Preparing method and medical application of optimized plant source recombination humanized bevacizumab

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a preparing method and medical application of optimized plant source recombination humanized bevacizumab. Heavy-chain and light-chain fusion protein of the recombinant antibody bevacizumab is expressed in plants, heavy chain and light chain are expressed in an proportion appropriate to 1:1 by adding 2A sequence to fusion protein, the proportion promotes assembly of a complete antibody obviously, and results show that the yield of antibodies expressed with the system is high. Meanwhile, due to the fact that a stable tetramer structure is formed through equal-proportion assembly of the heavy chain and light chain of the recombinant antibody, the number of unassembled polypeptides easy to degrade is reduced, and then pure and consistent monoclonal antibodies are obtained. The bevacizumab developed with a new method is applied to medicine to be used for treating breast cancer, lung cancer, spongioblastoma, kidney cancer, cervix uterus cancer, ovarian cancer, colon cancer and rectal cancer.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a preparation method and medical application of an optimized plant source recombinant humanized bevacizumab monoclonal antibody. Background technique [0002] Bevacizumab, referred to as (bevacizumab), its trade name is Avastin (Avastin), is a recombinant humanized monoclonal antibody, Roche's best-selling drug for cancer treatment (http: / / www. avastin.com / patient). Approved by the FDA on February 26, 2004, it is the first anti-tumor angiogenesis drug approved for marketing in the United States, and its sales in 2009 reached 5.9 billion US dollars. Bevacizumab is produced by a Chinese hamster ovary cell expression system and has a molecular weight of approximately 149,000 Daltons. Bevacizumab is a drug that hinders angiogenesis. It blocks the blood supply to tumors by inhibiting the effect of vascular endothelial growth factor, inhibits the spread of tumors in t...

AI Technical Summary

Problems solved by technology

The advantage of mammalian cell expression is that the antibody polypeptide chain can be correctly assembled, folded, and glycosylated into an active complete molecule, and a sufficient amount of antibody can be expressed, but the production cost is high

2) Escherichia coli system: Escherichia coli has been widely used to express antibody functional fragments, such as Fab, Fv and scFv, but currently it cannot express complete antibody molecules

3) Yeast expression system: Yeast can effectively express, assemble and secrete immunologically active antibodies or their functional fragments, but the glycosylation of polypeptides by yeast is different from that of mammalian cells, which affects the ACDC of antibodies (antibody-dependent complement-mediated cytotoxicity) potency

Or express the two genes on the same transcript through the IRES (internal ribosome entry site) system, but these methods cannot achieve equal expression of the heavy chain and light chain

For example, through different gene expression cassettes, although the same promoter and terminator sequences are used, due to the different insertion positions of the transgenes in the genome during the gene transformation process, their expression is affected by the surrounding genome sequences, resulting in positional effects, so that Different expression levels of heavy and light chain proteins

While using the IRES system, although the heavy chain and the light chain are on the same transcript, the expression levels of the heavy chain and the light chain are still different during protein translation because the translation efficiency of the gene behind the IRES is lower than that of the previous one.

For example, although the complete Bevacizumab antibody can be formed through the rice expression system in the past (Chen Lei, Yu Weichang, 2015), due to the poor ratio of heavy chain and light chain of the antibody in the expression system, the yield and antibody quality are affected. For example, the complete antibody 4 Polymer assembly efficiency is low, antibody degradation, etc.

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