Targeting the Extracellular Domain of Fibroblast ...

Cancer Therapy: Preclinical

Targeting the Extracellular Domain of Fibroblast Growth Factor Receptor 3 with Human Single-Chain Fv Antibodies Inhibits Bladder Carcinoma Cell Line Proliferation Jorge Martı ln ¤ ´ ez-Torrecuadrada,1 Gabriela Cifuentes,1 Paula Loo´¤ pez-Serra,1 Pilar Saenz,2 2 ¤ı Antonio Martlnez, ´ and J. Ignacio Casal1

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 therapeutic 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 ¨ e’’semi-synthetic human scFv libraries were used to select antil« v 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 resonance analysis and with RT112 bladder carcinoma cells by a fluorescence-activated cell sorter. The capacity of the selected scFvs to block RT112 cell 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 analysis was used to determine the affinity constants, which ranged from 12 to 40 nmol/L. Competition analysis showed that the FGF9 ligand was able to block the binding of two scFvs, 3C and 7D, to FGFR3, whereas FGF1 only 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 RT112 cells. Moreover, they recognized equally well wild-type and mutant FGFR3 containing the activating mutation S249C. Furthermore, they blocked proliferation of RT112 cells in a dose- and FGF-dependent manner. Conclusion: Our results suggest that these human anti-FGFR3 scFv antibodies may have potential applications as antitumoral agents in bladder cancer.

The fibroblast growth factors (FGF) represent one of the largest families of polypeptide growth and differentiation factors for mesodermal and epithelial cells (1). FGFs have been implicated in multiple biological processes during embryo development, wound healing, hematopoiesis, and angiogenesis. In addition, FGFs have been shown to increase the invasiveness of a variety of tumors from prostate, bladder, kidney, breast, and pancreas (2–5). Although more than 20 FGFs with different effects on various cells have been reported, only five FGF receptors (FGFR) have been described (6, 7). These receptors share 55% to 72% Authors’ Affiliations: 1Protein Technology Unit, Biotechnology Program, Centro Nacional de Investigaciones Oncolo o´¤ gicas, Madrid, Spain and 2Progenika, Zamudio, Bilbao, Spain Received 2/7/05; revised 6/6/05; accepted 6/10/05. Grant support: Spanish Ministry of Science and Technology grants BIO0200301481, FIT-010000-2003-86, and FIT-010000-2004-65. The Centro Nacional de Investigaciones Oncolo o´¤ gicas is partially supported by the Red Temo a´¤ tica de 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 charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Ignacio Casal, Biotechnology Program, Centro Nacional de Investigaciones Oncolo o´¤ gicas, Melchor Ferno a´¤ ndez Almagro 3, 28029 Madrid, Spain. Phone: 34-91-224-69-20; Fax: 34-91-224-69-72; E-mail: icasal@ cnio.es. F 2005 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-0282

Clin Cancer Res 2005;11(17) September 1, 2005

homology at the protein level. The FGFR structure consists of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular kinase domain. The ligand-binding domain contains three different immunoglobulin-like domains (called immunoglobulin I, immunoglobulin II, and immunoglobulin III). The ligand domain is followed by a single transmembrane domain and a cytoplasmic split tyrosine kinase domain in the intracellular domain. Alternative splicing of the mRNAs corresponding to FGFR1-3 gives place to two isoforms, a and 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. The two variants show different binding affinities: the IIIc variant is more promiscuous and binds a wide range of FGFs, including FGF1, FGF2, FGF4, and FGF9; on the other hand, the IIIb variant preferentially binds FGF1 and, at a lower extent, FGF8 and FGF9 (8). After binding to FGFRs in the presence of heparin, FGFs induce receptor dimerization, resulting in autophosphorylation of the intracellular kinase domain and a downstream activation of intracellular signaling cascades (9). Following activation of the ligand receptor complex, various signal transduction pathways are initiated by FGFs: elevation of intracellular calcium levels, induction of mitogen-activated protein kinase and protein kinase C pathways, stimulation of adenylate cyclase, and induction of the proto-oncogenes c-myc and c-fos (6).

