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

SIMPLE SUMMARY: Rhabdomyosarcoma (RMS) accounts for more than 50% of all soft tissue sarcomas in childhood and adolescence. Despite progress and intensified multimodality treatment, prognoses are extremely poor with an overall survival rate of approximately 20% in the advanced stage. Therefore, there is an urgent need for targeted treatment options to improve overall survival rates, and to limit long-term side effects. The fibroblast growth factor receptor 4 (FGFR4) is overexpressed in RMS and other tumors as well. The goal of this work was to select FGFR4 specific single-domain antibodies (sdAb) and to develop FGFR4-targeted therapies. We could show that FGFR4-targeted liposomes have the potential to deliver drugs specifically to FGFR4-positive tumor cells and that chimeric antigen receptor T cells built with the selected antibodies can kill specifically FGFR4-expressing RMS cells. ABSTRACT: The fibroblast growth factor receptor 4 (FGFR4) is overexpressed in rhabdomyosarcoma (RMS) and represents a promising target for treatments based on specific and efficient antibodies. Despite progress, there is an urgent need for targeted treatment options to improve survival rates, and to limit long-term side effects. From phage display libraries we selected FGFR4-specific single-domain antibodies (sdAb) binding to recombinant FGFR4 and validated them by flow cytometry, surface plasmon resonance, and fluorescence microscopy. The specificity of the selected sdAb was verified on FGFR4-wild type and FGFR4-knock out cells. FGFR4-sdAb were used to decorate vincristine-loaded liposomes and to generate chimeric antigen receptor (CAR) T cells. First, incubation of RMS cells with FGFR4-sdAb revealed that FGFR4-sdAb can block FGF19-FGFR4 signaling via the MAPK pathway and could therefore serve as therapeutics for FGFR4-dependent cancers. Second, FGFR4-targeted vincristine-loaded liposomes bound specifically to RMS cells and were internalized by the receptor, demonstrating the potential for active drug delivery to the tumor. Third, FGFR4-CAR T cells, generated with one sdAb candidate, demonstrated strong and specific cytotoxicity against FGFR4 expressing RMS cells. We selected novel FGFR4-sdAb with high specificity and nano- to picomolar affinities for FGFR4 which have the potential to enable multiple FGFR4-targeted cancer therapy approaches.