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.
Highlights
Integrins are important adhesion molecules that regulate tumor and endothelial cell survival, proliferation and migration
We have previously shown that this antibody elicits cell death in dividing endothelial cells, inhibits angiogenesis in a cynomolgus monkey model of choroidal neovascularization [21] and slows tumor growth in a rabbit VX2 carcinoma model [22]
Generation and characterization of rat anti-mouse integrin α5β1 antibodies To generate antibodies directed against murine integrin α5β1, Sprague-Dawley rats were immunized with mouse α5β1-Fc fusion protein or with affinity-purified integrin from mouse placenta
Summary
Integrins are important adhesion molecules that regulate tumor and endothelial cell survival, proliferation and migration. Tumors secrete multiple growth factors that drive activation, migration and proliferation of vascular endothelial cells (EC's), including TGF-α, bFGF and VEGF [9]. These agents bind their respective receptors on EC's to initiate signaling cascades that culminate in pro-angiogenic events. Tumor cells and tumor-associated macrophages are known to secrete matrix metalloproteinases, such as MMP-9 and MMP-2 [10] These enzymes degrade the basement membrane, exposing components of the extracellular matrix, including fibronectin. These exposed ECM proteins drive angiogenesis by ligating integrins, which play a central role in the angiogenic program [11,12,13,14]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
