An anti-bcma antibody or antigen-binding fragment thereof, and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses an anti-BCMA antibody or antigen binding fragment thereof, and a preparation method and application thereof. The amino acid sequences of CDR1-3 of the heavy chain variable region of the anti-BCMA antibody or antigen binding fragment thereof are respectively shown as SEQ ID NO:2-4, and the amino acid sequences of CDR1-3 of the light chain variable region are respectively shown as SEQ ID NO:6-8. The anti-BCMA antibody or antigen binding fragment thereof has excellent affinity to BCMA protein, especially the extracellular segment, can specifically recognize and combine the BCMA protein well, can be applied as a detection antibody of the BCMA protein to realize non-diagnostic purpose detection of BCMA. Meanwhile, the anti-BCMA antibody or antigen binding fragment thereof can realize effective killing of tumor cells overexpressing BCMA, such as pfeiffer cells, through complement-dependent cytotoxicity, and the cell killing rate is as high as 99%, and can be used as an inhibitor of abnormal bone marrow cells overexpressing BCMA protein, is an extremely potential active component for treating hematological malignancies, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an anti-BCMA antibody or an antigen-binding fragment thereof, and a preparation method and application thereof. Background Art

[0002] BCMA, or tumor necrosis factor receptor superfamily member 17 (TNFRSF17), also known as B cell maturation antigen (CD269), plays a key role in promoting B cell survival and regulating humoral immune responses. BCMA is primarily expressed on the surface of mature B lymphocytes and plasma cells and is a marker of B lymphocyte maturation. BCMA's agonist ligands include proliferation-inducing ligand (APRIL) and B cell activating factor (BAFF), which are primarily secreted paracrine-like in the bone marrow by bone marrow stromal cells, osteoclasts, and macrophages. BCMA is highly correlated with various hematologic malignancies, including multiple myeloma. Overexpressed BCMA in abnormal myeloid cells binds to agonist ligands, leading to overactivation of multiple signaling pathways within the cells, including NF-κβ, RAS / MAPK, and PI3K-PKB / Akt. This leads to overexpression of anti-apoptotic proteins, cell adhesion molecules, angiogenic factors, and immunosuppressive molecules, further enhancing the growth, proliferation, and spread of abnormal myeloid cells. Moreover, BCMA has similar expression levels in different stages of multiple myeloma, suggesting that BCMA has the potential to serve as an effective therapeutic target for hematological malignancies.

[0003] Therefore, obtaining antibodies with excellent specific recognition and binding capabilities for BCMA is of great significance for the development of immunotherapy for various hematological malignancies such as multiple myeloma. Summary of the Invention

[0004] In the first aspect, the present invention provides an anti-BCMA antibody or an antigen-binding fragment thereof, wherein the amino acid sequences of the heavy chain variable region CDR1 to 3 of the anti-BCMA antibody or the antigen-binding fragment thereof are shown as SEQ ID NOs: 2 to 4, respectively, and the amino acid sequences of the light chain variable region CDR1 to 3 are shown as SEQ ID NOs: 6 to 8, respectively, and the anti-BCMA antibody or the antigen-binding fragment thereof has excellent specific recognition and binding ability for BCMA.

[0005] Furthermore, in the anti-BCMA antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 5 or a variant sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO: 5 and retains the biological activity of the corresponding parent sequence, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 9 or a variant sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO: 9 and retains the biological activity of the corresponding parent sequence.

[0006] Furthermore, in the anti-BCMA antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 27 or a variant sequence that has at least 80% homology to the amino acid sequence shown in SEQ ID NO: 27 and retains the biological activity of the corresponding parent sequence, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 28 or a variant sequence that has at least 80% homology to the amino acid sequence shown in SEQ ID NO: 28 and retains the biological activity of the corresponding parent sequence.

[0007] Furthermore, the anti-BCMA antibody or antigen-binding fragment thereof comprises a signal peptide having a sequence as shown in SEQ ID NO: 26, and the signal peptide is connected to the N-terminus of the heavy chain variable region and / or the N-terminus of the light chain variable region.

