在肿瘤组织中,碱性成纤维细胞生长因子(bFGF)和血管内皮生长因子A(VEGFA)通过激活血管生成促进肿瘤发生,但靶向单因子可能产生耐药性和代偿性血管生成。设计具有bFGF/VEGFA的肽体以同时阻断这两种因子。我们的目标是大规模生产这种Fc融合蛋白。将肽体菌株的生物学特性鉴定为大肠杆菌,并在摇瓶和10-L生物反应器中优化了发酵模式。发酵扩大到100升,湿细胞重量(WCW)126g/L,产量1.41g/L,IPTG诱导和生产率0.35g/(L·h)。通过阳离子交换分离目标蛋白,疏水和蛋白A色谱,总回收率为60.28%,HPLC纯度为86.71%。宿主细胞的蛋白质,DNA,内毒素残留在阈值内。在小鼠模型中,Peptibody疫苗的免疫可以显著抑制肿瘤的生长和血管生成,抑制率分别为57.73和39.34%。肽体疫苗能引发高滴度的抗bFGF和抗VEGFA抗体,通过降低Akt/MAPK信号通路抑制Lewis肺癌细胞的增殖和迁移。因此,具有bFGF/VEGFA的肽体可用作治疗性肿瘤疫苗。我们开发的大规模工艺可以支持其未来的工业生产和临床前研究。
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.