Abstract 2488: FGFR4 as a potential therapeutic target for monoclonal antibody based intervention in rhabdomyosarcoma

Abstract

Background: Rhabdomyosarcoma (RMS) is most common soft tissue sarcoma in childhood and adolescence. There are two major subtypes of RMS - alveolar RMS (ARMS) and embryonal RMS (ERMS). With current treatment modalities the 5 year survival rate of patients with metastatic disease is only about 30%, thus necessitating development of novel targeted therapeutic strategies. Fibroblast Growth Factor Receptor 4 (FGFR4) is highly differentially expressed gene and activating mutations in FGFR4 is associated with increase in RMS metastasis. We hypothesize that FGFR4 can be exploited as a potential therapeutic target in RMS. Monoclonal antibodies (mAbs) against specific cancer cell surface antigens, have gained importance as therapeutic agents in other cancer types. Therefore, mAbs against FGFR4 may be developed either alone or conjugated with a chemotherapeutic agent for treatment of RMS. Methods: We have developed 15 mAbs that are of mouse, rabbit and human origin. The immunogen for rabbit antibodies is human FGFR4 extracellular domain (ECD), while human FGFR4-Fc chimeric protein or hFGFR4 transfected cells were used for mouse antibodies and were produced using hybridoma technology. Using recombinant DNA technology, human immunoglobulin library was selected using FGFR4 ECD to derive human anti-FGFR4 mAbs. All mAbs were purified using affinity chromatography and reactivity was confirmed by ELISA. These mAbs were tested for their ability to bind cell surface FGFR4 and internalization of mAb bound FGFR4 (upon incubation at 37°C) by flow cytometry using RMS cell lines. To further characterize these antibodies we have performed immunohistochemistry (IHC) on tissue microarrays (TMA) of normal tissue, xenografts of RMS cell lines, and primary tumor from RMS patients. Finally, to select a potential mAb candidate as therapeutic agent, we tested appropriate secondary antibody-drug conjugate (ADC) using cell based cytotoxic assay. Results: We observed that tested mAbs bind to both ARMS and ERMS cell lines. The level of FGFR4 expression is variable in both subtypes. Internalization assays demonstrated mAbs bound FGFR4 was internalized in RMS cell lines. Analysis by IHC on TMAs suggests that FGFR4 protein is expressed at a considerably higher level in RMS tumor tissue and xenografts compared to normal tissue. Based on these findings, we are currently screening the mAbs for their ability to elicit a cytotoxic response either alone or in conjunction with secondary ADC. Conclusion: These results suggest anti-FGFR4 mAbs can be used as therapeutic intervention for RMS. High expression of FGFR4 in other cancers such as prostate, melanoma, lung, breast, colorectal, and gastric cancers suggests the potential use of anti-FGFR4 mAbs and their derivatives in these cancers. Citation Format: Sivasubramanian Baskar, Nityashree Shivaprasad, Zhongyu Zhu, Dimiter Dimitrov, Mhairi Sigrist, Poul Sorensen, Marielle Yohe, John Shern, John Maris, Crystal Mackall, Javed Khan. FGFR4 as a potential therapeutic target for monoclonal antibody based intervention in rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2488. doi:10.1158/1538-7445.AM2015-2488

Novel FGFR4-Targeting Single-Domain Antibodies for Multiple Targeted Therapies against Rhabdomyosarcoma

“…Moreover, we could achieve effective cell-mediated cytotoxicity with FGFR4-CAR T cells in vitro. Therapies for RMS based on FGFR4 antibodies have been investigated pre-clinically with promising results, either as antibody–drug conjugates (ADC) [ 56 , 57 , 58 ], or with the antigen-binding domain grafted on chimeric antigen receptors (CARs) to generate CAR T cells [ 59 ]. With our work, we show here a novel FGFR4-targeting based on sdAb by active drug delivery and T cell recruitment.…”

Surfaceome Profiling of Cell Lines and Patient-Derived Xenografts Confirm FGFR4, NCAM1, CD276, and Highlight AGRL2, JAM3, and L1CAM as Surface Targets for Rhabdomyosarcoma

“…The advent of microarrays, and later of next-generation RNA sequencing led to the identification of several genes coding for surface proteins highly expressed in RMS tumors, FGFR4 being the most prominent [37][38][39]. Monoclonal antibodies [40], ADC [41], and CAR T cells are being developed with promising preclinical results [42,43]. Gene expression profiling has revealed that the gene coding for the cannabinoid receptor 1 (CB1) is highly upregulated in FP-RMS [44], but no targeting approaches have been developed yet.…”