[0008] Furthermore, the anti-BCMA antibody or antigen-binding fragment thereof is selected from one or more of Fab, Fab', F(ab')2, Fd, Fv, dAb, single-chain antibody, chimeric antibody, multispecific antibody, monoclonal antibody and polyclonal antibody.

[0009] In a second aspect, the present invention further provides a nucleic acid molecule encoding the above-mentioned anti-BCMA antibody or antigen-binding fragment thereof.

[0010] In a third aspect, the present invention further provides a cell line that expresses and secretes the above-mentioned anti-BCMA antibody or antigen-binding fragment thereof.

[0011] In a fourth aspect, the present invention also provides a method for preparing the above-mentioned anti-BCMA antibody or its antigen-binding fragment, which specifically comprises: culturing the above-mentioned cell line under suitable conditions, and recovering the anti-BCMA antibody or its antigen-binding fragment from the cell culture.

[0012] In a fifth aspect, the present invention also provides the use of the above-mentioned anti-BCMA antibodies or antigen-binding fragments thereof, nucleic acid molecules and / or cell lines in BCMA detection for non-diagnostic purposes.

[0013] In a sixth aspect, the present invention also provides the use of the above-mentioned anti-BCMA antibodies or antigen-binding fragments thereof, nucleic acid molecules and / or cell lines in the preparation of inhibitors of abnormal bone marrow cells.

[0014] In a seventh aspect, the present invention further provides an anti-tumor drug, which comprises the above-mentioned anti-BCMA antibody or antigen-binding fragment thereof, nucleic acid molecule and / or cell line.

[0015] Beneficial effects:

[0016] The anti-BCMA antibody or antigen-binding fragment thereof provided in the present invention has excellent affinity for BCMA protein, especially its extracellular segment, and can specifically recognize and bind to BCMA protein in different states. It can be used as a detection antibody for BCMA protein to achieve non-diagnostic detection of BCMA. At the same time, the anti-BCMA antibody or antigen-binding fragment thereof can also effectively kill tumor cells overexpressing BCMA, such as Pfeiffer cells, through complement-dependent cytotoxicity, with a tumor cell killing rate of up to 99%. It can be used as an inhibitor of abnormal bone marrow cells overexpressing BCMA protein, and is a highly promising active ingredient for treating hematological malignancies with good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the experimental results of the antibody titer test of the anti-BCMA antibody provided in Example 1 of the present invention;

[0018] Figure 2 This is a graph showing the experimental results of testing the affinity of the anti-BCMA antibody for tumor cells provided in Example 2 of the present invention;

[0019] Figure 3 This is a graph showing the experimental results of testing the tumor cell killing ability (concentration) of the anti-BCMA antibody provided in Example 2 of the present invention;

[0020] Figure 4 Figure 2 shows the experimental results of testing the tumor cell killing ability (concentration) of the anti-BCMA antibody provided in Example 2 of the present invention;

[0021] Figure 5 This is a graph showing the experimental results of testing the tumor cell killing ability (incubation time) of the anti-BCMA antibody provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0022] The nucleotide sequences and amino acid sequences involved in the present invention are specifically shown in Table 1.

[0023] Table 1.

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030] The present invention provides an anti-BCMA antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region of the anti-BCMA antibody or the antigen-binding fragment thereof is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 9.

[0031] In the present invention, the CDR1-3 on the heavy chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof pairs with the CDR1-3 on the light chain variable region and folds to form an antigen-binding site for the BCMA extracellular segment protein. This antigen-binding site has good binding ability to the BCMA extracellular segment protein and can specifically recognize and bind to free BCMA protein or BCMA protein on bone marrow cells. The amino acid sequences of the heavy chain variable region CDR1-3 are shown in SEQ ID NOs: 2-4, respectively, and the amino acid sequences of the light chain variable region CDR1-3 are shown in SEQ ID NOs: 6-8, respectively.

