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Clinical Cancer Research Vol. 11, 3897-3904, May 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

A Neutralizing Anti–Fibroblast Growth Factor 8 Monoclonal Antibody Shows Potent Antitumor Activity against Androgen-Dependent Mouse Mammary Tumors In vivo

Naoki Shimada1, Toshihiko Ishii2, Teruyoshi Imada2, Katsumi Takaba2, Yuka Sasaki1, Kumiko Maruyama-Takahashi1, Yoshimi Maekawa-Tokuda2, Hideaki Kusaka2, Shiro Akinaga2, Akira Tanaka3 and Kenya Shitara1

Authors' Affiliations: 1 Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan; 2 Drug Development Research Laboratories Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co., Ltd., Shizuoka, Japan; and 3 Department of Pathology, Jichi Medical School, Tochigi, Japan

Requests for reprints: Kenya Shitara, Tokyo Research Laboratories, Kyowa Hakko Kogyo, Co., Ltd., 3-6-6 Asahi-machi, Machida, Tokyo 194-8533, Japan. Phone: 81-42-725-0857; Fax: 81-42-725-2689; E-mail: kshitara{at}kyowa.co.jp.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Fibroblast growth factor 8b (FGF8b) has been implicated in oncogenesis of sex hormone–related malignancies. A murine monoclonal anti-FGF8 antibody, KM1334, has been raised against a FGF8b-derived peptide and shown to neutralize FGF8b activity in an androgen-dependent mouse mammary cell line (SC-3) in vitro growth. The purpose of this study was to evaluate KM1334 as a therapeutic agent for FGF8-dependent cancer.

Experimental Design: Specificity and neutralizing activity of KM1334 were examined in vitro. In vivo therapeutic studies were done in nude mice bearing SC-3 tumors s.c.

Results: KM1334 recognized FGF8b and FGF8f specifically out of four human FGF8 isoforms and showed little binding to other members of FGF family. Neutralizing activity of KM1334 was confirmed by both blocking of FGF8b binding to its three receptors (FGFR2IIIc, FGFR3IIIc, and FGFR4) and FGF8b-induced phosphorylation of FGFR substrate 2{alpha} and extracellular signal-regulated kinase 1/2 in SC-3 cells. The in vitro inhibitory effect could be extended to in vivo tumor models, where KM1334 caused rapid regression of established SC-3 tumors in nude mice. This rapid regression of tumors after KM1334 treatment was explained by two independent mechanisms: (a) decreased DNA synthesis, as evidenced by a decrease in uptake of 5-bromo-2'-deoxyuridine, and (b) induction of apoptosis as shown by the terminal deoxynucleotidyl transferase–mediated nick end labeling assay.

Conclusions: KM1334 possesses strong blocking activity in vitro and antitumor activity in vivo and therefore may be an effective therapeutic candidate for the treatment of cancers that are dependent on FGF8b signaling for growth and survival.

Key Words: Fibroblast growth factor 8, FGF8 • Monoclonal antibody • Shionogi carcinoma • Antitumor activity • Androgen-dependent

Since the discovery of monoclonal antibody (mAb) technology (1), attempts have been made to use their exquisite specificity and affinity for the treatment of human diseases, such as cancer. In recent years, significant advances have been made in the therapeutic use of antibodies as shown by the growing numbers that are gaining approval for clinical use (2). Many targets for therapeutic antibodies are membrane-bound antigens, which is necessary for antibodies to show effector functions, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. However, soluble molecules, which promote tumor progression directly or indirectly, can also be good targets for cancer therapy. For example, bevacizumab, an anti–vascular endothelial growth factor neutralizing antibody, showed clinical benefits for patients with metastatic colorectal cancer in phase III study (3). For the clinical application of mAbs against growth factors, candidate mAbs having high specificity, potent neutralizing activity, and in vivo antitumor activity should be selected for the further development of humanized antibodies.

The fibroblast growth factors (FGF) form a family of at least 24 growth-regulatory proteins. They share 35% to 50% amino acid sequence identity and induce proliferation and differentiation in a wide range of cells of epithelial, mesodermal, and neuroectodermal origin (4, 5). FGF8 was originally isolated from the conditioned medium of an androgen-dependent mouse mammary tumor cell line (SC-3) as an androgen-induced growth factor and was later classified as a member of the FGF family based on structural similarity (6). FGF8 has been found to have an important role in embryogenesis and morphogenesis (7). It is expressed during gastrulation, in brain development, and in the process of limb and facial morphogenesis of the developing mouse (8–13). FGF8 has been identified as an oncogene product based on its transforming activity in NIH3T3 cells (14). In addition, a high frequency of FGF8 mRNA overexpression, which is associated with decreased patient survival and persists in androgen-independent disease, was detected by tissue in situ hybridization in prostate cancer specimens (15).

