LAUSR

Translocations involving Fibroblast Growth Factor Receptor 2 (FGFR2) are found in up to 45% of Intrahepatic Cholangiocarcinoma (ICC). Treatment of patients bearing FGFR2 fusions with FGFR kinase inhibitors induces tumor regressions, though response rate and durability of response appear limited due to the emergence of resistance or to sub-optimal dosing which is limited by drug adverse effects. Thus, therapeutics that drive activity specifically towards FGFR2 are needed to enhance drug efficacy and reduce adverse effects. We first asked whether the extracellular domain (ECD) of FGFR2-BICC1 fusion is functional and important for receptor dimerization and signaling using NanoBiT system and western blotting. FGFR ECD consists of 3 Ig-fold domains D1, D2, and D3. Using focus transformation and viability assays, we found that deletions in D1, D2, D3, and D2-3 ECD FGFR2-BICC1 decreased growth and transformation of FGFR2 fusion driven cells. Next, we sought to investigate if antibodies against the ECD could perturb the growth of FGFR2 fusion transformed cells. We identified six FGFR2 specific antibodies that bind to epitopes in the ECD and confirmed the ligand blocking activity of these bivalent antibodies. In the absence of ligand, however, most of the bivalent antibodies were relatively inactive. As standard bivalent antibodies engage two receptors at a time, they are at risk for agonism or the maintenance of tonic signaling. Thus, to enhance efficacy and prevent the potential agonist or tonic activity of bivalent antibodies, all possible biparatopic antibodies (15), recognizing two distinct epitopes on the FGFR2 ECD, were engineered from the six parental antibodies. The biparatopic antibodies exhibited enhanced efficacies in FGFR2-fusion driven NIH3T3/BaF3 and ICC13-7 cancer cell lines and significantly improved binding affinities (~10-fold) as analyzed by Octet, MSD-SET, and SPR. Two biparatopic antibodies (BpAb-1, BpAb-2) showed the highest antiproliferative activity and induced apoptosis in FGFR2 fusion driven cells. BpAb-1, BpAb-2 were superior to their parental antibodies in reducing FGFR2-fusion driven growth, transformation and downstream signaling (pFGFR2, pFRS) in vitro and in reducing tumor growth in xenograft models. Importantly, BpAb-1 and BpAb-2 significantly induced FGFR2 internalization and lysosomal-mediated degradation as compared to their parental antibodies. Moreover, BpAb-1 and BpAb-2 had anti-growth activities against ICC patient-derived oncogenic FGFR2 ECD in-frame deletions, and FGFR kinase resistance mutations, including the gatekeeper mutation V565F. BpAb-1 and BpAb-2 were also active in FGFR2 WT amplified cells SNU-16. These results suggest that these FGFR2 biparatopic antibodies are a novel treatment option for ICC patients with tumors harboring FGFR2 fusions. Citation Format: Saireudee Chaturantabut, Sydney Oliver, John Kim, Dennie Frederick, Foxy Robinson, Alessandro Sinopoli, Diego J. Rodriguez, Liang Chang, Nabeel Bardeesy, William Sellers. Novel FGFR2 biparatopic antibodies for the treatment of cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2647.