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Anti-BCMA (B-cell Maturation Antigen) humanized single-chain antibody and application

A single-chain antibody, humanized technology, applied in the field of genetic engineering, can solve the problems of antibody stability and reliability that plague the antibody industry, and achieve the goal of enhancing sustainability and safety, reducing immunogenicity, and high degree of humanization Effect

Active Publication Date: 2019-08-02
CHONGQING PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In addition, the stability and reliability of antibodies has long plagued the antibody industry, and it is also a major bottleneck that needs to be broken through in the entire field of biological research.

Method used

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  • Anti-BCMA (B-cell Maturation Antigen) humanized single-chain antibody and application
  • Anti-BCMA (B-cell Maturation Antigen) humanized single-chain antibody and application

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Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1 Design the humanized single chain antibody of anti-human BCMA antigen

[0046] After molecular docking simulation, the murine antibody framework sequence was humanized and modified, and on this basis, the humanized ScFv was reverse-mutated and immunogenicity optimized to obtain ScFv with better activity and lower immunogenicity.

[0047]Wherein the amino acid sequence of the light chain of the antibody is shown in any one of SEQ ID NO: 14-18; the amino acid sequence of the heavy chain of the antibody is shown in any one of SEQ ID NO: 9-13.

[0048] The amino acid sequence of the humanized single-chain antibody is shown in any one of SEQ ID NO: 19-23; the nucleotide sequence is shown in any one of SEQ ID NO: 24-28.

Embodiment 2

[0049] Example 2 Expression and purification of humanized monoclonal antibody targeting BCMA

[0050] The plasmid carrying the monoclonal antibody was used to construct a lentiviral expression system, and the CHO expressing cells were infected by virus infection, and the monoclonal cell lines with high scFv expression were screened, and the cell expression supernatant was collected and purified every three days. The collected cell supernatant was purified using the AKTA Prime instrument. For specific experimental steps, refer to the AKTA Prime user manual. The purified protein was verified by SDS to verify the activity and purity of the scFv, such as figure 1 As shown, the far left is the protein maker (thermo26619ladder), m means maker, ScFv is the protein band, the antibody size is 25-35kDa, and the modified scFv has activity and high purity.

Embodiment 3

[0051] Example 3 Affinity Determination of Humanized Monoclonal Antibody Targeting BCMA

[0052] Flow cytometry was used to detect its affinity, and the expression abundance of ScFv was detected by using BCMA antigen (Acro-biosystems) to detect the expression abundance of ScFv after expressing ScFv in CHO cells by two-fold dilution of 20ug / ml once, and using GraphPad Prism to process the data and calculate the KD value. the following table and figure 2 As shown, KD=9.896.

[0053] Hyperbola(X is concentration) Best-fit values Bmax 21796 k 9.896

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Abstract

The invention belongs to the technical field of genetic engineering, and specifically relates to an anti-BCMA (B-cell Maturation Antigen) humanized single-chain antibody and application thereof. The amino acid sequence of the anti-BCMA humanized single-chain antibody is as show in SEQ ID NO: 19 or 20 or 21 or 22 or 23. The anti-BCMA humanized single-chain antibody is formed by sequentially connecting a light chain variable region, a Linker and a heavy chain variable region; the humanization degree is high; the immunogenicity of CAR (Chimeric Antigen Receptor) can be effectively reduced; the continuity and safety of CAR-T (Chimeric Antigen Receptor T-cell) in vivo can be enhanced.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an anti-BCMA humanized single-chain antibody and its application. Background technique [0002] The full name of BCMA is tumor necrosis factor receptor superfamily member 17, also known as B-cell maturation antigen (BCMA) or CD269 or TNFRSF13A. It is mainly expressed on the surface of mature B lymphocytes and plasma cells, and is highly expressed in MM tissues and cells. There are many suitable targets for blood diseases, and myeloma (MM) is one of the malignant blood diseases. [0003] Multiple myeloma is also known as multiple myeloma, abbreviated as MM. According to statistics, the high incidence age of MM is 65-74 years old. Complications include hypercalcemia, renal insufficiency, anemia and infection. Although initial treatment of MM is sensitive to a variety of cytotoxic drugs, due to high heterogeneity and high recurrence probability of drug-resis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C07K19/00A61P35/00
CPCA61P35/00C07K16/2878C07K2317/24C07K2317/33C07K2317/565C07K2317/622C07K2317/92C07K2319/33C07K2319/74
Inventor 张巍徐艳敏单娟娟赵文旭赵永春陈军黄霞
Owner CHONGQING PRECISION BIOTECH CO LTD
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