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FGFR3 Blocking scFvs

isoforms IIIc of the extracellular domains of FGFR3a or FGFR1a, Specific mutations have been found in FGFR3 that lead to the respectively, fused to the Fc region of human IgG1. FGF9, FGF1, and activation of its tyrosine kinase activities and to different epidermal growth factor (EGF) were provided by ReliaTech. syndromes related with bone development, multiple myeloma (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). Antipresence 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 was from DAKO (Glostrup, Denmark). R-phycoerythrin goat antiRecently, after gene expression analysis by using gene chip mouse IgG was purchased from Molecular Probes (Eugene, OR). Mouse technology, it was found that FGFR3 was overexpressed in 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 recombinant FGFR3s, the extracellular domain of the protein was fused to compared with the normal counterparts (20). The gene 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 mutathese data together suggest that FGFR3 might constitute an genesis using the primer 5V-CCCGCCTGCAGGATGGGCCGGTGCGGGCAGCGC-3V. The resulting plasmid, pcDNA3.1-FGFR3(IIIc)S249C-Fc, attractive target for therapy in urologic tumors. Bladder cancer was sequenced to confirm the presence of the S249C mutation. is the second most common malignancy of the genitourinary pcDNA3.1-FGFR3(IIIc)WT-Fc and pcDNA3.1-FGFR3(IIIc)S249C-Fc tract. About 40% to 50% of the bladder carcinoma showed were transfected into HEK293T cells with FuGENE 6 (Roche) and the FGFR3-activating gene mutations (17, 21); the incidence was expression and reactivity of the wild-type and mutant proteins were significantly bigger (80%) in superficial tumors than in invasive 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 Tomlinoverexpression 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 and the phages mixed 1:1 for selection. Phage selection and scFv by phage display. Phage antibody display is one of the best production were done on microtiter plates (Maxisorb, Nunc, Rockilde, alternatives for the production of human antibodies for Denmark) according to the scheme shown in Fig. 1. In the first round of research, clinical, and therapeutic applications (for reviews on selection, wells of the microplates were coated with 1 Ag of FGFR3 or this topic, see refs. 22 – 24). However, FGFR3 is a very elusive human IgG in PBS at 4jC overnight. Next, wells were washed with PBS molecule for antibody production given the high homology and blocked with 2% MPBS (skimmed milk in PBS) for 2 hours. Then, between FGFR3 from mouse and human species (92%). Only 1011 phages in 2% MPBS were incubated first for 1 hour with human IgG recently, and by using a very large commercial Fab library with to remove the reactivity against the Fc fragment of the recombinant a size of 2.1 1010, it has been described the production of Fab antigen, and then for 2 hours with FGFR3-coated wells. Plates were fragments against a nondisclosed FGFR3 isoform (25). In our washed 10 times with PBS-0.1% Tween 20 (20 times for the subsequent experiments, we have used two available public libraries of rounds) and treated with 100 AL of trypsin (Sigma) for elution of the bound phages. Elution and production of phages for other rounds of single-chain Fv (scFv) antibodies, Tomlinson I + J (MRC panning was carried out essentially as described by Goletz et al. (26). Geneservices, Cambridge, United Kingdom), which have been In other panning experiments and for a major Fc blocking efficiency, previously described (26). The size of both libraries is around 8 phage selection was carried out by mixing first the phage suspension 1.4 10 . scFvs penetrate tumors better than immunoglobulin with 50 Ag of human IgG in 2% MPBS. After 1-hour incubation at Gs (IgG) and Fabs, are cleared more rapidly, and have greater 37jC, this mixture was directly added to FGFR3-coated wells for specificity. In this report, we have developed a collection of 2 hours at room temperature. human scFv antibodies specific for FGFR3a isoform IIIc. These Phage ELISA. Flexible microtiter plates (Falcon, BD Biosciences, antibodies reacted with the bladder carcinoma cell line RT112 Franklin Lakes, NJ) were coated overnight with 0.3 Ag of FGFR3, FGFR1, by fluorescence-activated cell sorting (FACS) analysis and or human IgG in PBS. After washing thrice with PBS, plates were blocked inhibited cellular proliferation, showing promise for further with 2% MPBS for 2 hours at room temperature. Following additional therapeutic application. washing, different dilutions of the purified phage suspension coming

Materials and Methods Cell lines, proteins, and antibodies. RT112 cells were obtained from the German collection of Microorganisms and Cell Cultures (DSMZ; Braunschweig, Germany). Human embryonic kidney (HEK293T) and RT112 cells were cultured in RPMI 1640 (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum and antibiotics. The recombinant human FGFR3a(IIIc)/Fc and FGFR1a(IIIc)/Fc chimera were purchased from R&D Systems (Minneapolis, MN) and ReliaTech (Braunschweig, Germany). These proteins consist of the


from the selection steps, in the presence of 2% MPBS, were incubated for 1 hour at room temperature. After washing, a 1:5,000 dilution of antiM13 labeled with peroxidase in 2% MPBS was added for 1 hour and the color was developed with 3,3V,5,5V-tetramethylbenzidine substrate (Sigma). The reaction was stopped after 10 minutes by adding 1 mol/L sulfuric acid and the absorbance was read at 450 nm. Monoclonal phage ELISA. Usually groups of 96 colonies from the second or third round of selection were picked either manually or by using an automatic picker (Q-pix, Genetix, Hampshire, United Kingdom) and grown in 100 AL of 2 TY medium containing 100 Ag/mL ampicillin and 1% glucose in a 96-well plate (Sarstedt,

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Fig. 1. Flowchart to obtain FGFR3-specific scFvs by using phage display antibody libraries.

