Improved targeting of the αvβ3 integrin by multimerisation of RGD peptides

Purpose: The integrin alpha(v)beta(3) is expressed on sprouting endothelial cells and on various tumour cell types. Due to the restricted expression of alpha(v)beta(3) in tumours, alpha(v)beta(3) is considered a suitable receptor for tumour targeting. In this study the alpha(v)beta(3) binding characteristics of an In-111-labelled monomeric, dimeric and tetrameric RGD analogue were compared. Methods: A monomeric (E-c(RGDfK)), dimeric (E-[c ( RGDfK)] 2), and tetrameric ( E{E[ c( RGDfK)] 2} 2) RGD peptide were synthesised, conjugated with DOTA and radiolabelled with In-111. In vitro alpha(v)beta(3) binding characteristics were determined in a competitive binding assay. In vivo alpha(v)beta(3) targeting characteristics of the compounds were assessed in mice with SK-RC-52 xenografts. Results: The IC50 values for DOTA-E-c( RGDfK), DOTA-E[ c(RGDfK)](2), and DOTA-E{E[c(RGDfK)](2)}(2) were 120 nM, 69.9 nM and 19.6 nM, respectively. At all time points, the tumour uptake of the dimer was significantly higher as compared to that of the monomer. At 8 h p.i., tumour uptake of the tetramer (7.40 +/- 1.12% ID/g) was significantly higher than that of the monomer (2.30 +/- 0.34% ID/g), p< 0.001, and the dimer (5.17 +/- 1.22% ID/g), p< 0.05. At 24 h p.i., the tumour uptake was significantly higher for the tetramer (6.82 +/- 1.41% ID/g) than for the dimer (4.22 +/- 0.96% ID/g), p< 0.01, and the monomer (1.90 +/- 0.29% ID/g), p< 0.001. Conclusion: Multimerisation of c( RGDfK) resulted in enhanced affinity for alpha(v)beta(3) as determined in vitro. Tumour uptake of a tetrameric RGD peptide was significantly higher than that of the monomeric and dimeric analogues, presumably owing to the enhanced statistical likelihood for rebinding to alpha(v)beta(3).