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Anti-HER2/PD1 bispecific antibody

A bispecific antibody, antibody technology, applied in the direction of antibodies, specific peptides, anti-tumor drugs, etc.

Inactive Publication Date: 2020-05-26
SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with traditional monoclonal antibodies, bispecific antibodies have a unique mechanism of action: 1) bispecific antibodies can simultaneously bind to two or more different antigen molecules or different epitopes of the same molecule, while combined drugs often do not have this effect

Method used

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  • Anti-HER2/PD1 bispecific antibody
  • Anti-HER2/PD1 bispecific antibody
  • Anti-HER2/PD1 bispecific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1. Construction of anti-HER2 / PD1 double antibody molecule

[0116] In the present invention, the anti-HER2 / PD1 bispecific antibody a is constructed by using the HER2 monoclonal antibody IgG and the scFv of the PD1 monoclonal antibody in series. The light chain variable region VL (SEQ ID NO: 14) and the heavy chain variable region VH (SEQ ID NO: 13) of the anti-PD1 monoclonal antibody were connected through a peptide linker L1 (SEQ ID NO: 17) to obtain anti-PD1 The single-chain antibody fragment VL-L1-VH, that is, the anti-PD1 fragment scFv1 (SEQ ID NO: 19). Using L2 (SEQ ID NO: 18) to connect the single-chain antibody fragment with the heavy chain of the anti-HER2 monoclonal antibody, thereby obtaining the heavy chain of the bispecific antibody molecule anti-HER2 / PD1 biclonal antibody a (SEQ ID NO: 20 ), the light chain of HER2 monoclonal antibody (SEQ ID NO: 21) remains unchanged. In order to improve the expression efficiency of antibody molecules in CHO cell...

Embodiment 2

[0118] Example 2. Expression and purification of double antibodies

[0119] The heavy chain and light chain DNA fragments of the double antibody were subcloned into the pTT5 vector, and the recombinant plasmids were extracted and co-transfected into CHO cells and / or 293E cells. After the cells have been cultured for 5-7 days, the culture medium is subjected to high-speed centrifugation and vacuum filtration through a microporous membrane, then loaded onto a HiTrap MabSelectSuRe column, and the protein is eluted in one step with an eluent containing 100mM citric acid, pH 3.5, and the target is recovered Samples were dialyzed into PBS pH 7.4. The purified protein was detected by HPLC, and the HPLC detection patterns of anti-HER2 / PD1 double antibody a and b were as follows: Figure 2A , 2B As shown, the molecular state of the antibody is uniform, and the purity of the monomer reaches more than 97%. Add the purified anti-HER2 / PD1 double antibodies a and b to the non-reducing el...

Embodiment 3

[0120] Example 3. Enzyme-linked immunosorbent assay (ELISA) to measure the affinity of the double antibody to the antigen

[0121] In order to detect the affinity of anti-HER2 / PD1 double antibody a and b to HER2 antigen respectively, dilute HER2-ECD-His protein (manufactured by Sunshine Guojian) to 250 ng / ml with pH 7.4 PBS buffer, and then 100 μl / Add the wells to the ELISA plate; incubate overnight at 4°C; wash the plate twice with PBST the next day; add PBST+1% BSA to each well for blocking, block at 37°C for 1 hour; wash the plate twice with PBST; then add PBS+1% The antibody to be tested was serially diluted in BSA, and the HER2 monoclonal antibody was used as a positive control. The initial concentration was 100 nM, and 8 gradients were gradually diluted by 4 times. Incubate at 37°C for 1 hour; wash the plate twice with PBST, add HRP-labeled mouse anti-human Fab antibody, and incubate at 37°C for another 40 minutes; wash the plate three times with PBST and pat dry, add 1...

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Abstract

The invention belongs to the field of tumor treatment, and discloses an anti-HER2 / PD1 bispecific antibody, a preparation method and an application in tumor resistance. Specifically, the single-chain variable fragment scFv and the immunoglobulin antibody IgG are connected through a peptide joint to obtain a bispecific antibody, and the bispecific antibody can target a tumor cell surface molecule HER2 and a T lymphocyte surface molecule PD1. The experimental results show that the bispecific antibody provided by the invention can inhibit the growth of tumor cells, activate T lymphocytes and promote the killing of the T cells to the tumor cells.

Description

technical field [0001] The invention belongs to the field of tumor treatment and biotechnology, and relates to a preparation method and application of a bispecific antibody molecule against HER2 and PD1. Background technique [0002] HER2 (human epidermal growth factor receptor 2), which has receptor tyrosine protein kinase activity, is one of the members of the human epidermal growth factor receptor family, and is only expressed at a low level in a few normal tissues of adults. However, studies have shown that HER2 is overexpressed in a variety of tumors, such as overexpression in about 30% of breast cancer patients and 16% of gastric cancer patients. Overexpression of HER2 in tumors can significantly promote tumor angiogenesis, Tumor growth, and enhanced tumor invasion and metastasis capabilities, are important indicators of poor prognosis in such patients. Therefore, as early as 1998, Herceptin (Genentech / Roche), the first monoclonal antibody drug targeting HER2, was app...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/13A61K39/395A61P35/00
CPCC07K16/2818C07K16/2863A61P35/00C07K2317/31C07K2317/622C07K2317/565C07K2317/92C07K2317/76C07K2317/732A61K2039/505C07K16/32C07K2317/52C07K2317/56C07K2317/73C07K2317/94
Inventor 朱祯平黄浩旻顾昌玲
Owner SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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