Numbrecht, Germany) at 37jC. The next day, cultures were diluted 1:100 in the same medium and shaken for 2 hours at 37jC. Then, 25 AL of 2 TY medium containing 109 KM13 helper phages were added and the culture continued for 1 hour. Bacterial cells were pelleted, resuspended in 2 TY with 100 Ag ampicillin and 50 Ag/mL kanamycin, and grown overnight at 30jC. Finally, the culture was centrifuged and 50 AL of the supernatant were used for the monoclonal phage ELISA in the same conditions detailed above. Sequence analysis. Phagemid DNAs from individual colonies were amplified by PCR with the primers LMB3 5V-CAGGAAACAGCTATGAC3V and pHENseq 5V-CTATGCGGCCCCATTCA-3V. PCR products were purified with the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and directly sequenced by using fluorescent dideoxy chain terminators and by automated sequencing (ABI7002, Applied Biosystems, Foster City, CA). Production and purification of soluble single-chain Fv antibody and ELISA. The procedure for E. coli induction was identical to that described above except that at the exponential phase (A 600 = 0.9), E. coli HB2151 cells were induced with 2 TY containing 100 Ag/mL ampicillin and 1 mmol/L isopropyl-L-thio-h-D-galactopyranoside and incubated at 30jC overnight. For the preparation of periplasmic extracts, bacteria were harvested and resuspended in 1:50 of the culture volume of ice-cold TES buffer [0.5 mol/L sucrose in 200 mmol/L TrisHCl (pH 8.0), 0.5 mmol/L EDTA], containing 20 Ag/mL benzamidine (Sigma) and 10 Ag/mL soybean trypsin inhibitor (Sigma), and rapidly diluted in 0.2 TES. Cells were incubated on ice for 30 minutes and centrifuged at 16,250 g for 10 minutes at 4jC. The supernatant containing the periplasmic fraction was dialyzed overnight at 4jC against 50 mmol/L Tris-HCl (pH 7.0) and 300 mmol/L NaCl and subjected to affinity chromatography using TALON resin (BD Biosciences) following the instructions of the manufacturer. Fractions were analyzed by SDS-PAGE and Coomassie blue staining. For further


purification and to separate monomers from dimers and larger multimers, scFv-containing fractions obtained from immobilized metal affinity chromatography were pooled, dialyzed against PBS, applied to a HiLoad 26/60 Superdex 75 pg column equilibrated with the same buffer, and run in an A¨KTA-FPLC (GE Healthcare) at a flow rate of 0.75 mL/min. Fractions were analyzed by SDS-PAGE and those containing the monomeric form were pooled and concentrated using Vivaspin concentrators (Vivascience, Hannover, Germany). Immunoprecipitation-Western blot analysis. About 2 108 RT112 cells for each experiment were washed with ice-cold PBS and scraped in ice-cold lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 300 mmol/L NaCl, 5 mmol/L EDTA, 1% Triton X-100] supplemented with 1 protease inhibitor cocktail (Roche). The lysates were incubated for 30 minutes on ice and centrifuged at 16,000 g for 15 minutes at 4jC. In parallel, a precipitation complex of antibodies was made by incubating 300 AL of protein G-Sepharose (GE Healthcare) with 1 AL of anti – c-myc (Sigma) for 90 minutes at 4jC. After three washes with lysis buffer, 50 Ag of the purified scFvs were added and left to bind for 2 hours at 4jC followed by three washes with lysis buffer. For immunoprecipitation, the cell lysates were added to the preformed scFv precipitation complex and were incubated overnight at 4jC, pelleted by centrifugation, and washed thrice with lysis buffer. Proteins were released with 0.1 mol/L glycine-HCl (pH 2.7), precipitated with trichloroacetic acid (Sigma), and loaded onto 7.5 % SDS-PAGE for electrophoresis. They were then transferred to Hybond-C membrane (GE Healthcare) and incubated with anti-FGFR3 mAb overnight at 4jC. The filter was washed four times and bound antibodies were detected with Mouse IgG TrueBlot and SuperSignal Substrate (Pierce, Rockford, IL). Surface plasmon resonance analyses. The binding kinetics of soluble scFvs to FGFR3 was measured at 25jC on a Biacore X (Biacore, Uppsala, Sweden). For immobilization of the FGFR3-Fc, goat anti-human IgG (Fc fragment) antibodies (Sigma) were immobilized on CM5 chips

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Table 1. Effect of the blocking procedures in the phage selection process Selection

Panning

Picking

Tested clones

Positive, soluble scFv

Unique sequences

Selected clones

A1 A2 B Total

Third Second Third

Manual Picker Picker

96 96 96 288

5 3 7 15

2 1 3 6

3A, 3B 3C 1D, 2D, 7D

(Biacore) by using the amino coupling kit (Biacore). For each experiment, 1 Ag of purified FGFR3-Fc was first captured and then the different purified scFvs were added at various concentrations ranging from 500 to 10 nmol/L. After each experiment, the surface was regenerated with 0.1 mol/L glycine-HCl (pH 2.5). Sensorgrams were obtained at each concentration and evaluated using the BIA Evaluation 3.0 program to determine the rate constants K on and K off to calculate the K d. Experiments were made in duplicate. To determine the binding sites of the different scFvs, we carried out a competitive binding assay; 0.5 Ag FGFR3-Fc was immobilized and then a 1 Amol/L solution of each scFv was injected followed by the injection of the same amount of the competitor antibody. If there was an increase in the resonance units of the sensorgram, then the binding sites of both scFvs were different. If there was no difference, then the binding sites were the same or overlapping. The competitions for binding to FGFR3 between the ligand FGF9, FGF1, or control EGF and scFvs were also monitored by surface plasmon resonance. After capturing FGFR3 on the sensor chip, 1 Amol/L FGF9, FGF1, or EGF and 100 Ag/mL heparin were injected until saturation of binding. Then, 1 Amol/L of each scFv was subsequently injected and the binding curve of the antibody to the FGFR3-FGF ligand complex was compared with the binding to FGFR3 alone obtained with each scFv at the same concentration. Flow cytometry and confocal microscopy analyses. Phages and soluble scFvs were used for flow cytometry. Phages were prepared from 50 mL of culture, concentrated by polyethylene glycol precipitation, and resuspended in 2 mL of PBS. To determine binding of phage scFv to

