Engineering a synthetic ligand for tumor necrosis factor-alpha

One approach to prepare protein binding ligands is to join two low-affinity ligands that bind different sites on the target protein to create a high-affinity bivalent ligand. This typically requires some knowledge of the ligand binding site and requires exquisite orientation of the ligands in order to achieve maximum binding affinity. Here, we explored the limit of affinity improvement possible with no a priori knowledge of peptide binding site and with minimal effort spent in linking the lead peptides. We compared the affinity enhancement from linking two peptides with low affinity for tumor necrosis factor-α (TNFA) to the affinity enhancement from linking affinity improved versions of these peptides using several different scaffolds. We found that we achieved the highest affinity gain not by the precise positioning of the peptides, but rather by using affinity improved versions of the lead peptides to produce synbodies with apparent K_D's of 9 to 48 nM. Kinetic analysis showed that the binding kinetics of the synbody are strongly influenced by the kinetics of the starting peptide. This suggests that careful selection of peptides based on their kinetic profile prior to linking will influence the kinetics of the final binding agent.