Effect of Multimerization of a Linear Arg-Gly-Asp Peptide on Integrin Binding Affinity and Specificity

Abstract

Multivalent interactions are frequently used to enhance ligand-receptor binding affinity. In this study, mono-, di- and trimeric Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser-Tyr (AVTGRGDSY) peptides, labeled with ¹²⁵I or Cy5.5, were compared in vitro and in vivo. Using human embryonic kidney HEK293 (naturally α_V-positive and β₃-negative), HEK293(β₁) (β₁-transfected and α_Vβ₃-negative), HEK293(β₃) (β₃-transfected and strongly α_Vβ₃-positive), and human glioblastoma U87MG (naturally α_Vβ₃-positive) cell lines we evaluated their binding affinity and specificity. In vitro, the monomeric AVTGRGDSY showed specific binding to both HEK293(β₁) and HEK293(β₃) cells. Multimerization resulted in no change toward HEK293 cells, diminished binding with HEK293(β₁) cells, but substantially enhanced binding with α_Vβ₃-positive HEK293(β₃) and U87MG cells. Moreover, multimeric AVTGRGDSY peptides were found to be nearly comparable to the same molar concentration of a well-known α_Vβ₃-specific cyclo(RGDfV) (c(RGDfV)) peptide in specificity and affinity for targeting α_Vβ₃ integrin. Non-invasive in vivo optical imaging demonstrated that as compared to its monomeric analogue, the Cy5.5-labeled dimeric AVTGRGDSY peptide produced markedly enhanced tumor-to-background contrast in HEK293(β₃) tumor-bearing mice than in HEK293(β₁) tumor-bearing mice. In conclusion, the present study showed the difference of monomeric and multimeric linear Arg-Gly-Asp (RGD)-containing compound in integrin selectivity and affinity. Our data provide useful information for the design of novel RGD peptides.