native FGFR3 molecules, RT112 cells were gently detached from plastic plates by using a nonenzymatic cell dissociation solution (Sigma) followed by washing with PBS. Approximately, 1 106 cells and 1011 phage particles from fresh preparations were incubated in 2% MPBS for 1 hour on ice. Bound phages were detected using anti-M13 diluted 1:100 in 1% bovine serum albumin/PBS (1 hour on ice) and Rphycoerythrin goat anti-mouse diluted 1:100 in 1% bovine serum albumin/PBS (30 minutes on ice). After each incubation step, cells were washed twice with 1% bovine serum albumin/PBS. Finally, cells were resuspended in 500 AL of PBS and immediately analyzed in a FACScalibur with CellQuestPro software (BD Biosciences). For soluble scFvs, RT112 or transfected HEK293T cells (2 105) were incubated with the corresponding scFv at a concentration of 50 Ag/mL in FACS buffer (PBS containing 3% fetal bovine serum and 0.1% sodium azide) for 30 minutes at room temperature. After washing with FACS buffer, cells were fixed with 4% paraformaldehyde (Merck, Darmstadt, Germany) in PBS for 10 minutes. Bound scFvs were detected with anti-6xHis tag antibody diluted 1:50 in FACS buffer for 30 minutes at room temperature and then FITC-labeled anti-mouse antibody diluted 1:50 in FACS buffer. After each incubation step, cells were washed with FACS buffer. Cells were finally resuspended in 500 AL PBS and analyzed in a FACScalibur. Labeled RT112 cells were also examined with an ultraspectral confocal microscopy system (TCSSP2-AOBS-UV, Leica-Microsystems, Mannheim, Germany) after nucleus counterstaining with 4V,6-diamidino-2-phenylindole (Molecular Probes). Images were acquired with a 63 oil immersion objective using the Leica Confocal Software.

Fig. 2. Sequence analysis of the selected FGFR3-specific scFv. Amino acid sequence alignments of theVH (A) and VL (B) regions of the anti-FGFR3 3A, 3B, 3C, 1D, 2D, and 7D scFv clones. CDR2 and CDR3 are boxed. Dots, identical residues; asterisks, stop codons.


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Fig. 3. Purification of soluble scFvs. A, 3B, 3C, 2D, and 7D scFvs with 6xHis and c-myc tags at the COOH terminus were expressed in E. coli HB2151cells by induction with 1mmol/L isopropyl-L-thio-BD-galactopyranoside overnight at 30jC. After osmotic shock with sucrose, His-tagged scFvs were purified from the periplasmic fraction by usingTalon resin. Elution fractions (1-7) were examined by 10% SDS-PAGE and Coomassie staining. Arrowheads, full-length scFvs. Molecular weight markers in kilodaltons are indicated on the left. B, size exclusion chromatography on a HiLoad 26/60 Superdex 75 pg column of immobilized metal affinity chromatography ^ purified 3C scFv in PBS (pH 7.4). Arrows, elution volumes of the molecular weight markers bovine serum albumin (67 kDa) and glutathione S-transferase (26 kDa). Inset, fractions corresponding to dimeric (1-4) and monomeric (1-5) forms were collected, loaded onto a 10% SDS-PAGE, and stained with Coomassie brilliant blue.

Cell proliferation assays. RT-112 cells were plated onto 96-well tissue culture plates at a density of 2 103 cells per well in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics in duplicate. Once attached, cells were washed with PBS and cultured with different concentrations of anti-FGFR3 scFvs (ranging from 0.02 to 2 Amol/L) in RPMI 1640 containing 25 ng/mL FGF9, FGF1, EGF, or FGF9 plus 0.5 Ag/mL of recombinant FGFR3, together with 50 Ag/mL heparin (Sigma) and antibiotics for 48 hours at 37jC. Cell proliferation was scored by staining cells with the chromogenic dye 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. After removal of the medium, 100 AL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent (Sigma) were added to the culture wells at a final concentration of 1 mg/mL in RPMI 1640 and the cells were further incubated for 1 hour at 37jC. Then, 100 AL of DMSO (Sigma) were added to each well. Absorbance was read at 570 nm. All the experiments were done at least thrice. Cell viability is represented as the ratio of absorbance given by scFv-treated cells over absorbance of nontreated cells, expressed as a percentage.