[0032] It should be noted that in the non-CDR1 to 3 regions of the heavy chain variable region of the anti-BCMA antibody or its antigen-binding fragment, changes in some amino acid sequences have a certain effect on the ability of the antibody to bind to the BCMA extracellular segment protein, but the effect is relatively small compared to changes in amino acids in the CDR region. Therefore, variant sequences that have at least 90% homology to the amino acid sequence shown in SEQ ID NO: 5 and retain the biological activity of the corresponding parent sequence are also covered within the scope of protection of the present invention. In some specific embodiments, the homology of the above variant sequence to the amino acid sequence shown in SEQ ID NO: 5 can be 90%, 92.5%, 95%, 97.5%, 99.9999% or any value therebetween.

[0033] It should be noted that in the non-CDR1 to 3 regions of the light chain variable region of the anti-BCMA antibody or its antigen-binding fragment, changes in some amino acid sequences have a certain effect on the ability of the antibody to bind to the BCMA extracellular segment protein, but the effect is relatively small compared to changes in amino acids in the CDR region. Therefore, variant sequences that have at least 90% homology to the amino acid sequence shown in SEQ ID NO: 9 and retain the biological activity of the corresponding parent sequence are also covered by the protection scope of the present invention. In some specific embodiments, the homology of the above variant sequence to the amino acid sequence shown in SEQ ID NO: 9 can be 90%, 92%, 94%, 96%, 98%, 99.9999% or any value therebetween.

[0034] In the present invention, the amino acid sequence of the heavy chain constant region of the anti-BCMA antibody or antigen-binding fragment thereof is preferably as shown in SEQ ID NO: 27 or a variant sequence that has at least 80% homology to the amino acid sequence shown in SEQ ID NO: 27 and retains the biological activity of the corresponding parent sequence, and the amino acid sequence of the light chain constant region is preferably as shown in SEQ ID NO: 28 or a variant sequence that has at least 80% homology to the amino acid sequence shown in SEQ ID NO: 28 and retains the biological activity of the corresponding parent sequence.

[0035] In the present invention, the anti-BCMA antibody or antigen-binding fragment thereof preferably further comprises a signal peptide having a sequence as shown in SEQ ID NO: 26, and the signal peptide is linked to the N-terminus of the heavy chain variable region and / or the N-terminus of the light chain variable region.

[0036] In the present invention, the type of the anti-BCMA antibody or antigen-binding fragment thereof can be, but is not limited to, one or more of: Fab, F(ab')2, Fd, Fv, dAb, single-chain antibody, chimeric antibody, monoclonal antibody and polyclonal antibody.

[0037] It should be noted that Fab refers to the antigen-binding fragment obtained after papain hydrolysis of an antibody molecule, specifically the fragment formed by the heavy chain variable region and the light chain variable region linked by SS. F(ab')2 refers to the macromolecular fragment obtained after pepsin hydrolysis of an antibody molecule, specifically the fragment formed by the SS linkage between two Fab heavy chain variable regions. It has bivalent antibody activity and can bind to two corresponding BCMA protein antigenic determinants. The Fd fragment refers to the smallest antibody fragment containing complete antigen recognition and antigen binding sites. The Fv fragment contains a dimer formed by a heavy chain variable region domain and a light chain variable region domain in a tight non-covalent manner. The heavy chain variable region domain and the light chain variable region domain fold to produce six hypervariable loops. The amino acid residues in the hypervariable loops confer antigen-antibody binding specificity. The dAb fragment is a domain antibody, the smallest known antigen-binding fragment of an antibody, which can be prepared by culturing microbial cells and has good biological activity and stability.

[0038] Based on the purpose of obtaining the above-mentioned anti-BCMA antibody or antigen-binding fragment thereof, the present invention also provides a nucleic acid molecule encoding the above-mentioned anti-BCMA antibody or antigen-binding fragment thereof.

[0039] In the present invention, the nucleic acid molecule specifically includes nucleotide fragments encoding the light and heavy chains of an anti-BCMA antibody or antigen-binding fragment thereof. The specific nucleotide sequence of the heavy chain encoding gene is as shown in SEQ ID NO: 29 or a nucleotide fragment that is completely complementary to the sequence shown in SEQ ID NO: 29 or hybridizes under moderate to high stringency conditions. The specific nucleotide sequence of the light chain encoding gene is as shown in SEQ ID NO: 30 or a nucleotide fragment that is completely complementary to the sequence shown in SEQ ID NO: 30 or hybridizes under moderate to high stringency conditions.

