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Targeting the Extracellular Domain of Fibroblast Growth Factor Receptor 3 with Human Single-Chain Fv Antibodies Inhibits Bladder Carcinoma Cell Line Proliferation

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Targeting the Extracellular Domain of Fibroblast Growth Factor Receptor 3 with Human Single-Chain Fv Antibodies Inhibits Bladder Carcinoma Cell Line Proliferation Cancer Therapy: Preclinical TargetingtheExtracellularDomainofFibroblastGrowthFactor Receptor 3 with Human Single-Chain Fv Antibodies Inhibits Bladder Carcinoma Cell Line Proliferation 1 1 1 2 Jorge Martlı´nez-Torrecuadrada,¤ Gabriela Cifuentes, Paula Lo´o¤ pez-Serra, Pilar Saenz, 2 1 Antonio Martlnez,¤ı´ and J. Ignacio Casal Abstract Purpose: Previous gene expression studies have shown that fibroblast growth factor receptor 3 (FGFR3) is overexpressed in early stages of bladder cancer. To study the potential use of thera- peutic antibodies against FGFR3, we have produced a collection of human single-chain Fv (scFv) antibody fragments by using phage display libraries. Experimental Design:Two ‘‘naı¨l« ve’’semi-synthetic human scFv libraries were used to select anti- bodies against the extracellular domain of FGFR3a(IIIc).The reactivity of the selected scFvs with a recombinant FGFR3 was characterized by an enzyme immunoassay and surface plasmon reso- nance analysis and with RT112bladder carcinoma cells by a fluorescence-activated cell sorter. The capacity of the selected scFvs to block RT112cell proliferation was determined. Results: We have isolated six human scFv antibody fragments directed against FGFR3. These human scFvs specifically bound FGFR3, but not the homologous molecule FGFR1.Biacore anal- ysis was used to determine the affinity constants, which ranged from 12to 40 nmol/L. Competi- tion analysis showed that the FGF9ligand was able to block the binding of two scFvs, 3C and 7D, to FGFR3, whereas FGF1only blocked 7D. Immunoprecipitation and flow cytometric analysis confirmed the specificity of the antibodies to native membrane FGFR3. Two scFvs, 3C and 7D, gave an strong immunofluorescence staining of RT112cells. Moreover, they recognized equally well wild-type and mutant FGFR3 containing the activating mutation S249C. Furthermore, they blocked proliferation of RT112cells in a dose- and FGF-dependent manner. Conclusion: Our results suggest that these human anti-FGFR3 scFv antibodies may have poten- tial applications as antitumoral agents in bladder cancer. The fibroblast growth factors (FGF) represent one of the largest homology at the protein level. The FGFR structure consists of an families of polypeptide growth and differentiation factors for extracellular ligand-binding domain, a transmembrane domain, mesodermal and epithelial cells (1). FGFs have been implicated in and an intracellular kinase domain. The ligand-binding domain multiple biological processes during embryo development, wound contains three different immunoglobulin-like domains (called healing, hematopoiesis, and angiogenesis. In addition, FGFs have immunoglobulin I, immunoglobulin II, and immunoglobulin been shown to increase the invasiveness of a variety of tumors III). The ligand domain is followed by a single transmembrane from prostate, bladder, kidney, breast, and pancreas (2–5). domain and a cytoplasmic split tyrosine kinase domain in the Although more than 20 FGFs with different effects on various intracellular domain. Alternative splicing of the mRNAs cells have been reported, only five FGF receptors (FGFR) have corresponding to FGFR1-3 gives place to two isoforms, a and been described (6, 7). These receptors share 55% to 72% h. Only isoform a, which contains the three immunoglobulin domains, has been found for FGFR3. Alternative splicing of FGFR3 transcripts involving the COOH-terminal domain of the immunoglobulin III domain yields two isoforms, IIIb and IIIc. 1 Authors’ Affiliations: ProteinTechnology Unit, Biotechnology Program, Centro The two variants show different binding affinities: the IIIc variant Nacional de Investigaciones Oncolo´o¤ gicas, Madrid, Spain and 2Progenika, Zamudio, Bilbao, Spain is more promiscuous and binds a wide range of FGFs, including Received 2/7/05; revised 6/6/05; accepted 6/10/05. FGF1, FGF2, FGF4, and FGF9; on the other hand, the IIIb variant Grant support: Spanish Ministry of Science and Technology grants BIO02003- preferentially binds FGF1 and, at a lower extent, FGF8 and FGF9 01481, FIT-010000-2003-86, and FIT-010000-2004-65. The Centro Nacional (8). After binding to FGFRs in the presence of heparin, FGFs de Investigaciones Oncolo´o¤ gicas is partially supported by the Red Temo´a¤ tica de induce receptor dimerization, resulting in autophosphorylation Investigacio´o¤ n Cooperativa de Centros de Co´a¤ ncer (FIS C03/10). The costs of publication of this article were defrayed in part by the payment of page of the intracellular kinase domain and a downstream activation