Anti-human bcma monoclonal antibodies and uses thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to anti-human BCMA monoclonal antibody and its application. The present application provides a new anti-human BCMA monoclonal antibody 2B6, which can specifically recognize the BCMA protein expressed on the cell membrane, has high affinity and strong specificity. The anti-human BCMA monoclonal antibody provided by the present application can be used for flow cytometry detection and immunohistochemical staining, and can realize specific coloring in normal tissues such as tonsils, spleens and tumor tissues such as intestinal cancer. The anti-human BCMA monoclonal antibody provided by the present application can effectively block the combination of BCMA and its ligand BAFF in vitro, and provides a tool for studying the function of BCMA and developing related drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to anti-human BCMA monoclonal antibodies and their applications. Background Technology

[0002] B cell maturation antigen (BCMA), also known as TNFRSF17 or CD269, is a member of the tumor necrosis factor receptor superfamily. BCMA expression is lineage-specific, limited to B cell lines, and is selectively induced, especially during plasma cell differentiation. It is essential for the optimal survival of long-term surviving plasma cells in the bone marrow. BCMA has two agonist ligands: BAFF and APRIL. In normal human tissues, BCMA binds to these ligands to maintain B cell survival and differentiation.

[0003] Studies have found that BCMA is widely and highly expressed on malignant plasma cells in multiple myeloma, and its expression level increases as cells progress from a normal state to active multiple myeloma. BCMA is also expressed in other B-cell malignancies, including Waldenström macroglobulinemia, Burkitt lymphoma, and diffuse large B-cell lymphoma, making it an ideal target antigen for multiple myeloma treatment. Therefore, developing monoclonal antibodies that specifically recognize BCMA is of great significance for the diagnosis, detection, and functional study of BCMA-related diseases. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an anti-human BCMA monoclonal antibody and its application. The present invention provides a monoclonal antibody that can specifically recognize B cell maturation antigen and its preparation method, which has high affinity and strong specificity.

[0005] This invention provides an anti-human BCMA monoclonal antibody, comprising: a heavy chain variable region and a light chain variable region, wherein:

[0006] (1) The three CDR regions of the heavy chain variable region have amino acid sequences as shown in SEQ ID NO: 1, 2 and 3, and the three CDR regions of the light chain variable region have amino acid sequences as shown in SEQ ID NO: 4, 5 and 6: or

[0007] (2) The three CDR regions of the heavy chain variable region and / or the three CDR regions of the light chain variable region have amino acid sequences obtained by substitution, deletion or addition of one or more amino acids as described in (1), and amino acid sequences that are functionally identical or similar to the amino acid sequences shown in (1); or

[0008] (3) The three CDR regions of the heavy chain variable region and / or the three CDR regions of the light chain variable region have CDRs that have at least 85% sequence identity with the amino acid sequence shown in (1) or (2).

[0009] In some embodiments,

[0010] (4) Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO:7, and its light chain variable region has the sequence shown in SEQ ID NO:8; or

[0011] (5) Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequences described in (4), and which have the same or similar function as the amino acid sequences shown in (4); or

[0012] (6) An amino acid sequence that is at least 80% homologous to the sequence described in (4) or (5).

[0013] The present invention provides a nucleic acid molecule encoding the aforementioned anti-human BCMA monoclonal antibody.

[0014] This invention provides biological materials comprising at least one of the following (7) to (9):

[0015] (7) An expression vector containing the nucleic acid molecule;

[0016] (8) Transform or transfect the host cells of the expression vector;

[0017] (9) Culturing the host cells to obtain a culture containing the anti-human BCMA monoclonal antibody.

[0018] In some embodiments, the present invention also provides an expression module comprising the nucleic acid, the expression module comprising a promoter, a terminator, and the nucleic acid molecule described in the present invention.

[0019] In some embodiments, the expression module may further include an expression module formed by combining one or more nucleic acid molecules described in this invention in tandem, fusion expression or other feasible ways, and this invention does not limit this.

[0020] In some embodiments, the present invention also provides a transcription unit, which refers to a DNA sequence from the start of a promoter to the end of a terminator. Regulatory fragments may also be included flanking or between the promoter and terminator. These regulatory fragments may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, transmembrane signal peptides, and homologous recombination sites operatively linked to a nucleic acid sequence, such as enhancers of promoters, ITR sequences, polyA, MIS signal peptides, etc.