Results Selection of fibroblast growth factor receptor 3 – specific singlechain Fv antibodies. Two different phage selection procedures were carried out by using different blocking conditions. Selection process A was made by using a preblocking step with IgG-coated wells. In selection B, phages were mixed with the IgG for blocking. Three rounds of panning were done on each selection phase. Results are shown in Table 1. In total, 288


colonies were randomly picked and tested by ELISA after the different selection procedures, yielding 15 FGFR3-specific antibodies. The final selection efficiency for the blocking process A was 1.5% and for process B was 3.1%, showing that the inclusion of a high concentration of blocking molecule in the phage suspension is more efficient than a previous absorption of the phage on IgG-coated wells. Characterization of the selected single-chain Fv antibodies. After DNA sequencing, we found that 6 of 15 scFv sequences specific for FGFR3 were unique and different (Fig. 2). Two of the six clones (3A and 1D) contained a stop codon within the sequence; they were identical except for a mutation in the complementary-determining region (CDR) 2 of the VL region (DxA). The other four scFvs were different from each other. All the mutations were concentrated on the hypervariable regions CDR2 and CDR3. CDR3 was highly variable in both VH and VL, but CDR2 was mainly variable in VH. All the scFvs were expressed at high levels as soluble molecules. For scFv purification, the periplasmic fraction was purified by standard immobilized metal affinity chromatography procedures (Fig. 3A) followed by gel filtration on a Superdex 75 column (Fig. 3B). This step allowed for a good resolution and separation of monomers, dimers, and larger-size molecules. The purification of monomers is strictly necessary for determination of the kinetic variables of binding to the antigen. To assess the specificity of the selected antibodies, the periplasmic scFvs were tested in ELISA with FGFR3 and the

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highly homologous (>62%) FGFR1. The results of the ELISA analysis are shown in Fig. 4. Although different from clone to clone, there was a significant reactivity of all the scFvs against FGFR3. In contrast, the reactivity against FGFR1 was marginal in all the cases and about the same magnitude as the background response against human IgG. Given the lower reactivity of the 3A and 1D clones, besides the presence of stop codons in the scFv gene, they were discarded for further analysis. Fibroblast growth factor receptor-3 binding affinity of purified single-chain Fv antibodies. Monomeric forms of scFv were used for determination of the affinity constants by surface plasmon resonance on immobilized FGFR3 using a Biacore instrument. The results for K on, K off, and K d determinations are shown in Table 2. The affinities oscillated between 40.9 and 12.4 nmol/L, indicating a moderate to high affinity, which is rather good if we consider the medium size and complexity of the original libraries (1.6 108). However, the K off of two of the scFvs, 3C and 7D, are relatively poor: 16.28 seconds and 1.36 minutes, respectively. Single-chain Fv antibody and fibroblast growth factor competition analysis. To distinguish different binding sites, we did a competitive binding analysis by using FGFR3 immobilized on a sensorchip and consecutive binding of the two antibodies that we wanted to study by surface plasmon resonance. The first antibody was passed in a saturating amount, in such a way that signal could increase only if the binding sites of the antibodies were different. The results are shown in Fig. 5A. In summary, 2D and 3B should recognize similar or overlapping epitopes as they inhibit binding with each other. 7D and 3C must recognize different epitopes. However, the 7D epitope accessibility can be partially blocked by 2D and by previous binding of 3B, but not in the opposite way (data not shown). The ability of the natural ligands FGF9 and FGF1 to block the binding of scFvs was also tested by Biacore. As a negative control, we used EGF. The results for 3C and 7D are shown in Fig. 5B. Previous binding of FGF9 to FGFR3 significantly blocks the union of the two scFvs, indicating that FGF9 and the scFvs compete for the same binding site. In contrast, FGF1 was very

Fig. 4. Specificity of soluble scFvs from selected clones by ELISA. scFv-pIII fusion proteins from supernatant culture of scFv-expressing E. coli TG1strain were analyzed for binding to FGFR3-Fc (black columns), FGFR1-Fc (gray columns), and human IgG (white columns) immobilized on an ELISA plate. Bound scFvs were detected with horseradish peroxidase ^ labeled anti ^ c-myc using 3,3V,5,5Vtetramethylbenzidine as substrate. Results are expressed as absorbance at 450 nm.


Table 2. Affinity analyses of anti-FGFR3 scFvs by surface plasmon resonance scFv 2D 7D 3B 3C

K on (1/Ms) 2.29e+5 3.16e+5 3.66e+5 2.64e+6

K off (1/s) 5.65e 1.22e 4.66e 6.14e

K d (nmol/L)

