Anti-BCMA nano antibody and application thereof

Abstract

The invention relates to an anti-BCMA nano antibody and application thereof, and belongs to the technical field of biology. The anti-BCMA nano antibody comprises a heavy chain variable region which comprises three complementary determining regions CDR1, CDR2 and CDR3, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1. The nanometer antibody targeting BCMA is obtained through an alpaca nanometer antibody natural library and a phage display technology, the antibody can specifically recognize Human BCMA recombinant protein and HEK293 hBCMA overexpression cells, and the nanometer antibody can be used for treating or diagnosing various inflammatory diseases and autoimmune diseases which are characterized by abnormal activation of BCMA signals. In particular to BCMA high-expression related malignant tumors such as multiple myeloma, the BCMA high-expression related malignant tumors show good application prospects and development potential.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an anti-BCMA nanobody and its applications. Background Technology

[0002] BCMA (B-cell maturation antigen) is a member of the tumor necrosis factor receptor superfamily, with a molecular weight of approximately 34 kDa. Its extracellular domain consists of amino acids 1-54, with a theoretical molecular weight of 5.8 kDa. BCMA is mainly expressed on mature B cells and plasma cells. By binding to ligands such as BAFF (B-cell activating factor) and APRIL (proliferation-inducing ligand), it activates downstream signaling pathways (such as the NF-κB pathway), promoting B-cell survival, differentiation, and immunoglobulin secretion, which is crucial for maintaining B-cell homeostasis and humoral immunity. In tumors, high BCMA expression is closely associated with diseases such as multiple myeloma and is a key factor in tumor cell survival and proliferation. Targeted therapies for BCMA, such as CAR-T cell therapy and bispecific antibodies, have shown significant efficacy in the treatment of multiple myeloma, bringing new hope to patients. Furthermore, the role of BCMA in autoimmune diseases has also attracted attention, and it is expected to become a new target for the treatment of related diseases.

[0003] Phage display technology involves fusing a foreign protein gene with a phage coat protein gene, allowing the foreign protein to be expressed and displayed on the phage surface along with the phage coat protein. After incubation with a specific target molecule (such as an antibody), the bound phage is eluted and amplified. Through multiple rounds of screening, high-affinity phage clones can be enriched. This technology offers high screening efficiency, enabling the screening of a large number of clones in a short time; it is simple to operate, relatively low in cost, and directly provides the gene corresponding to the displayed protein, facilitating subsequent research and applications.

[0004] Nanobodies (single-domain antibodies) are antigen-binding fragments of heavy-chain antibodies, possessing unique advantages. Their small molecular weight (approximately 15 kDa) results in high affinity and specificity, enabling them to bind deeply to antigen sites that are difficult for conventional antibodies to reach. Nanobodies exhibit high stability, maintaining activity even under extreme conditions, and are easily produced through prokaryotic expression systems. Furthermore, nanobodies can display multiple valences, enhancing antigen-binding capabilities, and are widely used in diagnostics, therapy, and research, providing a powerful new tool for biomedical research and clinical applications. Summary of the Invention

[0005] The purpose of this invention is to provide an anti-BCMA nanobody that specifically recognizes the Human BCMA recombinant protein and HEK293 hBCMA-overexpressing cells, and also provides applications for this antibody. This invention also provides a method for preparing the anti-BCMA nanobody. This invention develops nanobodies using a natural alpaca nanobody library, which offers advantages over immune libraries, including lower cost, no antigenicity limitations, and the ability to enrich specific nanobodies without animal immunization, thus promoting the development of biomedical technology.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] An anti-BCMA nanobody, the nanobody comprising a heavy chain variable region including three complementarity-determining regions CDR1, CDR2, and CDR3;

[0008] The amino acid sequence of the complementarity-determining region CDR1 is shown in SEQ ID NO:3;

[0009] The amino acid sequence of the complementarity-determining region CDR2 is shown in SEQ ID NO:4;

[0010] The amino acid sequence of the complementarity-determining region CDR3 is shown in SEQ ID NO:5.

[0011] Preferably, the amino acid sequence of the variable region of the heavy chain of the nanobody is as shown in SEQ ID NO:1, or has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1.

[0012] The present invention also provides a nucleic acid molecule that encodes the above-mentioned anti-BCMA nanobody.

[0013] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:2.

[0014] The present invention also provides an expression vector containing the above-mentioned nucleic acid molecules, wherein the expression vector is a mammalian system expression vector.

[0015] Preferably, the expression vector for the mammalian system is pcDNA3.4.

[0016] The present invention also provides a host cell containing the above-described expression vector.

[0017] Preferably, the mammalian system expresses CHO-K1 in the host cell.