The structure of the FGF8 gene is more complicated than that of the other members of the FGF family. Alternative splicing of the FGF8 gene potentially gives rise to eight different protein isoforms (a-h) in mice and four isoforms (a, b, e, and f) in humans (6, 16, 17). The isoforms differ in the NH2 terminus. The biological function of these forms is not exactly known, but at least they differ in transforming potential. FGF8b has been found to have the highest NIH3T3 cell-transforming capacity (18, 19). Furthermore, transgenic mice overexpressing FGF8b in the mammary glands are found to develop mammary tumor and those in prostate epithelial cells develop prostatic intraepithelial neoplasia (20, 21). Overexpression of FGF8b gives a more aggressive phenotype, including increased growth rate to cultured human prostate and breast cancer cells (22, 23). Up-regulation of FGF8b was also observed in multiple human cancers, such as prostate, breast, and ovary carcinomas (24–28). The above findings strongly suggest that FGF8, especially FGF8b, is the potential target for antibody-based cancer therapy.

FGF signaling is transduced through the formation of a complex of a growth factor, a proteoglycan, and a high-affinity FGF receptor (FGFR), which is a transmembrane tyrosine kinase receptor (29). Four different high-affinity receptors (FGFR1, FGFR2, FGFR3, and FGFR4) bind FGF ligands and display varying patterns of expression (reviewed in refs. 1, 24). Extracellular domains of FGFRs consist of three immunoglobulin-like loops (loop I, II, and III). Alternative mRNA splicing of loop III of FGFR1 to FGFR3 leads to distinct functional variants (IIIb and IIIc) that have different ligand-binding specificities and affinities. FGFR4 does not have alternative splicing but is most similar to an IIIc-like domain (1, 30, 31). Differential expression of IIIb and IIIc variants is very important for determining FGF signaling specificity. For example, the expression of FGFR2 isoforms of IIIb and IIIc is restricted to the cells of epithelial and mesenchymal lineage, respectively. In addition, exon switching from FGFR2IIIb to FGFR2IIIc was observed in progressive prostate cancer (32). FGF8 preferentially activates FGFR2IIIc and FGFR3IIIc splice forms and FGFR4 (17, 33), although there are differences between the activation potential of various FGF8 isoforms. FGF8b also activates FGFR1IIIc but only at a very high concentration (17). The receptors are activated through dimerization and phosphorylation by the intracellular tyrosine kinase domains (34). This process activates the Ras signal transduction pathway via FGFR substrate 2 (FRS2), the key components of which are the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK) 1 (p44MAPK) and ERK2 (p42MAPK; for reviews, see refs. 35, 36).

We had already established a neutralizing monoclonal anti-FGF8b antibody, KM1334, by immunizing FGF8b-derived peptide. KM1334 was shown to inhibit androgen- and FGF8b-dependent growth of SC-3 cells in vitro (24). Furthermore, high frequency of FGF8 expression in clinical prostate cancers and breast diseases was immunohistochemically shown by KM1334 (24, 26), which is consistent with the results of immunohistochemistry and in situ hybridization reported by other groups (25, 27).

To investigate the potential of KM1334 as therapeutic agent for the treatment of cancers that are dependent on FGF8b signaling for growth and survival, we further evaluated its binding specificity, blocking activity, and in vivo antitumor activity. We show that KM1334 possesses high specificity and potent blocking activity in vitro. In addition, we show that this antibody exhibit strong antitumor activity in vivo mice model and its activity is mediated by both antiproliferative and proapoptotic activities.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Antibodies. A murine mAb against FGF8b, KM1334, was established as described previously (24). Briefly, the keyhole limpet hemocyanin–conjugated FGF8b peptide (FGF8b-1) from amino acid residues 23 to 46 of human and mouse FGF8b was used as an immunogen. A murine mAb, KM511, raised against a mutant of granulocyte colony-stimulating factor was described previously (37) and used as a negative control.

Anti-phosphospecific ERK1/2 rabbit polyclonal antibody and anti-phosphorylated FRS2{alpha} rabbit polyclonal antibody were obtained from Cell Signaling Technology (Beverly, MA). Anti-ERK2 mAb (clone 1B3B9) and anti-FRS2 rabbit polyclonal antibody (H-91) was purchased from Upstate Biotechnology (Lake Placid, NY) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Anti-ß-actin mouse mAb (ab8226) was from Abcam (Cambridge, United Kingdom).

Cells and animals. The SC-3 cell line used in the present study was derived from an androgen-responsive mouse mammary SC115 tumor (38). Adult male BALB/c nu/nu mice were purchased from Nippon Clea Co. (Tokyo, Japan).