3 2 3 2

26.8 40.9 12.4 25.0

effective for blocking the binding of 7D, but not the binding of 3C. No blocking effect was noticed for an irrelevant growth factor such as EGF. Reactivity of single-chain Fv antibodies to fibroblast growth factor receptor 3 – expressing RT112 cells. The binding of scFvs to native FGFR3 expressed by RT112 bladder carcinoma cells was assessed by flow cytometry using freshly prepared phages or soluble scFv. Either six scFv phages or four soluble scFvs were used for the cytometry experiments. They stained RT112 cells albeit at various levels. Flow cytometric analyses with phages displaying the six different scFvs revealed that all of them reacted at a similar significant level with viable bladder carcinoma RT112 cells (Fig. 6A). When purified scFv fragments corresponding to 3B, 3C, 2D, and 7D were used, two of them, 3C and 7D, gave an intense immunofluorescence staining with a significant fraction of viable RT112 cells. Some cells remained unstained, indicating some heterogeneity at the cellular level. 2D and 3B stained a lower percentage of cells, suggesting a different accessibility to the epitopes by the scFvs. The analysis of the 3C- and 7D-stained cells by confocal microscopy revealed the characteristic staining pattern of membrane proteins (Fig. 6A). None of the selected scFvs was functional by Western blot assays (data not shown). Therefore, to confirm the specific binding to FGFR3, an immunoprecipitation in nondenaturing conditions followed by Western blot assay was carried out with two purified scFvs. Results are shown in Fig. 6B. Immunoprecipitation of RT112 cell extracts with scFvs 3C and 7D yielded a prominent band with the correct size that was recognized by a FGFR3-specific mAb, confirming the ability of these two scFvs to recognize soluble and native FGFR3. Reactivity of single-chain Fv antibodies to fibroblast growth factor receptor 3 containing activating mutations. Because FGFR3 is frequently mutated in bladder cancer, we decided to test the binding ability of these two scFvs to a mutant form of FGFR3 containing a Cys in position 249 instead of a Ser. This activating mutation has been reported as the most frequent in bladder cancer (27). We prepared this mutant by site-directed mutagenesis using PCR with a modified oligonucleotide. The mutation was confirmed by DNA sequencing (Fig. 7A). The correct expression of the mutant form after transfection of HEK293T cells was assessed by Western blot analysis (Fig. 7B). The mutant displayed similar levels of expression when compared with the wild-type form. Tubulin expression was used for normalization of the loaded protein. Flow cytometric analysis showed that both scFvs, 3C and 7D, were able to recognize HEK293T cells transfected with the FGFR3 S249C mutant even more strongly than cells transfected with the wildtype form (Fig. 7C).

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Fig. 5. scFv competition analyses for FGFR3 binding. A, Biacore sensorgrams showing pairwise binding competition of scFvs on FGFR3. Purified monomers of a FGFR3binding scFv were first injected on CM5 chips containing immobilized FGFR3 until saturation; then a second scFv was loaded. ScFvs recognizing different epitopes should give additional increases in response units (RU), whereas an unchanged signal suggests the recognition of the same or overlapping epitope. Dashed lines, 3B; dashed lines, 3C; dotted lines, 2D; dashed-dotted lines, 7D. B, sensorgrams showing binding competition between FGF9, FGF1, or EGF and 3C or 7D scFvs to FGFR3. Purified 3C and 7D scFvs (1 Amol/L) were injected either to FGFR3 immobilized biosensor (dashed line) or to FGFR3 previously saturated with FGF9/heparin (solid line), FGF1/heparin (dotted line), or EGF/heparin (dashed-dotted line). Each sensorgram was overlaid and zeroed on the y-axis to the average baseline before scFv injection. The start injection time for each scFv was set to zero on the x-axis. Competition was estimated by subtracting the response units of a given scFv injected into a FGFR3-immobilized flow cell from the response units of the injected scFv into the FGFR3-Fc immobilized chip in the presence of bound FGF9, FGF1, or EGF.


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Blocking of RT112 proliferation. We examined the efficacy of the scFvs in inhibiting tumor cell proliferation in vitro. The RT112 tumor cell line was used in this study because it expresses significant levels of FGFR3 (20). They were cultured in the presence of FGF9 and heparin and in the absence of FCS. Three different concentrations of scFv were added to the cells for 48 hours. In two cases, 3C and 7D, there was a significant inhibition of the proliferation that was dependent on the scFv concentration added to the cultures (Fig. 8A). The highest inhibition was achieved at 2 Amol/L, but it was also significant at 0.2 Amol/L. Therefore, anti-FGFR3 scFvs 3C and 7D

significantly inhibited FGFR3-mediated proliferation of RT112 cells. 2D inhibition was at a much lower degree and 3B did not cause any inhibitory effect. A further confirmation of the specificity of the blocking was made by the addition of different growth factors. The scFv inhibition in the presence of different growth factors is shown in Fig. 8B. The inhibitory effect was factor specific. Both 3C and 7D inhibited the proliferation induced by FGF9, but only 7D was able to block FGF1. None of them was able to block the proliferation induced by EGF. Moreover, the addition of soluble FGFR3 to the cultures abolished the scFv blocking capability.

Fig. 6. Binding of phage and soluble scFvs to RT112 human bladder carcinoma epithelial cells by flow cytometry and confocal microscopy. A, Bound phages were detected with anti-M13 followed by R-phycoerythrin ^ conjugated goat anti-mouse IgG. For soluble scFvs, the bound scFv was fixed with paraformaldehyde and detected with sequential incubations with anti-6xHis mAb and FITC-labeled anti-mouse IgG. FACS histograms show the binding of each scFv clone (bold line) and the backgrounds of phycoerythrin- or FITC-conjugated secondary antibodies (gray). RT112 cells labeled with 3C or 7D scFvs and FITC-labeled anti-mouse IgG were counterstained with 4V,6-diamidino-2phenylindole and visualized with laser confocal microscopy. Representative micrographs show scFv membrane staining (green) and 4V,6-diamidino-2-phenylindole nuclear staining (red). Bar, 8 Am. B, FGFR3 immunoprecipitation (IP) of RT112 cellular extracts followed by Western blot (WB) analysis with FGFR3-specific mAb. A, Triton X-100 extracts of RT112 cells; B, scFv anti ^ glutathione S-transferase immunoprecipitation (negative control); C, scFv 3C immunoprecipitation; D, scFv 7D immunoprecipitation.


