in combination with drugs that promote MICA / B expression or enhance cellular immunity, can synergistically improve anti-tumor immune responses. It is expected to become a specific antibody drug for the clinical prevention and treatment of MICA / B+ cancer or MICA / B-related immune diseases.

Description

[0001] This application is a divisional application of the patent application filed on July 14, 2021, with application number CN202110794424.X, entitled "A monoclonal antibody against the human MICA / Bα3 region and its application". Technical Field

[0002] This invention relates to the field of biomedicine, and more particularly to an antibody against the human MICA / Bα3 region and its application. Background Technology

[0003] Tumor metastasis is a major challenge in the treatment of malignant tumors. During the metastatic process, the stage from detachment from the primary tumor to the formation of micrometastases presents the optimal window for effective intervention and prevention of metastatic lesion formation – the "window period" of tumor metastasis. However, this period is often insidious, and detection and early warning methods are lacking. In recent years, anti-tumor immunotherapy has developed rapidly, with the activation or modification of the body's own immune cells (mainly cytotoxic CD8+ T cells and NK cells) to monitor and eliminate tumor cells during the "window period" representing a promising treatment approach.

[0004] NK cells are core members of the innate immune system. When target cells undergo malignant transformation, NK cells can directly recognize stress-induced ligands without sensitization, initiating activation signals and exerting a powerful cytotoxic effect to kill tumor cells. They are important effector cells in the development of tumor immunotherapy. Unlike antigen-triggered T cells, which require co-stimulatory signals for optimal activation, NK cell activation is not governed by any single receptor, but rather by the integration of stimulatory and inhibitory signals. NKG2D is a major activating receptor for NK cells, expressed on the surface of almost all NK cells. MICA and MICB (major histocompatibility complex class I chain-associated proteins A and B) were the first discovered NKG2D ligands, which can bind with high affinity to NKG2D to activate an immune response, recruiting NK cells to effectively kill target cells expressing MICA / B. MICA / B proteins are generally absent in normal human tissues but are expressed on the surface of most epithelial tumor cells, including breast cancer, lung cancer, pancreatic cancer, colon cancer, ovarian cancer, kidney cancer, and prostate cancer. However, studies have shown that highly invasive tumor cells, especially those in the metastatic "window period," can release various proteases that hydrolyze the MICA / Bα3 region, causing MICA / B to detach from the tumor cell surface. This prevents NK cells from effectively recognizing and killing tumor cells that have invaded blood vessels, allowing tumor cells to "escape immunely" and promoting the formation of metastatic lesions. Therefore, a feasible strategy is to use specific antibodies to block the target sequence of hydrolytic enzymes on the MICA / Bα3 region, effectively blocking the detachment of MICA / B from the tumor cell membrane surface, thereby restoring the NK cell's killing effect on it.

[0005] Currently, there are no commercially available monoclonal antibodies targeting the human MICA / Bα3 region, either domestically or internationally. While existing literature reports that humanized anti-human MICA / Bα3 region 7C6 monoclonal antibodies increased MICA / B expression on tumor cell surface in a humanized NK cell-derived metastatic melanoma mouse model, inducing NK cell-mediated tumor immunity and inhibiting tumor metastasis, long-term monotherapy leads to antibody resistance, and antibodies recognizing different epitopes may have synergistic effects. Therefore, developing more sensitive and efficient anti-human MICA / Bα3 region monoclonal antibodies could provide alternatives after drug resistance develops, demonstrating significant clinical translational value and application prospects in the prevention or treatment of MICA / B+ cancers or MICA / B-related immune diseases. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an antibody against the human MICA / Bα3 region and its applications. The antibody of this invention can reduce the shedding of MICA / B from the cell membrane surface, enhance the anti-tumor killing ability of NK cells, effectively inhibit tumor growth and metastasis in vivo, and synergistically improve the anti-tumor immune response when used in combination with drugs that promote MICA / B expression or enhance cellular immunity. It is expected to become a specific antibody drug for the clinical prevention and treatment of MICA / B+ cancer or MICA / B-related immune diseases.

[0007] The specific technical solution of this invention is as follows:

[0008] First, the present invention provides a monoclonal antibody against the human MICA / Bα3 region, comprising a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region comprises SEQ ID NO:1 or SEQ ID NO:2, or comprises an amino acid sequence having similar or similar biological activity to SEQ ID NO:1 or SEQ ID NO:2; the amino acid sequence of the light chain variable region comprises SEQ ID NO:3 or SEQ ID NO:4, or comprises an amino acid sequence having similar or similar biological activity to SEQ ID NO:3 or SEQ ID NO:4.

[0009] Monoclonal antibodies possessing the aforementioned heavy chain variable region and light chain variable region can specifically bind to the α3 region of the MICA / B protein expressed on the surface of tumor cells, effectively blocking the shedding of MICA / B from the tumor cell membrane (inhibition rate can reach over 86%), and do not affect the binding between MICA / B and NKG2D (a major activating receptor of NK cells). Therefore, they can enhance the killing effect of NK cells on tumor cells, prevent tumor cells from "immune escape", and thus effectively inhibit the growth and metastasis of tumors in vivo.