Synthesis of fibroblast growth factor 8 fragment. Peptides with amino acid residues from 23 to 35 of human FGF8a (FGF8a-1), from 23 to 46 of human FGF8b (FGF8b-1), from 23 to 64 of human FGF8e (FGF8e-1), and from 23 to 75 of human FGF8f (FGF8f-1) were synthesized by an automated peptide synthesizer. For inhibition ELISA, FGF8b fragment (FGF8b-1) with additional cysteine residue in COOH terminus was synthesized and conjugated to bovine serum albumin (BSA).

Cross-reactivity of KM1334 to human fibroblast growth factor 8 variants (inhibition ELISA). The FGF8b-1 peptide conjugated to BSA was plated onto 96-well plate at a concentration of 1.0 µg/mL per 50 µL of each well. After overnight plating at 4°C, the plate was washed with PBS and blocked with 1% BSA in PBS. Then, the synthetic peptides, FGF8a-1, FGF8b-1, FGF8e-1, FGF8f-1, and a control peptide with a irrespective sequence (CGAGPKRRALAAPAAEEKEEA), were serially diluted and added into each well with KM1334 (final concentration, 0.08 µg/mL). After 2 hours of incubation at room temperature, the plate was interacted with horseradish peroxidase–labeled anti-mouse immunoglobulins (DAKO Corp., Carpinteria, CA) and 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid substances, WAKO, Osaka, Japan). The absorbance at 415 nm was measured using an E-max microplate reader (Molecular Devices Corp., Sunnyvale, CA).

Cross-reactivity of KM1334 to human fibroblast growth factor 17b and fibroblast growth factor 18 (binding ELISA). Recombinant human FGF17b (R&D Systems, Inc., Minneapolis, MN), human FGF18 (R&D Systems), mouse FGF8b (R&D Systems), and human FGF2 (PeproTech, London, United Kingdom) were plated onto 96-well plate at serial concentrations from 0.1 ng/mL to 10 µg/mL per 50 µL of each well. After overnight plating at 4°C, the plate was serially washed and blocked with 1% BSA, and KM1334 was added into each well at a concentration of 1 µg/mL per 50 µL of each well. After washing, the bound antibody was detected as described previously.

Binding activity of fibroblast growth factor receptor-Fc fusion proteins to fibroblast growth factor 8b (binding ELISA). FGFRs fused with human IgG1 Fc domain (FGFR-Fc fusion proteins; human FGFR1IIIc-Fc, human FGFR2IIIc-Fc, murine FGFR3IIIc-Fc, and human FGFR4-Fc) were purchased from R&D Systems. Production of a negative control, human interleukin (IL)-5 receptor (IL-5R)-Fc fusion protein, will be described elsewhere.4 FGF8b was coated onto 96-well plate at a concentration of 5 µg/mL. After washing, the wells were incubated with serial concentrations of the FGFR-Fc and IL-5R-Fc fusion proteins. Bound FGFR-Fc fusion proteins on FGF8b were detected with anti-human IgG labeled with horseradish peroxidase (American Qualex, San Clemente, CA) diluted 1:12,500 as a secondary antibody. Inhibition of the FGF8b binding to FGFR-Fc fusion proteins by KM1334 was evaluated in this system. Serially diluted KM1334 or KM511 were added with each FGFR-Fc solution (10 µg/mL FGFR2IIIc-Fc, 2 µg/mL FGFR3IIIc-Fc, and 1 µg/mL FGFR4-Fc) to the FGF8b-coated plate. After washing, the secondary antibody bound to human Fc was detected as described previously.

Western blot. SC-3 cells were plated into 10 cm dishes at 2 x 106 cells per dish in a serum-supplemented medium; 2S(–) [Ham's F-12:DMEM (1:1, v/v) containing 2% dextran-coated, charcoal-treated fetal bovine serum] and allowed to adhere. After 8 hours of incubation at 37°C, the medium was exchanged with a serum-free medium; B0.1 [Ham's F-12:DMEM (1:1, v/v) containing 2% BSA] and incubated for 16 hours. Then, the medium was changed to an experimental medium composed of B0.1 and incubated for 15 minutes. After wash with PBS, cells were lysed in 500 µL lysis buffer [50 mmol/L HEPES-NaOH (pH 7.4), 250 mmol/L NaCl, 1 mmol/L EDTA, 1% NP40, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride, 5 µg/mL leupeptin, 2 mmol/L Na3VO4, 1 mmol/L NaF, 10 mmol/L ß-glycerophosphate; ref. 39]. Cell lysates were clarified by centrifugation and total cell lysate (20 µg) from each sample was used for SDS-PAGE. After SDS-PAGE, the protein were transferred to polyvinylidene difluoride membranes and immunoblotted with appropriate secondary antibodies conjugated with horseradish peroxidase and developed using the enhanced chemiluminescence detection system according to the instructions of the manufacturer (Amersham Pharmacia Biotech, Piscataway, NJ).

