Antitumor activity of fibroblast growth factor receptor 3-specific immunotoxins in a xenograft mouse model of bladder carcinoma is mediated by apoptosis.
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 NH(2) terminus of rGel and expressed as a soluble protein in Escherichia coli. The 3C/rGel fusion construct showed an IC(50) 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.
Martínez-Torrecuadrada
Jorge L
JL
Protein Technology, Biotechnology Program, Centro Nacional de Investigaciones Oncológicas, Melchor Fernández Almagro 3, 28029 Madrid, Spain.
Cheung
Lawrence H
LH
López-Serra
Paula
P
Barderas
Rodrigo
R
Cañamero
Marta
M
Ferreiro
Sergio
S
Rosenblum
Michael G
MG
Casal
J Ignacio
JI
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects*; D002295:Carcinoma, Transitional Cell / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D051379:Mice; D008819:Mice, Nude; D016513:Mice, SCID; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000494:pharmacology; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000627:therapeutic use; D054789:Ribosome Inactivating Proteins, Type 1 / Q000627:therapeutic use*; D020349:Surface Plasmon Resonance; D015996:Survival Rate; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D023041:Xenograft Model Antitumor Assays
D000972:Antineoplastic Agents, Phytogenic; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C025674:GEL protein, Gelonium multiflorum; D051498:Receptor, Fibroblast Growth Factor, Type 3