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Fig. 7. Reactivity of the selected scFvs with FGFR3 mutated in the extracellular domain. A, electropherograms showing the mutation in the DNA sequence to change Ser (TCC) 249 to Cys (TGC). B, immunoblotting analysis. HEK293Tcells transfected with either wild-type or mutant (S249C) FGFR3 were resolved on SDS-PAGE, transferred to nitrocellulose filters, immunoblotted with mAb against FGFR3 or tubulin (bottom), and visualized by enhanced chemiluminiscence. C, FACS analysis of scFv binding to HEK293Tcells expressing either wild-type or mutant FGFR3 (S249C). Transfected HEK293Tcells were incubated with scFv 3C (left) and 7D (right). Bound antibodies were fixed with paraformaldehyde and detected with mAb against 6xHis and phycoerythrinconjugated goat anti-mouse IgG. Overlay histograms show the binding of the corresponding scFv to mock-transfected (gray), FGFR3(IIIc)WT-transfected (black line), and FGFR3(IIIc)S249Ctransfected (dashed line) cells.

These results correlate well with the competition analysis, flow cytometry, and the accessibility of the epitopes on the cell membrane. Finally, from the morphologic point of view, treatment with 3C and 7D scFvs resulted in changes in cellular morphology and slower growth (Fig. 8C), with a more prominent vacuolization in the case of 7D.

Discussion FGFR3 is a receptor tyrosine kinase implicated in cell growth and tumorigenesis through activation of different signal transduction pathways such as extracellular signal-regulated kinases 1 and 2, phosphatidylinositol 3 kinase, and phospholipase c-g pathways (9, 28, 29). Overexpression of receptor tyrosine kinase in tumors is generally associated with poor prognosis (30). Apart from bladder cancer, FGFR3 is mutated and overexpressed in a variety of cancers and, as a result, particular attention has been given to FGFR3 for therapeutic use. Blocking of the transduction pathways used by FGFR3 could be an appropriate way to inhibit cell growth and differentiation induced by this molecule. Thus, FGFR3 constitutes an excellent target for effective cancer intervention. In a previous report (20), it has been shown that murine mAbs specific for FGFR3 provoked an antiproliferative effect in the RT112 cell line. However, the use of mouse mAbs for human cancer therapy is not realistic due to the strong human antimouse antibody response, which has made almost impossible any therapeutic approach based on mouse mAbs. Currently available alternatives include ‘‘chimerization’’ and ‘‘humanization’’ of mouse antibodies. Both approaches are labor- and time-intensive and are unpredictable procedures. We have directly prepared human antibodies from a human phage display library using a soluble chimeric molecule composed of the extracellular domain FGFR3a(IIIc) coupled


to the Fc region of human IgG1 as antigen for antibody selection. Therefore, the specific reactivity obtained against FGFR3 must be targeted to the extracellular domains. After ELISA selection, we obtained six unique FGFR3-specific scFv sequences. The specificity of the antibodies was confirmed by immunoprecipitation, Biacore analysis, and flow cytometry. None of the selected scFvs was able to recognize FGFR3 by immunoblot analysis (data not shown). These results suggest that the recognized epitopes are conformational, nonlinear, and susceptible to denaturation, probably due to the use of soluble antigen for the panning procedures. These findings make difficult the identification of the amino acid sequence corresponding to these epitopes and its position into the FGFR3 molecule. Competition experiments have shown that the antibodies recognize different but, in some cases, overlapping epitopes. 2D and 3B probably recognize the same epitope. Biacore analysis was used for the determination of the kinetic constants of the scFv binding to the antigen. One of the problems associated with phage display is related to the generally low affinity shown by these scFv. As previously described, for selection of antigens immobilized on polystyrene plates, the resulting scFvs are a mixture of monomers and dimers (31). The resulting homodimers show a significant increase in apparent affinity (avidity; ref. 32). Due to this increase in avidity, phages displaying mixtures of monomeric and dimeric forms will be preferentially selected. To determine the kinetic constants of the scFvs, it was necessary to separate the monomers by gel filtration. In our case, the affinities of the selected scFvs oscillated between 40.9 and 12.4 nmol/L, indicating moderately high affinities, which is rather good if we consider the low size and complexity of the original libraries (1.6 108) and compare very favorably with the affinities obtained by using a much larger library (2 1010) of Fab fragments, which were approximately in the same range or 1