[0010] Preferably, the monoclonal antibody is a chimeric antibody, a humanized antibody, a nanobody, a multivalent antibody, or a functional fragment of an antibody.

[0011] Second, the present invention provides a nucleic acid molecule encoding the monoclonal antibody, comprising a nucleotide sequence encoding a heavy chain variable region and a nucleotide sequence encoding a light chain variable region; the nucleotide sequence encoding the heavy chain variable region includes SEQ ID NO:5 or SEQ ID NO:6; the nucleotide sequence encoding the light chain variable region includes SEQ ID NO:7 or SEQ ID NO:8.

[0012] Third, the present invention provides a cloning or expression vector comprising the following (a) and / or (b):

[0013] (a) A nucleotide sequence comprising the heavy chain variable region of the nucleic acid molecule;

[0014] (b) A nucleotide sequence comprising the light chain variable region of the nucleic acid molecule.

[0015] Preferably, in the vector, the DNA fragment encoding the heavy chain variable region and the light chain variable region can be effectively linked with the DNA fragment encoding the antibody constant region or the flexible linker.

[0016] Fourth, the present invention provides a host cell containing the nucleic acid molecule or the vector.

[0017] Preferably, the host cell is a CHO cell, a HEK-293 cell, or a PER.C6 cell.

[0018] Fifth, the present invention provides a method for preparing a monoclonal antibody, comprising the following steps: expressing and isolating the monoclonal antibody against the human MICA / Bα3 region in the host cells.

[0019] Sixth, the present invention provides a composition comprising the monoclonal antibody, the composition further comprising a drug that promotes MICA / B expression and / or a drug that enhances cellular immunity.

[0020] When the monoclonal antibody of the present invention is used in combination with drugs that promote MICA / B expression and / or drugs that enhance cellular immunity, it can be administered simultaneously or alone, and both can exert a synergistic effect of increasing the MICA / B level on the surface of tumor cell membranes and / or enhancing the anti-tumor immune response.

[0021] Preferably, the drug that promotes MICA / B expression is a histone deacetylase inhibitor (HDACi); and the drug that enhances cellular immunity is a PD-1 and / or CTLA-4 immune checkpoint inhibitor.

[0022] Sixth, the present invention provides the use of the monoclonal antibody, the nucleic acid molecule, the vector, the host cell, or the composition in the preparation of a drug, the drug being used to reduce the shedding of MICA / B from the cell membrane surface and / or enhance the killing ability of NK cells.

[0023] Preferably, the drug is used to prevent or treat MICA / B+ cancer or MICA / B-related immune diseases.

[0024] Furthermore, the MICA / B+ cancers are melanoma, breast cancer, lung cancer, pancreatic cancer, colorectal cancer, prostate cancer, stomach cancer, esophageal cancer, bladder cancer, ovarian cancer, kidney cancer, thyroid cancer, leukemia, myeloma, or lymphoma.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) The nucleotide sequence of the monoclonal antibody encoding the anti-human MICA / Bα3 region of the present invention is different from the nucleotide sequence of the monoclonal antibody against the anti-human MICA / Bα3 region reported in existing literature or patents.

[0027] (2) The monoclonal antibody against the human MICA / B α3 region of the present invention can specifically bind to the α3 region of the MICA / B protein expressed on the surface of tumor cells, reduce the shedding of MICA / B, and do not affect the binding between MICA / B and NKG2D, thus enhancing the killing ability of NK cells and exerting an anti-tumor effect.

[0028] (3) The mechanism of action of the monoclonal antibody against the human MICA / Bα3 region of the present invention in tumor treatment is mediated by NK cells, which can provide a potential new clinical option when the body develops tolerance to the cytotoxic CD8+ T cell immunity induced by immune checkpoint inhibitors.

[0029] (4) The monoclonal antibody against the human MICA / Bα3 region of the present invention can be used in combination with drugs that promote MICA / B expression, such as histone deacetylase inhibitors, or drugs that enhance cellular immunity, such as immune checkpoint inhibitors, to synergistically improve the anti-tumor immune response. It has good clinical translational value and application prospects. Attached Figure Description

[0030] Figure 1 Scatter plot for FCM detection of the inhibitory effects of MIA-2 and MIA-9 monoclonal antibodies on MICA / B shedding from cell membranes;

[0031] Figure 2 The recombinant protein MICA α3 was identified. Figure A shows the identification by SDS-PAGE electrophoresis (left: after renaturation, right: after purification); Figure B shows the identification by mass spectrometry that the recombinant protein is MICA α3 protein.

