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Construction and application of bispecific antibody CD133*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 problems such as application limitations, and achieve Improve efficacy and increase the effect of immunotherapy

Inactive Publication Date: 2015-08-12
WUHAN YZY BIOPHARMA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical coupling method is to link two different monoclonal antibodies together by chemical coupling to prepare bispecific monoclonal antibodies. This is the earliest concept of bispecific monoclonal antibodies. The disadvantages of this preparation method are Obvious
The hybrid-hybridoma method is to produce bispecific monoclonal antibodies by means of cell hybridization or ternary hybridomas. These cell hybridomas or ternary hybridomas are fused by established hybridomas, or established hybridomas and hybridomas derived from mice The fusion of the obtained lymphocytes can only produce mouse-derived bispecific antibodies, and its application is greatly limited

Method used

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  • Construction and application of bispecific antibody CD133*CD3
  • Construction and application of bispecific antibody CD133*CD3
  • Construction and application of bispecific antibody CD133*CD3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Construction of expression vectors for bispecific antibodies (CD133×CD3, MS133)

[0070] 1. Bispecific antibody sequence design

[0071] The bispecific antibody targeting CD133 and CD3 is named MS133 MSBODY, where the monovalent unit is the pair of heavy and light chains against CD133, and the amino acid sequence of the variable region refers to the sequence of monoclonal antibody AC133, see the sequence of US2010 / 0209439 No. 1-4, including anti-CD133 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, see the sequence number of US20070123479 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 IgG11) are modified by amino acid mutations. For the specific Fc modification process, see PCT / ...

Embodiment 2

[0097] Example 2: Expression and purification of bispecific antibodies

[0098] 1. Expression of bispecific antibody MS133

[0099] 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 Culture in a cell culture incubator. After preparing the cells, use the Maxcyte STX electroporator to co-transform the plasmid pCHO1.0-AC133-HL-KKW with HPCHO-L2K-ScFv-Fc-LDY according to the manufacturer's instructions (Maxcyte) Transfected into CHO-S cells, the anti-CD133×CD3 bispecific antibody MS133 was expressed. After 14 days of culture, the expression supernatant was harvested by centrifugation at 800×g.

[0100] 2. Purification of bispecific antibody MS133

[0101] The expression supernatant was filtered with a 0.22 μM filter membrane, and all Fc-containing str...

Embodiment 3

[0102] Example 3: Determination of the binding activity of bispecific antibodies to cells (FACS)

[0103] The bispecific antibody of the invention binds to the target antigen on the corresponding cell. In the present invention, HCT116 and HT29 (both purchased from China Center for Type Culture Collection) are used as CD133-positive cells, and human peripheral blood mononuclear cells (hPBMC) cells are used as CD3-positive cells, and the double antibody prepared by the present invention is used to determine the cells binding activity.

[0104] 1. Detection of binding activity of bispecific antibody to HCT116 and HT29 cells by flow cytometry

[0105] Sufficient HCT116 and HT29 cells were cultured, digested with 0.25% trypsin, and collected by centrifugation. At the same time dilute MS133, the concentration is 160nmol. 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 μl per wel...

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Abstract

The invention provides a bispecific antibody which is composed of a single-chain unit and a mono-valent unit, wherein the single-chain unit, aiming to surface antigen CD3 of immune cells, has a specifically combining capability while the mono-valent unit, aiming to surface antigen CD133 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. 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 bispecific antibodies to overcome the above obstacles and establish corresponding The imm...

Claims

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Application Information

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IPC IPC(8): C07K16/46C07K16/30C12N15/85A61K39/395A61P35/00
Inventor 张涛张敬赵磊杨玉丹王涛周鹏飞
Owner WUHAN YZY BIOPHARMA CO LTD
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