Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules
Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules
复制标题
DOI：
10.1093/protein/gzq118
复制
发表时间：
2011-04-01
影响因子：
2.4
通讯作者：
Lofblom, John
中科院分区：
文献类型：
--
作者：
Kronqvist, Nina;Malm, Magdalena;Lofblom, John
新的证据表明，催化失活的 ErbB3 (HER3) 蛋白在正常酪氨酸激酶受体信号传导以及这些信号传导途径的异常功能中发挥着重要作用，从而导致多种形式的人类癌症。 ErbB3 最近也被认为与 ErbB2 靶向疗法的耐药性有关。在这里，我们报告了高亲和力 ErbB3 特异性 Affibody 分子的生成，旨在用于未来的分子成像和生物治疗应用。使用高复杂性噬菌体展示的 Affibody 文库，分离了许多 ErbB3 结合物，并在免疫荧光显微镜研究中证明了特异性细胞结合活性。随后，根据噬菌体展示选择的候选序列构建了第二代文库。通过利用新型细菌表面展示技术的灵敏亲和力辨别能力，候选 Affibody 分子的亲和力进一步增加至亚纳摩尔亲和力。总之，天然 ErbB3 受体对人类癌细胞系的特异性靶向以及与调蛋白/ErbB3相互作用的竞争表明这些新型生物制剂可能成为针对表达 ErbB3 的癌症的诊断和治疗靶向的有用工具。我们的研究还强调了结合不同显示技术的优点来生成功能性高亲和力蛋白质结合物的强大方法。讨论了未来潜在的应用，例如基于放射性核素的人类癌症诊断和治疗。
Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.