Abstract 5427: Inducing tumor apoptosis by redirecting VEGF to activate cell death receptors

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. We previously demonstrated that a single intravenous dose of R1FasL induced widespread tumor apoptosis in a xenograft model of human renal cell carcinoma. We have now tested R1FasL in an intracranial xenograft model of human glioblastoma. Nude mice bearing U87 intracranial xenografts were treated with a single intratumoral injection of R1FasL (0.025 to 0.9 mg/kg in 10 microliters). Twenty-four hours later tumors were collected and analyzed for evidence of apoptosis by caspase-3 immunohistochemistry. Mice treated with a single intratumoral dose of R1FasL at 0.1 mg/kg demonstrated significant apoptosis within the tumor but not in surrounding brain. In a safety study, intracranial injection of R1FasL (0.1 or 0.8 mg/kg) into normal brains of nude mice did not cause increased weight loss or death versus PBS injection. In a subsequent survival study, mice treated with a single intratumoral dose of R1FasL (0.8 mg/kg) survived significantly longer than PBS-treated mice (39.5 versus 30 days, p=0.018). Non-invasive imaging demonstrated a median 59% reduction in tumor size 48 hours after R1FasL injection. 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, YJ Lu, Raquel Santos, Tomoko Ozawa, David James, Mary C. Nakamura. Inducing tumor apoptosis by redirecting VEGF to activate cell death receptors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5427. doi:10.1158/1538-7445.AM2014-5427