A function blocking anti-mouse integrin alpha5beta1 antibody inhibits angiogenesis and impedes tumor growth in vivo.
BACKGROUND
Integrins are important adhesion molecules that regulate tumor and endothelial cell survival, proliferation and migration. The integrin alpha5beta1 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 alpha5beta1, precluding its use in standard mouse xenograft models.


METHODS
We generated a panel of rat-anti-mouse alpha5beta1 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 alpha5beta1 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.


RESULTS
A panel of antibodies was characterized for specificity and potency. The affinity of antibody 339.1 for mouse integrin alpha5beta1 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.


CONCLUSIONS
The results herein demonstrate that 339.1, like volociximab, exhibits potent anti-alpha5beta1 activity and confirms that inhibition of integrin alpha5beta1 impedes angiogenesis and slows tumor growth in vivo.
Department of Research, PDL Biopharma, Inc,, Fremont, CA 94555, USA. vinay.bhaskar@pdl.com
Vinay
V
Bhaskar
Dong
D
Zhang
Melvin
M
Fox
Pui
P
Seto
Melanie H L
MH
Wong
Pauline E
PE
Wales
David
D
Powers
Debra T
DT
Chao
Robert B
RB
Dubridge
Vanitha
V
Ramakrishnan
D020533:Angiogenesis Inhibitors / Q000276:immunology*; D000818:Animals; D017304:Annexin A5 / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D000970:Antineoplastic Agents / Q000627:therapeutic use; D016923:Cell Death; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005353:Fibronectins / Q000037:antagonists & inhibitors / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000276:immunology; D039081:Integrin alpha5beta1 / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use; D051379:Mice; D016513:Mice, SCID; D010920:Placenta / Q000276:immunology; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000276:immunology; D014183:Transplantation, Heterologous
D020533:Angiogenesis Inhibitors; D017304:Annexin A5; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D005353:Fibronectins; D007141:Immunoglobulin Fc Fragments; D039081:Integrin alpha5beta1; D011993:Recombinant Fusion Proteins; C516631:volociximab