[0032] Figure 3 Serum titers of mice 1-5 were detected by ELISA;

[0033] Figure 4 Scatter plot of the binding ability of MIA-2 and MIA-9 monoclonal antibodies to MICA / B on the cell membrane surface of HCT116 (top) and LS-174T (bottom) cells for FCM detection;

[0034] Figure 5 The peak superposition histogram for FCM detection of the binding of MICA / B to NKG2D after binding of MIA-2 monoclonal antibody to cell membrane;

[0035] Figure 6The bar chart shows the MICA / B levels on the cell surface of DLD-1 cells after treatment with MIA-2 monoclonal antibody, SAHA, and MIA-2 monoclonal antibody + SAHA by FCM. *P<0.05;

[0036] Figure 7 The bar chart shows the LDH levels released into the supernatant after treatment with MIA-2 monoclonal antibody, Nivolumab, and MIA-2 monoclonal antibody + Nivolumab, respectively, on NK cells co-cultured with HCT116 cells. *P<0.05, **P<0.01;

[0037] Figure 8 To investigate the inhibition of mouse xenograft growth by MIA-2 monoclonal antibody, Figure A shows gross images of subcutaneous xenografts constructed in C57BL / 6 mice using B16F10-MICA cells, including the control group (top), the 250 μg MIA-2 monoclonal antibody group (middle), and the 750 μg MIA-2 monoclonal antibody group (bottom). Figure B shows the growth curves of subcutaneous xenografts in different treatment groups. Figure C is a bar chart comparing the weight of subcutaneous xenografts in different treatment groups. *P<0.05, **P<0.01;

[0038] Figure 9 A bar chart showing the MICA / B level on the surface of transplanted tumor cells in mice treated with MIA-2 monoclonal antibody (250 μg and 750 μg) and in the control group, as detected by FCM.

[0039] Figure 10 A bar chart showing the serum sMICA / B levels of mice in the MIA-2 monoclonal antibody treatment group (250 μg and 750 μg) and the control group as detected by ELISA;

[0040] Figure 11 Gross photographs of lung tissue in the control group (top) and the MIA-2 monoclonal antibody treatment group (bottom) to construct a lung metastasis model in C57BL / 6 mice using B16F10-MICA cells;

[0041] Figure 12 A bar chart showing the MICA / B level on the surface of lung metastatic tumor cells in mice in the FCM detection group and the control group. Detailed Implementation

[0042] The present invention will be further described below with reference to embodiments.

[0043] The terminology used in the embodiments of this invention is for describing specific implementations and not for limiting the scope of protection of this invention. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims and any equivalents thereof. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and biological materials described are commercially available unless otherwise specified.

[0044] Example 1: Preparation of monoclonal antibodies against human MICA / Bα3

[0045] (1) Recombinant expression of human MICA α3 protein

[0046] Using the MICA-pCMV6-XL5 plasmid (Origene) as a template, we designed upstream and downstream primers: 5'-ACTGTCAGGATCCAAATCCGGCGTAGTCCTGAGGAGAAC-3' and 5'-CTAGTCTTC TCGAGTTAATGGAATGTCTGCCAATGACTCTGAAGC-3', respectively. After PCR amplification, XhoI and BamHI double digestion, and ligation into the pET32a vector, we constructed the MICAα3-pET32a prokaryotic expression plasmid. This plasmid was transformed into competent BL21-DE3 *E. coli* cells, and expression was induced by IPTG. The recombinant protein was renatured using reduced / oxidized glutathione and purified using a Ni2+-NTA column. Finally, purity was identified by SDS-PAGE electrophoresis and authenticity was verified by LC-MS / MS. The results are shown below. Figure 2 As shown, we successfully expressed the human MICAα3 protein.

[0047] (2) Subcutaneous immunization in mice

[0048] Five 6-week-old Balb / c mice were prepared and, after acclimatizing within the barrier for one week, each group was labeled with picric acid on the head, back, tail, foreleg, and hind leg, respectively, and numbered 1-5. The first immunization used Freund's complete adjuvant, and subsequent immunizations used Freund's incomplete adjuvant. 100 μl of adjuvant was drawn into a 2 ml syringe and mixed 1:1 with 0.6 mg of the recombinant human MICA α3 protein expressed above. The two syringes were connected by an infusion tubing, and the syringes were pushed sequentially until the antigen-adjuvant mixture became a milky white water-in-oil emulsion, with no dripping when the syringe was inverted. Subcutaneous immunization was performed at 200 μg / mouse, taking samples from 4-5 points on the back of the neck. A second immunization was given 21 days after the first, and subsequent immunizations were spaced 14 days apart. After the sixth immunization, 50 μl of blood was collected from the orbital sinus of the mice, centrifuged at 10,000 rpm for 15 min, and the supernatant was used for subsequent titer determination.

