Selective Interaction of a Conformationally-constrained Arg-Gly- Asp (RGD) Motif with the Integrin Receptor αvβ3 Expressed on Human Tumor Cells

ABSTRACT: Two antigenized antibodies (AgAbs) were engineered to express peptidic Arg-Gly-Asp (RGD) motifs present in extracellular matrix molecules. The RGD tripeptide sequence was inserted in the third hypervariable loop of an immunoglobulin human/mouse chimeric heavy chain gene as a single or three repeat yielding two antibodies termed γ1RGD and γ1(RGD)3, respectively. The antibodies were used to target specific cell-surface receptors of the integrin type expressed by three human tumor cell lines, a melanoma (M21), an osteosarcoma (KRIB) and a fibroblastoma (WI-38). Based onin vitroadhesion assays and flow cytometric analysis, we found that all three cell lines interacted with γ1(RGD)3but not with γ1RGD. Binding of tumor cells to surface-immobilized γ1(RGD)3was inhibited in a dose-dependent manner by the RGD-containing synthetic peptides GdRGDSP and RGDS. These synthetic peptides, but not a GDR-containing control peptide, interfered with the binding of tumor cells to surface-immobilized human fibronectin. In their soluble form, neither fibronectin nor γ1(RGD)3inhibited tumor cell adhesion to surface-immobilized fibronectin. γ1(RGD)3specifically recognized integrin αvβ3 based on two criteria: reactivity with purified integrin receptors and binding to variants of M21 melanoma cells expressing αvβ3, αIIbβ3 or no β3 integrins, respectively. Collectively, our results indicate that the (RGD)3loop in the antigenized antibody mimics the ligand function of natural extracellular matrix proteins and has a restricted receptor specificity for the αvβ3 integrin which is not inherent to short RGD containing peptides.