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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Antagonistic VEGF variants engineered to simultaneously bind to and inhibit VEGFR2 and α_vβ_3 integrin
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Antagonistic VEGF variants engineered to simultaneously bind to and inhibit VEGFR2 and α_vβ_3 integrin

机译：拮抗VEGF变异体可同时结合和抑制VEGFR2和α_vβ_3整联蛋白

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摘要

Significant cross-talk exists between receptors that mediate angio-genesis, such as VEGF receptor-2 (VEGFR2) and a_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_(iib)β_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 en-dothelial 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.
机译：介导血管生成的受体（例如VEGF受体2（VEGFR2）和a_vβ_3整联蛋白）之间存在明显的串扰。因此，抑制两种受体的药物将具有重要的治疗潜力。在这里，我们使用拮抗性的VEGF配体作为分子支架来工程化双特异性蛋白，该蛋白与VEGFR2和α_vβ_3整联蛋白结合，具有类似抗体的亲和力，并在体内外抑制血管生成过程。将突变引入单链VEGF（scVEGF）配体中，该配体保留了VEGFR2结合，但阻止了受体二聚化和激活。创建酵母展示的scVEGF突变体文库，并通过高通量流式细胞术筛选以鉴定与VEGFR2和α_vβ_3整联蛋白均具有高亲和力的几种变体。这些工程改造的scVEGF突变体对α_vβ_3整联蛋白具有特异性，并且不与相关的整联蛋白α_vβ_5，α_（iib）β_3或α_5β_1结合。此外，表面等离振子共振和细胞结合试验表明，双特异性scVEGF蛋白可以同时与两个受体结合。与结合VEGFR2或α_vβ_3整联蛋白的单特异性scVEGF突变体相比，双特异性scVEGF蛋白更强烈地抑制VEGF介导的受体磷酸化，毛细管形成以及在基质胶或玻连蛋白包被的表面上培养的上皮细胞的增殖。此外，双重特异性在体内对基质胶塞中的VEGF介导的血管形成具有很强的抑制作用，而结合VEGFR2或α_vβ_3整联蛋白的单特异性scVEGF突变体则只是微不足道的。这项工作着重强调了一种创建双特异性蛋白质的方法，该方法将其他功能引入蛋白质配体中以补充其现有的生物学特性，而不是依赖于抗体缔合域或物理连接。

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