[0049] (3) ELISA detection of serum titer

[0050] MICAα3 recombinant protein was diluted to a concentration of 25 μg / ml with coating buffer (CBS, pH 9.6), coated into 96-well plates (JET BIOFIL), and incubated overnight at 4°C. The next day, the liquid in the wells was discarded, and the plates were washed four times with 1×TBST buffer and blotted dry. Each well was then filled with 5% BSA for blocking, incubated at 37°C for 1 h, and blotted dry. Immunized mouse serum was serially diluted 1:100, and 100 μl was added to each well. After incubation at 37°C for 1 h, the plates were washed four times and blotted dry. Then, 100 μl of goat anti-mouse secondary antibody-HRP diluted (1:3000) was added to each well, incubated at 37°C for 30 min, washed four times, and blotted dry. 50 μl of TMB solution A and 50 μl of TMB solution B were added to each reaction well, and the reaction was incubated at room temperature for approximately 5 min. The reaction was terminated by adding 50 μl of 2M stop solution. The absorbance at 450 nm was read using a microplate reader (Molecular Devices, USA).

[0051] The results are as follows Figure 3 As shown, mice 1, 3, and 4 had better titers, so we selected mouse 4, which had the highest titer, and injected it intraperitoneally with 100 μg of MICA α3 recombinant protein for antigen challenge.

[0052] (4) Hybridoma cell fusion

[0053] Mice were euthanized 3-5 days after antigen challenge by exsanguination of the eyeballs, cervical dislocation, and aseptic isolation of the spleen. The spleen cells were washed three times with IMEM culture medium containing 5× antibiotics. Splenic cells were collected using a syringe and transferred to 15ml centrifuge tubes. The tubes were centrifuged at 1,500 rpm for 10 min, the supernatant was discarded, and the cell pellet was washed twice before counting. SP2 / 0 cells in logarithmic growth phase were digested, centrifuged at 1,500 rpm for 5 min, and resuspended in 15ml of warm IMEM culture medium. After counting, the pellet was mixed with spleen cells at a 1:10 ratio and centrifuged at 1,500 rpm for 5 min. After 1 minute, discard the supernatant, drain the cells on sterile absorbent paper, carefully add 1 ml of 50% PEG (MW4000) along the wall of the centrifuge tube within 1 minute, and incubate at 37°C for 1 minute. Take 40 ml of IMEM culture medium and carefully add it to the cells along the wall of the centrifuge tube. Centrifuge at 1,500 rpm for 5 minutes, discard the supernatant, add DMEM culture medium, carefully resuspend the cells, and then spread the hybridoma cell suspension into a 96-well plate. After 6 hours, add 100 μl of 2×HAT culture medium to each well to complete cell fusion. After 8-10 days, collect the cell culture supernatant for screening.

[0054] (5) ELISA screening for positive clones

[0055] Dilute MICA α3 recombinant protein and MICA eukaryotic protein to a concentration of 10 μg / ml with coating buffer (CBS, pH 9.6), coat 96-well plates (JET BIOFIL) and incubate overnight at 4°C. Subsequent washing and blocking are performed as in step (3). After blocking, according to the cloning order of the 96-well plate, use a multichannel pipette to aspirate 30 μl of cell culture supernatant from the wells and add it to the sample wells of the ELISA plate. Add blocking buffer to each well to a total of 100 μl and incubate at 37°C for 60 min. Subsequent washing, adding secondary antibody, washing, and color development are performed as in step (3). Positive clones selected by MICA α3 recombinant protein are then screened a second time by MICA eukaryotic protein. After two limited clonings, single clones that can specifically bind to the eukaryotic conformation of MICA α3 recombinant protein are obtained. The corresponding hybridoma cells are then expanded and cryopreserved for seeding.

[0056] (6) Ascites fluid antibody collection and purification

[0057] Eight-week-old Balb / c mice were intraperitoneally injected with 500 μl of paraffin oil per mouse one week before the experiment. The hybridoma cell suspension was transferred to a 15 ml centrifuge tube and centrifuged at 1,000 rpm for 5 min. The cell pellet was resuspended in an appropriate amount of physiological saline, mixed by pipetting, and then aspirated into a syringe at a dose of 5 × 10⁻⁶. 5 One cell per mouse was injected intraperitoneally, approximately 1 ml per mouse. When mice showed obvious ascites, they were euthanized by cervical dislocation, and the ascites fluid was aseptically aspirated into a 50 ml centrifuge tube. The tube was centrifuged at 1,000 rpm for 30 min, the oil layer was discarded, and the pale yellow liquid layer, i.e., the ascites antibody, was collected. The ascites sample was purified by Protein G chromatography with a 3-5 fold dilution, and concentrated by centrifugation at 10,000 rpm for 60 min using an ultrafiltration tube (MW30000) to obtain two purified anti-human MICA α3 region monoclonal antibodies, named MIA-2 and MIA-9.

[0058] The amino acid sequences of the heavy chain variable region and light chain variable region of the MIA-2 monoclonal antibody are shown in SEQ ID NO:1 and SEQ ID NO:3, respectively, and the nucleotide sequences encoding the heavy chain variable region and light chain variable region are shown in SEQ ID NO:5 and SEQ ID NO:7, respectively. The amino acid sequences of the heavy chain variable region and light chain variable region of the MIA-9 monoclonal antibody are shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, and the nucleotide sequences encoding the heavy chain variable region and light chain variable region are shown in SEQ ID NO:6 and SEQ ID NO:8, respectively.

