Production of Novel VHH Nanobody Inhibiting Angiogenesis by Targeting Binding Site of VEGF

Vascular endothelial growth factor (VEGF) is one of the key players in angiogenesis and is considered as one of the major targets in cancer therapy. VEGF is secreted by the cancerous cells to form new vessels that carry oxygen and nutrients to the tumor, allowing it to grow beyond 1-2 mm. Cancerous cells spread using these veins and cause malignancy. Therefore, neutralization of VEGF could prevent tumor growth and malignancy, and nowadays, antibodies are widely used for such purpose. Among antibody fragments, nanobodies possess unique characteristics which make them appropriate tools for cancer therapy. In this study, the receptor-binding region of VEGF was used to produce a nanobody using phage display technology. A camel was immunized with the recombinant VEGF, and VHH fragments were amplified using nested PCR on lymphocyte complementary DNA (cDNA). The highest binding affinity was achieved after three rounds of panning. Twenty-four clones were tested by monoclonal phage ELISA, and the clone with the highest affinity (VA12) was selected for soluble expression of nanobody. VA12 was tested under various physicochemical conditions and showed considerable stability in extreme temperatures, pH, and various urea concentrations. Stability of VA12 under such conditions is considered as an advantage over the prevailing antibodies.