Targeting Tissue Factor to Tumor Vessels. Experimental Results and First-in-Man Experience.

Abstract 469

Activation of blood coagulation in tumor vessels with subsequent tumor infarction is an experimental strategy in cancer therapy. We have fused different targeting peptides, including GRGDSP (RGD), GNGRAHA (NGR) and 5 cyclic derivates to the C-terminus of truncated tissue factor (tTF) to preferentially target tumor vessel endothelial cell integrins such as αvβ3 or aminopeptidase (CD13). tTF fusion proteins were expressed in E. coli, purified and refolded. Molecular integrity of the fusion proteins was evaluated by SDS-PAGE, immunoblotting and mass spectrometry. Subsequently, the tTF-fusion proteins were tested for biological activity with the following results: They retained the thrombogenic activity of tTF as measured by factor × activation in vitro. When tested with their respective target molecules either in a purified preparation or present on growing endothelial cells, there was specific binding. In vivo studies with human tumor xenograft models in nude mice showed either significant inhibition of tumor growth or regression of established tumors of different histologies (e.g. lung, breast, melanoma, sarcoma) by systemic application of the tTF-fusion proteins in contrast to controls including non-targeted tTF. Histology of the tumors treated with tTF fusion proteins revealed thrombotic occlusion of vasculature and blood pooling. Contrast-enhanced magnetic resonance imaging (MRI) of the tumors in vivo before and shortly after application of tTF-RGD and tTF-NGR showed a significant reduction of tumor perfusion. Degree of reduction correlated with in vivo tumor response. Toxicity studies showed acceptable therapeutic range and at therapeutic dosage there was no thrombo-embolic event in histology of normal organs, such as lung, heart, liver, and kidney. After upscaling production to amounts sufficient for clinical use, we have treated the first cancer patients with tTF-NGR. MRI studies even at the lowest dose (1 mg/kg i.v.) given showed reduction of tumor perfusion with no side effects. Targeted infarction of tumor vasculature with tTF fusion proteins may be promising as cancer therapy and should be further studied.