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 gamma1RGD and gamma1(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), and
osteosarcoma (KRIB) and a fibroblastoma (WI-38). Based on in vitro adhesion assays and flow cytometric analysis, we found that all three cell lines interacted with gamma1(RGD)3 but not with gamma1RGD. Binding of
tumor cells to surface-immobilized gamma1(RGD)3 was inhibited in a dose-dependent manner by the RGD-containing synthetic
peptides GdRGDSP and RGDS. These synthetic
peptides, but no a GDR-containing control
peptide, interfered with the binding of
tumor cells to surface-immobilized human
fibronectin. In their soluble form, neither
fibronectin nor gamma1(RGD)3 inhibited
tumor cell adhesion to surface-immobilized
fibronectin. Gamma1(RGD)3 specifically recognized
integrin alphavbeta3 based on two criteria: reactivity with purified
integrin receptors and binding to variants of M21
melanoma cells expressing alphavbeta3, alphaIIbbeta3 or no beta3
integrins, respectively. Collectively, our results indicate that the (RGD)3 loop in the antigenized antibody mimics the
ligand function of natural
extracellular matrix proteins and has a restricted receptor specificity for the
alphavbeta3 integrin which is not inherent to short RGD containing
peptides.