Abstract 2574: Development and characterization of a potent immunotoxin targeting the Fn14 receptor on solid tumor cells

Abstract

Abstract TNF-like weak inducer of apoptosis (TWEAK) and FGF-inducible 14 (Fn14) are a TNF superfamily ligand-receptor pair involved in inflammation, oncogenesis, tumor invasion, migration, survival and resistance to chemotherapy. The Fn14 receptor is expressed at relatively low levels in normal tissues, but is known to be dramatically elevated in a number of tumor types, including brain and breast tumors. Thus, the TWEAK/Fn14 axis appears to be an excellent candidate for therapeutic intervention. We have developed an immunoconjugate designated ITEM4-rGel containing a high-affinity anti-Fn14 monoclonal antibody conjugated to recombinant gelonin (rGel), a highly cytotoxic, ribosome-inactivating n-glycosidase. The ITEM4-rGel conjugate was generated and purified and contained no contaminating free antibody or rGel. The final material contained both antibody + 1 rGel (major) and antibody + 2 rGel (minor) species. We analyzed Fn14 expression in human tumor cell lines using flow cytometry and Western blot analysis. Fn14 was expressed in a variety of tumor lines including breast, brain, bladder, skin, lung, ovarian, pancreatic, colon, prostate, and cervical tumor cell lines. Both ITEM4 and ITEM4-rGel were found to bind to cells to an equivalent extent. Confocal immunofluoresence studies showed that ITEM4-rGel specifically and rapidly (within 2 hrs) internalized into MDA-MB-231 breast cancer cells and T-24 bladder cancer cells but not into Fn14-deficient mouse embryonic fibroblasts. Cytotoxicity studies against 22 different tumor cell lines showed that ITEM4-rGel was highly cytotoxic to Fn14-expressing cells (IC50 ranged from 0.8 pM-25 nM) and was 50 to 0.5 ×106 fold more potent than free rGel. Minimum contact time studies showed that as little as 12 hr exposure achieved maximal cytotoxic effect. Mechanistic studies showed that ITEM4-rGel induced HMGB1 release following treatment of MDA-MB-231, T-24, AAB 527, and BxPC-3 cells. In addition, target cells showed induction of apoptosis, as measured by Annexin V staining and caspase-3 cleavage. ITEM4-rGel treatment also induced the non-canonical NF-κB pathway, up-regulated the tumor suppressor protein p53 and down-regulated survivin expression. Preliminary mouse xenograft tumor model studies are ongoing. These data suggest that the ITEM4-rGel construct may warrant further development as a novel therapeutic agent against a broad range of solid tumor types. Research conducted, in part, by the Clayton Foundation for Research (MGR), and supported by NIH grant NS55126 (JAW) and DOD Breast Cancer Concept Award BC086135 (JAW). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2574.