Abstract 2176: R1FasL: a recombinant fusion protein that converts VEGF to act as a cell death factor.

Abstract

Abstract Vascular endothelial growth factor (VEGF) is a key tumor angiogenesis factor that is overexpressed by many tumors; the Fas/CD95 death receptor induces apoptosis when activated by its ligand FasL. Agents that inhibit VEGF such as bevacizumab, or activate Fas receptor have been intensively developed as cancer therapeutics. Bevacizumab only modestly improves overall survival or has no effect on overall survival in most cancer patients. More effective VEGF-targeted agents are needed. We conceived of a new strategy that goes beyond VEGF inhibition and converts VEGF to act as a tumor cell death factor. Our aim is to co-opt overexpressed VEGF within the tumor microenvironment to initiate apoptosis via Fas death receptors expressed on tumor cells and/or stromal cells. We created a recombinant homotrimeric fusion protein designated R1FasL that combines the VEGF-binding domain of human VEGFR1 with the trimerization and death receptor-binding domains of human FasL ligand. R1FasL was produced in CHO cells and purified by FLAG antibody affinity chromatography to yield a 135 kD homotrimer. In vitro studies confirmed that VEGF binds to and crosslinks the R1FasL fusion protein to form higher-order complexes that efficiently aggregate and activate Fas death receptors to induce apoptosis. Jurkat cells were killed by R1FasL only when VEGF was added. R1FasL induced apoptosis in diverse human cancer cell lines in vitro, including renal cell, glioblastoma and breast cancers. When VEGF was sequestered by a VEGFR1-Fc trap protein, R1FasL did not induce apoptosis, demonstrating that R1FasL used the VEGF secreted by the cancer cells to kill them. R1FasL-induced apoptosis was inhibited by ZVAD caspase inhibitor and potentiated when combined with chemotherapeutic agents. R1FasL was tested in vivo in a xenograft model of human renal cell carcinoma. Nude mice bearing A498 human renal carcinoma xenografts (∼300 mm3)were treated with a single intravenous dose of R1FasL ranging from 0.08 mg/kg to 10 mg/kg. Twenty-four hours later tumors were collected and analyzed for evidence of apoptosis by histology and caspase-3 immunohistochemistry. Mice treated with a single dose of R1FasL at 2 mg/kg or 10 mg/kg demonstrated widespread tumor apoptosis 24 hours later. R1FasL therefore differs from numerous agents that target the VEGF/VEGFR system, such as bevacizumab, aflibercept and VEGFR kinase inhibitors, none of which induce acute tumor apoptosis. Serum ALT was mildly elevated at the 2 mg/kg dose, but in mice without tumors R1FasL at 4 mg/kg did not cause ALT elevation. These results suggest that R1FasL may not have significant toxicity. By converting VEGF to act as a Fas death receptor ligand, the R1FasL fusion protein may be an effective agent to simultaneously target VEGF and death receptors to selectively induce apoptosis in the tumor microenvironment. Citation Format: Timothy Quinn, Erene Niemi, Mary Nakamura. R1FasL: a recombinant fusion protein that converts VEGF to act as a cell death factor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2176. doi:10.1158/1538-7445.AM2013-2176