Immunocytochemistry for SC-3 cells. SC-3 cells were inoculated into eight-well chamber slides (Nalge Nunc International, Rochester, NY) at 1 x 104 cells per well suspended in 200 µL 2S(–) medium and allowed to adhere. After 16 hours of incubation at 37°C, the medium was changed to an experimental medium composed of B0.1 and incubated for 24 hours. After wash with PBS, the chambers were removed and the slides were air-dried for 24 hours. Then, the cells were fixed in a 1:1 mixture of acetone/methanol for 10 minutes at room temperature. The slides were immunocytochemically stained for incorporated 5-bromo-2'-deoxyuridine (BrdUrd; Sigma Chemical Co., St. Louis, MO) labeling and terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) method (40). For BrdUrd labeling, BrdUrd was added to the medium at 100 µmol/L concentration for 1 hour before PBS wash. Then, the slides were treated with 3% H2O2 and 2 N HCl. BrdUrd incorporated into SC-3 cells was detected with a mouse anti-BrdUrd antibody (DAKO A/S, Glostrup, Denmark), EnVision, and 3,3'-diaminobenzidine (Merck, Darmstadt, Germany). Mayer's hematoxylin (Muto Pure Chemicals Ltd., Tokyo, Japan) was used for counterstaining. Positive cells and total cells in the three independent areas of 370 x 500 µm were counted. TUNEL method was done using the ApopTag Apoptosis In situ Detection Kit (Intergen, Purchase, NY) according to the manufacturer's instruction. Briefly, the slides were treated with 3% H2O2 and the nick ends of DNA were labeled with digoxigenin by terminal deoxynucleotidyl transferase enzyme. Apoptotic cells were detected with an anti-digoxigenin peroxidase conjugate using 3,3'-diaminobenzidine (WAKO) as a substrate. Mayer's hematoxylin was used for counterstaining. Positive cells and total cells in the three independent areas of 370 x 500 µm were counted. Statistical significances in the ratio of BrdUrd- or TUNEL-positive cells between the KM1334 group and the KM511 group were determined by two-tailed unpaired t test.

Evaluation of antitumor activity. All of the in vivo experiments were done in conformity with institutional guidelines in compliance with national laws and policies. SC-3 cells were harvested, washed with PBS, and inoculated s.c. in the dorsal side of male athymic nude mice by 1 x 106 cells per head suspended with 100 µL PBS. Tumors were allowed to reach ~300 mm3 in size, and the mice were randomized into groups of five animals each. Mice were treated with KM1334 (50, 100, 200, and 400 µg in 200 µL PBS/mouse/shot) or PBS (200 µL/mouse/shot) by i.p. injection twice weekly. Treatment of animals was continued for the duration of the study (total of nine shots). Tumors were measured twice weekly with calipers, and tumor volumes were calculated by the following formula according to the methods of the National Cancer Institute (ref. 41; length and width of the tumors measured in mm): Tumor volume (mm3) = (Length x Width2) / 2.

Statistical significances in tumor size between the KM1334 group and the vehicle control (PBS) group were determined by two-tailed unpaired t test.

Immunohistochemistry for tumor sections. SC-3 cells were inoculated into athymic male nude mice according to the method written above. When SC-3 tumors reached 120 to 600 mm3 in size, the mice were randomized into seven groups of three animals each. KM1334 and KM511 were given into three groups each by 400 µg in 200 µL PBS/mouse/shot. Tumors were collected at timings as follows: before antibody injection and at 7, 24, and 72 hours after antibody injection. Tumor volume was also measured immediately before each collection of tumors. One hour before tumor collection, BrdUrd dissolved in saline (20 mg/mL) was given i.v. into the mice by 100 mg/kg. The collected tumors were cut into two aliquots and were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 3 µm. After deparaffinization and rehydration, sections were immunohistochemically stained for incorporated BrdUrd labeling and TUNEL method as described in immunocytochemistry of SC-3 cells (40). For BrdUrd labeling, sections were treated with 0.05% Pronase after neutralization with sodium borate. For TUNEL staining, sections were treated with proteinase K (Life Technologies, Rockville, MD) before digoxigenin labeling with terminal deoxynucleotidyl transferase enzyme. Positive cells in the area of 320 x 420 µm were counted. Statistical significances in BrdUrd- or TUNEL-positive cells between the KM1334 group and the KM511 group were determined by two-tailed unpaired t test.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Binding specificity of an anti–fibroblast growth factor 8b monoclonal antibody, KM1334. To evaluate the therapeutic potential of an anti-FGF8b mAb KM1334, we first analyzed its specificity. We generated four synthetic peptides derived from human FGF8 variants (a, b, e, and f) and examined their inhibitory effect on the binding of KM1334 to its epitope-containing conjugate (FGF8b-1-BSA conjugate) in ELISA. Figure 1A and B show the structure of human FGF8 protein isoforms and the amino acid sequences of the synthetic peptides. Among the peptides examined, FGF8b-1 and FGF8f-1 significantly inhibited the binding of KM1334 to its antigen, indicating that KM1334 recognizes both FGF8b and FGF8f isoforms (Fig. 1C). On the other hand, FGF8a-1 and FGF8e-1 did not inhibit the binding. The lack of binding of KM1334 to FGF8a-1 is consistent with previously reported results (24). We next examined the cross-reactivity of KM1334 to FGF17b and FGF18, which are the closest members of FGF8 in FGF family, and their sequences corresponding to FGF8b-1 have significant homology with FGF8b (Fig. 2A). KM1334 showed strong binding to FGF8b; however, KM1334 did not bind to FGF17b, FGF18, and FGF2 (Fig. 2B). These results show that KM1334 possesses the high specificity to FGF8b.