[0021] In some embodiments, the host cells described in this invention may be derived from plants, animals, microorganisms, or viruses, and this invention is not limited thereto. This invention uses vectors constructed using recombinant DNA technology to transform or transfect host cells, thereby enabling the transformed host cells to replicate the protein-encoding vector or express the desired protein.

[0022] In some embodiments, the transformation method includes chemical transformation and electrotransformation; the transfection method includes calcium phosphate coprecipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0023] The present invention provides labeled antibodies, including a marker and the aforementioned anti-human BCMA monoclonal antibody.

[0024] In some embodiments, the markers include chemical markers and biomarkers;

[0025] The chemical markers include isotopes and / or chemical drugs;

[0026] The biomarkers include biotin, avidin, or enzymes.

[0027] In some embodiments, the enzyme includes horseradish peroxidase or alkaline phosphatase.

[0028] The present invention provides a conjugate comprising a conjugation medium and the aforementioned anti-human BCMA monoclonal antibody.

[0029] In some embodiments, the coupling medium includes a solid medium or a semi-solid medium.

[0030] In some embodiments, the coupling medium is selected from colloidal gold, polystyrene sheets, or beads.

[0031] The present invention provides the use of at least one of the following (10) to (14) in the preparation of a product for detecting and / or treating BCMA-positive diseases:

[0032] (10) The anti-human BCMA monoclonal antibody;

[0033] (11) The aforementioned nucleic acid molecule;

[0034] (12) The aforementioned biomaterials;

[0035] (13) The labeled antibody;

[0036] (14) The aforementioned coupling.

[0037] In some embodiments, BCMA-positive diseases include at least one of multiple myeloma, Waldenström macroglobulinemia, Burkitt lymphoma, and diffuse large B-cell lymphoma.

[0038] This invention provides products for detecting BCMA-positive cells and / or treating diseases associated with BCMA-related tumors, comprising at least one of the following (15) to (19):

[0039] (15) The anti-human BCMA monoclonal antibody;

[0040] (16) The aforementioned nucleic acid molecule;

[0041] (17) The aforementioned biomaterials;

[0042] (18) The labeled antibody;

[0043] (19) The aforementioned coupling.

[0044] In some embodiments, BCMA-positive diseases include at least one of multiple myeloma, Waldenström macroglobulinemia, Burkitt lymphoma, and diffuse large B-cell lymphoma.

[0045] This invention provides a method for detecting BCMA-positive tumors, including testing samples using the product described herein.

[0046] This invention provides a method for treating BCMA-related tumors, including administering the product.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] 1. This invention provides a novel anti-human BCMA monoclonal antibody 2B6, which can specifically recognize BCMA protein expressed on the cell membrane, and has high affinity and strong specificity.

[0049] 2. The anti-human BCMA monoclonal antibody provided by this invention can be used for flow cytometry detection and immunohistochemical staining, and can achieve specific staining in normal tissues such as tonsils and spleen, as well as tumor tissues such as colorectal cancer.

[0050] 3. The anti-human BCMA monoclonal antibody provided by this invention can effectively block the binding of BCMA to its ligand BAFF in vitro, providing a tool for studying BCMA function and developing related drugs. Attached Figure Description

[0051] Figure 1 The electrophoresis diagram of the BCMA monoclonal antibody in Example 1 is shown;

[0052] Figure 2 The image shows the results of Western blot analysis of the BCMA monoclonal antibody in Example 3, where the left lane is the marker and the right lane is 293T-BCMA.

[0053] Figure 3 The image shows the results of immunohistochemical detection of different tissue samples using the BCMA monoclonal antibody in Example 4. The samples in the image, from left to right, are tonsils, spleen, and colorectal cancer.

[0054] Figure 4 The flow cytometry results of BCMA monoclonal antibody binding to different target cells in Example 5 are shown in Figure A, where Figure B shows the detection results of 293T-BCMA cells and Figure B shows the detection results of H929 cells.

