Construction method and application of bispecific antibody EpCAM*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 achieve improved efficacy, increase the effect of immunotherapy

Active Publication Date: 2015-05-06
WUHAN YZY BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, immunotherapy can only remove a small amount of scattere

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  • Construction method and application of bispecific antibody EpCAM*CD3
  • Construction method and application of bispecific antibody EpCAM*CD3
  • Construction method and application of bispecific antibody EpCAM*CD3

Examples

Experimental program
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Effect test

Embodiment 1

[0072] Example 1: Construction of expression vectors for bispecific antibodies (EpCAM×CD3, M701)

[0073] 1. Bispecific antibody sequence design

[0074] The bispecific antibody targeting EpCAM and CD3 was named M701, as figure 2 , the anti-EpCAM side is in the form of IgG, including anti-EpCAM 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 KKW transformation on one side of Fc, and the side of ScFv-Fc undergoes LDY 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, namely EpCAM×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

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

[0100] 1. Expression of bispecific antibodies

[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 CHO medium (Gibco, 10743-029) at 37°C in 5% CO according to the instructions provided by the manufacturer. 2 The cells were cultured in the incubator. After the cells were prepared, the plasmid pCHO1.0-anti-EpCAM-HL-KKW and pCHO1.0-hygromycin-L2K- ScFv-Fc-LDY were co-transfected together into CHO-S cells, and the co-transfection of these two plasmids was designed to express the bispecific antibody M701 against EpCAM×CD3.

[0102] 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 harve...

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, HCT116 (purchased from ATCC, CCL-247) is used as EpCAM-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.

[0107] 1. Detection of binding activity of bispecific antibody to HCT116 cells by flow cytometry

[0108] Sufficient HCT116 cells were cultured, digested with 0.25% trypsin, and collected by centrifugation. Simultaneously dilute the bispecific antibody, the concentration starts from 10ug / ml, and 10-fold gradient dilution is obtained to obtain 12 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, 50...

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Abstract

The invention provides a bispecific antibody. The bispecific antibody is composed of a single-chain unit and a univalent unit, wherein the single-chain unit has specific binding capacity for a surface antigen CD3 of an immune cell; the univalent unit has specific binding capacity for a surface antigen EpCAM of a tumor cell; the single-chain unit contains a single-chain variable fragment (scFv) fused with a Fc fragment; and the univalent unit contains a light and heavy chain pair. The invention also provides a preparation method of the bispecific antibody and pharmaceutical application of the bispecific antibody.

Description

technical field [0001] The present invention relates to the technical field of immunology. Specifically, it relates to a method for constructing and preparing a bispecific antibody, as well as a method for detecting the function and properties of the bispecific antibody. 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. However, in the process of developing bispecific antibody drugs, there are many obstacles such as difficult expression, low yield, difficult purification, and poor stability. Therefore, it is necessary to construct new bisp...

Claims

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Application Information

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IPC IPC(8): C07K16/46C07K16/30C12N15/85A61K39/395A61P35/00
Inventor 王涛方丽娟胡柳刘杨张雨范克索周鹏飞
Owner WUHAN YZY BIOPHARMA CO LTD
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