Construction and application of bispecific antibody HER2*CD3

A bispecific antibody, -CD3 technology, applied in the direction of antibodies, anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the limited efficacy of solid tumors, etc. problem, to achieve the effect of improving the efficacy

Active Publication Date: 2015-08-12
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 scatt...

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
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Construction of expression vectors for bispecific antibodies (HER2×CD3, S802)

[0073] 1. Bispecific antibody sequence design

[0074]The bispecific antibody targeting HER2 and CD3 is named HER2×CD3SMBODY, in which the monovalent unit is the anti-CD3 heavy chain and light chain pair, and the amino acids of the variable region refer to the sequence of monoclonal antibody L2K, and refer to sequence 2 of US20070123479, including The anti-CD3 heavy chain and light chain contain Fab and Fc domains; the single chain unit is the ScFv-Fc form of anti-HER2, and the amino acid sequence of the variable region refers to the sequence of the monoclonal antibody Herceptin (PDB database No.1N8Z), including VH, VL, Fc domains of Herceptin monoclonal antibody. 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 IgG11) are modified by amino acid mutations. For the specific Fc modification process, see P...

Embodiment 2

[0099] Example 2: Expression and purification of bispecific antibodies

[0100] 1. Expression of bispecific antibody S802

[0101] 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-L2K-HL-KKW and pCHO1.0-hygromycin-Herceptin- ScFv-Fc-LDY were co-transfected into CHO-S cells to express anti-HER2×CD3 bispecific antibody S802. After 14 days of culture, the expression supernatant was harvested by centrifugation at 800×g. The expression experiment procedure of S801 is the same as that of S802.

[0102] 2. Purification of bispecific antibody S802

[0103] The expression supernatant was filtered with a 0.22 μM filter membrane, and all Fc-containing structures we...

Embodiment 3

[0104] Example 3: Determination of the binding activity of bispecific antibodies to cells (FACS)

[0105] 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.

[0106] 1. Detection of binding activity of bispecific antibody to SK-BR-3 cells by flow cytometry

[0107]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 which is composed of a mono-valent unit and a single-chain unit, wherein the mono-valent unit, aiming to surface antigen CD3 of immune cells, has a specifically combining capability while the single-chain unit, aiming to surface antigen HER2 of tumor cells, has a specifically combining capability. The single-chain unit includes a single-chain variable fragment (ScFv) fused with an Fc fragment. The mono-valent unit includes a light chain-heavy chain pair. The application also provides a preparation of the bispecific antibody and medicinal applications of these antibodies.

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 张敬胡伶俐王瑞周祥范克索周鹏飞
Owner WUHAN YZY BIOPHARMA CO LTD
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