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Anti-bcma humanized single chain antibody and its 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 enhanced sustainability and safety, reduced immunogenicity, and a high degree of humanization. Effect

Active Publication Date: 2022-04-08
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 humanized single chain antibody and its application
  • Anti-bcma humanized single chain antibody and its application
  • Anti-bcma humanized single chain antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 Design the humanized single chain antibody of anti-human BCMA antigen

[0047]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.

[0048] The light chain amino acid sequences of the antibodies are respectively shown in SEQ ID NO:9-12; the heavy chain amino acid sequences of the antibodies are respectively shown in SEQ ID NO:13-16.

[0049] The amino acid sequence of the humanized single-chain antibody is shown in any one of SEQ ID NO:17-20; the nucleotide sequence is shown in any one of SEQ ID NO:21-24.

Embodiment 2

[0050] Example 2 Expression and purification of humanized monoclonal antibody targeting BCMA

[0051] 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 leftmost and rightmost m is the protein maker (thermo26619 ladder), the size of each band marked on the left side of m is the maker, the 1st channel is the protein band of ScFv1, and the 2nd channel is the protein band of ScFv2 band, the antibody size is 25-35kDa, and the engineered scFv has high purity.

Embodiment 3

[0052] Example 3 Affinity Determination of Humanized Monoclonal Antibody Targeting BCMA

[0053] Fortebio Octet K2 and Nicoya OpenSPR were used to determine the affinity of monoclonal antibodies. The selected sensors were ProA biosensor and Ni+NTA. The concentration gradient of the detection sample was: 100nM, 50nM, 25nM, 12.5nM, 6.25nM; and 250nM, 125nM, 62.5nM , 31.25nM, 15.625nM, see the instrument operating procedures for specific experimental procedures. The result is as figure 2 and image 3 As shown, ScFv1 and ScFv2 have better affinity, and the affinity constants KD=4.65nM and 16.4nM.

[0054] Sample ID Loading Sample ID KD(M) KD(nm) kon(1 / Ms) kdis(1 / s) ScFv1 hBCMA-hFc 4.65E-09 4.65 9.42E+04 4.38E-04 ScFv2 hBCMA-hFc 1.64E-08 16.4 2.66E+05 4.37E-03

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Abstract

The invention belongs to the technical field of genetic engineering, and in particular relates to an anti-BCMA humanized single-chain antibody and an application thereof. The amino acid sequence of the anti-BCMA humanized single-chain antibody is such as SEQ ID NO: 17-20 or 34-41 shown in either sequence. The anti-BCMA humanized single-chain antibody is formed by connecting the light chain variable region, the Linker and the heavy chain variable region in turn, and has a high degree of humanization, which can effectively reduce the immunogenicity of CAR and enhance the In vivo persistence and safety.

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 Patents(China)
IPC IPC(8): C07K16/28C12N15/13C07K19/00A61P35/00
CPCC07K16/2878A61P35/00C07K2317/24C07K2317/565C07K2317/622C07K2317/92C07K2319/33C07K2319/74
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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