Abstract LB042: A potent FGFR4-targeted antibody-drug conjugate therapy for patients with rhabdomyosarcoma and other cancers expressing FGFR4
Authors: Tian Meijie , Jia Katrina , Wu Jerry T. , Wei Jun S. , Cheuk Adam T. , Zhang Zhongmei , Pope Eleanor G. , Shor Boris , Tumey L. Nathan , Khan Javed 
Published: April 2024

Abstract
Background Rhabdomyosarcoma (RMS) is the most common pediatric sarcoma, representing 3-4% of childhood and adolescent cancers. Although multimodal therapies have significantly improved the overall survival of patients with localized disease, 5-year overall survival for patients with relapsed and metastatic disease has remained dismal. Therefore, new therapies are desperately needed for this deadly disease. Antibody-drug conjugates (ADCs) combine the specificity of monoclonal antibodies with the cytotoxic potency of chemotherapy drugs to selectively deliver potent anticancer agents to tumor cells while sparing healthy tissues. FGFR4 is a cell-surface receptor tyrosine kinase highly expressed in RMS and other human cancers such as hepatocellular carcinoma (HCC) and some breast cancers. It is expressed at much lower levels in normal tissues, making it a promising immune target. We hypothesize that an FGFR4-targeted ADC would have potent activity against RMS and other cancers expressing FGFR4 with minimal toxicity toward normal tissues.
Methods We developed high-affinity binders to human FGFR4 and determined their internalization ability in RMS cancer cell lines. Our top candidate 3A11, a murine monoclonal antibody, was conjugated to monomethyl auristatin E (MMAE), a potent cytotoxic agent, using a valine-citrulline-p-aminocarbamate linker (VC-PABC), to generate an FGFR4-targeted ADC (3A11-vc-MMAE). We tested the in-vitro efficacy of 3A11-vc-MMAE against multiple cell lines of RMS, triple-negative breast cancer, HCC, and FGFR4 negative controls using a live-cell analysis system Incucyte. Western blots were performed to validate 3A11-vc-MMAE’s mechanism of action. We finally tested the in vivo efficacy of 3A11-vc-MMAE in subcutaneous xenograft models of a fusion-positive RMS (RH4) and a fusion-negative RMS with an FGFR4 V550L activating mutation (RMS559).
Results We showed that 3A11 was internalized by FGFR4-expressing cell lines and trafficked to lysosomes. Cell viability assays demonstrated that 3A11-vc-MMAE selectively killed FGFR4-expressing cells with minimal effect on low or non-expression cells. Western blot confirmed that 3A11-vc-MMAE induced apoptosis specifically in FGFR4-expressing RMS cell lines. Mice treated with one cycle of 3A11-vc-MMAE (3mg/kg twice a week for 2 weeks) demonstrated a significantly increased survival rate of 30% for the RH4 model with a median survival of 43 days for 3A11-vc-MMAE and 23 days for control, vehicle-treated mice (p-value = 0.0038), and 70% for the RMS559 model, with a median survival not reached for of 3A11-vc-MMAE treated compared with 23 days for control mice (p-value &lt; 0.0001). Of note, tumors were eradicated in 4 out of 10 mice bearing RMS559 xenografts.
Conclusions and Future Directions Our results demonstrate that the ADC 3A11-vc-MMAE provides a potent and specific precision immunotherapy for rhabdomyosarcoma and other cancers with a high expression of FGFR4, which warrants further development for clinical translation.
Citation Format: Meijie Tian, Katrina Jia, Jerry T. Wu, Jun S. Wei, Adam T. Cheuk, Zhongmei Zhang, Eleanor G. Pope, Boris Shor, L. Nathan Tumey, Javed Khan. A potent FGFR4-targeted antibody-drug conjugate therapy for patients with rhabdomyosarcoma and other cancers expressing FGFR4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB042.