Abstract 2576: Novel adnectin/rGelonin fusion constructs targeting the insulin-like growth factor(IGF1) receptor on tumor cells
Authors: Zhigang Liu, Weihe Zhang, Lee M. Ellis, Fan Fan, Yu Cao, Lawrence H. Cheung, John W. Marks, Hong Zhou, Raymond Camphausen, Michael G. Rosenblum
Journal: Cancer Research (2010)

Abstract The insulin-like growth factor (IGF) signaling pathway is a major regulator of cellular proliferation, stress response, apoptosis and transformation in mammalian cells. IGFIR is the signaling receptor for both IGFI and IGFII ligands and is overexpressed in many different human tumor types(eg, breast, pancreatic, ovarian, prostate and sarcoma). Therefore, the IGF/IGFIR pathway is an attractive tumor target. Adnectins™ are a novel class of targeted biologics derived from human fibronectin, an abundant extracellular protein that naturally binds to other proteins. They can be rapidly developed to bind target proteins of interest with high affinities. An Adnectin was developed for its high affinity for the extracellular domain of IGFIR(designated Adn-IGF1R). Adn-IGF1R alone can inhibit growth of some tumor cell lines in vitro and xenograft tumor growth in vivo by interfering with IGFR1 signaling, but its effects are modest. Recombinant gelonin toxin (rGel) is a highly cytotoxic, ribosome-inactivating n-glycosidase. Two Adnectin/rGel fusions with orientations containing rGel at either the N-terminus or C-terminus of the Adn-IGF1R (rGel-Adn-IGF1R and Adn-IGF1R-rGel respectively) were constructed by PCR, expressed in E. coli and purified. Both Adnectin/rGel fusions demonstrated equivalent cell-binding (ELISA) to MCF7 and BXPC3 cells. Reticulocyte lysate translation assays showed that both constructs retained enzymatic activity vs free rGel. Confocal immunofluoresence studies showed that the rGel-Adn-IGF1R internalized rapidly into MCF7 and BXPC3 cells while the Adn-IGF-rGel did not. Testing against 17 tumor cell lines showed that rGel-Adn-IGF1R was 50-100 fold more potent (IC50 ranged from 5pM-300nM) than the Adn-IGF1R-rGel. The rGel-Adn-IGF1R construct was 3-60,000 fold more active than free rGel. Both Adn-IGF1R-rGel constructs and Adn-IGF1R alone effectively inhibited the phosphorylation of IGFIR and AKT. The rGel-AND-IGF1R construct was more cytotoxic to 4 cell lines resistant to oxaliplatin than the parental cells. Treatment of target cells with rGel-Adn-IGF1R showed induction of apoptosis (AnnexinV staining and PARP cleavage). Preliminary xenograft studies show that rGel-Adn-IGF1R was more potent in inhibiting the growth of BXPC3 xenograft tumors than Adn alone. This data demonstrates that Adnectins, in general, may be effective in delivering cytotoxic payloads to target cells and rGel-Adn may be an effective novel agent for therapy of patients with IGFIR overexpressing malignancies. Further development of this material is warranted. Research conducted, in part, by the Clayton Foundation for Research. 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 2576.