[0059] Example 2: Flow cytometry detection of the binding ability of MIA-2 and MIA-9 monoclonal antibodies to MICA / B on the cell membrane surface. Based on the identification of two MICA / B-overexpressing cell lines, HCT116 and LS-174T, using a commercially available anti-human MICA / B flow cytometry antibody (6D4), we used flow cytometry (FCM) to detect the binding ability of MIA-2 and MIA-9 monoclonal antibodies to MICA / B on the cell membrane surface. HCT116 cells and LS-174T cells, after incubation in 96-well plates for 24 h, were digested with 0.02% EDTA solution and transferred to EP tubes. The cells were washed once with PBS, and the cell pellet was resuspended in 100 μl of FACS Buffer. Control groups, secondary antibody-only groups, isotype control antibody groups, MIA-2 monoclonal antibody experimental groups, and MIA-9 monoclonal antibody experimental groups were set up. The isotype control antibody, MIA-2 monoclonal antibody, and MIA-9 monoclonal antibody were added to each EP tube at a concentration of 10 μg / ml. The tubes were incubated at 4°C for 30 min, the supernatant was discarded, and the cells were washed twice with FACS Buffer. The cell pellet was resuspended in 100 μl of FACS Buffer, and PE-goat anti-mouse secondary antibody was added at 5 μl / tube. The tubes were incubated at 4°C in the dark for 30 min, 1 ml of FACS Buffer was added to each tube, and the tubes were centrifuged at 1,500 rpm for 5 min. The supernatant was discarded, and the cells were washed twice with FACS Buffer. Finally, the cell pellet was resuspended in 300 μl of FACS Buffer. After resuspending in buffer, the mixture was transferred to flow cytometry tubes and analyzed using a flow cytometer (Beckman Coulter, Inc., USA).

[0060] We used FlowJo V10.0 software to analyze streaming data, and the results are as follows: Figure 4 As shown, both MIA-2 and MIA-9 monoclonal antibodies showed significant binding to HCT116 and LS-174T colorectal cancer cell lines that highly expressed MICA / B on their cell membranes.

[0061] Example 3: Flow cytometry detection of the inhibitory rates of MIA-2 and MIA-9 monoclonal antibodies on MICA / B cell membrane shedding. PANC-1 cells in logarithmic growth phase were digested, counted, and analyzed at a ratio of 1×10⁻⁶. 5Cells were seeded per well in a 24-well plate, with 150 μl of DMEM culture medium added to each well. After 2 hours of cell attachment, 100 μl of 100 nM MIA-2 or MIA-9 monoclonal antibody was added to one well. A separate well contained a mouse IgG control antibody at the same concentration. Cells were incubated at 37°C for 24 hours. Cells were then digested with 0.02% EDTA solution and transferred to EP tubes. The cells were centrifuged at 1,500 rpm for 5 minutes, the supernatant was discarded, and the pellet was resuspended in PBS. After resuspending, the pellet was centrifuged at 1,500 rpm for 5 minutes. The cell pellet was resuspended in 100 μl of FACS Buffer. 5 μl of PE anti-human MICA / B flow cytometry antibody was added to each sample tube, and the tubes were incubated at 4°C in the dark for 30 minutes. 1 ml of FACS Buffer was added to each tube, and the tubes were centrifuged at 1,500 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was washed twice. After the final wash, FACS Buffer was added. Resuspend in 200 μl and analyze using a flow cytometer (Beckman Coulter, Inc., USA).

[0062] We used FlowJo V10.0 software to analyze streaming data, and the results are as follows: Figure 1 As shown, compared with the control group, MIA-2 monoclonal antibody and MIA-9 monoclonal antibody significantly inhibited the shedding of MICA / B from the cell membrane, with inhibition rates of 86.03% and 87.83%, respectively.

[0063] Example 4: Flow cytometry verification of whether the MIA-2 monoclonal antibody competitively inhibits the binding of MICA / B and NKG2D. PANC-1 cultured cells in logarithmic growth phase were digested with 0.02% EDTA solution, counted, and seeded into 96-well plates (1×10⁻⁶). 5 After tumor cells adhered to the well ( / well) for 1-2 hours, MIA-2 monoclonal antibody and isotype control antibody were added at concentration gradients of 0.1 nM, 1 nM, 10 nM, 100 nM, and 1 μM, respectively, and incubated in a 37°C CO2 incubator for 24 h. The cells were then digested with 0.02% EDTA solution and collected into EP tubes. The cells were washed twice with PBS, and the cell pellet was resuspended in 100 μl of PBS. Then, 10 μg / ml NKG2D recombinant protein was added, and the cells were incubated at 4°C for 1 h. The cells were washed three times with PBS, and the cells were divided into an NKG2D antibody group with PE fluorescence and an isotype control antibody group with PE fluorescence. After resuspending in 100 μl of PBS, 5 μl of flow cytometry antibody was added to each tube according to the group, and the cells were incubated at 4°C in the dark for 30 min. The cells were washed three times with PBS. After the last wash, the cells were resuspended in 300 μl of FACS Buffer and detected using a flow cytometer (Beckman Coulter, USA).