[0018] This invention also provides a method for preparing anti-BCMA nanobodies, comprising the following steps:

[0019] S1. PBMCs were isolated from alpaca peripheral blood, and total RNA was extracted and cDNA was obtained by reverse transcription. The nanobody encoding gene was amplified by PCR using cDNA as a template. The amplified product was digested with enzymes and ligated into a phage vector for phage packaging to obtain a natural library of nanobodies.

[0020] S2. The above-mentioned natural library of nanobodies was subjected to pressure panning with BCMA antigen as the target to enrich phages that can bind to BCMA antigen;

[0021] S3. Select the panned single clones for culture and induced expression. After screening to obtain positive clones, perform sequencing analysis to determine the target antibody coding sequence.

[0022] S4. Construct a recombinant expression vector, transfect mammalian host cells, collect the culture supernatant, purify the expression product by affinity chromatography, and obtain anti-BCMA nanobodies.

[0023] The present invention also provides a pharmaceutical composition comprising the above-described anti-BCMA nanobody and a pharmaceutically acceptable carrier.

[0024] The present invention also provides a reagent or kit for detecting BCMA, which comprises the above-mentioned anti-BCMA nanobody.

[0025] The present invention also provides the use of the above-mentioned anti-BCMA nanobody in the preparation of diagnostic reagents for detecting BCMA-positive cells.

[0026] The present invention has the following technical effects:

[0027] This invention obtains a nanobody targeting BCMA from a natural library of alpaca nanobodies using phage display technology. This antibody can specifically recognize recombinant Human BCMA protein and HEK293 hBCMA-overexpressing cells. It shows good application prospects and development potential in the treatment or diagnosis of various inflammatory diseases and autoimmune diseases characterized by abnormal activation of BCMA signaling, especially BCMA-high expression-related malignancies such as multiple myeloma. Attached Figure Description

[0028] Figure 1 This is a diagram showing the results of ELISA screening using Human BCMA / His protein as an antigen in Example 1.

[0029] Figure 2 This is a diagram showing the results of FACS screening using HEK293 hBCMA-overexpressing cells as antigens in Example 1.

[0030] Figure 3 This is a map of the antibody-mammal system expression vector in Example 2.

[0031] Figure 4 The image shows the SDS-PAGE results of the purified antibody in Example 2, where R represents the reduction condition, NR represents the non-reduction condition, and M represents the marker.

[0032] Figure 5 The image shows the SEC-HPLC results of the purified antibody in Example 2, where A is the result of BCMA-LP1R3-C12 at 214 nM and B is the result at 280 nM.

[0033] Figure 6 This refers to the binding of the purified antibody to the Human BCMA protein in Example 3.

[0034] Figure 7 This refers to the binding of the purified antibody from Example 3 to the HEK293 hBCMA overexpressing cell line. Detailed Implementation

[0035] The present invention will be further explained below with reference to specific embodiments. However, it should be noted that the following embodiments are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions that are the same as or similar to the present invention are within the protection scope of the present invention.

[0036] For any techniques or conditions not specified in this embodiment, the operation shall be carried out in accordance with conventional technical methods and instrument manuals in this field; for reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained commercially.

[0037] Example 1: Screening anti-BCMA nanobodies from a natural library of alpaca nanobodies

[0038] Phage display technology was used to screen anti-BCMA nanobodies from a natural library of alpaca nanobodies.

[0039] Preparation and blocking of magnetic beads: Take streptavidin magnetic beads (Thermo Fisher Scientific, 11206D) into EP tubes, wash three times with 1 mL PBS, add 1 mL of 5% NON-fat Powdered Milk (Sangon Biotech, A600669-0250), mix by inversion, and incubate at 25°C for 1 h.

[0040] Take the sealed magnetic beads, add 5% NON-fat Powdered Milk (Sangon Biotech, A600669-0250), then add phage to a total volume of 1 mL, and incubate at 25°C for 1 h.

[0041] Separately, after sealing the magnetic beads, add 100 nM biotin-labeled hBCMA / His recombinant protein (Biointron, catalog number: B21846208) in the first round, and add 30 nM biotin-labeled hBCMA / His recombinant protein and 10 nM biotin-labeled hBCMA / His recombinant protein in the second and third rounds, respectively, and incubate at 25°C for 1 h.

[0042] Take the magnetic beads that have bound the positive screening antigen from the previous step, add the phage supernatant (excluding background), and incubate at 25°C for 1 hour. Remove the phage supernatant that has not bound to the magnetic beads. For the first round, wash the remaining magnetic beads 3 times each with 1 mL of 0.05% PBST and 1 mL of PBS. For the second and third rounds, wash 5 times each with 1 mL of 0.05% PBST and 1 mL of PBS. After resuspending, add the beads to the logarithmic phase of TG1 bacterial culture for amplification, and use them for the next round of selection.

[0043] After three rounds of pressure screening, the bacterial culture after the third round of amplification was plated. The next day, 94 clones were selected from one plate for culture, and the supernatant of each clone was collected for screening and testing.