[0055] Figure 5 The following figures show the results of the competitive binding experiment between the cellular level antibody and ligand protein in Example 6. Figure A shows the competitive binding results of 293T-BCMA cells, and Figure B shows the competitive binding results of H929 cells. Detailed Implementation

[0056] This invention provides an anti-human BCMA monoclonal antibody and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0057] The purpose of this invention is to provide an anti-human BCMA monoclonal antibody 2B6 and its applications. Specifically, it includes: providing the amino acid sequences and CDR sequences of the heavy and light chain variable regions of the antibody; providing a nucleic acid molecule encoding the antibody, an expression vector, and a host cell; providing a detection kit containing the antibody; and providing the use of the antibody in flow cytometry detection, immunohistochemical staining, blocking the binding of BCMA to BAFF, and diagnosing BCMA-positive diseases.

[0058] To achieve the above objectives, the present invention provides the following technical solution:

[0059] This invention involves extracting the heavy and light chain variable region sequences from hybridoma cells secreting anti-human BCMA monoclonal antibodies, constructing heavy and light chain expression vectors, and co-transfecting them into CHO eukaryotic expression cell lines to obtain recombinant anti-human BCMA monoclonal antibodies, named 2B6.

[0060] Specifically:

[0061] In a first aspect, the present invention provides an anti-human BCMA monoclonal antibody 2B6, the antibody comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being as shown in SEQ ID NO:7, specifically as follows:

[0062] VQLQQSGPELVKTGASVKISCKASGYSFTGYYMHWVKQSHGKSLEWIGYISCYNGATSYNQKFKGKATFTVDTSSSTAYMQFNSLTSEDSAVYYCARYGYDYAMDYWGQGTSVTVSS; The amino acid sequence of the light chain variable region is shown in SEQ ID NO:8, and is as follows:

[0063] DVLMTQTPLSLFVSLGDQASISCRSSQSIIQSNGNTYLEWYLQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKITRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK.

[0064] Furthermore, the heavy chain variable region has three complementarity-determining regions (CDRs), whose amino acid sequences are as follows:

[0065] HCDR1:GYYMH (SEQ ID NO:1);

[0066] HCDR2:YISCYNGATSYNQKFKG (SEQ ID NO:2);

[0067] HCDR3: YGYDYAMDY (SEQ ID NO: 3).

[0068] The light chain variable region has three complementarity-determining regions (CDRs), whose amino acid sequences are as follows:

[0069] LCDR1:RSSQSIIQSNGNTYLE(SEQ ID NO:4);

[0070] LCDR2: TVSNRFS (SEQ ID NO:5);

[0071] LCDR3: FQGSHVPWT (SEQ ID NO:6).

[0072] Secondly, the present invention also provides a nucleic acid molecule encoding the above-mentioned anti-human BCMA monoclonal antibody 2B6, as well as an expression vector and a host cell containing the nucleic acid molecule.

[0073] Furthermore, the host cell is a eukaryotic cell, preferably a CHO cell.

[0074] Thirdly, the present invention provides a detection kit comprising the above-mentioned anti-human BCMA monoclonal antibody 2B6.

[0075] Fourthly, the present invention provides the application of the above-mentioned anti-human BCMA monoclonal antibody 2B6 in the preparation of reagents for detecting BCMA protein, wherein the detection includes flow cytometry detection and / or immunohistochemical staining detection; the tissue sample for immunohistochemical staining detection is tonsillar tissue, spleen tissue or tumor tissue.

[0076] Fifthly, the present invention provides the use of the above-mentioned anti-human BCMA monoclonal antibody 2B6 in the preparation of reagents for blocking the binding of BCMA to its ligand BAFF.

[0077] In a sixth aspect, the present invention also provides the use of anti-human BCMA monoclonal antibody 2B6 in the preparation of a kit for diagnosing BCMA-positive diseases.

[0078] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0079] Example 1: Preparation of anti-human BCMA monoclonal antibody

[0080] The process for preparing anti-human BCMA monoclonal antibodies provided by this invention mainly consists of mouse immunogen inoculation, hybridoma cell preparation, positive monoclonal cell screening and amplification, and the operation steps are as follows:

[0081] 1. Antigen protein preparation: Dissolve the required antigen protein in PBS buffer and adjust to an appropriate concentration (100 μg dissolved in 150 μL of PBS).

[0082] Shake the Complete Freund's Adjuvant (CFA) well and set aside. Mix the prepared antigen solution and CFA at a 1:1 volume ratio. Gently shake to further emulsify the antigen protein, forming a stable water-oil emulsion.

