Identification of potent biparatopic antibodies targeting FGFR2 fusion–driven cholangiocarcinoma
Saireudee Chaturantabut, Sydney Oliver, Dennie T. Frederick, Jiwan J. Kim, Foxy P. Robinson, Alessandro Sinopoli, Tian-Yu Song, Yao He, Yuan-Chen Chang, Diego J. Rodriguez, Liang Chang, Devishi Kesar, Meilani Ching, Ruvimbo Dzvurumi, Adel Atari, Yuen-Yi Tseng, Nabeel Bardeesy, William R. Sellers
Saireudee Chaturantabut, Sydney Oliver, Dennie T. Frederick, Jiwan J. Kim, Foxy P. Robinson, Alessandro Sinopoli, Tian-Yu Song, Yao He, Yuan-Chen Chang, Diego J. Rodriguez, Liang Chang, Devishi Kesar, Meilani Ching, Ruvimbo Dzvurumi, Adel Atari, Yuen-Yi Tseng, Nabeel Bardeesy, William R. Sellers
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Research Article Oncology

Identification of potent biparatopic antibodies targeting FGFR2 fusion–driven cholangiocarcinoma

Abstract

Translocations involving FGFR2 gene fusions are common in cholangiocarcinoma and predict response to FGFR kinase inhibitors. However, response rates and durability are limited due to the emergence of resistance, typically involving FGFR2 kinase domain mutations, and to suboptimal dosing, relating to drug adverse effects. Here, we develop biparatopic antibodies targeting the FGFR2 extracellular domain (ECD) as candidate therapeutics. Biparatopic antibodies can overcome drawbacks of bivalent monospecific antibodies, which often show poor inhibitory or even agonist activity against oncogenic receptors. We show that oncogenic transformation by FGFR2 fusions requires an intact ECD. Moreover, by systematically generating biparatopic antibodies targeting distinct epitope pairs in FGFR2 ECD, we identified antibodies that effectively block signaling and malignant growth driven by FGFR2 fusions. Importantly, these antibodies demonstrate efficacy in vivo, synergy with FGFR inhibitors, and activity against FGFR2 fusions harboring kinase domain mutations. Thus, we believe that biparatopic antibodies may serve as an innovative treatment option for patients with FGFR2-altered cholangiocarcinoma.

Authors

Saireudee Chaturantabut, Sydney Oliver, Dennie T. Frederick, Jiwan J. Kim, Foxy P. Robinson, Alessandro Sinopoli, Tian-Yu Song, Yao He, Yuan-Chen Chang, Diego J. Rodriguez, Liang Chang, Devishi Kesar, Meilani Ching, Ruvimbo Dzvurumi, Adel Atari, Yuen-Yi Tseng, Nabeel Bardeesy, William R. Sellers

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Figure 1

The extracellular domain is necessary for full transformation by FGFR2 fusions.

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The extracellular domain is necessary for full transformation by FGFR2 f...
(A) Transformation assays showing cumulative population doublings in BaF3 cells expressing FGFR2-PHGDH (12 days) and FGFR2-AHCYL1 (15 days) with or without FGF10 (100 ng/mL) or IL-3 (10 ng/mL), as indicated (n = 3). (B) Growth of BaF3 cells expressing FGFR2-PHGDH and FGFR2-AHCYL1 analyzed by CellTiter-Glo at 5 days after IL-3 removal (n = 5). (C) Illustration of the dimerization assay using FGFR2-fusion NanoBiT constructs. Large BiT and Small BiT subunits are fused to the C-terminus of FGFR2 fusions. SP, signal peptide; TM, transmembrane; KD, kinase domain; FP, fusion partner;PM, plasma membrane. (D) HEK-293T cells expressing FGFR2-WT and FGFR2-AHCYL1 fused to LgBiT alone or fused to LgBiT and SmBiT were used to quantify the receptor dimerization in the presence or absence of FGF10. Shown is the fold increase over FGFR2-LgBiT activity alone (n = 5). (E) Illustration of FGFR2-BICC1 constructs with D1 (Ig1), D2 (Ig2), D3 (Ig3), or D2+D3 (Ig2+Ig3) deletions in the ECD. (F) Representative images of focus formation assays of NIH-3T3 cells expressing FGFR2 WT or the indicated ECD deletion variants. Scale bar: 250 μm. (G) Quantification of number of colonies from F (n = 6). (H) Growth of NIH3T3 cells overexpressing FL, D1, D2, D3, and D2+3–deleted FGFR2-BICC1 constructs as measured by Incucyte at 5 days after plating (n = 5). (I) Dimerization of FGFR2-BICC1 D1, D2, D3, or D2+D3 ECD–deleted constructs in HEK-293T cells compared with full-length FGFR2-BICC1. Fold change in luminescence over FGFR2-WT–LgBiT is shown (n = 5). (J) Immunoblotting of FGFR2 downstream pathway effectors in HEK-293 cells expressing FGFR2-BICC1 ECD deletion constructs. All data are mean ± SEM. Data are representative of 1 out of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 1-way ANOVA multiple comparisons.