Fermentation, Purification, and Tumor Inhibition of a Disulfide-Stabilized Diabody Against Fibroblast Growth Factor-2.

Angiogenesis is considered one of the hallmarks of cancer and plays a critical role in the development of tumor. Fibroblast growth factor 2 (FGF-2) is a member of the FGF family and participates in excessive cancer cell proliferation and tumor angiogenesis. Thus, targeting FGF-2 was considered to be a promising anti-tumor strategy. A disulfide-stabilized diabody (ds-Diabody) against FGF-2 was produced in Pichia pastoris (GS115) by fermentation and the anti-tumor activity was analyzed. The novel 10-L fed batch fermentation with newly designed media was established, and the maximum production of the ds-Diabody against FGF-2 reached 210.4 mg/L. The ds-Diabody against FGF-2 was purified by Ni2+ affinity chromatography and DEAE anion exchange chromatography. The recombinant ds-Diabody against FGF-2 could effectively inhibit proliferation, migration, and invasion of melanoma and glioma tumor cells stimulated by FGF-2. Furthermore, xenograft tumor model assays showed that the ds-Diabody against FGF-2 had potent antitumor activity in nude mice by inhibiting tumor growth and angiogenesis. The tumor growth inhibition rate of melanoma and glioma was about 70 and 45%, respectively. The tumor angiogenesis inhibition rate of melanoma and glioma was about 64 and 51%, respectively. The results revealed that the recombinant ds-Diabody against FGF-2 may be a promising anti-tumor drug for cancer therapy.

Large-scale production, purification, and function of a tumor multi-epitope vaccine: Peptibody with bFGF/VEGFA.

In tumor tissue, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA) promote tumorigenesis by activating angiogenesis, but targeting single factor may produce drug resistance and compensatory angiogenesis. The Peptibody with bFGF/VEGFA was designed to simultaneously blockade these two factors. We were aiming to produce this Fc fusion protein in a large scale. The biological characterizations of Peptibody strains were identified as Escherichia coli and the fermentation mode was optimized in the shake flasks and 10-L bioreactor. The fermentation was scaled up to 100 L, with wet cell weight (WCW) 126 g/L, production 1.41 g/L, and productivity 0.35 g/(L·h) of IPTG induction. The target protein was isolated by cation-exchange, hydrophobic and Protein A chromatography, with total recovery of 60.28% and HPLC purity of 86.71%. The host cells protein, DNA, and endotoxin residues were within the threshold. In mouse model, immunization of Peptibody vaccine could significantly suppressed the tumor growth and angiogenesis, with inhibition rate of 57.73 and 39.34%. The Peptibody vaccine could elicit high-titer anti-bFGF and anti-VEGFA antibodies, which inhibited the proliferation and migration of Lewis lung cancer cell cells by decreasing the Akt/MAPK signal pathways. Therefore, the Peptibody with bFGF/VEGFA might be used as a therapeutic tumor vaccine. The large-scale process we developed could support its industrial production and pre-clinical study in the future.

Monensin inhibits proliferation, migration, and promotes apoptosis of breast cancer cells via downregulating UBA2.

Breast cancer is a malignant tumor that occurs in the epithelial tissue of the breast gland, the morbidity, and mortality of which continue to increase. Therefore, it is crucial to find new drugs to treat breast cancer. Monensin is a carrier antibiotic that has been reported to inhibit the growth of cancer cells; however, its impacts on breast cancer cells have not been reported. In this article, the cell survival rate was measured by CCK-8. Colony formation assay was utilized to detect the level of cell proliferation. Transwell was used to measure the ability of cell invasion, and wound healing was used to measure the ability of cell migration. RT-qPCR and western blot were, respectively, used to detect the expression of related genes and proteins. The level of apoptosis was detected by flow cytometry. Cell transfection technique was used for overexpressing UBA2. We found that Monensin inhibited the proliferation and migration of breast cancer cells and inhibited the expression of MMP-2 and MMP-9. In addition, Monensin promoted the apoptosis accompanied by the increase of Bax, caspase3, caspase7, and caspase9 and the decreased of bcl-2 of breast cancer cells. Monensin was also found to inhibit UBA2 expression in breast cancer cells. Subsequently, after overexpression of UBA2, the impacts of Monensin on proliferation, migration, and apoptosis of breast cancer cells was inhibited. In conclusion, Monensin can inhibit the proliferation and migration and activate apoptosis of breast cancer cells via downregulating the expression of UBA2.