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  		Fig. 1. Specificity of KM1334 to FGF8 variants. A, human premature FGF8a, FGF8b, FGF8e, and FGF8f protein isoforms, depicted NH2 terminus (left) to COOH terminus (right). Solid bars above each columns, region used for synthetic peptides (right). B, alignment of amino acid sequences belonging to human FGF8 isoforms. FGF8a-1, FGF8b-1, FGF8e-1, and FGF8f-1 correspond to partial sequences of FGF8a, FGF8b, FGF8e, and FGF8f, respectively. KM1334 was raised against FGF8b-1 (bold) that includes unique sequence for FGF8b and FGF8f (VTVQSPPNFTQ). All four synthetic peptides were designed to contain the shared sequence (HVREQSLVTDQL) of all FGF8 isoforms. FGF8b-1 and FGF8f-1 contained unique sequence for FGF8b and FGF8f. FGF8e-1 and FGF8f-1 contained unique sequence for FGF8e and FGF8f. Gaps were introduced to facilitate understanding of the identity among each sequence. C, inhibition ELISA to analyze binding specificity of FGF8 variants: FGF8b-1 ({circ}; positive control), FGF8f-1 ({bullet}), FGF8a-1 ({triangleup}), FGF8e-1 (x), and peptide for negative control ({square}). Points, mean for A415-490; bars, SD.

 


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  		Fig. 2. Specificity of KM1334 to other members of FGF family with high homology to FGF8. A, alignment of amino acid sequences of FGF17b and FGF18 with FGF8b. Gaps were introduced to optimize the homology. Boxed area, shared residues in more than two FGFs. B, binding ELISA to detect cross-reactivity of KM1334 to other FGFs. A ELISA plate was coated for FGFs [i.e., FGF8b ({circ}), basic-FGF ({blacklozenge}; negative control), FGF17b ({triangleup}), and FGF18 ({square})] at various concentrations and KM1334 was reacted to them at 1.0 µg/mL. Points, mean for A415-490; bars, SD.

 
Neutralizing activity of KM1334. Since Tanaka et al. reported that KM1334 inhibited SC-3 growth in vitro (24), direct evidence of blocking activity of KM1334 has not been shown yet. We first examined the binding of FGF8b to FGFR-Fc fusion proteins (FGFR1IIIc-Fc, FGFR2IIIc-Fc, FGFR3IIIc-Fc, and FGFR4-Fc) in ELISA. Among FGFR-Fc fusion proteins tested, FGFR2IIIc-Fc, FGFR3IIIc-Fc, and FGFR4-Fc showed significant binding activity to FGF8b; however, FGFR1IIIc-Fc showed little binding to FGF8b (Fig. 3A). The order of affinity was as follows: FGFR4-Fc greater double equals FGFR3IIIc-Fc > FGFR2IIIc-Fc. The apparent affinities of FGFR4-Fc and FGFR3IIIc-Fc with FGF8b were 32.2- and 11.3-fold higher than that of FGFR2IIIc-Fc, respectively. Then, the ability of KM1334 to block the FGF8b binding to FGFR-Fc fusion proteins was assessed in this ELISA. KM1334 blocked the binding of FGF8b to all the FGFR-Fc fusion proteins examined: FGFR2IIIc-Fc (IC50, 0.31 µg/mL; Fig. 3B), FGFR3IIIc-Fc (IC50, 0.86 µg/mL; Fig. 3C), and FGFR4-Fc (IC50, 0.95 µg/mL; Fig. 3D). Control antibody KM511 did not show any blocking activity.



