A novel monoclonal antibody to fibroblast growth factor 2 effectively inhibits growth of hepatocellular carcinoma xenografts

Abstract

Expression of fibroblast growth factor 2 (FGF2) is believed to be a contributing factor to the growth of a number of tumor types, including hepatocellular carcinoma (HCC). However, the potential of monoclonal antibodies that neutralize FGF2 for treatment of patients with cancer has not yet been explored in clinical trials. We therefore generated a novel monoclonal antibody (mAb), GAL-F2, specific for FGF2 and characterized its properties in vitro and in vivo. GAL-F2 binds to a different epitope than several previous anti-FGF2 mAbs tested. This novel epitope was defined using chimeric FGF1/FGF2 proteins and alanine scanning mutagenesis and was shown to comprise amino acids in both the amino and carboxy regions of FGF2. GAL-F2 blocked binding of FGF2 to each of its four cellular receptors, strongly inhibited FGF2-induced proliferation and downstream signaling in human umbilical vein endothelial cells, and inhibited proliferation and downstream signaling in two HCC cell lines. Moreover, GAL-F2, administered at 5 mg/kg i.p. twice weekly, potently inhibited growth of xenografts of the SMMC-7721, HEP-G2, and SK-HEP-1 human HCC cell lines in nude mice, and in some models, had a strong additive effect with an anti-VEGF mAb or sorafenib. Treatment with GAL-F2 also blocked angiogenesis and inhibited downstream cellular signaling in xenografts, indicating its antitumor mechanism of action. Our report supports clinical testing of a humanized form of the GAL-F2 mAb for treatment of HCC and potentially other cancers.

Figure 1

A, Binding of GAL-F2, other anti-FGF2 mAbs and negative control mAb mIgG to human FGF2 measured by ELISA; B, Competitive binding ELISA of anti-FGF2 mAbs against biotinylated GAL-F2; C, Binding ELISA of GAL-F2 to human FGF2 (hFGF2) and mouse FGF2 (mFGF2). In C, the curves for control mIgG binding superimpose and cannot be distinguished.

Figure 2

Inhibition of binding of FGF2 to each FGFR by GAL-F2 and other anti-FGF2 mAbs, measured by ELISA. A, FGFR1IIIc; B, FGFR2IIIc; C, FGFR3IIIc; D, FGFR4.

Figure 3

A, Inhibition of FGF2-induced proliferation of HUVEC by GAL-F2 and other anti-FGF2 mAbs. The error bars are S.D. B, Western blot of HUVEC incubated with or without FGF2 and GAL-F2. The blot was stained with mAbs detecting p-Akt (1), Akt (2), p-Erk1/2 (3), Erk1/2 (4). By densitometry, the relative intensities of the p-Akt bands, normalized to FGF2+ with no mAb, were from left to right: 32, 100, 35, 87, 14, 23. C, RT-PCR analysis of FGFR mRNA expression in HCC cell lines HEP-G2 (1), SMMC-7721 (2) and SK-HEP-1 (3), using primers specific for each of the FGFRs. D, E, Western blot of HEP-G2 and SMMC-7721 cells incubated with or without GAL-F2. The blot was stained with mAbs detecting p-Erk1/2 (1), Erk1/2 (2) and Hsp70 (3).

Figure 4

Relative binding of GAL-F2, bFM-1 and 3H3 to FGF2 or FGF1 or chimeric FGF2-FGF1 proteins (A), and of GAL-F2 to alanine mutants of FGF2 (B), measured by ELISA. Binding of each mAb to wildtype (WT) FGF2 is set as 100%. The means of triplicate values are shown; there was little variation between triplicates. C, Ribbon diagram of the crystallographic structure of FGF2 complexed with the extracellular D2 and D3 domains of FGFR2 (PDB ID 1IIL, ref. 28), with the indicated amino acids shown in space-filling form (R31, Y33, K55 and S152 in yellow; F40L and M151, which eliminate GAL-F2 binding, in red). The leucine mutant was used at F40 because the alanine mutant could not be expressed.

Figure 5

Inhibition of growth of SMMC-7721 (A – D), HEP-G2 (E – G) and SK-HEP-1 (H) HCC xenografts by the indicated agents compared to negative control mAb mIgG. In the legends, “Both” indicates that GAL-F2 and the other listed agent were both administered. The means of groups of 5–7 mice are shown; the error bars are S.E.M. GAL-F2 and A.4.6.1 were administered i.p. at 5 mg/kg twice per week, cisplatin at 6 mg/kg i.p. once per week, and sorafenib orally at 20 mg/kg five times per week. Panels A and D are from the same experiment but shown separately for greater visual clarity.

Figure 6

A, Western blot of whole cell lysates from SMMC-7221 xenografts in individual mice treated with GAL-F2 mAb or control mouse mAb mIgG, stained with antibodies to the indicated proteins. The relative intensities of the p-Erk1/2 bands determined by densitometry, normalized to the highest value (mouse #43), are shown above the blot. B, Representative fields from cryostat sections of HEP-G2 xenografts from mice treated with GAL-F2 or mIgG, stained with anti-CD31 mAb to detect blood vessels, counterstained with Mayer's hematoxylin, and photographed at 100×.