title: Antagonistic VEGF variants engineered to simultaneously bind to and inhibit VEGFR2 and alphavbeta3 integrin.
abstract: Significant cross-talk exists between receptors that mediate angiogenesis, such as VEGF receptor-2 (VEGFR2) and α(v)β(3) integrin. Thus, agents that inhibit both receptors would have important therapeutic potential. Here, we used an antagonistic VEGF ligand as a molecular scaffold to engineer dual-specific proteins that bound to VEGFR2 and α(v)β(3) integrin with antibody-like affinities and inhibited angiogenic processes in vitro and in vivo. Mutations were introduced into a single-chain VEGF (scVEGF) ligand that retained VEGFR2 binding, but prevented receptor dimerization and activation. Yeast-displayed scVEGF mutant libraries were created and screened by high-throughput flow cytometric sorting to identify several variants that bound with high affinity to both VEGFR2 and α(v)β(3) integrin. These engineered scVEGF mutants were specific for α(v)β(3) integrin and did not bind to the related integrins α(v)β(5), α(iib)β(3), or α(5)β(1). In addition, surface plasmon resonance and cell binding assays showed that dual-specific scVEGF proteins can simultaneously engage both receptors. Compared to monospecific scVEGF mutants that bind VEGFR2 or α(v)β(3) integrin, dual-specific scVEGF proteins more strongly inhibited VEGF-mediated receptor phosphorylation, capillary tube formation, and proliferation of endothelial cells cultured on Matrigel or vitronectin-coated surfaces. Moreover, dual specificity conferred strong inhibition of VEGF-mediated blood vessel formation in Matrigel plugs in vivo, whereas monospecific scVEGF mutants that bind VEGFR2 or α(v)β(3) integrin were only marginally effective. Instead of relying on antibody associating domains or physical linkage, this work highlights an approach to creating dual-specific proteins where additional functionality is introduced into a protein ligand to complement its existing biological properties.
authors.affiliation: Department of Bioengineering, Cancer Institute, Bio-X Program, Stanford University, Stanford, CA 94305, USA.
authors.forename: Niv
authors.initials: N
authors.lastname: Papo
authors.forename: Adam P
authors.initials: AP
authors.lastname: Silverman
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Lahti
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Cochran
mesh_terms: D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D050505:Mutant Proteins / Q000378:metabolism / Q000494:pharmacology*; D018919:Neovascularization, Physiologic / Q000187:drug effects; D019151:Peptide Library; D011485:Protein Binding / Q000187:drug effects; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D042442:Vascular Endothelial Growth Factors / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D039302:Integrin alphaVbeta3; D008024:Ligands; D050505:Mutant Proteins; D019151:Peptide Library; D011994:Recombinant Proteins; D042442:Vascular Endothelial Growth Factors; D040301:Vascular Endothelial Growth Factor Receptor-2