[0083] 2. Antigen inoculation:

[0084] Day 1: Initial immunization (Frederick complete adjuvant + 100 μg antigen protein)

[0085] Day 21: Boost immunization (French incomplete adjuvant + 100 μg antigen protein)

[0086] Day 42: Boost immunization (French incomplete adjuvant + 100 μg antigen protein)

[0087] Day 108: Booster immunization (100μg antigen protein without adjuvant), sample collection will be prepared 3 days after the fourth booster immunization.

[0088] 3. Preparation of mouse spleen single-cell suspension

[0089] The spleen was cut into small pieces with sterile scissors, and single cells were filtered out using a cell sieve. The separated single cell solution was filtered again, and 300g of the resulting single cell suspension was centrifuged for 5 minutes. The waste liquid was discarded, and the suspension was then resuspended in sterile PBS and washed once more.

[0090] 4. Cell Fusion: Centrifuge spleen cells and myeloma cells separately at 300g for 5 min, discard the waste liquid, and resuspend in fusion diluent. Mix 10⁷ spleen cells and 10⁶ myeloma cells at a spleen cell:myeloma cell ratio of 10:1 and add to a 50ml centrifuge tube. Centrifuge at 300g for 5 min, discard the waste liquid. Add 1 ml of polyethylene glycol (PEG) dropwise over 1 min, followed by 6 ml of serum-free RPMI 1640 medium dropwise over 10 min at the same pace. Finally, add 7 ml of serum-free RPMI 1640 medium, centrifuge at 300g for 5 min, and discard the supernatant.

[0091] 5. Hybridoma cell culture: Prepare RPMI 1640 medium + L-Gln + 10% FBS + antibiotics + 1% ULtroser G, and hybridoma medium containing HAT: RPMI 1640 medium + L-Gln + 10% FBS + antibiotics + 1% ULtroser G + 1X HAT supplement. Seed cells in 96-well plates, and replace with hybridoma medium containing HAT after 48 hours. Replace the medium with fresh hybridoma medium containing HAT every 2 days.

[0092] Positive well screening: After 10-14 days, cell supernatant was collected for ELISA and flow cytometry to screen for cell wells capable of secreting antibodies. To further screen for monoclonal antibodies, positive wells were limitedly diluted and identified using the same methods (ELISA, flow cytometry). Antibody-producing hybridoma cells were then amplified and cryopreserved.

[0093] Purification and identification: The obtained positive hybridoma cell lines were inoculated into the peritoneal cavity of BALB / c mice, and the ascites fluid was collected. The monoclonal antibodies were then purified using Protein G affinity chromatography. Figure 1 As shown, the protein gel electrophoresis results indicate that the purified anti-human BCMA monoclonal antibody 2B6 exhibits clear and single heavy and light chain bands without any contaminating proteins, suggesting that the obtained antibody has high purity.

[0094] Anti-human BCMA monoclonal antibody 2B6 includes a heavy chain variable region and a light chain variable region, wherein:

[0095] The amino acid sequence of the heavy chain variable region is shown below:

[0096] EVQLQQSGPELVKTGASVKISCKASGYSFTGYYMHWVKQSHGKSLEWIGYISCYNGATSYNQKFKGKATFTVDTSSSTAYMQFNSLTSEDSAVYYCARYGYDYAMDYWGQGTSVTVSS (SEQ ID NO:7)

[0097] The amino acid sequence of the light chain variable region is shown below:

[0098] DVLMTQTPLSLFVSLGDQASISCRSSQSIIQSNGNTYLEWYLQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKITRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK (SEQ ID NO: 8)

[0099] The heavy chain variable region has three complementarity-determining regions (CDRs), whose amino acid sequences are as follows:

[0100] HCDR1:GYYMH (SEQ ID NO:1);

[0101] HCDR2:YISCYNGATSYNQKFKG (SEQ ID NO:2);

[0102] HCDR3: YGYDYAMDY (SEQ ID NO: 3).

[0103] The light chain variable region has three complementarity-determining regions (CDRs), whose amino acid sequences are as follows:

[0104] LCDR1:RSSQSIIQSNGNTYLE(SEQ ID NO:4);

[0105] LCDR2: TVSNRFS (SEQ ID NO:5);

[0106] LCDR3: FQGSHVPWT (SEQ ID NO:6).

