Abstract 1766: Development of single chain immunotoxins targeting the fibroblast growth factor-inducible 14 (Fn14) receptor on solid tumor cells
Authors: Zhou Hong , Marks John W. , Ekmekcioglu Suhendan , Hittelman Walter N. , Tran Nhan , Yagita Hideo , Winkles Jeffrey A. , Rosenblum Michael G. 
Published: August 2014

Abstract
Previous studies have indicated that Fn14, the cell surface receptor for the cytokine TWEAK, is over-expressed in multiple human solid tumor types, including brain, breast, pancreatic, esophageal, colon and lung cancers. Using a melanoma tumor array, we recently found that Fn14 expression is elevated in 177/190 (93%) primary melanoma specimens and in 87/150 (58%) melanoma metastases specimens. This suggests that Fn14 may be a novel therapeutic target in this disease setting as well. We developed an immunoconjugate designated ITEM4-rGel composed of a high-affinity anti-Fn14 monoclonal antibody (ITEM-4) conjugated to recombinant gelonin (rGel), a highly cytotoxic, ribosome-inactivating n-glycosidase. To develop Fn14-targeted immunotoxins more suitable for clinical use, a humanized single-chain version of ITEM-4 (designated hscFvIT4) specific for Fn14 was successfully produced and characterized. BiaCore analysis of ITEM-4, ITEM4-rGel and hscFvIT4 binding to the Fn14 extracellular domain indicated that these proteins bound to Fn14 with similar affinity, with Kds of ∼ 1.1 nM, ∼ 0.7 nM, and ∼ 6.2 nM, respectively. Using hscFvIT4 and rGel toxin, we next engineered a bivalent immunotoxin (hscFvIT4/rGel/29) by adding a COOH-terminal dimerization domain. Cytotoxicity studies showed that ITEM4-rGel and its humanized counterpart, hscFvIT4/rGel/29, were highly cytotoxic to Fn14-expressing cells (IC50 ranged from 0.8 pM-25 nM) and were 50 to 0.5 x106 fold more potent than free rGel. Minimum contact time studies showed that as little as 8 hr exposure achieved maximal cytotoxic effect. Both ITEM4-rGel and hscFvIT4/rGel/29 were shown to specifically bind to Fn14-expressing tumor cells as analyzed by ELISA. Confocal immunofluoresence studies showed that ITEM4-rGel and hscFvIT4/rGel/29 specifically and rapidly (within 2 hrs) internalized into MDA-MB-435 melanoma cells but not into Fn14-deficient mouse embryonic fibroblasts. Mechanistic studies showed that ITEM4-rGel induced apoptosis in antigen-positive T24 human bladder carcinoma cells as measured by Annexin V staining. Treatment of MDA-MB-435 cells with hscFvIT4/rGel/29 induced the non-canonical NF-κB pathway and down-regulated survivin expression. Preliminary mouse xenograft tumor model studies are ongoing. We propose that the hscFvIT4/rGel/29 construct may warrant further development as a novel therapeutic agent against a broad range of Fn14-positive 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 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1766. doi:10.1158/1538-7445.AM2011-1766