Anti-Her2/PD-1 bispecific antibody and preparation method thereof

A bispecific antibody, PD-1 technology, applied in the direction of antibodies, chemical instruments and methods, specific peptides, etc., to achieve the effect of reducing the formation of homodimers

Inactive Publication Date: 2018-12-18
苏州塞恩塔生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing methods for preparing bispecific antibod

Method used

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  • Anti-Her2/PD-1 bispecific antibody and preparation method thereof
  • Anti-Her2/PD-1 bispecific antibody and preparation method thereof
  • Anti-Her2/PD-1 bispecific antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Construction of Anti-Her2 Antibody and Anti-PD-1 Antibody Expression Vectors

[0030]The sequence of the full-length gene (light chain and heavy chain) of the fully human anti-Her2 monoclonal antibody adopts the sequence published by Trastuzumab (IMGT INN 7637_H, INN7637_L), see SEQ ID NO.1 for the heavy chain, and SEQ ID NO.2 for the light chain . The Fab (light chain and heavy chain) sequence of the fully human anti-PD-1 monoclonal antibody adopts the sequence published by Pembrolizumab (IMGT INN 9798_H, INN9798_L), see SEQ ID NO.3 for the heavy chain, and SEQ ID NO.4 for the light chain , Fc adopts the Fc sequence of Trastuzumab. Carry out charge analysis on the Fc amino acid ammonium sequences of Trastuzumab and Pembrolizumab, and carry out amino acid modification on the CH3 region. The modified Trastuzumab heavy chain amino acid sequence is SEQ ID NO.5, and the modified Pembrolizumab heavy chain amino acid sequence is SEQ ID NO.6 . Amino acid modificat...

Embodiment 2

[0034] Example 2. Construction of anti-HER2 antibody and anti-PD-1 antibody cell lines

[0035] The anti-PD-1 antibody and anti-Her2 antibody expression plasmids were transferred into CHO-S host cells by electroporation kit (AmaxaTM cell line NucleofectorTM Kit V) and single-well cell electroporation instrument (NucleofectorTM 2b, Lonza), respectively, CHO-S cells expressing anti-PD-1 antibody and CHO-S cells expressing Her2 antibody were obtained respectively.

[0036] Specifically, 24 hours before transfection, 0.6×10 6 CHO-S cells were subcultured into freshly prepared CD CHO (Gibco) medium at a cell density of 1 / ml, and placed at 37°C, 8% CO 2 , 130r / min shaker culture. During transfection, preheat the Nucleofector Kit and medium, and prepare 800 μl electrotransfer solution according to the Nucleofector solution:supplement ratio of 4.5:1, add 50 μg plasmid to the electrotransfer solution, and mix well. 1000r / min, 5min centrifugation 3×10 7 cells, discard the supernatan...

Embodiment 3

[0039] Example 3 Respective expression of anti-HER2 antibody and anti-PD-1 antibody

[0040] The seed cells of anti-Her2 antibody and anti-PD-1 antibody were divided into 0.6×10 6 The inoculum density of cells / ml was diluted into a 3L cell culture reactor, and the initial culture volume was 1L; the stirring speed was set to 250r / min; the pH was set to 7.00, DeadBand was set to 0.10, and the associated sodium bicarbonate solution (NaHCO 3 90.7g / L) and CO 2 Adjustment is made; the dissolved oxygen is set to 40%; the culture temperature is set to 36.5° C. for 1 to 4 days; the temperature is lowered to 32.0° C. on the 5th day of cultivation;

[0041] (1) Two cell reactors were fed with feed medium on days 3, 5, 7, 9, and 11. Add feed medium A and feed medium B, and feed medium A The feed volume is 4% of the initial culture volume, and the feed volume of feed medium B is 0.4% of the initial culture volume. Sample 3 mL every day to detect the viable cell density, cell viability a...

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Abstract

The invention discloses an anti-Her2/PD-1 bispecific antibody and a preparation method thereof, and belongs to the field of biotechnology. The anti-Her2/PD-1 bispecific antibody can bind to Her2 and bind to PD-1, and can effectively enhance immune response by blocking a Her2 and/or PD-1 signaling pathway, so that the anti-Her2/PD-1 bispecific antibody is possible to have a good effect of enhancingthe anti-tumor immune response.

Description

technical field [0001] The invention relates to an anti-Her2 / PD-1 bispecific antibody and a preparation method thereof, belonging to the field of biotechnology. 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. [0003] A large number of studies have shown that tumors can escape the surveillance of the immune system through multiple mechanisms during the formation process. We call this process immune escape or immune editing of tumor tissues. PD-1 is a protein on the surface of T cells and one of the proteins known as "immune checkpoint" i...

Claims

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Application Information

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IPC IPC(8): C07K16/46A61K39/395A61P35/00
CPCC07K16/468A61K2039/505A61P35/00C07K16/2818C07K16/32C07K2317/31
Inventor 刘金涛宋莉
Owner 苏州塞恩塔生物技术有限公司
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