[0107] Example 2: Heavy and light chain expression plasmids for anti-human BCMA monoclonal antibody

[0108] The expression plasmids for the heavy and light chains of the anti-human BCMA monoclonal antibody in this invention are prepared as follows:

[0109] 1. Resuscitate CHO cells: Prepare 125 mL cell culture shake flasks and preheated CD CHO + 4 mM Glutamine medium, and simultaneously set the water bath temperature to 37°C to resuscitate the cells. Count the resuscitated cells and record the cell density. Culture conditions: 36.5°C, 8% CO2, 120 rpm.

[0110] 2. CHO cell passage culture: at 0.5 × 10⁻⁶ cells / cell. 6 Cells were seeded at a density of 125 mL into 125 mL shake flasks and cultured in 20 mL of CD CHO + 4 mM glutamine. After 2-3 days of culture, cell density and viability were counted using a cell counter. Cells were passaged and the culture medium was replaced with fresh medium every 3-4 days.

[0111] 3. Cell expression: On the day of expression, cells in the logarithmic growth phase were collected, and cell density was counted; according to 1×10⁻⁶ cells / year... 6 Expression culture was performed at a cell density of 1000 / mL. The required cell suspension volume was calculated. The cell suspension was transferred to a 50mL centrifuge tube, centrifuged at 800 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 20mL of culture medium and transferred to a 125mL shake flask. The heavy and light chain plasmids were mixed with the transfection reagent PEI and slowly added to the cell suspension. The shake flask was placed in a shaker incubator at 37 ℃, 8% CO2, and 120 rpm for suspension culture.

[0112] 4. Cell status monitoring: Cells were counted daily after inoculation, and glucose and lactate levels were measured using a biochemical analyzer starting on the third day. Feed or glucose was added based on daily glucose consumption (maintaining glucose concentration at approximately 2-6 g / L).

[0113] 5. Cell harvesting: When the cell viability is ≤80% or the total culture time reaches 7 days or more, the cell expression supernatant is collected. The cell suspension is transferred to a centrifuge tube, centrifuged at 4000 rpm for 20 min, and the supernatant is collected into a clean container. The positive rate of the culture supernatant is detected by ELISA and flow cytometry.

[0114] Example 3: Western blot detection of anti-human BCMA monoclonal antibody

[0115] 1. Extraction of total cellular protein: 293T-BCMA cells in logarithmic growth phase were used, 1×10⁻⁶ 7 Add RIPA lysis buffer to the cell count / mL, incubate on ice for 30 min, gently pipette the lysis buffer every 10 min, transfer the lysis buffer to an enzyme-free centrifuge tube, centrifuge at 12000 r / min for 15 min at 4℃, carefully aspirate the supernatant, and keep the total protein extract on ice for later use.

[0116] 2. The BCA protein quantification kit was used to determine the concentration of the extracted total protein. After quantification, an appropriate amount of protein sample was taken, thoroughly mixed with 5×SDS loading buffer, boiled in a 95℃ constant temperature metal bath for 5 min, and the cooled protein sample was used for SDS-PAGE electrophoresis.

[0117] 3. Protein loading: Add 20 μg of protein sample to the loading well, add 5 μL of pre-stained protein marker to the leftmost well, and set the electrophoresis parameters: after electrophoresis at 90 V for 30 min, adjust the voltage to 120 V until the marker bands are clearly separated and the target protein migrates to the lower part of the separating gel.

[0118] 4. Transfer: Lay the filter paper, gel, PVDF membrane and filter paper layer by layer in the transfer holder in the order of filter paper → gel → PVDF membrane → filter paper. Transfer the membrane at a constant current of 200 mA for 75 min. Wash the PVDF membrane with TBST buffer. Repeat twice, 5 min each time.

[0119] 5. Sealing: Place in a 5% skim milk powder sealing solution and seal at room temperature for 1 hour.

[0120] 6. Antibody incubation: Add primary antibody solution to the surface of the PVDF membrane and incubate overnight at 4°C with a shaker. After primary antibody incubation, wash the membrane 5 times with TBST buffer for 5 min each time, then add fluorescent secondary antibody and incubate at room temperature for 1 h.