[0040] Based on the purpose of obtaining the above-mentioned anti-BCMA antibody or antigen-binding fragment thereof, the present invention also provides a cell line, which can secrete and express the above-mentioned anti-BCMA antibody or antigen-binding fragment thereof.

[0041] Based on the purpose of obtaining the above-mentioned anti-BCMA antibody or antigen-binding fragment thereof, the present invention also provides a method for preparing the anti-BCMA antibody or antigen-binding fragment thereof, which specifically comprises: culturing the above-mentioned cell line under suitable conditions, collecting the cell culture, and obtaining the anti-BCMA antibody or antigen-binding fragment thereof after purification.

[0042] Based on the excellent affinity of the above-mentioned anti-BCMA antibodies or antigen-binding fragments thereof for BCMA protein, the present invention also provides the use of the above-mentioned anti-BCMA antibodies or antigen-binding fragments thereof, nucleic acid molecules and / or cell lines in BCMA detection for non-diagnostic purposes.

[0043] Based on the excellent killing ability of the above-mentioned anti-BCMA antibodies or antigen-binding fragments thereof on abnormal bone marrow cells that overexpress BCMA protein, the present invention also provides the use of the above-mentioned anti-BCMA antibodies or antigen-binding fragments thereof, nucleic acid molecules and / or cell lines in the preparation of inhibitors of abnormal bone marrow cells.

[0044] Based on the potential of the above-mentioned anti-BCMA antibody or its antigen-binding fragment as an inhibitor of abnormal bone marrow cells, the present invention also provides an anti-tumor drug, which comprises the above-mentioned anti-BCMA antibody or its antigen-binding fragment, a nucleic acid molecule and / or a cell line.

[0045] The embodiments of the present invention are described in detail below. The examples of the embodiments are intended to explain the present invention and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0046] The reagents and their sources involved in the following examples specifically include:

[0047] Recombinant human BCMA extracellular domain / hFc fusion protein (Jiangsu Biotechnology Co., Ltd., catalog number B528001 20201230A42); water-soluble adjuvant (Jiangsu Biotechnology Co., Ltd., catalog number BMA1001); recombinant human BCMA extracellular domain-His protein (Jiangsu Biotechnology Co., Ltd., catalog number B21846208 / 20230210ZA05); coating buffer (Shanghai Yuanye Biotechnology Co., Ltd., catalog number R20934); blocking solution (Shanghai Yuanye Biotechnology Co., Ltd., catalog number R22840); diluent (Shanghai Yuanye Biotechnology Co., Ltd., catalog number R40019); Goat Anti-Mous IgG-HRP (Shanghai Sigma, Catalog No. XGK3996); color development solution (Beijing Meikewande, Catalog No. 1001); stop solution (Biyuntian, Catalog No. P025); 1640 culture medium (Sigma, Catalog No. R8758); FBS (Sigma, Catalog No. F8687); Biotin-Ter119 (BioLgend, Catalog No. 116204); PE fluorescent-labeled antigen Recombinant Human X / His Protein (Jiangsu Baiying, Catalog No. B21846208); Alexa 647 Goat anti-mouse IgG (BioLegend, Catalog No. 405322); Bioiotron Human IgG1 plasmid (Jiangsu Baiying, Catalog No. BIHH1X); CHO-S cells (Gibco, Catalog No. A29129); Anti-human Fc specific HRP (Southern Biotech, Catalog No. 2040-05); Anti-human IgG-PE (Biolegend, Catalog No. 410708); Aqua Fixable Dead Cell Stain Kit (Thermo Fisher, Cat. No. L34965).

