Construction and application of bispecific antibody EGFR*CD3

A bispecific antibody, antibody technology, applied in the direction of antibodies, anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problem of limited efficacy in solid tumors , to increase the effect of immunotherapy and improve the efficacy

Active Publication Date: 2015-07-15
WUHAN YZY BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, immunotherapy can only remove a small amount of scattered tumor cells, and has limited efficacy for advanced solid tumors

Method used

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  • Construction and application of bispecific antibody EGFR*CD3
  • Construction and application of bispecific antibody EGFR*CD3
  • Construction and application of bispecific antibody EGFR*CD3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Construction of expression vectors for bispecific antibodies (EGFR×CD3, M1001, M1003)

[0067] 1. Bispecific antibody sequence design

[0068] The bispecific antibody targeting EGFR and CD3 is named EGFR×CD3MSBODY, in which the monovalent unit is the anti-EGFR heavy chain and light chain pair, and the amino acid sequence of the variable region refers to the sequence of the monoclonal antibody Erbitux (PDB database sequence number 1YY8 ) and the sequence of Vectibix (the source is the sequence number 38 and 49 in the patent US6235883), including the anti-EGFR heavy chain and light chain, containing Fab and Fc domains; the single-chain unit is the ScFv-Fc form of anti-CD3, the variable region Amino acids refer to the sequence of monoclonal antibody L2K (refer to US20070123479 sequence number 2), including anti-CD3 VH, VL, and Fc domains. Among them, the heavy chain Fc of the monovalent unit and the Fc of the single chain unit (same as the heavy chain Fc of hum...

Embodiment 2

[0100] Example 2: Expression and purification of bispecific antibodies

[0101] 1. Expression of bispecific antibodies

[0102] The endotoxin-free large-scale extraction kit (Qiagen, 12391) was used for large-scale extraction of plasmids, and the specific operation was performed according to the instructions provided by the manufacturer. CHO-S cells were cultured in CD FortiCHO medium (Invitrogen, Cat. 2 The cells were cultured in the incubator. After the cells were prepared, the plasmid pCHO1.0-Erbitux-HL-KKW and pCHO1.0-Hygromycin-L2K-ScFv- Fc-LDY were co-transfected into CHO-S cells to express anti-EGFR×CD3 bispecific antibody M1001; the plasmid pCHO1.0-Vectibix-HL-KKW and pCHO1.0-hygromycin -L2K-ScFv-Fc-LDY were co-transfected into CHO-S cells in the same way as above to express the anti-EGFR×CD3 bispecific antibody M1003. After culturing for 14 days, the expression supernatant was harvested by centrifugation at 800×g.

[0103] 2. Purification of bispecific antibodies ...

Embodiment 3

[0105] Example 3: Determination of the binding activity of bispecific antibodies to cells (FACS)

[0106] The bispecific antibody of the invention binds to the target antigen on the corresponding cell. In the present invention, HT29 (purchased from China Center for Type Culture Collection) is used as EGFR-positive cells, Jurkat (ATCC, TIB-152) is used as CD3-positive cells, and the cell binding activity is determined by the diabody prepared in the present invention.

[0107] 1. Detection of binding activity of bispecific antibody to HT29 cells by flow cytometry

[0108] Sufficient HT29 cells were cultivated, digested with 0.25% trypsin, and collected by centrifugation. Simultaneously dilute the bispecific antibody, starting from 160nM, 4-fold serial dilution to obtain 6 concentration gradients for future use. Wash the collected cells twice with PBS+1%FBS, then resuspend the cells in PBS+1%FBS to 4×10 6 cells / ml, the cells were plated in a 96-well plate, 50ul per well (2×10 ...

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Abstract

The invention provides a bispecific antibody. The bispecific antibody is composed of a single brand unit and a univalent unit, wherein the single brand unit has specific binding ability on surface antigen CD3 of immune cells, and the univalent unit has specific binding ability on surface antigen EGFR of tumor cells; and the single brand unit includes a single brand variable fragment (ScFv) fused with an Fc fragment, and the univalent unit includes a light strand and heavy strand pair. The invention also provides a preparation method and a pharmaceutical use of the bispecific antibody.

Description

technical field [0001] The present invention relates to the technical field of immunology. Specifically, it relates to the construction and preparation methods of bispecific antibodies. Background technique [0002] Bispecific antibody (bispecific antibody, BiAb) is an artificial antibody containing two specific antigen-binding sites, which can build a bridge between target cells and functional molecules (cells) to produce oriented effector functions. BiAb has broad application prospects in biomedicine, especially in tumor immunotherapy. Killing tumor cells through BiAb-mediated cytotoxicity is a hot topic in current immunotherapy application research. Sexual killing. The following is an introduction to the background technology of the studied immune cell antigens and tumor cell antigens, as well as the development of related technologies. [0003] 1. CD3 [0004] The CD3 molecule consists of four subunits: δ, ε, γ, and ζ, with molecular masses of 18.9 kDa, 23.1 kDa, 20...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C07K16/30C12N15/85A61K39/395A61P35/00
Inventor 王涛方丽娟杨锦霞戴晴刘勇周鹏飞
Owner WUHAN YZY BIOPHARMA CO LTD
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