[0121] 6. Imaging: Add an appropriate amount of ECL chemiluminescent reagent to the washed PVDF membrane, ensuring it evenly covers the entire membrane surface. Allow the reaction to proceed in the dark for 1-2 minutes. Then, expose the membrane for imaging.

[0122] The results of Western blot analysis of the 2B6 monoclonal antibody are as follows: Figure 2 As shown, the target antibody exhibits a clear BCMA-specific band at approximately 43 kDa, without tailing or interference from other bands, confirming that monoclonal antibody 2B6 has good specific binding ability to BCMA molecules at the protein level.

[0123] Example 4: Immunohistochemical detection of pathological tissues using anti-human BCMA monoclonal antibody

[0124] The anti-human BCMA monoclonal antibody of this invention is used for immunohistochemical detection of pathological tissues, including the following steps:

[0125] 1. Reagent preparation and setup:

[0126] 3% H2O2: 3% H2O2 + 9 parts pure water;

[0127] Repair solution: 0.01M citrate buffer, diluted 50 times;

[0128] Blocking solution: prepared with 3% BSA and PBS.

[0129] 2. Tissue section processing:

[0130] Dewaxing: Tissue sections were baked in an oven at 70°C for more than 2 hours, and then immersed in 100% xylene solution for 5 minutes in a first vat, xylene solution for 5 minutes in a second vat, and xylene solution for 5 minutes in a third vat.

[0131] Dexylene removal: Immerse in 100%, 95%, and 75% alcohol solutions for 2 minutes in sequence;

[0132] Hydration: Remove the slices and soak them in distilled water for 1 minute. Then, immerse them in 3% H2O2 for 10 minutes and rinse with distilled water.

[0133] 3. Antigen retrieval

[0134] Place the slides in a container filled with citrate buffer, heat to boiling in a microwave oven, and maintain the temperature at 98°C for 20 minutes. Remove the container and cool in water for 10 minutes. Remove the slides, rinse once with distilled water, and wash three times with PBS for 5 minutes each time.

[0135] 4. Blocking non-specific proteins

[0136] Wipe the tissue section on the slide dry, delineate the tissue area with an immunohistochemical pen, add 50-100 μL of 3% BSA blocking solution, and block at room temperature for 60-120 min.

[0137] 5. Primary antibody incubation

[0138] Discard the blocking solution, add 50-100 μl of primary antibody depending on the size of the tissue block to completely cover the tissue, and incubate overnight at 4°C.

[0139] 6. Washing

[0140] The next day, remove the slides and incubate at room temperature for 15 minutes. Wash three times with PBS. Add 50-100 μL of enzyme-labeled secondary antibody (goat anti-mouse / rabbit IgG polymer) to completely cover the tissue, depending on the size of the tissue block. Incubate at 37°C for 30 minutes in a humidified chamber. Wash off the secondary antibody and rinse three times with PBS for 8 minutes each.

[0141] 7. Color Development

[0142] Add 50-100 μL of DAB chromogenic reagent (prepare fresh and protect from light) depending on the size of the tissue block. React at room temperature for 5-10 minutes. Observe the color development of the positive control under a microscope. Stop the color development when the ideal positive color is achieved and there is no obvious background. Rinse thoroughly with distilled water.

[0143] 8. Staining cell nuclei

[0144] Place the slide on a slide holder, immerse it in hematoxylin for counterstaining, and allow it to react at room temperature for 30 seconds to 1 minute. Rinse with PBS or tap water.

[0145] 9. Dehydrated and transparent:

[0146] Immerse the tissue sections in 85% alcohol for 5 minutes, then transfer them to 100% alcohol for 5 minutes, and finally to 100% xylene for 5 minutes. After air-drying, mount them with neutral resin.

[0147] Immunohistochemistry was used to stain and analyze paraffin sections of human tonsils, spleen, and colorectal cancer, and microscopic observation was performed as follows. Figure 3 As shown: In tonsil, spleen tissue and colorectal cancer samples, monoclonal antibody 2B6 can clearly label BCMA-positive cells and has obvious brown-yellow specific staining with clear background.