[0048] Example 1

[0049] This example illustrates a method for constructing an anti-BCMA antibody. In this method, mice are immunized with a human BCMA extracellular domain protein comprising an amino acid sequence as shown in SEQ ID NO: 1 as an antigen. The method then undergoes steps such as serum antibody titer determination, cell sorting, sequencing, and humanized recombinant expression, as shown below:

[0050] 1. Mouse immunization: The above-mentioned recombinant human BCMA extracellular segment / hFc fusion protein was mixed with a water-soluble adjuvant and mice were immunized multiple times. The immunization process was as follows: 60 μg of recombinant human BCMA extracellular segment / hFc fusion protein was mixed with a water-soluble adjuvant and injected subcutaneously into mice for the first immunization. Ten days after the first immunization, 60 μg of recombinant human BCMA extracellular segment / hFc fusion protein was mixed with a water-soluble adjuvant and injected subcutaneously into mice for the second immunization. Twelve days after the second immunization, 60 μg of recombinant human BCMA extracellular segment / hFc fusion protein was mixed with a water-soluble adjuvant and injected subcutaneously into mice for the third immunization. The mouse serum antibody titer was detected 6 days after the third immunization.

[0051] 2. Mouse serum antibody titer determination: (1) Dilute the recombinant human BCMA extracellular domain-His protein to 2 μg / mL with coating buffer, add 100 μL to each well, incubate at 37°C for 1 h, wash the plate 6 times, and pat dry;

[0052] (2) Add 200 μL of blocking solution to each well and incubate in a 25°C incubator for 1 h. Wash the plate 6 times and pat dry.

[0053] (3) Dilute the mouse serum with blocking solution at a dilution factor of 1:1000, and perform 10 serial dilutions with blocking solution at a dilution factor of 2. Add 100 μL of the mouse serum obtained after the final dilution to each well, and use the mouse serum before immunization as a negative control, and the dilution solution as a blank control. Incubate in a 25°C incubator for 1 hour, wash the plate 6 times, and pat dry.

[0054] (4) Dilute Goat Anti-Mous IgG-HRP 10,000-fold with PBS, add 100 μL of diluted Goat Anti-Mous IgG-HRP to each well, incubate in a 25°C incubator for 1 h, wash the plate 6 times, and pat dry;

[0055] (5) Add 100 μL of color development solution to each well. After color development for 3 min at room temperature in the dark, add 50 μL of stop solution to each well to terminate the reaction. Read the result at 450 nm using a microplate reader.

[0056] 3. B cell sorting and sequencing: (1) Select the mouse with the highest antibody titer, remove the spleen and bone marrow, grind the spleen and bone marrow, add 1640 medium, filter through a mesh, centrifuge at 1000 rpm, remove the supernatant, add a certain volume of 1640 medium to resuspend the cells, and take Biotin-Ter119 and remove the red blood cells according to the instructions to obtain a single cell suspension;

[0057] (2) Take PE fluorescent labeled antigen Recombinant Human X / His Protein and Alexa 647Goat anti-mouse IgG and the single cell suspension were co-incubated and stained according to the instructions. After washing twice, the cells were analyzed using BD FACS Melody TM B cells were sorted by M flow cytometry to obtain B cells that were double positive for IgG and PE antigens;

[0058] (3) A single-cell cDNA library was constructed based on the obtained IgG and PE antigen double-positive B cells, and the cDNA library was sequenced and analyzed by NGS. The obtained antibodies and their sequences are shown in Table 2.

[0059] Table 2.

[0060]

[0061] 3. Humanized recombinant expression of antibodies: (1) Based on the amino acid sequences of each antibody shown in Table 2, codon optimization was performed to obtain the coding sequences of the heavy chain variable region and light chain variable region of each antibody, which were respectively connected to the signal peptide coding gene and the human IgG1 constant region coding gene on the Bioiotron Human IgG1 plasmid to construct a recombinant plasmid capable of expressing the corresponding humanized chimeric antibody light chain or heavy chain. The specific coding genes are shown in Table 3.

[0062] Table 3.

