Ranibizumab Biosimilar (Beijing Jingyitaixiang Technology Development)

To improve the production yield of N-glycosylated anti-VEGFR2 (vascular endothelial growth factor receptor 2) monobody (FN3_VEGFR2-Gly) in lpp knockout Escherichia coli cells harboring Campylobacter jejuni N-glycosylation pathway.

The leaky CLM37-Δlpp strain efficiently secreted FN3_VEGFR2-Gly into culture medium. The extracellular levels of glycosylated FN3_VEGFR2-Gly in CLM37-Δlpp culture medium were approximately 11 and 15 times higher compared to those in CLM37 cells via IPTG and auto-induction, respectively. In addition, the highest level of total glycosylated FN3_VEGFR2-Gly (70 ± 3.4 mg/L) was found in culture medium via auto-induction. Furthermore, glycosylated FN3_VEGFR2-Gly was more stable than unglycosylated FN3_VEGFR2-Gly in this expression system, but their bioactivities were relatively similar.

Lpp knockout leaky E. coli strain combined with auto-induction method can enhance the extracellular production of homogenous N-glycosylated FN3_VEGFR2-Gly, and facilitate the downstream protein purification. The findings of this study may provide practical implications for the large-scale production and cost-effective harvesting of N-glycosylation proteins.

In human, the interaction between vascular endothelial growth factor (VEGF) and its receptor (VEGFR2) is critical for tumor angiogenesis. This is a vital process for cancer tumor growth and metastasis. Blocking VEGF/VEGFR2 conjugation by antibodies inhibits the neovascularization and tumor metastasis. This investigation designed to use a transient expression platform for production of recombinant anti-VEGFR2 nanobody in tobacco plants. At first, anti-VEGFR2-specific nanobody gene was cloned in a Turnip mosaic virus (TuMV)-based vector, and then, it was expressed in Nicotiana benthamiana and Nicotiana tabacum cv. Xanthi transiently. The expression of nanobody in tobacco plants were confirmed by reverse transcription-polymerase chain reaction (RT-PCR), dot blot, enzyme-linked immunosorbent assays (ELISA), and Western blot analysis. It was shown that tobacco plants could accumulate nanobody up to level 0.45% of total soluble protein (8.3 µg/100 mg of fresh leaf). This is the first report of the successful expression of the camelied anti-VEFGR2 nanobody gene in tobacco plants using a plant viral vector. This system provides a fast solution for production of pharmaceutical and commercial proteins such as anti-cancer nanobodies in tobacco plants.