Antibody fusion protein, preparation method thereof and application thereof in tumor resistance

A fusion protein and antibody technology, which is applied in the direction of anti-tumor drugs, anti-enzyme immunoglobulin, hybrid immunoglobulin, etc., can solve the problems of slow tumor growth and inability to achieve metastasis

Inactive Publication Date: 2021-03-26
SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

If there is no angiogenesis within the tumor, the prima

Method used

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  • Antibody fusion protein, preparation method thereof and application thereof in tumor resistance
  • Antibody fusion protein, preparation method thereof and application thereof in tumor resistance
  • Antibody fusion protein, preparation method thereof and application thereof in tumor resistance

Examples

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Embodiment 1

[0093] Example 1. Molecular Construction of Antibody Fusion Protein HD2

[0094] In the present invention, the anti-HER2 monoclonal antibody IgG and the D2 domain of VEGFR1 are connected in series to construct the antibody fusion protein HD2. The D2 domain of VEGFR1 (SEQ ID NO: 14) and the heavy chain (SEQ ID NO: 7) of the anti-HER2 monoclonal antibody are connected through the peptide linker Linker (SEQ ID NO: 9) to obtain the heavy chain of the fusion protein (SEQ ID NO: 9). ID NO: 10). The light chain of the HER2 mAb (SEQ ID NO: 11) remained unchanged. In order to improve the expression efficiency of this molecule in 293E cells, Jinweizhi Company was entrusted to optimize the codons of the nucleic acid sequence of HD2 molecule. Optimization mainly considers factors such as codon preference, GC content, mRNA secondary structure, repeat sequence, etc., and then entrusts Jinweizhi Company to synthesize. After splicing, the HD2 heavy chain nucleic acid sequence is SEQ ID NO:...

Embodiment 2

[0095] Example 2. Expression and purification of antibody fusion protein HD2

[0096] The heavy chain and light chain DNA fragments of HD2 were respectively cloned into 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-SEC detection patterns of HD2 were as follows: Figure 2A As shown, the molecular state of the antibody is uniform, and the purity of the monomer reaches more than 98%.

[0097] The purified antibody fusion protein HD2 was added to non-reducing electrophoresis buffer respectively, an...

Embodiment 3

[0098] Example 3. Enzyme-linked immunosorbent assay (ELISA) to measure the affinity of HD2 to HER2 antigen and VEGF

[0099] In order to detect the affinity of HD2 antibody fusion protein and HER2 antigen, the HER2-ECD-His protein produced by Sunshine Guojian was diluted to 250ng / ml with PBS buffer solution of pH 7.4, then 100μl / well was added to the ELISA plate, and incubated at 4°C overnight. The next day, wash the plate twice with PBST, add PBST+1% BSA to each well for blocking, block at 37°C for 1 hour, and wash the plate twice with PBST. Then, the antibody fusion protein HD2 to be detected was diluted with PBS+1% BSA, and the anti-HER2 monoclonal antibody was used as a positive control. The initial concentration was 100 nM, and 12 gradients were gradually diluted 3 times. Incubate at 37°C for 1 hour, wash the plate twice with PBST, add HRP-labeled mouse anti-human Fab antibody, incubate at 37°C for another 40 minutes, wash the plate with PBST three times and pat dry, add...

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Abstract

The invention belongs to the field of tumor treatment, and discloses an antibody fusion protein, a preparation method thereof and the application thereof in tumor resistance. The antibody fusion protein specifically comprises an anti-HER2 monoclonal antibody IgG and a D2 domain of VEGFR1, and the D2 domain of VEGFR1 is connected to the C-terminus of an IgG heavy chain through a peptide connector L. The antibody fusion protein can block HER2 and VEGFR2 signal channels at the same time, has a tumor proliferation inhibition effect superior to that of the monoclonal antibody, provides a candidatedrug with a better treatment effect for anti-tumor treatment, and has a wide application prospect in treatment of tumor diseases.

Description

technical field [0001] The invention belongs to the field of tumor treatment and biotechnology, and relates to an antibody fusion protein composed of an anti-HER2 monoclonal antibody IgG and a D2 domain of VEGFR1, a preparation method and application thereof. 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. 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 growth and enhance tumor growth. The ability of tumor invasion and metastasis is an important indicator of the poor prognosis of these patients. Therefore, as early as 1998, Herceptin, the first monoclonal antibody ...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/62C12N5/10C12N15/85A61K39/395A61P35/00
CPCC07K16/2863C07K16/30C07K16/40A61P35/00C07K2317/92C07K2317/31C07K2317/73C07K2317/94A61K2039/507A61K39/395C07K16/46C12N5/10C12N15/62C12N15/85C12P21/02C07K19/00C12N15/63
Inventor 朱祯平黄浩旻顾昌玲
Owner SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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