[0063] name sequence signal peptide SEQ ID NO:26 Human IgG1 CH domain SEQ ID NO:27 Human IgG1 CL domain SEQ ID NO:28 B-52 antibody heavy chain encoding gene SEQ ID NO:29 B-52 antibody light chain encoding gene SEQ ID NO:30 B-96 antibody heavy chain encoding gene SEQ ID NO:31 B-96 antibody light chain encoding gene SEQ ID NO:32 B-181 antibody heavy chain encoding gene SEQ ID NO:33 B-181 antibody light chain encoding gene SEQ ID NO:34

[0064] (2) After transfecting CHO-S cells with the recombinant plasmid according to the instructions, the cells were cultured at 37°C, 115 rpm, and 8% CO2 for 7 days, and the supernatant products were collected and purified using affinity columns to obtain humanized chimeric antibodies, i.e., anti-BCMA antibodies.

[0065] 4. Anti-BCMA antibody titer determination: (1) Dilute the human BCMA extracellular segment protein to 2 μg / mL with coating buffer, add 100 μL to each well, adsorb and coat overnight at 4°C, wash the plate, pat dry, add 200 μL / well of PBS containing 5% skim milk powder, and incubate in a 37°C incubator for 30 minutes, wash the plate 3 times, and pat dry;

[0066] (3) Anti-BCMA antibody was diluted with PBST buffer, and anti-BCMA antibody was added to each well at final concentrations of 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM, 0.0001 nM, and 0.00001 nM, respectively. PBST buffer was used as a blank control, and the plate was incubated in a 37°C incubator for 1 h. The plate was washed three times and patted dry.

[0067] (4) Dilute Anti-human Fc specific HRP 16,000-fold with PBST buffer, add 100 μL of diluted Anti-human Fc specific HRP to each well, incubate in a 37°C incubator for 30 min, wash the plate 5 times, and pat dry;

[0068] (5) Add 100 μL of color development solution to each well, develop the color at room temperature in the dark for 3 minutes, then add 50 μL of stop solution to each well to terminate the reaction. Read the results at 450 nm using a microplate reader. Figure 1 shown.

[0069] Depend on Figure 1 It can be seen that compared with the anti-BCMA antibodies B-96 and B-181, the anti-BCMA antibody B-52 has a much better affinity for the BCMA protein.

[0070] Example 2

[0071] This example is used to illustrate the ability of the anti-BCMA antibodies provided in the above examples to kill cells that highly express BCMA. The test specifically includes:

[0072] 1. Affinity of anti-BCMA antibody to tumor cells with high BCMA expression: 2×10 5 Human diffuse large cell lymphoma B lymphocytes (Pfeiffer cells) with high BCMA expression were added to a flow tube, centrifuged at 300g for 5 minutes, and the supernatant was removed; 100 μL of anti-BCMA antibody solution with a concentration gradient (1nM, 5nM, 10nM, 25nM, 50nM and 100nM) was added to the flow tube and incubated at 4°C for 30 minutes; 2 mL of PBS was added to the flow tube, centrifuged at 300g for 5 minutes, and the supernatant was removed. Then, Anti-human IgG-PE was diluted 100 times with PBS buffer, and 100 μL of diluted Anti-human IgG-PE was added to each tube, and the tube was incubated at 4°C for 30 minutes; 2 mL of PBS was added to the flow tube, centrifuged at 300g for 5 minutes, and the supernatant was removed; then, 500 μL of PBS was used to resuspend the cells, and the fluorescence signal of each tube was detected by flow cytometry, and a concentration curve was drawn. The results are shown in FIG. Figure 2 and as shown in Table 3.

[0073] Table 3.

[0074] Monoclonal antibodies Median effective dose (nM) B-52 antibody 8.104 B-96 antibody 28.300 B-181 antibody 24.980

[0075] Depend on Figure 2 As shown in the test results in Table 3, compared with the anti-BCMA antibodies B-96 and B-181, the anti-BCMA antibody B-52 has a very good affinity for Pfeiffer cells that highly express BCMA, and its half-effective dose is only 8.104 nM.

