Integrin-targeted imaging and therapy with RGD4C-TNF fusion protein

This study used integrin alpha(v)beta(3) as a target for tumor-specific delivery of tumor necrosis factor-alpha (TNF). The fusion protein RGD4C-TNF bound specifically to alpha(v)beta(3) as evidenced by cell receptor binding assay and noninvasive micro-positron emission tomography imaging. Cu-64-DOTA-RGD4C-TNF had significantly higher activity accumulation in integrin-positive tumors (U87MG and MDA-MB-435) but not in integrin-negative tumors (C6) compared with Cu-64-DOTA-TNF. The magnitude of tumor uptake of Cu-64-DOTA-RGD4C-TNF correlated well with the alpha(v)beta(3) level (U87MG > MDA-MB-435 > C6). Tumor accumulation of Cu-64-DOTA-RGD4C-TNF could be effectively blocked by c(RGDyK) peptide in alpha(v)beta(3)-positive tumor models, suggesting alpha(v)beta(3) specificity of RGD4C-TNF fusion protein in vivo. Furthermore, although the fusion of RGD4C moiety to TNF had little effect on the bioactivity and cytotoxicity of RGD4C-TNF compared with TNF in cell culture, RGD4C-TNF was significantly more potent than TNF in inhibiting orthotopic MDA-MB-435 tumor growth. Ex vivo tissue staining confirmed specific cytotoxicity of RGD4C-TNF against integrin-positive tumor cells and tumor vasculature.