A synthetic humanized camelid single-domain antibody targeting VEGF exhibits in vitro antiangiogenic effects.

Vascular endothelial growth factor (VEGF) is a key player in the development and progression of several diseases, most notably cancer and retinal disorders. Over the last twenty years, VEGF has emerged as a significant therapeutic target for these conditions. This study reports the isolation and characterization of a fully synthetic, humanized, affinity-matured single-domain antibody fragment (VHH) designed to target VEGF. Our approach involved a two-step strategy to construct a phage-displayed library: first, diversifying the CDR3 region of the NbBCII10 nanobody and then diversifying the CDR1 and CDR2 regions. The selection process involved three rounds of biopanning against VEGF isoform 121. The resulting antibody fragment, NbH1, showed a four-fold increase in affinity for human VEGF compared to the original VHH. Significantly, this enhanced affinity did not compromise the solubility or thermal stability of NbH1. Molecular modeling indicated that NbH1 recognizes a unique epitope on VEGF. Functional assays, including competitive and conventional ELISAs, demonstrated that NbH1 effectively disrupts the interaction between VEGF and its main receptor VEGFR2, and with bevacizumab, making it particularly promising for therapeutic applications. This novel molecule demonstrates cross-species activity and does not interfere with VEGFR1 binding. Furthermore, NbH1 inhibits VEGF-induced phosphorylation of VEGFR2 in human endothelial cells and hampers their sprouting in a 3D spheroid angiogenesis assay, highlighting its potential for treating pathological angiogenesis in vivo.