Identification of potent biparatopic antibodies targeting FGFR2 fusion–driven cholangiocarcinoma
Saireudee Chaturantabut, … , Nabeel Bardeesy, William R. Sellers
Saireudee Chaturantabut, … , Nabeel Bardeesy, William R. Sellers
Published February 27, 2025
Citation Information: J Clin Invest. 2025;135(8):e182417. https://doi.org/10.1172/JCI182417.
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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 6

The biparatopic antibodies promote receptor internalization and lysosomal degradation.

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The biparatopic antibodies promote receptor internalization and lysosoma...
(A) Flow cytometry histograms of surface FGFR2-PHGDH in BaF3 cells at 4°C (blue) and 37°C (red) upon treatment with bpAb-B/C or bpAb-B/D from 60–960 minutes. (B) Quantification of the histograms demonstrating the percentage of internalized FGFR2 at 60, 120, 180, 240, and 960 minutes after bpAb-B/C or bpAb-B/D incubation. (C) Quantification of histograms showing percent internalized FGFR2 in ICC13-7 cell line at 4°C and 37°C after 5 hours of treatment with parental antibody B, D, C or biparatopic antibodies bpAb-B/C or bpAb-B/D (n = 3). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 1-way ANOVA multiple comparisons. Data are representative of 1 out of 2 independent experiments. (D) Illustrations of Fabfluor-pH antibody labeling assay. The pH sensitive dye-based system exploits the acidic environment of the lysosomes to quantify internalization of the labeled antibody. Fluorescent signals that indicate the internalization/degradation events were tracked using Incucyte. (E) Representative images of detected fluorophore in NIH3T3 cells expressing FGFR2-PHGDH treated with parental antibody B, D, C, or biparatopic antibody bpAb-B/C and bpAb-B/D at 15 hours after incubation. Scale bars: 300 μm. (F–H) Quantification of internalization/degradation signals in FGFR2-AHCYL1 (F), FGFR2-BICC1 (G), and FGFR2-PHGDH (H) expressing NIH3T3 cells treated with parental antibodies B, D, C, or biparatopic antibody bpAb-B/C and bpAb-B/D from 24 hours after incubation. Data are representative of 1 out of 2 independent experiments. (I) Quantification of internalization/degradation signals in ICC13-7 cells treated with parental antibodies B, D, C, or biparatopic antibody bpAb-B/C and bpAb-B/D at 4 hours after incubation. Data are representative of 1 out of 2 independent experiments. (J) Immunoblot of ICC13-7 cells treated with IgG1, bpAb-B/C,or bpAb-B/D antibodies alone or cotreated with bafilomycin A1 (BafA1) for 24 hours. BafA1 was preincubated for 1 hour prior to antibody treatments. Data are representative of 1 independent experiment.