We previously reported generation of high affinity peptide binders (HAPs) to the external domain of VEGFR-2 that inhibit its kinase activity using a multi-step process comprising phage-display selection, identification of peptide pairs suitable for hetero-dimerization (non-competing), and chemical synthesis of heterodimers. In order to test the generality of this approach, we decided to create HAPs against another receptor tyrosine kinase (RTK), c-Met. Using peptide phage display, we first identified a number of low-affinity phage binders to the human c-Met/Fc fusion protein. Subsequently we chemically synthesized the binding peptides coded for by the phage and determined their KD values by fluorescence polarization (FP). This was followed by cross-competition studies using FP to identify a pair of peptides that did not compete with each other. Finally, we chemically synthesized a HAP against c-Met (HAPcm) comprising a peptide with a KD of 0.2 μM and a second peptide with a KD of 0.8 μM. Testing HAPcm for binding affinity toward c-Met revealed that HAPcm had at least a 250-fold lower KD (0.8 nM) than the best-binding monomeric component peptide. Duplicating the identification of a subnanomolar affinity peptide to another receptor suggests that our strategy can be used as a general approach to generate HAPs to the extracellular domains of receptor tyrosine kinases. In fact, this method may possibily be applied to any protein for generating high affinity peptide (HAP) binders.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]