Fully humanized single-chain antibody of targeted BCMA as well as preparation method and application thereof

A single-chain antibody and antibody technology, applied in the field of biomedicine, can solve problems such as human anti-mouse antibody reaction, and achieve the effect of strong specificity, high expression, and low immunogenicity

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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  • Fully humanized single-chain antibody of targeted BCMA as well as preparation method and application thereof
  • Fully humanized single-chain antibody of targeted BCMA as well as preparation method and application thereof
  • Fully humanized single-chain antibody of targeted BCMA as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: Screening of specific antibodies targeting BMCA using a fully human phage display library

[0085] 1. Construction of fully human phage antibody library

[0086] (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 is as figure 1 As shown, the results indicated good RNA purity. Reverse transcription was performed using the First-Strand Synthesis System for RT-PCR Kit to obtain the cDNA template required for the construction of the phage display library. 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 fra...

Embodiment 2

[0102] Example 2: Identification and sequence analysis of specific antibodies targeting BMCA

[0103] 1. Screening and identification of positive clones

[0104] (1) Polyclonal Phage ELISA (Polyclonal Phage ELISA)

[0105] 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:

[0106] 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;

[0107] 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;

[0108] 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 ...

Embodiment 3

[0129] Example 3: Prokaryotic expression vector construction and soluble expression of antibody

[0130] (1) Source and analysis of B48 single-chain antibody sequence

[0131] According to the sequence analysis results of the BCMA-targeting 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. In combination with the sequence name, the anti-BCMA single-chain antibody was named B48. 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: 2 ; The heavy chain variable region CDR-H1 nucleotide sequence is shown in SEQ ID NO: 5, the amino acid sequence is shown in SEQ ID NO: 8; the heavy chain variable region CDR-H2 nucleotide sequence is shown in SEQ ID NO: 6 As show...

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Abstract

The invention discloses a fully humanized single-chain antibody of targeted BCMA as well as a preparation method and application thereof. The single-chain antibody is formed by connecting a heavy chain variable region and a light chain variable region through a flexible connecting peptide, and the sequences of skeleton regions and CDR regions of the light chain and the heavy chain are derived from human, so that compared with murine and humanized antibodies, the single-chain antibody has higher specificity and lower immunogenicity; the specificity on BCMA is good; and a theoretical basis can be provided for treating diseases related to BCMA expression, and the method is particularly suitable for diagnosis and treatment of tumors such as multiple myeloma.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a fully human single-chain antibody targeting BCMA and its preparation method and application. 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 consists 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 plasma cells, and hardly expressed in other tissue cells. However, it is highly expressed in malignantly proliferating B lymphocytes (such as myeloma cells), and i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/62C12N15/867C12N7/01C12N5/10A61K39/00A61K47/68A61P35/00A61P35/02
CPCC07K16/2878C07K14/7051C12N15/86C12N7/00A61K39/0011A61K47/6801A61P35/00A61P35/02C07K2319/02C07K2319/03C07K2319/33C07K2317/622C07K2317/56C07K2317/565C07K2317/92C12N2740/15043C12N2740/15021
Inventor 龙飞杨文娟
Owner XIAN YUFAN BIOTECH CO LTD
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