Dimer Peptide Ligands of Vascular Endothelial Growth Factor: Optimizing Linker Length for High Affinity and Antiangiogenic Activity

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 PEG25-dimer D6, the binding affinity was improved 40-fold compared to a monomer
control, resulting in a single-digit nanomolar Kd 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).