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  		Fig. 3. Binding activity of FGFRs to FGF8b and their inhibition by KM1334. A, FGF8b was coated to a ELISA plate at concentration of 5 µg/mL. Then, FGFR extracellular domains fused to human Fc region [FGFR1IIIc-Fc ({lozenge}), FGFR2IIIc ({square}), FGFR3IIIc ({triangleup}), and FGFR4 ({circ})] were reacted to the FGF8b at various concentrations. IL5R-human Fc fusion protein was used as a negative control ({bullet}). B-D, effect of KM1334 ({circ}) and KM511 ({square}), negative control, on the binding of FGF8b to FGFR-Fc fusion proteins [FGFR2IIIc-Fc (B), FGFR3IIIc-Fc (C), and FGFR4-Fc (D)]. Points, mean for A415-490; bars, SD.

 
To identify FGF8b-stimulated signal transduction pathway in SC-3 cells, phosphorylation of FRS2{alpha} and MAPK (ERK1/2) was examined in Western analysis. Phosphospecific antibodies against FRS2{alpha} and ERK1/2 gave stronger signal to FGF8b-stimulated cells than nonstimulated control cells. The expression level of ERK2 was almost the same in all samples (Fig. 4A). On the other hand, FRS2 expression seemed to be up-regulated slightly in the FGF8-stimulated cells. KM1334 inhibited FGF8b-induced phosphorylation of both FRS2{alpha} and ERK1/2 in a dose-dependent manner (Fig. 4A).



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  		Fig. 4. In vitro effect of KM1334 on SC-3 cells stimulated by FGF8b. A, Western blot analysis. SC-3 cells were stimulated by 50 ng/mL FGF8b with KM1334 or KM511 (isotype control). After 15 minutes of incubation at 37°C, cells were collected and cell lysates were prepared. Western blot analyses were done using the indicated phosphospecific antibodies to FRS2{alpha} (Tyr196) or ERK1/2 (Thy202/Tyr204), and the blots were reprobed with antibodies to total FRS2 or total ERK2. A representative ß-actin reprobed blot is shown as a loading control. B and C, immunocytochemical analysis of SC-3 cells. SC-3 cells were stimulated by 50 ng/mL FGF8b with KM1334 or KM511. After 24 hours of incubation, BrdUrd was added and incubated for additional 1 hour. Uptake of BrdUrd was examined by anti-BrdUrd antibody with an immunocytochemical method (B). Results of TUNEL staining were shown in (C). Labeling index shows the number of 3,3'-diaminobenzidine-positive cells divided by the total number of nuclear pixels (hematoxylin-positive cells + 3,3'-diaminobenzidine-positive cells) multiplied by 100. Points, mean for three independent areas of 370 x 500 µm; bars, SD. Statistics were done by two-tailed Student's t test. *, P < 0.05, relative to control (B and C).

 
Furthermore, immunocytochemical analysis revealed that FGF8b promotes uptake of BrdUrd and rescues apoptosis in SC-3 cells, which show that FGF8b is critical for the growth and survival of SC-3 cells. KM1334 also inhibited those effects in a dose-dependent manner (Fig. 4B and C), which was in agreement with a previous report demonstrating that KM1334 inhibited FGF8b- and androgen-dependent growth of SC-3 cells (24). These results indicate that KM1334 possesses the potent in vitro neutralizing activity.

In vivo effect of KM1334 on SC-3 tumors in mice. We next examined the potential of antitumor activity of KM1334 on the growth of SC-3 tumor implanted on nude mice. SC-3 cells were injected into male nude mice s.c. and allowed to grow to ~300 mm3 in size. Antibody treatment of 50, 100, 200, and 400 µg/injection was started 10 days after tumor inoculation and the tumor sizes were monitored until day 38. As shown in Fig. 5, treatment with KM1334 resulted in dose-dependent suppression of SC-3 tumor growth. On day 38, at the end of the experiment, the treated versus control values (i.e., relative volume of treated tumors divided by the relative volume of control tumors x 100) of 50, 100, 200, and 400 µg treated groups were 60%, 53%, 19%, and 4.3%, respectively. Rapid regression of SC-3 tumors were observed in the maximum dose group of KM1334 (400 µg/injection).



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  		Fig. 5. Antitumor activity of KM1334 against SC-3 tumors established on nude mice. SC-3 cells were transplanted s.c. into adult male nude mice. SC-3 tumors were allowed to grow ~300 mm3; then, KM1334 [50 µg/head (x), 100 µg/head ({lozenge}), 200 µg/head ({triangleup}), or 400 µg/head ({circ})] and PBS ({bullet}), a vehicle control, were given into the mice twice weekly (total of nine times). Tumor volume was measured until day 38. Arrows below the transverse axis, days for antibody treatment. Points, mean for tumor volume (n = 5 each group); bars, SD. *, P < 0.01, relative to control, two-tailed Student's t test.