[0064] The results are as follows Figure 5As shown, as the antibody concentration gradually increases, the expression level of MICA / B on the cell surface increases, and the fluorescence intensity of the NKG2D binding site to MICA / B also increases proportionally, indicating that the MIA-2 monoclonal antibody specifically recognizes the MICA / B α3 region, but has no competitive inhibitory effect on the binding of MICA / B to NKG2D.

[0065] Example 5: MIA-2 monoclonal antibody combined with histone deacetylase inhibitor increases MICA / B levels on cell membrane surface. DLD-1 cells in logarithmic growth phase were digested, counted, and analyzed at a ratio of 1×10⁻⁶. 5 Cells were seeded per well in 24-well plates, and DMEM culture medium was added to each well to a final volume of 150 μl. After 2 hours of cell adhesion, the following groups were established: isotype control antibody group, MIA-2 monoclonal antibody treatment group, histone deacetylase inhibitor (SAHA) treatment group, and MIA-2 monoclonal antibody + SAHA combination therapy group. In this study, 100 μl of 50 nM MIA-2 monoclonal antibody was added to the MIA-2 monoclonal antibody treatment group, 100 μl of 100 nM SAHA was added to the SAHA treatment group, and 50 μl of 50 nM MIA-2 monoclonal antibody and 50 μl of 100 nM SAHA were added to the combination treatment group. The mixture was incubated at 37°C for 24 h in a CO2 incubator. Cells were digested with 0.02% EDTA solution and transferred to EP tubes. The cells were centrifuged at 1,500 rpm for 5 min, the supernatant was discarded, and the pellet was resuspended in PBS. After mixing, the pellet was centrifuged at 1,500 rpm for 5 min. The cell pellet was resuspended in 100 μl of FACS Buffer. 5 μl of PE anti-human MICA / B flow cytometry antibody was added to each sample tube, and the mixture was incubated at 4°C in the dark for 30 min. 1 ml of FACS Buffer was added to each tube, and the mixture was centrifuged at 1,500 rpm for 5 min. The supernatant was discarded, and the cell pellet was washed twice. After the final wash, FACS Buffer was added. Resuspend in 200 μl of buffer and analyze using a flow cytometer (Beckman Coulter, Inc., USA).

[0066] The results are as follows Figure 6 As shown, the combined use of SAHA and MIA-2 monoclonal antibody increased the MICA / B level on the surface of DLD-1 cells compared with the use of SAHA or MIA-2 antibody alone, and the difference was statistically significant (P < 0.05).

[0067] Example 6: MIA-2 monoclonal antibody combined with PD-1 inhibitor enhances NK cell killing activity. We used freshly extracted PBMCs at a ratio of 2×10 5 Seed cells per well into a 96-well plate with a round bottom. Add 1000 U / ml of recombinant human IL-2 to each well, mix well, and incubate overnight at 37°C using a CO2 incubator. The next day, collect HCT116 cells in the logarithmic growth phase, digest and count them, wash once with PBS, resuspend the cell pellet in PBS, and dilute to 1×10⁻⁶.5 Cells / ml; simultaneously, PBMC cells incubated in the 96-well plate were transferred to 1.5 ml centrifuge tubes, centrifuged at 1,000 rpm for 5 min, and the resulting PBMC cell pellet was resuspended in PBS and diluted to 1×10⁻⁶ cells / ml. 6 Cells / ml; then PBMC cells and HCT116 cells were mixed at a ratio of 10:1, and the resulting cell suspension was added to 200 μl / well of a round-bottom 96-well plate. Subsequently, control IgG, MIA-2 monoclonal antibody, PD-1 inhibitor (Nivolumab), or MIA-2 monoclonal antibody + Nivolumab were added to the experimental wells, mixed well, and incubated at 37°C in a CO2 incubator for 4 h. After that, the cell suspension was transferred to a 1.5 ml centrifuge tube, centrifuged at 1,000 rpm for 10 min, and the cell supernatant was collected. The LDH content was measured on a fully automated biochemical analyzer to determine the effect of the antibody on NK cell killing activity.

[0068] The results are as follows Figure 7 As shown, the addition of either Nivolumab or MIA-2 monoclonal antibody increased the LDH content in the cell supernatant compared to the control antibody (P < 0.01), with the effect of MIA-2 monoclonal antibody being more pronounced. When Nivolumab and MIA-2 monoclonal antibody were used in combination, the LDH content in the cell supernatant further increased (P < 0.05), suggesting that the two can synergistically enhance the killing activity of NK cells.

