Construction and application of bispecific antibody CD20*CD3

A bispecific antibody, -CD20 technology, applied in the direction of antibody, anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the limited curative effect of solid tumors, etc. problem, 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 scattered tumor cells, and has limited efficacy for advanced solid tumors

Method used

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  • Construction and application of bispecific antibody CD20*CD3
  • Construction and application of bispecific antibody CD20*CD3
  • Construction and application of bispecific antibody CD20*CD3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Construction of expression vectors for bispecific antibodies (CD20×CD3, M903)

[0069] 1. Bispecific antibody sequence design

[0070] The bispecific antibody targeting CD20 and CD3 is named M903, such as figure 2 , the anti-CD20 side is in the form of IgG, including anti-CD20 heavy chain and light chain, containing Fab and Fc domains; the anti-CD3 side is in the form of ScFv-Fc, including anti-CD3 VH, VL, and Fc domains. Among them, the IgG form undergoes LDY transformation on one side of Fc, and the side of ScFv-Fc undergoes KKW transformation on Fc. For the specific Fc transformation process, refer to PCT / CN2012 / 084982, so that they are not easy to form homodimers, but easy to form hybrid dimers, that is CD20×CD3 bispecific antibody. At the same time, in order to express the diabody in CHO cells and secrete it into the culture medium, the leader peptide sequence of the mouse kappa chain was selected as the secretion signal peptide. The amino acid seque...

Embodiment 2

[0094] Example 2: Expression and purification of bispecific antibodies

[0095] 1. Expression of bispecific antibodies

[0096] 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 CHO medium (Gibco, 10743-029) at 37°C in 5% CO according to the instructions provided by the manufacturer. 2The cells were cultured in the incubator. After the cells were prepared, the plasmid pCHO1.0-anti-CD20-HL-LDY and pCHO1.0-hygromycin-L2K- ScFv-Fc-KKW were co-transfected together into CHO-S cells, and the co-transfection of these two plasmids was designed to express the bispecific antibody M903 against CD20×CD3.

[0097] On the second day after transfection, the culture temperature was lowered to 32° C., and 3.5% FeedA was added every day. After 14 days of culture, the expression supernatant was harveste...

Embodiment 3

[0100] Example 3: Determination of the binding activity of bispecific antibodies to cells (FACS)

[0101] The bispecific antibody of the invention binds to the target antigen on the corresponding cell. In the present invention, Raji (purchased from ATCC, CCL-86) is used as CD20-positive cells, Jurkat (Jurkat, TIB-152) is used as CD3-positive cells, and the cell-binding activity is determined by the diabody prepared in the present invention.

[0102] 1. Detection of binding activity of bispecific antibody to Raji cells by flow cytometry

[0103] Sufficient Raji cells were cultured, and the cells were collected by centrifugation. Dilute the bispecific antibody at the same time, the concentration starts from 1000nmol, and 3-fold gradient dilution is obtained to obtain 12 concentration gradients for later 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 5 ...

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Abstract

The invention provides a bispecific antibody. 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 CD20 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 methods for constructing and preparing bispecific antibodies, as well as methods for detecting the functions and properties of the 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 is ...

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 周鹏飞王涛刘杨黄谧李娜刘登念范克索
Owner WUHAN YZY BIOPHARMA CO LTD
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