Antagonistic VEGF variants engineered to simultaneously bind to and inhibit VEGFR2 and α vβ 3 integrin

Niv Papo, Adam P. Silverman, Jennifer L. Lahti, Jennifer R. Cochran

Research output: Contribution to journalArticlepeer-review


Significant cross-talk exists between receptors that mediate angiogenesis, such as VEGF receptor-2 (VEGFR2) and α vβ 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 vβ 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 vβ 3 integrin. These engineered scVEGF mutants were specific for α vβ 3 integrin and did not bind to the related integrins α vβ 5, α iibβ 3, or α 5β 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 vβ 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 vβ 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.

Original languageAmerican English
Pages (from-to)14067-14072
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number34
StatePublished - 23 Aug 2011
Externally publishedYes


  • Bispecific protein
  • Directed evolution
  • Protein engineering
  • Yeast surface display

All Science Journal Classification (ASJC) codes

  • General


Dive into the research topics of 'Antagonistic VEGF variants engineered to simultaneously bind to and inhibit VEGFR2 and α vβ 3 integrin'. Together they form a unique fingerprint.

Cite this