Background: We designed the Icon as a high affinity and specificity targeting molecule for Tissue Factor (TF), a transmembrane receptor expressed on vascular endothelial cells of solid tumors but not of normal tissues. TF also is expressed on metastasized tumor cells, which are accessible because the tumor vasculature is leaky. The Icon functions as a human antibody for tumor-specific immunotherapy, and it also can be conjugated to a photosensitizer for targeted-photodynamic therapy (PDT). Methods: The complete Icon (Icon-1) is a 210 kD homodimeric molecule, each chain composed of Factor VII, the natural ligand for TF, as the targeting domain which is conjugated to the Fc effector domain of an IgG1 immunoglobulin. Because the Icon is synthesized by recombinant DNA technology, both the targeting and effector domains can be derived from the cDNAs of various species including human. Icon-1 binds to its natural receptor TF with high affinity (Kd in the picomolar range) and specificity, activating a cytolytic immune attack against accessible cells that express TF. The monomeric Icon (Icon-2) is a 50 kD molecule composed of a single Factor VII domain conjugated to a (His)8 affinity tag. For immunotherapy, the experiments were done in a SCID mouse xenograft model of human breast cancer. One procedure involved intratumoral injection of a replication-incompetent adenoviral vector encoding Icon-1. The vector-infected tumor cells synthesize and secrete Icon-1, which circulates systemically and binds to TF on tumor vascular endothelial cells and tumor cells. Another procedure involved intravenous injection of Icon-1 protein. For PDT, Icon-2 was covalently conjugated with the photosensitizer Visudyne (Verteporfin), which can be activated with a 689 nm laser beam. Endocytosis of the Icon was determined by confocal microscopy. The PDT tests were done with cultured tumor cells incubated with Icon-2 conjugated with Visudyne or free Visudyne as a control. The cultures were irradiated with different doses of laser energy. The cell-lethal effect of PDT was determined by a cell viability assay, and the killing mechanism was determined by a Caspase 3/7 assay. Results: 1. Immunotherapy mediated by injections of vector-encoded Icon-1 into human breast tumor xenografts, or intravenous injections of Icon-1 protein, resulted in tumor regression. 2. Icon molecules were endocytosed into human breast tumor cells and VEGF-induced human umbilical vein endothelial cells (HUVEC). 3. Icon-targeted PDT of human and mouse tumor cells showed dose-dependent cell lethality caused by apoptosis. Conclusions: 1. Icon-targeted immunotherapy results in regression of human breast tumor xenografts in a SCID mouse model. 2. PDT of cultured human and mouse tumor cells is more efficacious with Icon-targeted Visudyne than with free Visudyne. Acknowledgements: The project is supported in part by the Yale Cancer Center/Swebilius Translational Cancer Research Award (Z. H.).

[Proc Amer Assoc Cancer Res, Volume 46, 2005]