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CN-121991228-A - Anti-BCMA nano antibody and application thereof

CN121991228ACN 121991228 ACN121991228 ACN 121991228ACN-121991228-A

Abstract

The invention relates to an anti-BCMA nano antibody and application thereof, belonging to the field of biotechnology. The anti-BCMA nanobody of the invention comprises a heavy chain variable region which comprises 3 complementarity determining regions CDR1, CDR2 and CDR3, and the amino acid sequence of the heavy chain variable region is SEQ ID NO. 1. The invention obtains the targeted BCMA nanobody through the alpaca nanobody natural library and phage display technology, can specifically identify the Human BCMA recombinant protein and HEK293 hBCMA over-expression cells, and has good application prospect and development potential in the aspect of treating or diagnosing various inflammatory diseases and autoimmune diseases which are characterized by abnormal activation of BCMA signals, especially BCMA high-expression related malignant tumors such as multiple myeloma and the like.

Inventors

  • WANG WEI
  • LI YIXIAN
  • TANG YUJIE
  • XIANG YANG
  • SHI LEI

Assignees

  • 江苏百英生物科技有限公司

Dates

Publication Date
20260508
Application Date
20260206

Claims (10)

  1. 1. An anti-BCMA nanobody comprising a heavy chain variable region comprising 3 complementarity determining regions CDR1, CDR2, CDR3; The amino acid sequence of the CDR1 of the complementarity determining region is shown as SEQ ID NO. 3; The amino acid sequence of the CDR2 is shown in SEQ ID NO. 4; The amino acid sequence of the CDR3 of the complementarity determining region is shown in SEQ ID NO. 5.
  2. 2. The anti-BCMA nanobody according to claim 1 wherein the nanobody heavy chain variable region amino acid sequence 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 to SEQ ID No. 1.
  3. 3. A nucleic acid molecule encoding the anti-BCMA nanobody according to claim 1 or 2.
  4. 4. The nucleic acid molecule of claim 3, wherein the nucleotide sequence of said nucleic acid molecule is set forth in SEQ ID NO. 2.
  5. 5. An expression vector comprising the nucleic acid molecule of claim 3, wherein the expression vector is a mammalian expression vector.
  6. 6. A host cell comprising the expression vector of claim 5.
  7. 7. A method of preparing the anti-BCMA nanobody according to claim 1 or 2 comprising the steps of: S1, separating PBMC from alpaca peripheral blood, extracting total RNA, performing reverse transcription to obtain cDNA, amplifying a nanobody coding gene by PCR (polymerase chain reaction) with the cDNA as a template, connecting an amplified product to a phage vector after enzyme digestion, and performing phage packaging to obtain a nanobody natural library; S2, pressurizing and panning the nano antibody natural library by taking BCMA antigen as a target spot, and enriching bacteriophage capable of combining with the BCMA antigen; S3, selecting the elutriated monoclonal to culture and induce expression, screening to obtain positive clone, and then carrying out sequencing analysis to determine a target antibody coding sequence; S4, constructing a recombinant expression vector, transfecting a mammalian host cell, collecting culture supernatant, and purifying an expression product by an affinity chromatography method to obtain the anti-BCMA nano antibody.
  8. 8. A pharmaceutical composition comprising an anti-BCMA nanobody according to claim 1 or 2 and a pharmaceutically acceptable carrier.
  9. 9. A reagent or kit for detecting BCMA comprising the anti-BCMA nanobody according to claim 1 or 2.
  10. 10. Use of an anti-BCMA nanobody as claimed in claim 1 or 2 for the preparation of a diagnostic reagent for detecting BCMA positive cells.

Description

Anti-BCMA nano antibody and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to an anti-BCMA nano antibody and application thereof. Background BCMA (B cell maturation antigen) is a member of the tumor necrosis factor receptor superfamily, having a molecular weight of about 34 kDa and an extracellular domain consisting of amino acids 1-54, having a theoretical molecular weight of 5.8 kDa. BCMA is expressed primarily in mature B cells, plasma cells, etc., and by binding to ligands such as BAFF (B cell activating factor) and APRIL (proliferation-inducing ligand), activates downstream signaling pathways (such as NF- κb pathway), promotes B cell survival, differentiation and immunoglobulin secretion, and is critical to maintaining B cell homeostasis and humoral immunity. In tumors, BCMA high expression is closely related to diseases such as multiple myeloma and the like, and is a key factor for tumor cell survival and proliferation. BCMA-targeted therapeutic strategies, such as CAR-T cell therapies, bispecific antibodies, etc., have shown significant efficacy in the treatment of multiple myeloma, bringing new promise for patients. In addition, BCMA has also received attention in autoimmune diseases, and is expected to be a new target for the treatment of related diseases. The phage display technology is to fuse the exogenous protein gene with the phage coat protein gene to make the exogenous protein expressed along with the phage coat protein and displayed on the phage surface. By incubating with a specific target molecule (e.g., an antibody), the bound phage is eluted and amplified, and through multiple rounds of screening, high affinity phage clones can be enriched. The technology has high screening efficiency, can screen a large number of clones in a short time, is simple and convenient to operate and relatively low in cost, can directly obtain genes corresponding to the display proteins, and is convenient for subsequent research and application. Nanobodies (single domain antibodies) are antigen-binding fragments of heavy chain antibodies, with unique advantages. The molecular weight is small (about 15 kDa), the affinity and the specificity are high, and the antigen can be deeply combined with antigen sites which are difficult to reach by conventional antibodies. The nano antibody has high stability, can keep activity under extreme conditions, is easy to produce, and can be efficiently prepared by a prokaryotic expression system. In addition, the nano antibody can be displayed in a multivalent mode, the antigen binding capacity is enhanced, and the nano antibody is widely applied to the fields of diagnosis, treatment and research and provides a novel powerful tool for biomedical research and clinical application. Disclosure of Invention The invention aims to provide an anti-BCMA nano antibody which can specifically identify a Human BCMA recombinant protein and HEK293 hBCMA over-expression cells, and also provides application of the antibody. The invention also provides a preparation method of the anti-BCMA nano antibody. Compared with an immune library, the nano antibody is lower in cost, is not limited by antigenicity of an antigen, can be enriched without animal immunity to obtain the specific nano antibody and the like, and plays a role in promoting development of biomedical technology. Aiming at the purposes, the invention adopts the technical scheme that: an anti-BCMA nanobody comprising a heavy chain variable region comprising 3 complementarity determining regions CDR1, CDR2, CDR3; The amino acid sequence of the CDR1 of the complementarity determining region is shown as SEQ ID NO. 3; The amino acid sequence of the CDR2 is shown in SEQ ID NO. 4; The amino acid sequence of the CDR3 of the complementarity determining region is shown in SEQ ID NO. 5. Preferably, the nanobody heavy chain variable region amino acid sequence 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. The invention also provides a nucleic acid molecule which codes for the anti-BCMA nanobody. Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 2. The invention also provides an expression vector containing the nucleic acid molecule, and the expression vector is a mammalian expression vector. Preferably, the mammalian system expression vector is pcDNA3.4. The invention also provides a host cell containing the expression vector. Preferably, the mammalian system expression host cell is CHO-K1. The invention also provides a preparation method of the anti-BCMA nano antibody, which comprises the following steps: S1, separating PBMC from alpaca peripheral blood, extracting total RNA, performing reverse transcription to obtain cDNA, amplifying a nanobody coding gene by PCR (polymerase chain reaction) with the cDNA as a template, connecting an amplified product to a