[0148] Example 5: Flow cytometry verification of anti-human BCMA monoclonal antibody

[0149] The anti-human BCMA monoclonal antibody in this invention has been verified by flow cytometry to specifically bind to H929 human myeloma cells and 293T-BCMA transgenic cells. The cell surface staining procedure includes the following steps:

[0150] 1. Cell counting: Collect the cultured cells into 15mL centrifuge tubes and count them.

[0151] 2. Preparation of single-cell suspension: Resuspend cells in 1 mL of pre-cooled 0.5% BSA / PBS solution, centrifuge at 300g for 5 min, discard the supernatant, and repeat twice; resuspend cells in 0.5% BSA / PBS solution to a concentration of 1x10⁻⁶. 7 Cell count / mL: Take 100 μL of cell suspension and place it in a flow cytometer.

[0152] 3. Fc receptor blocking: The cells can be thoroughly mixed with the antibody homologous serum full IgG antibody and incubated at room temperature for 10-20 minutes.

[0153] 4. Primary antibody incubation: Add 100 μL of diluted primary antibody solution to each tube and incubate at 4°C for 30 min. Shake the reaction tube every 10 min during incubation to ensure sufficient reaction between cells and antibody. Directly labeled antibodies should be reacted in the dark.

[0154] 5. Washing: Centrifuge at 300g for 5 min, discard the supernatant, add 1 mL of 0.5% BSA / PBS solution to resuspend, centrifuge to remove the supernatant, and repeat twice.

[0155] 6. Secondary antibody incubation: Add 100 μL of fluorescent secondary antibody to resuspend the antibody and react at 4°C in the dark for 30 min.

[0156] 7. Washing: Centrifuge at 300g for 5 min, remove the supernatant, add 1 mL of 0.5% BSA / PBS solution to resuspend, centrifuge to remove the supernatant, and repeat twice.

[0157] 8. Resuspend the cells in 500 μL of 0.5% BSA / PBS solution and analyze them.

[0158] In this study, hybridoma cell fusion and antibody screening were carried out in immunized mice. In the first round of ELISA screening, hybridoma supernatant with OD value greater than 2.0 was selected, and 6 positive BCMA monoclonal antibodies were screened out with OD values ​​of 6H11 (3.008), 2D5 (2.746), 2B6 (3.255), 4E4 (3.021), 2A8 (2.122), and 2B7 (3.028).

[0159] Further flow cytometry validation was performed using the stable BCMA-expressing cell line 293T-BCMA and the H929 tumor cell line as flow cytometry selection cells. This successfully yielded the anti-human BCMA monoclonal antibody 2B6, which specifically recognizes these cells, as well as a hybridoma cell line that stably secretes the antibody. Flow cytometry results are as follows: Figure 4 As shown, the antibodies in the hybridoma cell culture supernatant exhibited significant positive binding peaks for 293T-BCMA and H929. Figure 4 Figure A shows 293T-BCMA cells. Figure 4 Figure B shows H929 cells. Gray peak: blank control; dashed peak: single or secondary antibody; solid peak: BCMA monoclonal antibody.

[0160] Example 6: Anti-human BCMA monoclonal antibody blocks BAFF protein recognition of cell surface sites

[0161] Validation of the blocking effect of anti-human BCMA monoclonal antibody on BAFF protein recognition of cell surface sites includes the following steps:

[0162] 1. Cell preparation: Collect target cells (adherent cells digested / suspension cells centrifuged), count them, and adjust the cell concentration to 1×10⁻⁶. 6 Cell count / mL, resuspend cells in pre-chilled PBS. Perform the entire procedure on ice to minimize non-specific binding.

[0163] 2. Set up grouping and control:

[0164] No blocking antibody added + ligand protein added (positive binding control)

[0165] Blocking monoclonal antibodies (1 μg, 0.5 μg, 0.25 μg, 0.125 μg, at different concentration gradients) + ligand protein

[0166] No ligand added (negative blank control)

[0167] Adding a secondary antibody (negative background)

[0168] 3. Ligand incubation group: Add biotin-labeled ligand protein biotin-BAFF, incubate at 4℃ for 20 min, and wash twice with PBS;

[0169] 4. Monoclonal antibody blocking incubation group

[0170] Add the blocking monoclonal antibody to each group first, mix gently, and incubate at 4°C for 20-30 min;

[0171] The antibody was washed away with PBS, and the biotin-labeled ligand protein biotin-BAFF was added. The mixture was incubated at 4°C for 20 min and washed twice with PBS. Finally, streptavidin labeled with fluorescein was added.

