Anti-cancer Research Of Recombination Human Interferonα-2b And Endostatin Peptide Fusion Proteins

Interferon α2b is an important tumor immunotherapy drug, as first-line drugs arewidely used in cancer treatment. It has been approved by FDA for the treatment ofvarious viral diseases and cancer. However, IFNα2b in the clinical treatment oflong-term administration of large doses required, as so often lead to acute and chronictoxicity, damage to the nervous system and blood system and other significant sideeffects, has seriously hampered its wide application in clinical practice.Human endostatin is an inhibitor of angiogenesis, the inhibition of tumorangiogenesis drugs in outstanding performance in2009, approved by SFDA for thetreatment of tumors. After a number of studies confirm that human endostatinN-terminal27amino acids is the core functional areas. It on the basis of the replacementand addition, the introduction of series of RGD (Arg-Gly-Asp) sequence, formationendostatin peptide (29amino acid, EP29), while maintaining the endostatin inhibition oftumor angiogenesis activity and given by the endostatin27peptide-RGD targetingtumor vascular endothelial cells, inhibit tumor cell growth, metastasis and enhancing theeffectiveness of its anti-tumor activity. Laboratory studies have confirmed, EP29in vitroanti-tumor activity was significantly higher than the natural endostatin, in vivoanti-tumor activity of endostatin is also slightly higher than the natural, pathologicalstudies have shown that a large area of tumor necrosis and shrinkage. Therefore, it wasexpected to become a new generation of small molecule anti-cancer drugs.In this study, we used genetic engineering techniques to integrate the IFNα2b andEP29formation IFNα2b-endostatin29peptide (IEP29) fusion protein in E. coli forexpression. Expect that the RGD sequence of fusion protein can specific bindintegrin-αvβ3/αvβ5of endothelial cells within tumor angiogenesis. IEP29enriched attumor tissue to play targeted inhibition tumor angiogenesis of EP29and anti-tumor roleof IFNα2b, but also can inhibit integrin-mediated angiogenesis, thereby reducing theIFNα2b clinical dosage, increased dose-effect ratio. Through a series of in vivo and invitro analysis confirmed that, IEP29protein with high biological activity, can inhibittumor growth and tumor angiogenesis, but also has good tumor targeting. Therefore,according with the original design, with a high clinical application development value,is expected to become a new generation of anticancer drugs. Research details are as follows:1. IEP29fusion protein upstream researchIn this study, we successfully constructed IEP29prokaryotic expression vectorpET3a-IEP29by DNA recombinant technology, the vector was transfected into BL21engineering bacteria and induced by IPTG as inclusion bodies after expression, IEP29expression about10%of total bacterial protein. IEP29can be specific binding withmonoclonal antibodies of anti-IFNα2b. Re-engineering of bacteria using5L NBSfermentor cultivation, collection of bacteria by lysis, inclusion body wash, dissolve thedenaturation and refolding dialysis, and finally by affinity chromatography andion-exchange column purification the purity greater than95%of the recombinantprotein, in full compliance with USP standards for toxin detection.In order to meet the needs of pre-clinical study, we optimized the fermentation andpurification process to enlarge and pilot scale. Continuous fermentation using NBS5LFermentation batches, each batch of wet biomass harvest of about150g, IEP29proteinfusion protein is about the total amount of10%. Consecutive batches of IEP29purifiedfusion protein, more than95%purity, yield30~50mg/batch scale of production toachieve the basic test requirements. IEP29by physical and chemical properties, purity,biological activity, endotoxin content, residual impurities and other aspects of control,confirm IEP29protein gene engineering drugs meet quality standards.2. Activity analsis of IEP29proteinIn order to verify that both the biological activity of IFNα2b and EP29, the tumorcell growth inhibition and invasion assay were adopted. The experimental validation invitro fusion protein can effectively inhibit tumor cell invasion. Endothelial cell adhesionassay verificated the fusion protein in vitro effective adhesion endothelial cells andtumor-specific high affinity. Results indicate that IEP29in vitro inhibition of tumor cellproliferation and migration was significantly higher than IFNα2b and EP29, whilespecific adhesion to endothelial cells, statistically significant higher.3. IEP29fusion protein anti-cancer researchExperiments of drug biodistribution studies, tumor pathology biopsy study, chickchorioallantoic membrane test, etc shows the IEP29protein inhibitory effect of tumor.The other hand, some assaies study the mechanism of inhibition of tumor growth. Theresults showed that IEP29anti-tumor effective than IFNα2b and EP29. It is more significant and effective inhibition of tumor angiogenesis, targeting both the tumor. Tofurther study if the IEP29is maintained IFNα2b and EP29of the biological function, weconducted a series of studies, help to reveal the pathway-targeted drug, while the fusionprotein drug development is also instructive.In tumor inhibition assay, IEP29was significantly reduced tumor weight thattumor-bearing nude mice was injected tumor cells H22and Lewis's, and the controlgroup was significant difference between the rate of tumor weight in a dose-dependentinhibition. IEP29group were significantly higher than the same dose of the EP29group,and the same dose IFNα2b significantly different. In vivo distribution studies show that30min after administration of tumor tissue the concentration of IEP29is IFNα2b5times,60min after administration of tumor tissue the concentration of IEP29is1.8times IFNα2b. IEP29can effectively gather in tumor tissue in mice.In the chick embryo chorioallantoic membrane experiments, EP29, IFNα2b andIEP29protein without affecting the blood vessels under the premise of the original,there is a certain degree of inhibition of the growth of new blood vessels. EP29andIEP29inhibition of angiogenesis was significantly stronger than IFNα2b. The resultscan be seen from the control group grew well in blood vessels, capillaries clearly visible,the main blood vessel stout, branch moderate. IFNα2b group shows part of the capillaryto reduce, but not obvious. EP29group significantly reduced the number of capillaries,blood vessels become thin and blurred. IEP29group was more obvious, a large numberof capillaries disappear, the main blood vessels as well as some disappear.In summary, we have established a relatively stable IEP29protein productionprocess, the establishment of an optimized expression and purification system andquality control standards. In vivo and in vitro experiments have confirmed that, IEP29and EP29fusion protein compared with IFNα2b, is a highly effective anti-tumorangiogenesis and inhibit tumor growth, results of this study shows IEP29is hopefulindustrial production and laid the foundation for clinical trials in the future.