The vascular endothelial growth factor (VEGF) family of cytokines plays a key role in vasculogenesis, angiogenesis, and lymphangiogenesis. VEGF-A is the main member of this family, alongside placental growth factor (PlGF), VEGF-B/C/D in mammals, and VEGF-E/F in other organisms. To study the activities of these growth factors under physiological and pathological conditions, resulting in therapeutic applications in cancer and age-related macular degeneration, blocking ligands have been developed. These have mostly been large biomolecules like antibodies. Ligands with high affinities, at least in the nanomolar range, and accurate structural data from X-ray crystallography and NMR spectroscopy have been described. They constitute the main focus of this overview, which evidences similarities and differences in their binding modes. For VEGF-A ligands, and to a limited extent also for PlGF, a transition is now observed towards developing smaller ligands like nanobodies and peptides. These include unnatural amino acids and chemical modifications for designed and improved properties, such as serum stability and greater affinity. However, this review also highlights the scarcity of such small molecular entities and the striking lack of small organic molecule ligands. It also shows the gap between the rather large array of ligands targeting VEGF-A and the general absence of ligands binding other VEGF members, besides some antibodies. Future developments in these directions are expected in the upcoming years, and the study of these growth factors and their promising therapeutic applications will be welcomed.

Macromolecular ligands targeting vascular endothelial growth factor A (VEGF) to inhibit pathological angiogenesis are used in the clinic for the treatment of cancers and ocular diseases. To develop smaller ligands retaining high affinity through an avidity effect, here we design homodimer peptides targeting the two symmetrical binding sites of the VEGF homodimer. A series of 11 dimers were synthesized with flexible poly(ethylene glycol) (PEG) linkers of increasing lengths. The binding mode was determined by size exclusion chromatography, and analytical thermodynamic parameters were measured by isothermal titration calorimetry and compared to the antibody bevacizumab. The effect of linker length was qualitatively correlated to a theoretical model. With the optimal length in PEG 25 -dimer D6, the binding affinity was improved 40-fold compared to a monomer control, resulting in a single-digit nanomolar K d value. Finally, we validated the benefit of the dimerization strategy by evaluating the activity of control monomers and selected dimers in cell-based assays with human umbilical vein endothelial cells (HUVECs).