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

<p>Abstract</p> <p>Background</p> <p>Integrins 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 <it>in vitro</it>, independent of the growth factor milieu, and inhibits tumor growth <it>in vivo </it>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.</p> <p>Methods</p> <p>We 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 <it>in vitro</it>. One antibody that encompassed all of these attributes, 339.1, was selected from this panel and tested in xenograft models.</p> <p>Results</p> <p>A 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% (<it>p </it>< 0.05) and this inhibition correlates with a concomitant decrease in vessel density.</p> <p>Conclusion</p> <p>The 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 <it>in vivo</it>.</p

A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth -5

<p><b>Copyright information:</b></p><p>Taken from "A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth "</p><p>http://www.translational-medicine.com/content/5/1/61</p><p>Journal of Translational Medicine 2007;5():61-61.</p><p>Published online 27 Nov 2007</p><p>PMCID:PMC2235829.</p><p></p>reated with 339.1 (10 mg/kg, intraperitoneally, thrice or twice weekly, respectively) or vehicle control and tumor volume was monitored using vernier calipers. 339.1 inhibits tumor growth relative to control. Results were statistically significant in both settings. A673 tumors were resected from 339.1- and control-treated mice and frozen sections were assessed for vessel density by immunohistochemical staining for CD31, (C). Vessel density was significantly reduced in tumors from animals treated with 339.1, (D)

A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth -1

<p><b>Copyright information:</b></p><p>Taken from "A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth "</p><p>http://www.translational-medicine.com/content/5/1/61</p><p>Journal of Translational Medicine 2007;5():61-61.</p><p>Published online 27 Nov 2007</p><p>PMCID:PMC2235829.</p><p></p> Fc fusion proteins, (A). A subset of competitive antibodies cross-reacted with human integrin. Antibodies were tested by immunohistochemistry for staining of sections from C32 melanoma (α5β1 negative) or MDA-MB-231 breast carcinoma (α5β1 positive) xenografts, (B). The majority of antibodies tested stained murine α5β1 on tumor vasculature, but only antibodies found to cross-react with human α5β1 by ELISA specifically stained MDA-MB-231 xenograft cells as well. IIA1, the mouse parent antibody of volociximab, which recognizes only human integrin, anti-mouse CD31, which stains mouse vessels, and pooled rat IgG are shown as controls

A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth -3

<p><b>Copyright information:</b></p><p>Taken from "A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth "</p><p>http://www.translational-medicine.com/content/5/1/61</p><p>Journal of Translational Medicine 2007;5():61-61.</p><p>Published online 27 Nov 2007</p><p>PMCID:PMC2235829.</p><p></p> assessed for Annexin V-Alexa Fluor 488 binding by flow cytometry. Cells were counterstained with propidium iodide (PI) to follow non-specific death and the percentage of cells that stained positive for Annexin V and negative for PI was plotted. Representative results from three individual experiments are shown, (A). Only antibody 339.1, which does not cross-react with human α5β1, elicited cell death in these cells. A dose response curve for 339.1 in this assay using SVR murine angiosarcoma cells revealed an ECof 5.3 nM, (A)

A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth -2

<p><b>Copyright information:</b></p><p>Taken from "A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth "</p><p>http://www.translational-medicine.com/content/5/1/61</p><p>Journal of Translational Medicine 2007;5():61-61.</p><p>Published online 27 Nov 2007</p><p>PMCID:PMC2235829.</p><p></p> the opposite side of transwell membranes in the presence or absence of anti-integrin antibodies. Cells were stained with Calcein AM and visualized by fluorescence microscopy. Migration in quintiplicate wells was quantified using the Discovery-1 system, (B). Antibodies that inhibited binding of α5β1 binding to fibronectin also inhibited migration towards fibronectin