 
To understand the mechanism by which tumors treated by 400 µg KM1334 regressed rapidly in vivo, histologic analysis was done on tumor sections from SC-3 tumor model, and tumor sections were stained for markers of cell proliferation and apoptosis. Figure 6A shows the effect of KM1334 on cumulative BrdUrd incorporation at three time points (7, 24, and 72 hours). Twenty-four hours after treatment with KM1334, there was a remarkable reduction in BrdUrd incorporation persisting until 72 hours, which is suggestive of arrest in G0-G1. We next examined the effect of KM1334 on apoptosis over the same time course via TUNEL labeling (Fig. 6B). The number of apoptotic cells increased markedly between 7 and 24 hours and decreased to the level of control by 72 hours. These results suggested that inhibition of cell proliferation and induction of apoptosis were responsible for the dramatic regression of tumor size in vivo.



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  		Fig. 6. Histologic analysis of SC-3 tumors treated with KM1334. A, uptake of BrdUrd. Mice carrying SC-3 tumors were treated with KM1334 ({bullet}) or KM511 ({circ}), negative control. SC-3 tumors were collected before and at 7, 24, and 72 hours after antibody injection. Uptake of the BrdUrd was detected by anti-BrdUrd antibody. B, TUNEL staining. The same sample used for the BrdUrd uptake experiment was used for TUNEL staining. Labeling index indicates the number of 3,3'-diaminobenzidine-positive cells per mm2. Points, mean for three independent areas; bars, SD. *, P < 0.05, relative to control, two-tailed Student's t test (A and B).

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
To our knowledge, this is the first report that describes mAb against FGF8 for therapeutic use. We showed that an anti-FGF8b murine mAb, KM1334, suppressed SC-3 tumor growth dose-dependently and the maximum dose injection of the antibody caused rapid regression of SC-3 tumors established in nude mice accompanied by inhibition of cell proliferation and induction of apoptosis. KM1334 showed very high specificity to FGF8b and FGF8f out of FGF family members examined and potent blocking activity of FGF8b binding to its three receptors (FGFR2IIIc, FGFR3IIIc, and FGFR4) and FGF8b-induced downstream signaling. These results provide the rationale for further development of KM1334 as a candidate anticancer therapeutic agent against FGF8b-dependent tumors.

Contribution of FGFs, including FGF2, FGF5, FGF8, etc., as autocrine and/or paracrine growth factors in tumorigenesis have been widely studied (31, 42); however, there are only limited reports that showed the potential of anti-FGF ligand neutralizing antibodies as candidates of therapeutic agents. Although several groups reported that anti-FGF2 antibodies showed antitumor activities in vivo (43, 44), they were not so potent as observed in this study. We show for the first time that FGF ligands are very potent targets for antibody-based cancer therapy.

Many studies reported up-regulation of FGF8 in various clinical cancers, including prostate and breast cancers, and the strongest mitogenic isoform FGF8b among human FGF8 variants (a, b, e, and f) is considered to be the most important for the progression of those diseases (22, 23, 25, 27). FGF8f has the second strongest mitogenic activity in human FGF8 isoforms (17, 31) and coexpression of FGF8f with FGF8b resulted in worse prognosis than single expression of FGF8b in esophageal carcinoma (45). Therefore, inhibition of both FGF8b and FGF8f activity might be desirable for the therapy of FGF8-associated cancers. It was already shown that KM1334 specifically recognizes FGF8b among murine FGF8 variants (a-c); however, it was unclear which human FGF8 variants KM1334 recognizes. Among synthetic peptides derived from four human FGF8 variants, FGF8b-1 (derived from FGF8b and used for the immunogen of KM1334) and FGF8f-1 (derived from FGF8f) were proven to inhibit the binding of KM1334 to its antigen, indicating that KM1334 recognize both FGF8b and FGF8f isoforms (Fig. 1C). Human FGF17 and FGF18 have been isolated recently and form a subfamily with FGF8 in FGFs. Their amino acid sequences corresponding to FGF8b-1 have significant identity to FGF8b (FGF17, 54.1%; FGF18, 20.0%; Fig. 2A); however, KM1334 did not bind to FGF17b and FGF18 (Fig. 2B). These results indicate that KM1334 possesses high specificity enough to target FGF8b and FGF8f selectively.