[0044] The monoclonal ELISA screening process is as follows: 2 μg / mL hBCMA / His protein and 100 μL blocking buffer (3% NON-fat Powdered Milk in PBS) are coated onto the microplate and incubated overnight at 4°C. The next day, the plate is washed three times with 0.1% PBST, blocked with blocking buffer for 1 h, washed three times with 0.1% PBST, and the prepared sample is added. 10 μg / mL purified antibody Anti-Human BCMA (Belantamab) (Abinvivo, B564601) is used as a positive control, and Anti-HEL IgG1 hFc (Biointron, B117901) is used as a negative control. 100 μL / well is incubated at 25°C for 1 h. After washing five times with 0.1% PBST, 100 μL of Mouse anti-M13 mAb HRP (Sino Biolo, 11973-MM05T-H) diluted 1:10000 was added, and the mixture was incubated at 25°C for 1 h. The control antibody secondary antibody was Goat Anti-Human IgG-Fc, HRP (Sigma, A0170), diluted 1:10000. After washing five times with 0.1% PBST, ABTS (Thermo, 002024) was added, and the mixture was incubated at room temperature in the dark. The absorbance was read at 415 nm using a microplate reader. Positive clone criteria: BCMA > 3 × NC and Milk < 3 × NC, BCMA / Milk > 2.5. The detection results are as follows: Figure 1As shown, 67 out of 94 single clones were positive clones that bound the hBCMA / His recombinant protein.

[0045] The FACS screening process for monoclonal phages was as follows: HEK293 cells overexpressing hBCMA and GFP were mixed 1:1 and seeded into 96-well V-type plates. The cells were centrifuged at 400 g for 5 min, and the supernatant was discarded. The cells were resuspended in the monoclonal phage supernatant in the 96-well V-type plates. Purified antibody Anti-Human BCMA (Belantamab) (Abinvivo, B564601) was used as a positive control, and Anti-HEL IgG1 hFc (Biointron, B117901) was used as a negative control. The cells were incubated at 4℃ for 0.5 h. The cells were centrifuged at 400 g for 5 min, and the supernatant was discarded. 200 μL / well of PBS buffer was added and mixed thoroughly. The cells were centrifuged at 400 g for 5 min, and the supernatant was discarded. THE™ DYKDDDDK Tag Antibody (Thermo, A-21281) was added to each well at a 1:1000 ratio, 100 μL / well, and incubated at 4℃ for 0.5 h. Add Goat Anti-hIgG (Fcγ Specific) pAb [Alexa Fluor 647] (Jackson, 109-605-190), 1:800, 100 μL / well, and incubate at 4°C for 0.5 h. Centrifuge at 400 g for 5 min and discard the supernatant. Add 200 μL / well of PBS buffer, mix well, centrifuge at 400 g for 5 min, and discard the supernatant. Resuspend cells in 100 μL / well of PBS buffer and analyze by flow cytometry. Positive clone determination: Sample / Negative control Median APC-H (≥3). Results are as follows. Figure 2 As shown, 53 out of 94 clones bound to HEK293 hBCMA-overexpressing cells.

[0046] Based on the combined screening results of the two antigens, 37 clones that simultaneously bound to Huamn BCMA recombinant protein and Cynomolgus BCMA were sequenced. The final obtained single clone was BCMA-LP1R3-C12, whose amino acid sequence is shown in SEQ ID NO: 1. The amino acid sequence of CDR1 in its heavy chain variable region is shown in SEQ ID NO: 3, the amino acid sequence of CDR2 is shown in SEQ ID NO: 4, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 5. The corresponding nucleotide sequence is SEQ ID NO: 2.

[0047] Example 2: Expression and purification of anti-BCMA nanobodies in a lactational system

[0048] The mammalian system expression vector pcDNA3.4 for the BCMA-LP1R3-C12 nanobody in Example 1 was constructed (see diagram). Figure 3 Then, plasmids were prepared. CHO-K1 cells were selected as the host cells for antibody expression, with an expression volume of 40 mL. The supernatant after expression was purified using a Protein A affinity chromatography column to obtain high-purity expressed antibodies. Purity was determined by SDS-PAGE and SEC-HPLC, and the results are shown below. Figure 4 and Figure 5 As shown, the antibody purity all reached over 95%, indicating high purity.

[0049] Example 3: Detection of the binding ability of the antibody from Example 2 to the hBCMA antigen.

