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Research Articles: Therapeutics, Targets, and Development

Antitumor activity of fibroblast growth factor receptor 3–specific immunotoxins in a xenograft mouse model of bladder carcinoma is mediated by apoptosis

¹ Protein Technology, ² Comparative Pathology, and ³ Animal Facility Units, Biotechnology Program, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain and ⁴ Immunopharmacology and Targeted Therapy Laboratory, M. D. Anderson Cancer Center, Houston, Texas

Requests for reprints: J. Ignacio Casal, Protein Technology, Biotechnology Program, Centro Nacional de Investigaciones Oncológicas, Melchor Fernández Almagro 3, 28029 Madrid, Spain. Phone: 34-91-224-69-20; Fax: 34-91-224-69-72. E-mail: icasal{at}cnio.es

Abstract

Human single-chain Fv directed against fibroblast growth factor receptor 3 (FGFR3) have been shown to block proliferation of RT112 bladder carcinoma cells in vitro. Here, we examined the ability of the recombinant gelonin toxin (rGel) to enhance this inhibitory effect in vitro and in vivo on the bladder cancer cell line RT112 and the corresponding xenografts. Immunotoxins were genetically engineered by fusing FGFR3-specific Fv fragments (3C) to the NH₂ terminus of rGel and expressed as a soluble protein in Escherichia coli. The 3C/rGel fusion construct showed an IC₅₀ of 200 nmol/L against log-phase RT112 cells compared with 1,500 nmol/L for free rGel. Immunofluorescence studies showed that the 3C/rGel construct internalized rapidly into the cytoplasm of RT112 cells within 1 h of exposure. The mechanism of immunotoxin-induced cell death was found to be mediated by apoptosis. RT112 tumor xenografts in severe combined immunodeficient mice treated with 50 mg/kg 3C/rGel exhibited considerable growth delay relative to control tumors and a significant reduction of 55% to 70% in mean tumor size. Immunohistochemical analysis showed that tumors from mice treated with 3C/rGel displayed considerable apoptotic damage compared with control groups. Subcellular location of FGFR3 in immunotoxin-treated tumors indicated a translocation of FGFR3 to the nuclear membrane in contrast to tumors from saline-treated controls. These results show that FGFR3-driven immunotoxins may be an effective therapeutic agent against human bladder and other tumor types overexpressing FGFR3. [Mol Cancer Ther 2008;7(4):862–73]

Grant support: Spanish Ministry of Education and Science grant PTR1995-0849-OP, Comunidad Autónoma de Madrid grant S-BIO/0236/2006 and CDTI grant "CDTEAM."

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Research was conducted in part by the Clayton Foundation for Research.

Received 6/12/07; revised 1/17/08; accepted 2/22/08.