[0172] 5. Data Acquisition and Analysis:

[0173] Filter before collection (40 µm) to avoid tube blockage.

[0174] Analysis workflow: FSC / SSC cell selection → Single-cell phylogenetic analysis → Live-cell phylogenetic analysis → Target cell population delineation → Detection of ligand fluorescence signals.

[0175] Using 2B6 monoclonal antibody as the primary antibody, 293T-BCMA-transfected cells and H929 human multiple myeloma cells were reacted with the target antibody. After incubation, the primary antibody was washed off, and biotin-labeled BAFF protein was added for a second round of reaction; after washing, fluorescein-labeled streptavidin was added. Figure 5 As shown, anti-human BCMA monoclonal antibody can block the binding of BCMA to its ligand BAFF, and there is competitive inhibition between the antibody and the ligand protein. Figure 5 Figure A shows 293T-BCMA cells. Figure 5 Figure B shows H929 cells. Gray peak: blank control; long dashed peak: single or secondary antibody; dotted peak: BCMA monoclonal antibody + biotin-labeled BAFF protein; solid peak: biotin-labeled BAFF protein binding peak to target cells.

[0176] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Anti-human BCMA monoclonal antibody, characterized in that, include: Heavy chain variable region and light chain variable region, wherein: (1) The three CDR regions of the heavy chain variable region have amino acid sequences as shown in SEQ ID NO: 1, 2 and 3, and the three CDR regions of the light chain variable region have amino acid sequences as shown in SEQ ID NO: 4, 5 and 6: or (2) The three CDR regions of the heavy chain variable region and / or the three CDR regions of the light chain variable region have amino acid sequences obtained by substitution, deletion or addition of one or more amino acids as described in (1), and amino acid sequences that are functionally identical or similar to the amino acid sequences shown in (1); or (3) The three CDR regions of the heavy chain variable region and / or the three CDR regions of the light chain variable region have CDRs that have at least 85% sequence identity with the amino acid sequence shown in (1) or (2).

2. The anti-human BCMA monoclonal antibody as described in claim 1, characterized in that, (4) Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO:7, and its light chain variable region has the sequence shown in SEQ ID NO:8; or (5) or (4) amino acid sequences obtained by substitution, deletion or addition of one or more amino acids, and which have the same or similar function as the amino acid sequence shown in (4); or (6) An amino acid sequence that is at least 80% homologous to the sequence described in (4) or (5).

3. A nucleic acid molecule encoding the anti-human BCMA monoclonal antibody as described in claim 1 or 2.

4. Biomaterials characterized in that, Includes at least one of the following (7) to (9): (7) An expression vector comprising the nucleic acid molecule of claim 3; (8) Transform or transfect the host cells of the expression vector; (9) Culturing the host cells to obtain a culture containing the anti-human BCMA monoclonal antibody.

5. A labeled antibody characterized in that, Includes markers and the anti-human BCMA monoclonal antibody as described in claim 1 or 2.

6. The labeled antibody of claim 5, wherein, The markers include chemical markers and biological markers; The chemical markers include isotopes and / or chemical drugs; The biomarkers include biotin, avidin, or enzymes.

7. Conjugate characterized in that, Includes a conjugation medium and the anti-human BCMA monoclonal antibody as described in claim 1 or 2.

8. The conjugate of claim 7, wherein, The coupling medium includes a solid medium or a semi-solid medium.

9. Use of at least one of the following (10) to (14) in the preparation of a product for the detection and / or treatment of BCMA-positive diseases: (10) The anti-human BCMA monoclonal antibody according to claim 1 or 2; (11) The nucleic acid molecule according to claim 3; (12) The biomaterial as described in claim 4; (13) The labeled antibody according to claim 5 or 6; (14) The coupling as described in claim 7 or 8.

10. Products for the detection and / or treatment of BCMA-positive diseases, characterized in that, Includes at least one of the following (15) to (19): (15) The anti-human BCMA monoclonal antibody according to claim 1 or 2; (16) The nucleic acid molecule according to claim 3; (17) The biomaterial as described in claim 4; (18) The labeled antibody according to claim 5 or 6; (19) The coupling as described in claim 7 or 8.

Images