A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth in vivo

Abstract

BackgroundIntegrins are important adhesion molecules that regulate tumor and endothelial cell survival, proliferation and migration. The integrin α5β1 has been shown to play a critical role during angiogenesis. An inhibitor of this integrin, volociximab (M200), inhibits endothelial cell growth and movement in vitro, independent of the growth factor milieu, and inhibits tumor growth in vivo in the rabbit VX2 carcinoma model. Although volociximab has already been tested in open label, pilot phase II clinical trials in melanoma, pancreatic and renal cell cancer, evaluation of the mechanism of action of volociximab has been limited because this antibody does not cross-react with murine α5β1, precluding its use in standard mouse xenograft models.MethodsWe generated a panel of rat-anti-mouse α5β1 antibodies, with the intent of identifying an antibody that recapitulated the properties of volociximab. Hybridoma clones were screened for analogous function to volociximab, including specificity for α5β1 heterodimer and blocking of integrin binding to fibronectin. A subset of antibodies that met these criteria were further characterized for their capacities to bind to mouse endothelial cells, inhibit cell migration and block angiogenesis in vitro. One antibody that encompassed all of these attributes, 339.1, was selected from this panel and tested in xenograft models.ResultsA panel of antibodies was characterized for specificity and potency. The affinity of antibody 339.1 for mouse integrin α5β1 was determined to be 0.59 nM, as measured by BIAcore. This antibody does not significantly cross-react with human integrin, however 339.1 inhibits murine endothelial cell migration and tube formation and elicits cell death in these cells (EC50 = 5.3 nM). In multiple xenograft models, 339.1 inhibited the growth of established tumors by 40–60% (p < 0.05) and this inhibition correlates with a concomitant decrease in vessel density.ConclusionThe results herein demonstrate that 339.1, like volociximab, exhibits potent anti-α5β1 activity and confirms that inhibition of integrin α5β1 impedes angiogenesis and slows tumor growth in vivo.

Fibronectin Induces Endothelial Cell Migration through β1 Integrin and Src-dependent Phosphorylation of Fibroblast Growth Factor Receptor-1 at Tyrosines 653/654 and 766

“…Indeed, genetic deletion of FN leads to embryonic death due to the indispensible role of FN in developmental branching morphogenesis (42)(43)(44). Prior studies in endothelial cells have revealed that the FN-integrin complex is requisite for FN-induced chemotaxis and angiogenesis (45)(46)(47)(48), with both ␣v␤3 and ␣5␤1 implicated in this process (35,36,39). Another study on IGF-I receptor also found FN may transactivate this receptor via ␤3 integrin to protect cells from apoptosis (49).…”

Integrins and metastasis

“…Volociximab is found to inhibit angiogenesis and hinder tumor growth. 102,103 Clinical efficacy and tolerance of volociximab was found satisfactory in phase I trial. 104 This observation led this to phase II trial for solid tumor.…”

α2β1 integrin controls association of Rac with the membrane and triggers quiescence of endothelial cells

“…Genetic deletion of 5 integrin in mice leads to embryonic death owing to extensive vascular and mesodermal defects (Francis et al, 2002). Consistently, 5 integrin antagonists inhibit angiogenesis induced by tumors or growth factors in adult mice (Bhaskar et al, 2007;Kim et al, 2000). Furthermore, 51 integrin was shown to influence endothelial cell survival in vivo (Kim et al, 2002) and induces endothelial cell proliferation (Mettouchi et al, 2001).…”