Long-acting low-toxicity recombinant anti-VEGF humanized monoclonal antibody and production method thereof

A technology of humanization and antibodies, which is applied in chemical instruments and methods, biochemical equipment and methods, antibodies, etc., can solve the problems of patient burden, short vitreous half-life, etc., to prolong the treatment cycle and improve the physiological and psychological conditions of patients , highly stable effect

Active Publication Date: 2019-07-12
BIO THERA SOLUTIONS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this smaller antibody fragment results in a shorter intravitreal half-life (due to faster diffusion into the serum) and must be administered more frequently, placing a mental and physical burden on the patient on treatment

Method used

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  • Long-acting low-toxicity recombinant anti-VEGF humanized monoclonal antibody and production method thereof
  • Long-acting low-toxicity recombinant anti-VEGF humanized monoclonal antibody and production method thereof
  • Long-acting low-toxicity recombinant anti-VEGF humanized monoclonal antibody and production method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1 Research on the Inhibitory Mechanism of Recombinant Humanized Anti-VEGF Antibody on Neovascularization

[0092] The heavy chain and light chain amino acid sequences of the recombinant anti-VEGF humanized monoclonal antibody (antibody BAT5906) are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0093] Laser photocoagulation was performed on the eyes of mice to destroy the choroidal structure and form an angiogenesis model. After injecting human VEGFA antigen into this model, the choroidal angiogenesis will be enhanced; then the recombinant humanized anti-VEGF monoclonal antibody BAT5906, Ranibizumab (Ran), Fab domain of BAT5906 antibody (BAT5906-Fab), Fc Domain (BAT5906-Fc) and human IgG1 (hIgG1), compared with the control group (PBS), choroidal angiogenesis were inhibited, presumably there are two different mechanisms of angiogenesis inhibition. Among them, BAT5906-Fc and human IgG1 showed non-target-dependent inhibition, Ranibizumab and BAT5906-Fab sho...

Embodiment 2

[0095] Embodiment 2 Recombinant plasmid and stable cell line construction of recombinant anti-VEGF monoclonal antibody (antibody BAT5906)

[0096] The recombinant expression plasmid pBAT5906 was constructed according to the amino acid sequence, which contains GS cDNA elements for the synthesis of glutamine synthetase gene, which is used as an amplification and screening target for stable cell lines, so that a certain amount of L-methionine subunit can be added to the culture medium. Sulfone (methionine sulphoximine, MSX) was used to screen stable cell lines. The constructed recombinant expression plasmid pBAT5906 was verified by restriction endonuclease Pvu I / Not I digestion, which was consistent with the expected design results, proving that the pBAT5906 recombinant expression vector was successfully constructed.

[0097] The host cell line used for antibody expression is a derivative cell line of CHO-K1 cells, grown in suspension in CD-CHO medium. The construction process o...

Embodiment 3

[0098] Embodiment 3 Expression and purification of monoclonal antibody

[0099] The expression and purification process of the monoclonal antibody is as follows: After the cells are cultured on a large scale for 2 weeks, the cells and the medium are separated by low-speed centrifugation, and the harvested supernatant is further centrifuged at high speed to obtain a clarified feed solution. The recombinant antibody was purified by two-step affinity chromatography (Protein A) and ion exchange. The media used in the purification were MabSelect SuRe LX produced by GE, Giga Cap Q-650M produced by TOSOH and POROS XS produced by ABI. . The purified antibody was verified by SDS-PAGE to verify its correct size ( figure 2 and image 3 ), the results showed that the BAT5906 band was of the correct size under both reduced and non-reduced conditions.

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Abstract

The invention relates to a full-length humanized monoclonal antibody that can specifically bind to a vascular endothelial growth factor (VEGF). The antibody can inhibit binding of the VEGF with VEGF-1and VEGF-2, thereby inhibiting signal transduction of the VEGF. The antibody has strong affinity to the VEGF, has long half-life period, and has high safety after vitreous injection. The antibody caneffectively treat diseases related to over-expression of the VEGF, especially diseases related to abnormal angiogenesis caused by over-expression of the VEGF.

Description

technical field [0001] The invention belongs to the field of biomedicine; more specifically, the invention relates to an antibody for reducing human vascular endothelial growth factor (vascular endothelial growth factor, VEGF / VEGF-A), and also relates to the preparation method and application of the antibody . Background technique [0002] Angiogenesis is the proliferation and reorganization of vascular endothelial cells from a preexisting vascular network into new blood vessels. Angiogenesis is essential for normal proliferative processes in humans, including wound healing and organ development and differentiation. Angiogenesis is also involved in the formation of various pathological diseases, such as age-related macular degeneration, tumors, rheumatoid arthritis and psoriasis. Given their important physiopathological importance, it has been suggested that the angiogenic process is regulated by a balance between pro-angiogenic and anti-angiogenic molecules and is dysregu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22C12N15/13A61K39/395A61P27/02G01N33/53G01N33/68
CPCC07K16/22C07K2317/51C07K2317/515A61K2039/505A61P27/02A61P9/10C07K2317/24C07K2317/92C07K2317/76C07K2317/732C07K2317/734A61K39/39591G01N33/74
Inventor 吴晓云徐臣超王志刚李胜峰
Owner BIO THERA SOLUTIONS LTD
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