Construction and application of bispecific antibody HER2*CD3

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

Active Publication Date: 2015-04-29
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 scattere

Method used

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  • Construction and application of bispecific antibody HER2*CD3
  • Construction and application of bispecific antibody HER2*CD3
  • Construction and application of bispecific antibody HER2*CD3

Examples

Experimental program
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Effect test

Embodiment 1

[0076] Example 1: Construction of expression vectors for bispecific antibodies (HER2×CD3, M802)

[0077] 1. Bispecific antibody sequence design

[0078] The bispecific antibody targeting HER2 and CD3 is named HER2×CD3 MSBODY, in which the monovalent unit is the pair of heavy and light chains against HER2, and the amino acid sequence of the variable region refers to the sequence of the monoclonal antibody Herceptin (PDB database No. .1N8Z), including anti-HER2 heavy chain and light chain, containing Fab and Fc domains; the single chain unit is in the form of anti-CD3 ScFv-Fc, and the amino acids of the variable region refer to the sequence of monoclonal antibody L2K (refer to US20070123479 sequence number 2) , including anti-CD3 VH, VL, 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 human IgG1) are modified by amino acid mutations. For the specific Fc modification process, see PCT / CN2012 / 0849...

Embodiment 2

[0102] Example 2: Expression and purification of bispecific antibodies

[0103] 1. Expression of bispecific antibodies

[0104] 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 CDFortiCHO medium (Invitrogen, Cat. 2 Culture in a cell culture incubator. After the cells were prepared, the plasmid pCHO1.0-Herceptin-HL-KKW and pCHO1.0-Hygromycin-L2K-ScFv were transferred using a MaxcyteSTX electroporator according to the manufacturer's instructions (Maxcyte). -Fc-LDY were co-transfected into CHO-S cells to express the anti-HER2×CD3 bispecific antibody M802. After culturing for 14 days, the expression supernatant was harvested by centrifugation at 800×g.

[0105] 2. Purification of bispecific antibodies

[0106] The expression supernatant was filtered with a 0.22uM filter membrane, and all ...

Embodiment 3

[0107] Example 3: Determination of the binding activity of bispecific antibodies to cells (FACS)

[0108] The bispecific antibody of the invention binds to the target antigen on the corresponding cell. The present invention uses SK-BR-3 (purchased from China Type Culture Collection Center) as HER2-positive cells, Jurkat (American Type Culture Collection (ATCC), TIB-152) as CD3-positive cells, and uses this The diabodies prepared by the invention were tested for their cell binding activity.

[0109] 1. Detection of binding activity of bispecific antibody to SK-BR-3 cells by flow cytometry

[0110] Cultivate enough SK-BR-3 cells, digest with 0.25% trypsin, and collect the cells by centrifugation. Dilute the bispecific antibody at the same time, the concentration starts from 160nmol, and 4-fold gradient dilution is obtained to obtain 6 concentration gradients, which are ready for use. Wash the collected cells twice with PBS+1%FBS, then resuspend the cells in PBS+1%FBS to 4×10 ...

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Abstract

The invention provides a bispecific antibody, and particularly relates to construction and application of a bispecific antibody HER2*CD3. The bispecific antibody comprises a single-chain unit and a monovalent unit, wherein the single-chain unit has a specific binding capability against the surface antigen CD3 of an immune cell, and the monovalent unit has a specific binding capability against the surface antigen HER2 of a tumor cell; and the single-chain unit comprises a single-chain variable fragment (ScFv) which is fused with an Fc fragment, and the monovalent unit comprises light chain and heavy chain pairs. The invention further provides a method for preparing the bispecific antibody and the medicinal application of the 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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IPC IPC(8): C07K16/46C07K16/30C12N15/85A61K39/395A61P35/00
Inventor 周鹏飞王涛方丽娟杨锦霞马莹莹李娜
Owner WUHAN YZY BIOPHARMA CO LTD
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