Abstract 526: Alternative format bispecific antibodies targeting multiple oncogenic FGFR3 mutations in bladder cancer through different mechanisms

FGFR3 is a clinically validated driver for muscle invasive bladder cancer (MIBC). 15-20% of MIBC patients express FGFR3 mutations, including point mutations in the extracellular domain (S249C, R248C, G370C, Y373C) and kinase domain (K650E) as well as in-frame fusions with dimerization domains (e.g., coiled-coil domain of TACC3). Generating an antibody-based therapeutic that potently inhibits multiple FGFR3 oncogenic mutants, with different activation mechanisms, remains as a challenge. Therefore, regular IgG-like and alternative format (AF) bispecific antibodies were explored to examine whether enhanced FGFR3 degradation can serve as a “pan-inhibition” solution for tackling multiple FGFR3 genetic aberrations. APLP2 has been identified as a cell surface receptor that undergoes rapid internalization and degradation. Cell surface CD63 plays a more complex role in intracellular trafficking by itself or with its associated surface protein, including both endocytosis and surface retention. Bispecific antibodies co-targeting APLP2 and HER2 have been shown to enhance HER2 internalization and lysosomal trafficking (Bay AP et al 2019). IgG-like and AF bispecific antibodies targeting FGFR3xAPLP2 or FGFR3xCD63 with varied valency and geometry were engineered, produced and characterized. AF bispecific antibodies showed significant enhancement (compared to parental antibody) of in vitro growth inhibition in bladder cancer cell lines expressing FGFR3 S249C mutation while maintaining potent inhibitory activity in cells expressing FGFR3-TACC3 mutant. Both FGFR3 and APLP2/CD63 binding sites contribute to enhanced bispecific antibody potency. Interestingly, different activities were observed in antibody-induced receptor degradation indicating that APLP2 and CD63 regulate FGFR3 trafficking via distinct mechanisms. FGFR3xAPLP2 AF bispecifics enhanced receptor degradation compared to parental FGFR3 antibody, while FGFR3xCD63 AF bispecifics increased growth inhibition without receptor degradation. Our proof-of-concept case study suggests that AF bispecific antibodies can be used as a single novel modality to offer more potent inhibition against diverse mechanisms of oncogenic receptor activation. Citation Format: Yan Yang, Arif Wibowo, Suhasini Avvaru, Zaoli Jiang, Antonio Luz, Ashique Rafique, Prasad Sarangapani, Ryan McKay, Michael Amatulli, Meghana Vootlakrishna, Rachel Edwards, Terra Potocky, Aynur Hermann, Christopher Daly, Gavin Thurston, John Chia-Yang Lin, Yang Shen. Alt