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Preparation method and application of BCMA antibody

An antibody and identity technology, applied in the field of preparation and application of BCMA antibodies, can solve the problems that stem cell transplantation is not suitable for patients and rarely achieve complete remission.

Active Publication Date: 2021-07-02
EXCYTE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because of advanced age, the presence of other serious medical conditions, or other physical limitations, stem cell transplantation may not be an option for most patients
Chemotherapy only partially controls multiple myeloma and rarely achieves complete remission

Method used

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  • Preparation method and application of BCMA antibody
  • Preparation method and application of BCMA antibody
  • Preparation method and application of BCMA antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Screening of anti-human BCMA antibody from natural human antibody phage surface display library

[0066] Antibody library technology is a method of cloning all antibody variable region genes of a certain animal (including humans) into plasmids or phages. After the latter infects E. coli, antibody fragments are expressed on the surface of phage particles, or the periplasm of E. coli, including in the pulp. Then use the target antigen to screen the clones carrying specific antibody genes from the antibody library, so as to obtain the corresponding specific antibody technology. Various antibodies needed in basic research and clinical development have been screened from the antibody library, such as tumor-associated membrane protein antigens, autoantigens related to autoimmune diseases, and antibodies against viral antigens of viral diseases. These show the great application potential of antibody library technology in basic research and antibody drug development....

Embodiment 2

[0072] Example 2 Antibody function verification

[0073] In order to verify whether the obtained BCMA antibody clone binds to the purified BCMA antigen and BCMA expressed on the cell membrane surface, the gene of the B10 single-chain antibody was cloned into the eukaryotic expression vector pFH (manufactured by Ekest) to obtain the plasmid pFH-B10. In this vector, the scFv gene is fused with the Fc gene of human IgG4 to express the protein in the form of scFv-Fc, which can be affinity purified with Potein-A or labeled with (HRP or fluorescein) anti-human Fc antibody detection.

[0074] After obtaining the scFv-Fc protein of B10, the binding of this antibody to BCMA was detected by Octet RED, which confirmed the specific binding of B10 to BCMA.

[0075] B10 was detected by flow cytometry on the BCMA-expressing cell line H929 (NCI-H929, CRL-9068 TM ), indicating that B10 specifically binds to the BCMA antigen on the cell membrane surface (see figure 1 ). In this FACS anal...

Embodiment 3

[0076] Example 3 Antibody affinity maturation

[0077] Antibody affinity refers to the interaction strength between an antibody and its specific antigen, more precisely, the binding strength between the antibody and its corresponding antigenic determinant (or epitope) on the specific antigen. This interaction is non-covalent in nature, and the antibody-antigen is tightly bound by hydrogen bonds, hydrophobic forces, ionic bonds or / and Van der Waals forces by direct contact of amino acids at some positions on both sides of the antibody-antigen to form an antibody-antigen Antigen complex. Affinity in the strict sense (also known as intrinsic affinity, KD) refers to the strength of antibody-antigen 1:1 monovalent binding, such as antigen-binding fragment (Fab) or single-chain antibody (scFv) to monomeric antigen Interaction. Due to the Y-shaped symmetrical structure of the classical antibody, the binding strength exhibited when the antibody recognizes and binds to the correspond...

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Abstract

The invention provides a preparation method and application of a BCMA antibody. The specific single-chain antibody resisting the human B cell surface antigen BCMA is screened from a single-chain antibody library of a non-immune fully human source sequence through genetic engineering and phage surface display library technology. A plurality of Fab clones with remarkably improved binding capacity are obtained through affinity maturation modification, and the affinity of the Fab clones with human BCMA is between 0.26 nM and 0.31 nM. The apparent affinity measured by a flow cytometry is improved by 61-97 times compared with the apparent affinity of a parent antibody, and it is proved that the anti-human BCMA antibody and the optimized mutant have good BCMA binding capacity. The invention provides a human BCMA specific antibody candidate molecule for research and development of an anti-tumor antibody drug aiming at a BCMA target, development of a CAR-T reagent and prevention and treatment of other diseases such as B cell related inflammation and autoimmune diseases.

Description

technical field [0001] The invention relates to the preparation and application of human gene engineering antibody for treatment, and mainly relates to the preparation method and application of the antibody specific for BCMA. Background technique [0002] BCMA (i.e., B-cell maturation antigen, TNFRSF17, CD269) is an aglycosylated type I transmembrane protein that is preferentially expressed in differentiated plasma cells and belongs to the tumor necrosis (TNF) receptor superfamily member, which is involved in B Cells mature, grow and survive. BCMA is an integral membrane protein originally reported as an integral membrane protein in the Golgi of human mature B lymphocytes, ie as an intracellular protein, suggesting that BCMA appears to have an important role in B-cell development and homeostasis. [0003] Expression of BCMA is restricted to the B-cell lineage and is mainly present on plasma cells and plasmablasts, and to some extent on memory B-cells, but virtually absent o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C07K19/00G01N33/68A61K39/395A61P35/00A61P37/02
CPCC07K16/2878G01N33/68A61P35/00A61P37/02C07K2317/565C07K2319/00A61K2039/505
Inventor 袁清安孟庆武白丽莉赵立坤李艺嘉李延虎
Owner EXCYTE LLC
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