Antibody-based targeting of FGFR3 in bladder carcinoma and t(4;14)-positive multiple myeloma in mice
Jing Qing, … , Christian Wiesmann, Avi Ashkenazi
Jing Qing, … , Christian Wiesmann, Avi Ashkenazi
Published April 20, 2009
Citation Information: J Clin Invest. 2009;119(5):1216-1229. https://doi.org/10.1172/JCI38017.
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Research Article

Antibody-based targeting of FGFR3 in bladder carcinoma and t(4;14)-positive multiple myeloma in mice

Abstract

Overexpression of FGF receptor 3 (FGFR3) is implicated in the development of t(4;14)-positive multiple myeloma. While FGFR3 is frequently overexpressed and/or activated through mutations in bladder cancer, the functional importance of FGFR3 and its potential as a specific therapeutic target in this disease have not been elucidated in vivo. Here we report that inducible knockdown of FGFR3 in human bladder carcinoma cells arrested cell-cycle progression in culture and markedly attenuated tumor progression in xenografted mice. Further, we developed a unique antibody (R3Mab) that inhibited not only WT FGFR3, but also various mutants of the receptor, including disulfide-linked cysteine mutants. Biochemical analysis and 2.1-Å resolution crystallography revealed that R3Mab bound to a specific FGFR3 epitope that simultaneously blocked ligand binding, prevented receptor dimerization, and induced substantial conformational changes in the receptor. R3Mab exerted potent antitumor activity against bladder carcinoma and t(4;14)-positive multiple myeloma xenografts in mice by antagonizing FGFR3 signaling and eliciting antibody-dependent cell-mediated cytotoxicity (ADCC). These studies provide in vivo evidence demonstrating an oncogenic role of FGFR3 in bladder cancer and support antibody-based targeting of FGFR3 in hematologic and epithelial cancers driven by WT or mutant FGFR3.

Authors

Jing Qing, Xiangnan Du, Yongmei Chen, Pamela Chan, Hao Li, Ping Wu, Scot Marsters, Scott Stawicki, Janet Tien, Klara Totpal, Sarajane Ross, Susanna Stinson, David Dornan, Dorothy French, Qian-Rena Wang, Jean-Philippe Stephan, Yan Wu, Christian Wiesmann, Avi Ashkenazi

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Figure 3

R3Mab inhibits Ba/F3 cell proliferation driven by WT and mutated FGFR3.

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R3Mab inhibits Ba/F3 cell proliferation driven by WT and mutated FGFR3. ...
(A) Inhibitory effect of R3Mab on the viability of Ba/F3 cells expressing WT human FGFR3-IIIb. Cells were cultured in medium without FGF1 (No FGF1), in the presence of 10 ng/ml FGF1 plus 10 μg/ml heparin alone (FGF1), or in combination with a control antibody (Ctrl) or R3Mab. Cell viability was assessed with CellTiter-Glo (Promega) after 72-hour incubation with antibodies. (B) Inhibition of FGFR3 and MAPK phosphorylation by R3Mab in Ba/F3–FGFR3-IIIbWT stable cells. Cells were treated with 15 ng/ml FGF1 and 10 μg/ml heparin (+) or heparin alone (–) for 10 minutes, following preincubation with a control Ab, decreasing amounts of R3Mab (1, 0.2, and 0.04 μg/ml) in PBS, or PBS alone (Mock) for 3 hours. Lysates were immunoblotted to assess phosphorylation of FGFR3 and p44/42 MAPK with antibodies against pFGFRY653/654 and p-MAPKThr202/Tyr204, respectively. (C) Schematic representation of FGFR3 mutation hot spots and frequency in bladder cancer based on published data (32). TM, transmembrane domain; TK1 and TK2, tyrosine kinase domains 1 and 2. (D–H) Inhibitory effect of R3Mab on the viability of Ba/F3 cells expressing cancer-associated FGFR3 mutants. G372C is derived from the IIIc isoform, and the other mutants are derived from the IIIb isoform. Cell viability was assessed after 72-hour incubation with antibodies as described in A. Error bars represent SEM.