Abstract 3777: Development of potent FGFR4-targeted antibody-drug conjugate therapies for rhabdomyosarcoma and other cancers expressing FGFR4

Abstract Background: Rhabdomyosarcoma (RMS) is the most common pediatric sarcoma, representing 3-4% of childhood and adolescent cancers. While multimodal therapies improved the outcomes for localized disease, 5-year survival for relapsed or metastatic RMS cases remains poor. Antibody-drug conjugates (ADCs) use the specificity of monoclonal antibodies to selectively deliver potent anticancer chemotherapy agents to tumor cells while sparing healthy tissues. FGFR4, a cell-surface receptor tyrosine kinase highly expressed in RMS and other cancers such as some breast cancers, but minimally in normal tissues, is a promising immune target. We hypothesize that FGFR4-targeted ADCs could effectively treat RMS and other FGFR4-positive cancers with limited systemic toxicity. Methods: High-affinity human FGFR4-specific binders were developed and evaluated for their internalization in RMS cell lines. The top candidate 3A11, a murine monoclonal antibody, was conjugated to either monomethyl auristatin E (MMAE) via a cathepsin cleavable mcValCit-PABC linker (3A11-MMAE), or an Exatecan derivative via a legumain-cleavable mpGlyAsnAsn linker (3A11-Exatecan). ADCs’ in-vitro efficacy was assessed in FGFR4-positive or FGFR4-negative cells using a live-cell analysis system. Western blots were performed to validate their action mechanisms. Finally, ADCs' in-vivo efficacy was tested in 3 subcutaneous xenograft models: fusion-positive RMS (RH4), fusion-negative RMS with an FGFR4 V550L activating mutation (RMS559), and FGFR4-positive breast cancer (MDA-MB-453). Results: 3A11 was internalized by FGFR4-positive cells and the internalization efficiency was significantly correlated with FGFR4 surface expression levels. Both ADCs selectively killed FGFR4-expressing cells, with their potency correlating with FGFR4 expression and 3A11 internalization. Western blot confirmed that 3A11-MMAE induced specific apoptosis and 3A11-Exatecan induced cell death after DNA damage in FGFR4-expressing RMS cells. In vivo, 3A11-MMAE (3 mg/kg, twice weekly for two weeks) delayed RH4 tumor growth, improving survival by 30%, whereas a single dose of 3A11-Exatecan (10 mg/kg) eradicated RH4 tumors, achieving 100% survival. In the aggressive RMS559 model, 40% of 3A11-MMAE treated mice achieved tumor clearance, with a 70% survival rate. While two doses of 3A11-Exatecan completely eradicated RMS559 tumors. Furthermore, both ADCs effectively controlled MDA-MB-453 breast cancer growth. Conclusions and Future Directions: Our results demonstrate an unprecedented efficacy of these FGFR4-targeting ADCs specifically against human cancers expressing FGFR4 including aggressive rhabdomyosarcoma and breast cancers. We plan to humanize the 3A11 binder and perform pharmacokinetic and toxicology studies in non-human primates for preparation in clinical trials. Citation Format: Meijie Tian, Katrina Jia, Jerry T. Wu, Jun S. Wei, Adam T. Cheuk, Siteng Fang, Victor Ojo, Eleanor G. Pope, Yong Yean Kim, Shyam K. Sharan, Zoe Weaver Ohler, Ludmila Szabova, Simone Difilippantonio, Borys Shor, L. Nathan Tumey, Javed Khan. Development of potent FGFR4-targeted antibody-drug conjugate therapies for rhabdomyosarcoma and other cancers expressing FGFR4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3777.