Antibody-based targeting of FGFR3 in bladder carcinoma and t(4;14)-positive multiple myeloma in mice
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
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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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 1

FGFR3 knockdown in RT112 bladder cancer cells inhibits proliferation and induces G1 cell-cycle arrest in vitro, and it suppresses tumor growth in vivo.

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FGFR3 knockdown in RT112 bladder cancer cells inhibits proliferation and...
Three different FGFR3 shRNAs were cloned into a Tet-inducible expression vector. RT112 cells stably expressing FGFR3 shRNAs or a control shRNA were established with puromycin selection. (A) Representative blots showing FGFR3 expression in selected clones treated with or without doxycycline (Dox; 0, 0.1, and 1 μg/ml, left to right). (B) [3H]thymidine incorporation by stable RT112 clones. Selected RT112 stable clones, namely clone 4 for FGFR3 shRNA2 (sh2-4), clone 1 for FGFR3 shRNA4 (sh4-1), and clone 16 for FGFR3 shRNA6 (sh6-16), were cultured with or without 1 μg/ml doxycycline for 3 days prior to 16-hour incubation with [3H]thymidine (1 μCi per well). Counts of incorporated [3H]thymidine (in cpm) were normalized to those from cells without doxycycline induction. Error bars represent SEM. (C) DNA fluorescence flow cytometry histograms of RT112 stable cells. RT112 clones expressing control shRNA or FGFR3 shRNA4 were cultured with or without 1 μg/ml doxycycline for 72 hours, and the nuclei were stained with propidium iodide (PI). Similar results were obtained for FGFR3 shRNA2 and -6 (Supplemental Figure 2). (D) The growth of RT112 cells expressing control shRNA (n = 9 per treatment group) or FGFR3 shRNA4 (n = 11 per treatment group) in mice. Mice were given 5% sucrose alone or supplemented with 1 mg/ml doxycycline, and tumor size was measured twice a week. Error bars represent SEM. Similar results were obtained for FGFR3 shRNA2 and -6 (Supplemental Figure 2). P < 0.0001. Lower panel: Expression of FGFR3 protein in tumor lysates extracted from control shRNA or FGFR3 shRNA4 stable cell xenograft tissues.