Construction of a disulfide-stabilized diabody against fibroblast growth factor-2 and the inhibition activity in targeting breast cancer.

Angiogenesis plays a pivotal role in tumor growth, progression and metastasis.1 Strategies targeting angiogenesis have been extensively studied, providing substantial data supporting the potential of angiogenesis targeting for cancer therapy and prevention.2 Among many angiogenic factors overexpressed in tumors, the fibroblast growth factor 2 (FGF‐2) and the vascular endothelial growth factor (VEGF) are the most important factors.3 Agents that target VEGF and its receptors have shown promising activity in clinical trials and clinic therapy. The anti‐VEGF monoclonal antibody (mAb) bevacizumab (Avastin; Genentech, South San Francisco, CA, USA) approved by the Food and Drug Administration of America (2004) has demonstrated good therapeutic effects in cancer therapy.4 However, when used for a long time, patients may develop resistance to this antibody drug. One proposed mechanism of tumor escape from anti‐VEGF therapy is the compensatory upregulation of fibroblast growth factor‐2 (FGF‐2) and platelet derived growth factor (PDGF).5 Furthermore, as stated above, FGF‐2 blockade could impair tumor progression in the evasion phase of anti‐VEGF therapy.6 Fibroblast growth factor‐2 (also named basic fibroblast growth factor, bFGF) is a pleiotropic angiogenesis inducer belonging to the family of the heparin‐binding FGF growth factors.7 FGF‐2 is highly expressed in numerous tumors and exerts its proangiogenic activity by interacting with tyrosine kinase receptors, heparin‐sulfate proteoglycans and integrins expressed on the endothelial cells.8 FGF‐2/FGFR interaction leads to complex signal transduction pathways (including MAPK/ERK and PI3K/AKT) and activation of a “proangiogenic phenotype” in endothelium, which regulates proliferation, migration and survival of tumor cells.9, 10 Blocking FGF‐2/FGFR activity with antibodies should be a good therapeutic strategy, especially in tumor therapy.11 The anti‐FGF‐2 murine mAbs can strongly inhibit tumor growth.11, 12, 13, 14, 15 An FGF‐2‐neutralizing mAb was reported to inhibit angiogenesis induced by FGF‐2 both in vitro and in vivo,13 and block vascularization and growth of chondrosarcoma in the rat.14 Li (2010) reports that anti‐bFGF‐neutralizing mAbs could inhibit the growth of melanoma and tumor angiogenesis in vivo.16 In our previous study, high affinity human antibodies of single chain Fv fragment (scFv) against FGF‐2 were selected from a phage display library, and a full‐length human antibody against FGF‐2 was reconstructed.17 The anti‐FGF‐2 human antibody could inhibit the proliferation and migration of glioma cells, and tube formation of vascular endothelia cells.17 Many attempts to reduce the size of the heterotetrameric IgG molecule (MW: 160 kDa) while retaining its antigen‐binding properties have been made in relation to some critical factors for therapeutic antibodies (such as high‐yield production, solubility, stability and small size).18 This resulted in a series of antibody fragment constructs, such as diabody, which is a non‐covalently associated bivalent molecule, created from scFv by shortening the polypeptide linker between the VH and VL domains.19 Some diabodies were unstable and some showed lower affinity than full‐length antibodies or Fabs because the diabody was non‐covalently associated or the linker may have interfered with the antigen binding.20 The best way to stabilize the diabody is to introduce the disulphide bond in the framework of VH and VL domains.21, 22, 23, 24 Here, we report on the construction of the ds‐Diabody against FGF‐2 based on an scFv, the secretory expression of the target protein in Picha pastoris, and the effects on tumor angiogenesis and tumor growth of breast cancer.