Abstract 3866: TWEAK receptor (Fn14) Is a novel target in melanoma: Characterization of unique targeted therapeutics
Authors: Zhou Hong , Ekmekcioglu Suhendan , Marks John W. , Asrani Kaushal , Phillips Keeley K. , Brown Sharron , Cheng Emily , Weiss Michele B. , Hittelman Walter N. , Tran Nhan L. , Yagita Hideo , Winkles Jeffrey A. , Rosenblum Michael G. 
Published: August 2012

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, and lung cancers, and overexpression can be a negative prognostic indicator. We analyzed a series of melanoma cell lines and tumor tissue microarrays and detected Fn14 expression in ∼60% of the melanoma cell lines, including both B-Raf WT and B-Raf V600E lines. Fn14 expression was elevated in 178/190 (93.6%) of primary melanoma specimens and in 87/150 (58%) of melanoma metastases tested. Fn14 expression was not elevated in normal skin tissues. Initial development and characterization of an immunoconjugate designated ITEM4-rGel targeting Fn14 receptor has been published (Zhou et al., Mol. Cancer Ther. 10:1276 (2011)). We have now developed an Fn14-targeted immunotoxin more suitable for long-term clinical use. Specifically, we generated a humanized, dimeric single-chain version of ITEM-4 and fused this scFv to rGel. The resulting anti-Fn14 immunotoxin, designated hSGZ, bound to Fn14 with a Kd of ∼1.4 nM as determined by Biacore analysis. Confocal immunofluoresence studies showed that hSGZ specifically and rapidly (within 2 hrs) internalized into Fn14-expressing MDA-MB-435 melanoma cells. Cytotoxicity studies showed that hSGZ was highly cytotoxic to a panel of different melanoma cell lines (IC50 ranged from 0.1 pM to 1.1 nM) and was 2.2 to 2.8 ×105 fold more potent than free rGel. Treatment of cells expressing the multidrug resistance protein MDR1 showed no cross-resistance to the fusion construct in vitro. When hSGZ was combined with 5-FU, cisplatin, doxorubicin, etoposide or dacarbazine, we found an additive effect on melanoma cell growth inhibition. Mechanistic studies showed that hSGZ induced melanoma cell death consistent with a necrotic mechanism. Additionally, Fn14-targeted immunotoxins increased Fn14 expression and triggered cell signaling events similar to those induced by the TWEAK ligand. Finally, treatment of mice bearing human melanoma MDA-MB-435 and breast MDA-MB-231 xenografts with either ITEM4-rGel or hSGZ showed significant tumor growth inhibition compared to controls (P&lt; 0.05). Fn14 appears to be an excellent new target for melanoma and the Fn14-targeting construct hSGZ appears to warrant further development as a novel therapeutic agent against Fn14-positive tumors. Melanoma lines appear to be the most sensitive tumor type tested but the reasons for this are unclear. Additional studies are in progress to investigate the biodistribution and pharmacokinetics of hSGZ in tumor-bearing mice. This work was conducted, in part, by the Clayton Foundation for Research (MGR); and supported by NIH grant NS055126 (JAW) and DOD Breast Cancer Concept Award BC086135 (JAW).
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3866. doi:1538-7445.AM2012-3866