charges. This article must therefore be hereby marked advertisement in accordance of intracellular signaling cascades (9). Following activation of with 18 U.S.C. Section 1734 solely to indicate this fact. the ligand receptor complex, various signal transduction path- Requests for reprints: Ignacio Casal, Biotechnology Program, Centro Nacional ways are initiated by FGFs: elevation of intracellular calcium de Investigaciones Oncoloo´¤ gicas, Melchor Ferno´a¤ ndez Almagro 3, 28029 Madrid, Spain. Phone: 34-91-224-69-20; Fax: 34-91-224-69-72; E-mail: i [email protected]. levels, induction of mitogen-activated protein kinase and protein F 2005 American Association for Cancer Research. kinase C pathways, stimulation of adenylate cyclase, and doi:10.1158/1078-0432.CCR-05-0282 induction of the proto-oncogenes c-myc and c-fos (6). Clin Cancer Res 2005;11(17) September 1, 2005 6280 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. FGFR3 Blocking scFvs Specific mutations have been found in FGFR3 that lead to the isoforms IIIc of the extracellular domains of FGFR3a or FGFR1a, activation of its tyrosine kinase activities and to different respectively, fused to the Fc region of human IgG1. FGF9, FGF1, and syndromes related with bone development, multiple myeloma epidermal growth factor (EGF) were provided by ReliaTech. (10, 11), cervical carcinomas (12, 13), and bladder carcinomas (12, Mouse anti-FGFR3 monoclonal antibody (mAb) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-human IgG (Fc (12, 14–17). There are recent observations that FGFR signaling specific), mouse anti–c-myc mAb (clone 9E10), and antitubulin were is absolutely necessary for the survival of prostate cancer cells from Sigma. Horseradish peroxidase–conjugated anti–c-myc (clone in vitro (18). Recently, FGFR3 has been proposed as a possible 9E10) was purchased from Roche (Mannheim, Germany). Anti-6xHis therapeutic target for multiple myeloma (10, 19). Although the was provided by NeoMarkers (Westinghouse Dr, Fremont, CA). Anti- presence of activating mutations is well documented, little was M13 and horseradish peroxidase–conjugated anti-M13 mAbs were known about the expression of FGFR3 in tumoral tissues. obtained from GE Healthcare. FITC-conjugated rabbit anti-mouse IgG Recently, after gene expression analysis by using gene chip was from DAKO (Glostrup, Denmark). R-phycoerythrin goat anti- technology, it was found that FGFR3 was overexpressed in mouse IgG was purchased from Molecular Probes (Eugene, OR). Mouse IgG TrueBlot was provided by eBioscience (San Diego, CA). tumoral samples corresponding to stages pTa,pT1 (fold change FGFR3 cDNA cloning and cell transfection. For cellular expression of >8), and pT2 (fold change >2) of bladder carcinoma when compared with the normal counterparts (20). The gene recombinant FGFR3s, the extracellular domain of the protein was fused to the COOH-terminal Fc region of human IgG1. Vector pcDNA3.1-Fc, expression levels were confirmed at the protein level by which contains the human Fc region, was used to clone the cDNA Western blot and immunohistochemical analysis. In fact, this fragment encoding the extracellular domain of FGFR3-IIIc (amino acids protein overproduction seems to occur more frequently in 1-370) to generate pcDNA3.1-FGFR3(IIIc)WT-Fc. This plasmid was used transitional carcinomas than activating mutations (20). All as template to prepare the mutant FGFR3 S249C by site-directed muta- these data together suggest that FGFR3 might constitute an genesis using the primer 5V-CCCGCCTGCAGGATGGGCCGGTGCGGGC- attractive target for therapy in urologic tumors. Bladder cancer AGCGC-3V. The resulting plasmid, pcDNA3.1-FGFR3(IIIc)S249C-Fc, is the second most common malignancy of the genitourinary was sequenced to confirm the presence of the S249C mutation. tract. About 40% to 50% of the bladder carcinoma showed pcDNA3.1-FGFR3(IIIc)WT-Fc and pcDNA3.1-FGFR3(IIIc)S249C-Fc FGFR3-activating gene mutations (17, 21); the incidence was were transfected into HEK293T cells with FuGENE 6 (Roche) and the significantly bigger (80%) in superficial tumors than in invasive expression and reactivity of the wild-type and mutant proteins were analyzed 2 days after transfection by immunoblotting with anti-FGFR3 tumors. mAb and by FACS with the specific scFvs as described below. With the increasing interest in FGFR3 as a therapeutic target Selection of fibroblast growth factor receptor 3–specific single-chain Fv in different neoplasias and the recent findings about its antibodies from phagemid libraries. The human scFv libraries Tomlin- overexpression in transitional cell carcinoma, we have started son I + J, helper phage KM13, and Escherichia coli TG1 and HB2151 were the development of human antibodies for therapeutic purposes obtained from MRC Geneservices. Both libraries were grown separately by phage display. Phage antibody display is one of the best and the phages mixed
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