Anti-BCMA antibody or antigen binding fragment thereof as well as preparation method and application thereof

A technology that combines fragments and antigens, applied in botany equipment and methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as human anti-mouse antibody reaction, achieve strong specificity, simple operation, and immunogen Sexually low effect

Active Publication Date: 2021-05-28
XIAN YUFAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the application of single-chain antibodies or humanized antibodies prepared by traditional methods for diagnosis or treatment can cause human anti-mouse antibody reactions (HAMA) to a certain extent.

Method used

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  • Anti-BCMA antibody or antigen binding fragment thereof as well as preparation method and application thereof
  • Anti-BCMA antibody or antigen binding fragment thereof as well as preparation method and application thereof
  • Anti-BCMA antibody or antigen binding fragment thereof as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: Screening of specific antibodies targeting BMCA using a fully human phage display library

[0088] 1. Construction of fully human phage antibody library

[0089] (1) Collect 60 samples of peripheral blood (about 2500 mL) from healthy people, and separate peripheral blood mononuclear cells (PBMC) according to the instructions of the lymphocyte separation medium. Total RNA from PBMCs was extracted using a blood total RNA extraction kit. Take 1 μg of RNA for electrophoresis and check the purity of RNA, the result shows that the purity of RNA is good (see figure 1 ). Reverse transcription was performed using the First-Strand Synthesis System for RT-PCR kit to obtain the cDNA template required for phage display library construction. Then, use the designed and synthesized specific primers to amplify the light chain full-length fragment VL by PCR, obtain the specific VHH fragment by two rounds of nested PCR, and obtain the full-length VH fragment by Overlap PCR a...

Embodiment 2

[0105] Embodiment 2: Anti-BMCA specific antibody identification and sequence analysis

[0106] 1. Screening and identification of positive clones

[0107] (1) Polyclonal Phage ELISA (Polyclonal Phage ELISA)

[0108] After four rounds of panning, the phages concentrated in each round of precipitation were subjected to polyclonal phage ELISA, and the specific steps were as follows:

[0109] 1) Antigen coating: Dilute BCMA protein and BSA protein (as control protein) with PBS buffer solution of pH 7.4 to a final concentration of 4 μg / mL, coat the microtiter plate at 100 μL / well, and coat overnight at 4°C;

[0110] 2) Blocking: Discard the coating solution, wash 3 times with PBST, add 200 μL blocking solution PBSM (PBS+3% Milk) to each well and block at 37°C for 1 hour;

[0111] 3) Phage incubation: Discard the blocking solution, wash 3 times with PBST, dilute the phage after each round of concentration with the blocking solution at 1:10, add 100 μL / well, and incubate at 37°C fo...

Embodiment 3

[0132] Example 3: Prokaryotic expression vector construction and soluble expression of anti-BCMA antibody

[0133] (1) Sequence source and analysis of anti-BCMA antibody

[0134] According to the sequence analysis results of the anti-BCMA antibody in Example 2, one of the sequences was selected for the next step of antibody expression, and the antibody form was determined as a single-chain antibody form. The selected sequence heavy chain variable region and light chain variable region were analyzed by IMGT, and the nucleotide sequence of the heavy chain variable region was found to be shown in SEQ ID NO: 1, and the amino acid sequence was shown in SEQ ID NO: 15 The nucleotide sequence of heavy chain variable region CDR-H1 is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 9; the nucleotide sequence of heavy chain variable region CDR-H2 is shown in SEQ ID NO: 4, the amino acid sequence is shown in SEQ ID NO: 10; the heavy chain variable region CDR-H3 ...

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Abstract

The invention provides an anti-BCMA antibody or an antigen binding fragment thereof, a chimeric antigen receptor containing the anti-BCMA antibody or the antigen binding fragment thereof, an immune cell and a drug. The invention also provides a preparation method and application of the anti-BCMA antibody or the antigen binding fragment thereof. The anti-BCMA antibody or the antigen binding fragment thereof prepared by the invention is a fully humanized single-chain antibody, has stronger specificity and lower immunogenicity, can specifically recognize and bind to BCMA positive target cells, and can provide a theoretical basis for treating diseases related to BCMA expression, so that the anti-BCMA antibody or the antigen binding fragment thereof can be applied to treatment and diagnosis of the diseases related to BCMA expression.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to an anti-BCMA antibody or an antigen-binding fragment thereof and a preparation method and application thereof, wherein the anti-BCMA antibody or an antigen-binding fragment thereof is a high-affinity fully human single-chain antibody targeting BCMA . Background technique [0002] BCMA (B cell maturation antigen, B cell maturation antigen) is a member of the tumor necrosis factor receptor (TNF-receptor) superfamily, also known as CD269, which is a marker protein of B lymphocyte maturation edited by the TNFRSF17 gene. BCMA is composed of 184 amino acids, its intracellular region can interact with TRAF1, 2, 3 and activate TRAF-dependent TNF-KB, JNK and P38 MAPK pathways, the extracellular region is rich in cysteine ​​domain, and can pass Protein-protein interactions to transmit cell stimulatory signals. [0003] BCMA is mainly expressed on the surface of B lymphocytes and plas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00A61P35/02
CPCC07K16/2878C07K14/7051C12N15/86A61K39/0011A61P35/00A61P35/02C07K2319/02C07K2319/03C07K2319/33C07K2317/622C07K2317/56C07K2317/565C07K2317/92C12N2740/15043
Inventor 龙飞封金云
Owner XIAN YUFAN BIOTECH CO LTD
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