Abstract 652: Development and characterization of anti-FGFR4 monoclonal antibodies as therapeutic agents for human rhabdomyosarcoma
Authors: Baskar Sivasubramanian , Zhu Zhongyu , Labitigan Ramon Lorenzo , Ovanesian Michelle , Orentas Rimas J. , Li Samuel Q. , Marielle Yohe E. , Shern John , Dimitrov Dimiter S. , Maris John , Mackall Crystal , Javed Khan 
Published: May 2015

Abstract
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood with two major subtypes embryonal (ERMS) and alveolar (ARMS), and current treatment modalities have yielded event free 5-year survival in only 30% of the patients with high-risk disease. Therefore, there is an absolute need for novel strategies and to identify and validate clinically relevant targets for the treatment of RMS. The fibroblast growth factor receptor 4 (FGFR4) is a very attractive therapeutic target because: 1) the FGFR4 gene is over expressed in RMS, 2) it is crucial for survival, proliferation, metastasis and drug resistance, 3) activating mutations in the kinase domain lead to aggressive growth and poor survival in patients with alveolar RMS and 4) genetic or pharmacologic inhibition of FGFR4 signaling inhibited tumor growth in vitro and in vivo. Monoclonal antibodies (mAbs) against specific antigens expressed on cancer cell surface have gained importance as potential therapeutic agents that may be used either alone or in combination with chemotherapeutic drugs. We have developed several mAbs against human FGFR4 protein from rabbit and mouse (by hybridoma technology), and from human immunoglobulin libraries (by recombinant DNA technology). In the present study, we report the development and characterization of some of these mAbs. Biacore analysis of these antibodies showed low nM affinity to purified extracellular domain of FGFR4 (FGFR4-ECD). The ability of these mAbs to bind the native molecule was demonstrated by specific binding to RMS cell lines of both subtypes, and dose response curves exhibited higher binding in ARMS cells than ERMS cells. More importantly, significant binding was noticed in freshly isolated tumor cells from a breast metastatic nodule of a patient with ARMS. The anti-FGFR4 mAbs also bound to transfected cell line expressing FGFR4 on the surface, but not the vector control (FGFR4 negative) cell line indicating the specificity of the reaction. Furthermore, cell surface FGFR4 can mediate internalization of the bound antibody upon incubation at 37C for as little as 30 min and maximum internalization was observed at 2 h. Receptor mediated internalization of the bound mAb was inhibited by a chemical inhibitor, and ARMS cells showed more internalization than ERMS. Together, these observations support the contention that anti- FGFR4 mAb can be used as a vehicle to deliver a cytotoxic payload in the form of small molecule drugs and toxins. Ongoing investigations are aimed at developing anti-FGFR4 mAbs and their derivatives as potential therapeutic agents for the treatment of patients with RMS.
This study is supported in part by the intramural research program of the National Cancer Institute, National Institutes of Health, grants from St. Baldrick's Foundation and Stand Up To Cancer - St. Baldrick's Pediatric Dream Team Translational Cancer Research Grant, Grant Number SU2C-AACR-DT11-13.
Citation Format: Sivasubramanian Baskar, Zhongyu Zhu, Ramon Lorenzo Labitigan, Michelle Ovanesian, Rimas J. Orentas, Samuel Q. Li, Yohe E. Marielle, John Shern, Dimiter S. Dimitrov, John Maris, Crystal Mackall, Khan Javed. Development and characterization of anti-FGFR4 monoclonal antibodies as therapeutic agents for human rhabdomyosarcoma. [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 652. doi:10.1158/1538-7445.AM2014-652