[0069] Example 7: MIA-2 monoclonal antibody inhibits the growth of xenografts in mice

[0070] We transfected the packaged MICA-GFP lentiviral vector into adherent B16F10 melanoma cells in logarithmic growth phase. GFP+ cells were sorted using a flow cytometry system (Beckman Coulter, USA) to obtain a stable B16F10 cell line expressing MICA. After routine culture and passage to the third generation, the cells were digested and counted. The digested cells were washed twice with PBS, and the resulting cell pellet was resuspended in an appropriate amount of serum-free culture medium according to the required cell volume. After mixing thoroughly by pipetting, the cells were placed on ice and seeded into the left axillary fat pad of 5-week-old C57BL / 6 mice at a volume of 200 μl per mouse (approximately 1 × 10⁶ cells). 6 (cells / animal); MIA-2 monoclonal antibody treatment group (250μg / animal and 750μg / animal) and isotype antibody control group were set up. One day before subcutaneous cell inoculation, MIA-2 monoclonal antibody or isotype control antibody was injected intraperitoneally at a dose of 250μg / 200μl. The drugs were administered on days 4, 7, and 10 after inoculation. Tumor growth was monitored twice weekly, and the long and short diameters of the tumor were measured using electronic calipers. The volume was calculated as long diameter × long diameter × short diameter / 2. When the tumor volume reached 2000 mm², the tumor growth was monitored. 3Mice were euthanized when tumor necrosis occurred, survival rates were calculated, tumors were removed, weighed, photographed, and tumor volume growth curves and survival curves were plotted using GraphPad Prism software.

[0071] The results are as follows Figure 8 As shown, compared with the control, the tumor volume and weight of mice treated with intraperitoneal injection of MIA-2 monoclonal antibody were significantly reduced, and the difference was statistically significant (P<0.01), indicating that MIA-2 monoclonal antibody can inhibit tumor growth in vivo.

[0072] Example 8: MIA-2 monoclonal antibody inhibits the shedding of MICA / B xenograft cells in mice.

[0073] The subcutaneous xenografts from C57BL / 6 mice in Example 7 were dissected, washed 2-3 times with sterile PBS, and the tumor tissue was minced with sterile ophthalmic scissors. Approximately 5 ml of serum-free RPMI 1640 culture medium containing 1 mg / ml type IV collagenase and 0.5 mg / ml hyaluronidase was added, and the mixture was transferred to a 15 ml centrifuge tube. The tube was incubated at 37°C on a shaker for 60 min. The digested tissue suspension was passed through a 40 μm cell filter, and the filtered cell suspension was collected. The suspension was centrifuged at 1,500 rpm for 10 min, and the cell pellet was washed once with PBS. A single-staining 6D4 antibody group and a single-staining isotype control group were set up. The tumor cell pellet was resuspended in FACS Buffer, and 100 μl was aliquoted into each EP tube. 5 μl of PE anti-human MICA / B flow cytometry antibody (6D4) or isotype control antibody was added, and the tubes were incubated at 4°C in the dark for 30 min. 1 ml of FACS Buffer was added, and the tubes were centrifuged at 1,500 rpm for 5 min. Wash twice more with buffer. After the final wash, resuspend the precipitate in 300 μl of FACS buffer and use a flow cytometer (Beckman Coulter, USA) to detect the expression of MICA / B on the surface of GFP+ tumor cells.

[0074] The results are as follows Figure 9 As shown, in the mouse xenograft model constructed from B16F10-MICA cells, the mean fluorescence intensity of MICA / B on the surface of tumor cells in the low-dose (250 μg) MIA-2 monoclonal antibody treatment group was 21961.33±2467.34, and the mean fluorescence intensity of MICA / B on the surface of tumor cells in the high-dose (750 μg) MIA-2 monoclonal antibody treatment group was 32291.33±7406.68, which were significantly higher than the mean fluorescence intensity of MICA / B on the surface of tumor cells in the control IgG treatment group (5147±28.30).

[0075] Furthermore, we enucleated the eyeballs of the mice and collected peripheral blood in EP tubes. After centrifugation at 5,000 rpm for 10 min, we carefully aspirated the supernatant serum into a new EP tube and measured the serum sMICA / B concentration of the mice according to the instructions of the Human sMICA / B ELISA kit (Thermo Fisher Scientific, USA).

[0076] The results are as follows Figure 10 As shown, the serum sMICA / B level in the low-dose (250 μg) MIA-2 monoclonal antibody treatment group was 5.57 ± 3.19 pg / ml, and the serum sMICA / B level in the high-dose (750 μg) MIA-2 monoclonal antibody treatment group was 14.26 ± 7.12 pg / ml, both lower than the serum sMICA / B level of 66.04 ± 29.68 pg / ml in the control IgG treatment group. This indicates that the MIA-2 monoclonal antibody can inhibit the shedding of MICA / B from the tumor cell membrane and reduce the release of sMICA / B in vivo.