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order of magnitude better (25). In terms of application to cancer therapy, the K d of these scFvs might prove appropriate taking into account that recent reports have shown that a very high affinity can restrict the tumor penetration of the scFvs (33). Currently available antibodies for therapeutic use have a K d range of 10 to 0.1 nmol/L (34). Flow cytometric analysis confirmed the specific binding of the 3C and 7D scFvs to the native membrane FGFR3. More interestingly, 3C and 7D were able to recognize even better a mutant FGFR3 containing one of the most abundant activating mutations present in bladder cancer, S249C, stressing the ability of these two scFvs to bind tumoral tissues. This enhanced recognition could be explained by the natural tendency to dimerize the mutant forms, which might stabilize the antibody binding. Previous results have shown that secreted soluble, extracellular ligand-binding domains of the native FGFRs play an inhibitory effect on cell proliferation, mopping up the ligand binding to the receptor (35). The antibody fragments may mimic this capability of the secreted extracellular domains. In fact, several results presented in this study suggest that the mechanism of RT112 cell growth inhibition by 3C and 7D scFvs is probably due to a direct block of FGF-FGFR3 interaction, resulting in the inhibition of FGFR3 activation. This is based on the observation that 3C and 7D specifically bind to both soluble and cell-surface expressed FGFR3 on RT112 cells. Moreover, 3C and 7D were blocked by FGF9 from binding to FGFR3, but only 7D competed with FGF1.

This result indicates the capacity of these antibodies to use the same binding site as FGF9, blocking its natural effector function. Furthermore, 3C and 7D inhibit FGF9-induced proliferation in a significant and concentration-dependent manner. Both 7D and 3C have a very rapid off rate (1.22 10 2 and 6.14 10 2, respectively), which might lead to a very short association with the tumoral cells due to the rapid turnover and internalization. A reduction in K off is typically the major kinetic mechanism resulting in higher affinity. Thus, it would be convenient to improve this affinity to slow the off rate. Because of the relatively poor K off of 3C and 7D when compared with other scFvs, it is very likely that an improvement in the affinity of these molecules might suppose an improvement in their blocking ability. The 3C and 7D scFv sequences will serve as a template for affinity maturation studies according to well-established technologies such as complementary-determining region mutagenesis (36) or somatic hypermutation mimicry (37). There are a number of important human or humanized antibodies in clinical trials against different molecules implicated in cancer progression (38). Antibodies are showing a great promise in cancer treatment. Compared with small molecules, antibody therapy may provide higher specificity to tumoral cells and better safety profile. The moderate affinity of the scFv fragments is sufficient to react only with a population of cells expressing significant levels of FGFR3, but not towards cell populations that express only low levels of the receptor. The

Fig. 8. Inhibition of RT112 cell proliferation by FGFR3-specific scFvs. A, inhibitory activity of 3B, 3C, 2D, and 7D scFvs on RT112 growth was tested at various concentrations: 2 Amol/L (black columns), 0.2 Amol/L (gray columns), and 0.02 Amol/L (white columns). After 48 hours of incubation, cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay at 450 nm. Absorbance of the untreated control cells was taken as 100% and absorbance of treated cells was taken as percentage of relative growth for each concentration used. Columns, mean of three independent cell proliferation experiments (each concentration tested in duplicate); bars, SD. B, the inhibitory effects of 2 Amol/L 3C (dashed columns) and 7D (dotted columns) scFvs were assayed as described above in the presence of FGF9, FGF1, EGF, or 0.5 Ag/mL of recombinant FGFR3 in the media (FGF9 + FGFR3 columns). C, the number and morphology of cells, either untreated or treated with 2 Amol/L 3C and 7D scFvs, after 48 hours of incubation were visualized before the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay at 20 and 40 magnifications by using an inverted Leica DM IL microscope equipped with a Leica DFC350 FX digital camera.


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scFvs could be used as therapeutic agents either alone or combined with other molecules such as immunotoxins or radionuclides that can selectively kill the tumoral cells displaying FGFR3, as previously shown for angiogenic molecules and growth factor receptors, such as vascular endothelial growth factor or erbB2 (neu/HER2; ref. 24), or other overexpressed proteins (39). The small size of the scFvs considerably increases their tumor penetration capabilities (33, 40) and pharmacokinetic properties (41). Moreover, the use of antibody fragments confers a high specificity to the inhibitory effect, which allows the discrimination between the different FGFRs. This is particularly relevant given the opposite effects they play. Thus, whereas FGFR3 presents an oncogenic activity, FGFR2 shows a tumor suppressor activity (42). Apart from its overexpression in bladder or cervical cancer, dysregulated FGFR3 has been show to play a major role in multiple myeloma tumorigenesis and has already been tested as

a molecular target for therapy (19). Specific inhibitors of FGFR3 induced cell cycle arrest and apoptosis in human myeloma cells. It will be interesting to test the antiproliferative effect of these antibodies on multiple myeloma cell lines. Our data, together with previous results of gene expression analysis and polyclonal anti-FGFR3 antibodies, support further evaluation of 3C and 7D scFv antibodies as antitumor agents. Previous findings (20) support that FGFR3 inhibition should be introduced at early disease stages (when FGFR3 overexpression is more abundant) before progressive loss of expression.

Acknowledgments We thank Arancha Garcia, Diego Megı´as, and Maria Montoya of the Centro Nacional de Investigaciones Oncolo¤gicas confocal and cytometry unit for their help and collaboration in the flow cytometric and confocal microscopy analysis.We thank Silvia Romero for her excellent technical assistance and Giovanna Roncador for her help in the transfection experiments.

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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 Jorge Martínez-Torrecuadrada, Gabriela Cifuentes, Paula López-Serra, et al. Clin Cancer Res 2005;11:6280-6290.

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