Although colocalization of FGF8 and its receptors was identified in various neoplastic tissues (27, 28, 45), it was still unknown which type of FGFRs are activated by FGF8. Therefore, an anti-FGF8 neutralizing mAb, which has the ability to inhibit FGF8-induced activation via all possible FGFRs, is desired. Relative mitogenic activities of FGF8 isoforms to each FGFR have already been described (17, 33); however, direct comparison of mitogenic activity or binding activity of FGF8b to FGFRs has never been reported. In this study, we could establish for the first time the ELISA system to detect direct binding of FGF8b to FGFR-Fc fusion proteins. In this system, FGFR4-Fc showed the most sensitive detection limit; however, FGFR3IIIc-Fc gave the highest value of the absorbance indicative of binding activity (Fig. 3A). The affinity of FGFR2IIIc-Fc to FGF8b was lower than that of FGFR3IIIc-Fc and FGFR4-Fc. These results were consistent with previous reports (17, 33). KM1334 inhibited the binding of FGF8b to all FGF8b receptors examined (FGFR2IIIc-Fc, FGFR3IIIc-Fc, and FGFR4-Fc) completely in the ELISA (Fig. 3B-D), which indicates that KM1334 has an ideal character as an inhibitor of FGF8b. It has already been shown that FGF8b-dependent growth of SC-3 cells is mediated by FGFR1IIIc (14, 46). However, in this study, the specific binding of FGFR1IIIc-Fc to FGF8b was not detected in the ELISA. Several explanations of this discrepancy were possible. One explanation is that mutations in FGFR1IIIc (47) facilitate the binding between FGFR1IIIc and FGF8b in SC-3 cells. Another explanation, which is considered as more likely than the preceding one, is that concentration of FGF8b has significant effects on the detection of FGFR1IIIc-FGF8b binding. In previous reports, FGF8b could activate FGFR2IIIc, FGFR3IIIc, and FGFR4 at nanomolar concentrations (17, 33). However, FGFR1IIIc needs micromolar range of FGF8b concentration for its activation (17). Therefore, the binding between FGFR1IIIc and FGF8b in the ELISA might be enhanced by increasing the amount of FGF8b coated on the ELISA plate.

FRS2{alpha} is essential for the FGF-induced MAPK response (48) and ERK is a member of the MAPK implicated in the regulation of cell proliferation and differentiation. FGF8b treatment with SC-3 cells induced phosphorylation of both FRS2{alpha} and ERK1/2 (Fig. 4A), indicating that the FGF8b-induced proliferation of SC-3 cells is mediated by FGFR(s)-FRS2{alpha}-ERK1/2 signal transduction pathway. Together with the fact that KM1334 blocked this FGF8b-induced phosphorylation of FRS2{alpha} and ERK1/2 in SC-3 cells (Fig. 4A), FGF8b-neutralizing activity of KM1334 on SC-3 cell growth could be explained by the inhibition of FGF8b binding to its receptors and subsequent blockade of FGFR-mediated signal transduction.

Regression of SC-3 tumors by KM1334 treatment was explained by inhibition of cell proliferation and induction of apoptosis (Fig. 6A and B), which were well consistent with in vitro results shown in Fig. 4B and C. Together with the fact that that the antitumor effect of KM1334 was equal to castration (data not shown), it was suggested that FGF8b is critical to the androgen-dependent growth of SC-3 cells in vivo as well as in vitro. These results indicate that KM1334 might become a good therapeutic reagent against FGF8b-dependent tumors. In clinical prostate carcinoma in which androgen ablation therapy was effective, increase of the apoptotic index and decrease of mitotic index of tumors were detected (49, 50). Although the period of treatment was different, the same phenomenon was observed in the regressing SC-3 tumors treated with KM1334 (Fig. 6). Because the expression of FGF8 was identified in hormone-refractory prostate cancer as well as in hormone-responsive cancer (15), KM1334 may also be effective against hormone-refractory prostate cancer. As additional supportive evidence of this possibility, we have recently shown that overexpression of FGF8b in human prostate cancer LNCaP cells gave growth advantage to the cells in vitro and in vivo, and KM1334 could inhibit tumor growth in xenograft models of FGF8b-transfected LNCaP cells under androgen-dependent and androgen-independent conditions.5 Further analysis, including additional in vivo xenograft studies and future clinical studies, might be needed to fully address the importance and contribution of FGF8b in human cancers.

Because amino acid sequences of mouse and human FGF8b are 100% identical (16), KM1334 could block endogenous FGF8b in mice. Administration of KM1334 caused no apparent toxicity, like weight loss or death to mice (data not shown), suggesting that targeting FGF8b in adult human might not cause severe side effects, although further studies are needed to fully analyze the side effects.


   Footnotes
 
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.

4 M. Koike et al. Anti-human IL-5R{alpha} neutralizing monoclonal antibodies, manuscript in preparation. Back

5 K. Maruyama-Takahashi et al. An anti–fibroblast growth factor (FGF) 8 monoclonal antibody, KM1334, inhibits the growth of FGF8b-overexpressing LNCaP xenografts under androgen-dependent and -independent conditions, submitted for publication. Back

Received 11/18/04; revised 2/16/05; accepted 2/17/05.


   References
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 Abstract
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 References
 

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