[0076] 2. Anti-BCMA antibody killing ability of tumor cells: (1) Pfeiffer cells were diluted to 5×10 5 100 μL, and anti-BCMA antibody was added to make the final concentration of the solution antibody 0.1nM, 0.25nM, 0.5nM, 1nM, 5nM, 10nM, 25nM and 50nM, and placed in a cell culture incubator at 37°C and 5% CO2 for 2h; 2mL of PBS was added to the flow tube, centrifuged at 300g for 5min, the supernatant was removed, and then the tube was centrifuged at 300g for 5min. The cells were resuspended using the Aqua Fixable Dead Cell Staining Kit according to the instructions and incubated at room temperature for 30 minutes in the dark. 2 mL of PBS was added to the flow cytometry tube and centrifuged at 300 g for 5 minutes. The supernatant was discarded. The pellet was then resuspended in 500 μL of PBS and the fluorescence signal of each tube was detected by flow cytometry. The results are shown in Figure 2. Figure 3 and 4 shown.

[0077] Depend on Figure 3 and 4 The test results shown in the figure show that compared with the anti-BCMA antibodies B-96 and B-181, the anti-BCMA antibody B-52 can kill Pfeiffer cells more efficiently through complement-dependent cytotoxicity, and its IC 50 The value is 0.3903nM, which has excellent ability to kill Pfeiffer tumor cells.

[0078] (2) Pfeiffer cells were diluted to 5×10 5 100 μL, and added B-52 antibody to make the final concentration of the solution antibody 10 nM, and placed in a cell culture incubator at 37 ° C, 5% CO2 for 2h, 4h, 6h, 8h and 12h; took 2mL of PBS and added it to the flow tube, centrifuged at 300g for 5min, removed the supernatant, and then used The cells were resuspended using the Aqua Fixable Dead Cell Staining Kit according to the instructions and incubated at room temperature for 30 minutes in the dark. 2 mL of PBS was added to the flow cytometry tube and centrifuged at 300 g for 5 minutes. The supernatant was discarded. The pellet was then resuspended in 500 μL of PBS and the fluorescence signal of each tube was detected by flow cytometry. The results are shown in Figure 2. Figure 5 shown.

[0079] The test results shown in Table 5 show that the anti-BCMA antibody B-52 provided by the present invention has a killing rate of up to 99% for Pfeiffer tumor cells after being co-incubated with Pfeiffer tumor cells for 12 hours, and has excellent tumor cell killing ability.

[0080] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. An anti-BCMA antibody or an antigen-binding fragment thereof, characterized in that: In the anti-BCMA antibody or antigen-binding fragment thereof, the amino acid sequences of the heavy chain variable region CDR1 to 3 are shown in SEQ ID NOs: 2 to 4, respectively, and the amino acid sequences of the light chain variable region CDR1 to 3 are shown in SEQ ID NOs: 6 to 8, respectively.

2. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 1, wherein In the anti-BCMA antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 5 or a variant sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO: 5 and retains the biological activity of the corresponding parent sequence, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 9 or a variant sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO: 9 and retains the biological activity of the corresponding parent sequence.

3. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 1, wherein In the anti-BCMA antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 27 or a variant sequence that has at least 80% homology to the amino acid sequence shown in SEQ ID NO: 27 and retains the biological activity of the corresponding parent sequence, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 28 or a variant sequence that has at least 80% homology to the amino acid sequence shown in SEQ ID NO: 28 and retains the biological activity of the corresponding parent sequence.

4. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 1, wherein The anti-BCMA antibody or antigen-binding fragment thereof includes a signal peptide whose sequence is shown in SEQ ID NO: 26, and the signal peptide is connected to the N-terminus of the heavy chain variable region and / or the N-terminus of the light chain variable region.

5. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 1, wherein The anti-BCMA antibody or antigen-binding fragment thereof is selected from one or more of Fab, F(ab')2, Fv, single-chain antibody, chimeric antibody and monoclonal antibody.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. A cell line, characterized in that The cell line expresses and secretes the anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

8. The method for preparing the anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that: The cell line of claim 7 is cultured under appropriate conditions, and the anti-BCMA antibody or antigen-binding fragment thereof is recovered from the cell culture.

9. Use of the anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, and / or the cell line according to claim 7 in BCMA detection for non-diagnostic purposes.

10. Use of the anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, and / or the cell line according to claim 7 in the preparation of an inhibitor of human diffuse large cell lymphoma.

11. An anti-tumor drug, characterized in that: The anti-tumor drug comprises the anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6 and / or the cell line according to claim 7.

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