[0077] Example 9: MIA-2 monoclonal antibody inhibits the growth of mouse metastatic tumors

[0078] After resuscitation, the B16F10 cell line stably expressing MICA from Example 7 was routinely cultured. At passage 3, the cells were digested and counted. The digested cells were washed twice with PBS. The resulting cell pellet was resuspended in an appropriate amount of physiological saline according to the required cell volume, mixed thoroughly by pipetting, and placed on ice. Using a 1ml syringe, 200μl of the pellet was seeded into the tail vein of 5-week-old C57BL / 6 mice (approximately 5 × 10⁶ cells / mL). 5 (cells / mouse); set up a MIA-2 monoclonal antibody treatment group and an isotype antibody control group. The MIA-2 antibody or isotype control antibody was injected intraperitoneally at a dose of 250μg / 200μl. The drugs were administered on days 2, 5, 7 and 10 after inoculation. On day 21 after inoculation, the mice were sacrificed, the thoracic cavity was opened, the lung lobes of the mice were separated and photographed.

[0079] The results are as follows Figure 11 As shown, after treatment with MIA-2 monoclonal antibody, the number of melanoma metastatic nodules in the lungs of mice inoculated with melanoma cells was significantly reduced, indicating that MIA-2 monoclonal antibody has an anti-tumor metastasis effect in vivo.

[0080] Example 10: MIA-2 Monoclonal Antibody Inhibits the Shedding of MICA / B Metastatic Tumor Cells in Mice. We washed the fresh mouse lung tissue from Example 9 2-3 ​​times with sterile PBS. After mincing the lung tissue with sterile ophthalmic scissors, we added approximately 7 ml of serum-free RPMI 1640 culture medium containing 1 mg / ml type IV collagenase, 0.5 mg / ml hyaluronidase, and 20 U / ml DNase. The mixture was transferred to a 15 ml centrifuge tube and digested on a shaker at 37°C for 60 min. The digested tissue suspension was passed through a 40 μm cell filter, and the filtered cell suspension was collected. The cells were centrifuged at 1,500 rpm for 10 min, and the cell pellet was washed once with PBS to obtain a mixture of lung epithelial cells and tumor cells. We set up a single-staining 6D4 antibody and a single-staining isotype control group. The tumor cell pellet was resuspended in FACS Buffer, and 100 μl was dispensed into each EP tube. PE anti-human antibodies were added. 5 μl of MICA / B flow cytometry antibody (6D4) or isotype control antibody was incubated at 4°C in the dark for 30 min. 1 ml of FACS Buffer was added, and the cells were centrifuged at 1,500 rpm for 5 min. The cells were washed twice with FACS Buffer. After the last wash, the cells were resuspended in 300 μl of FACS Buffer. The expression of MICA / B on the surface of GFP+ tumor cells was detected using a flow cytometer (Beckman Coulter, USA).

[0081] The results are as follows Figure 12 As shown, the average fluorescence intensity of MICA / B on the surface of tumor cells in the MIA-2 monoclonal antibody treatment group was 53067.75±5122.81, which was significantly higher than that in the control group (17782.25±5266.35). This indicates that the MIA-2 monoclonal antibody can stably metastasize MICA / B on the surface of tumor cell membranes in vivo.

[0082] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields.

[0083] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody against the human MICA / B α3 region, characterized in that, It includes the heavy chain variable region and the light chain variable region, as shown in SEQ ID NO:2 and SEQ ID NO:4, respectively.

2. A nucleic acid molecule encoding the monoclonal antibody as described in claim 1, characterized in that, This includes nucleotide sequences encoding the variable region of the heavy chain and nucleotide sequences encoding the variable region of the light chain.

3. The nucleic acid molecule as described in claim 2, characterized in that, The nucleotide sequence encoding the variable region of the heavy chain is SEQ ID NO:

6.

4. The nucleic acid molecule as described in claim 2, characterized in that, The nucleotide sequence encoding the variable region of the light chain is SEQ ID NO:

8.

5. A cloning or expression vector, characterized in that, Includes the following (a) and (b): (a) A nucleotide sequence comprising the heavy chain variable region in a nucleic acid molecule as described in any one of claims 2 to 4; (b) A nucleotide sequence encoding a light chain variable region in a nucleic acid molecule as described in any one of claims 2 to 4.

6. A host cell, characterized in that, It includes the nucleic acid molecule as described in any one of claims 2 to 4.

7. A host cell, characterized in that, It includes the carrier as described in claim 5.

8. A method for preparing a monoclonal antibody, characterized in that, Includes the following steps: The monoclonal antibody against the human MICA / B α3 region is expressed and isolated in the host cells as described in claim 6 or 7.

9. The use of the monoclonal antibody as described in claim 1, or the nucleic acid molecule as described in any one of claims 2 to 4, or the vector as described in claim 5, or the host cell as described in claim 6 or 7, in the preparation of a drug, characterized in that, The drug is used to reduce the shedding of MICA / B from the surface of pancreatic cancer cell membranes.

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