[0050] The ELISA detection procedure for antibody binding to hBCMA / His protein is as follows: 2 μg / mL of recombinant hBCMA / His protein was coated onto an ELISA plate (Corning, 3590) and incubated overnight at 4°C. The next day, the plate was washed three times with 0.1% PBST, blocked with blocking buffer (3% non-fat powdered milk in PBS) for 1 h, and washed three times with 0.1% PBST. The purified BCMA-LP1R3-C12 antibody and control antibody from Example 2 were diluted to 100 nM as the initial concentration, serially diluted 3-fold, with the last well being a blank, at 100 μL / well. After incubation at 25°C for 1 h, the plate was washed five times with 0.1% PBST. The secondary antibody was Goat Anti-Human IgG-Fc, HRP (Sigma, A0170), diluted 1:10000, at 100 μL / well, and incubated at 25°C for 1 h. After washing five times with 0.1% PBST, ABTS (Thermo, 002024) was added and the sample was developed at room temperature in the dark. The absorbance was read at 415 nm using a microplate reader. The positive control was Anti-Human BCMA (Belantamab) (Abinvivo, B564601), and the negative control was Anti-HEL IgG1 hFc (Biointron, B117901). The test results are as follows: Figure 6 As shown, BCMA-LP1R3-C12 binds to the hBCMA / His protein with an EC50 value of 2.625 nM.

[0051] Example 4: Detection of the binding ability of the antibody from Example 2 to the HEK293 hBCMA overexpressing cell line.

[0052] The FACS detection procedure is as follows: Seed an appropriate amount of HEK293 hBCMA cells into a 96-well V plate, centrifuge at 400 g for 5 min, and discard the supernatant. Dilute the purified BCMA-LP1R3-C12 antibody and control antibody from Example 2 to 100 nM as the initial concentration, perform a 3-fold serial dilution, with the last well being a blank, 100 μL / well, incubate at 4℃ for 0.5 h, centrifuge at 400 g for 5 min, and discard the supernatant. Add Goat Anti-hIgG (Fcγ Specific) pAb [Alexa Fluor 647], 1:800, 100 μL / well, incubate at 4℃ for 0.5 h, centrifuge at 400 g for 5 min, and discard the supernatant. Add 200 μL / well of PBS buffer, mix well, centrifuge at 400 g for 5 min, and discard the supernatant. Resuspend the cells in 100 μL / well of PBS buffer, and analyze using flow cytometry to generate FACS binding curves. The positive control was Anti-Human BCMA (Belantamab) (Abinvivo, B564601), and the negative control was Anti-HEL IgG1hFc (Biointron, B117901). The results of the binding of BCMA-LP1R3-C12 antibody to HEK293 hBCMA cells are as follows: Figure 7 As shown, BCMA-LP1R3-C12 can bind to the HEK293 hBCMA overexpressing cell line, with an EC50 of 2.846 nM.

[0053] Finally, it should be noted that the above embodiments are merely illustrative of the principles, performance, and effects of the present invention, and are not intended to limit the present invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. An anti-BCMA nanobody, characterized in that, The nanobody contains a heavy chain variable region, which includes three complementarity-determining regions CDR1, CDR2, and CDR3. The amino acid sequence of the complementarity-determining region CDR1 is shown in SEQ ID NO:3; The amino acid sequence of the complementarity-determining region CDR2 is shown in SEQ ID NO:4; The amino acid sequence of the complementarity-determining region CDR3 is shown in SEQ ID NO:

5.

2. The anti-BCMA nanobody according to claim 1, characterized in that, The amino acid sequence of the variable region of the heavy chain of the nanobody is as shown in SEQ ID NO:1, or has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

1.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-BCMA nanobody as described in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

5. An expression vector containing the nucleic acid molecule as described in claim 3, characterized in that, The expression vector is a mammalian system expression vector.

6. A host cell containing the expression vector as described in claim 5.

7. A method for preparing the anti-BCMA nanobody as described in claim 1 or 2, characterized in that, Includes the following steps: S1. PBMCs were isolated from alpaca peripheral blood, and total RNA was extracted and cDNA was obtained by reverse transcription. The nanobody encoding gene was amplified by PCR using cDNA as a template. The amplified product was digested with enzymes and ligated into a phage vector for phage packaging to obtain a natural library of nanobodies. S2. The above-mentioned natural library of nanobodies was subjected to pressure panning with BCMA antigen as the target to enrich phages that can bind to BCMA antigen; S3. Select the panned single clones for culture and induced expression. After screening to obtain positive clones, perform sequencing analysis to determine the target antibody coding sequence. S4. Construct a recombinant expression vector, transfect it into mammalian host cells, collect the culture supernatant, purify the expression product by affinity chromatography, and obtain anti-BCMA nanobodies.

8. A pharmaceutical composition, characterized in that, It includes the anti-BCMA nanobody as described in claim 1 or 2 and a pharmaceutically acceptable carrier.

9. A reagent or kit for detecting BCMA, characterized in that, It includes the anti-BCMA nanobody as described in claim 1 or 2.

10. Use of the anti-BCMA nanobody according to claim 1 or 2 in the preparation of a diagnostic reagent for detecting BCMA-positive cells.

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