Albumin-binding fusion partners are frequently used as a means for the in vivo half-life extension of small therapeutic molecules that would normally be cleared very rapidly from circulation. However, in applications where small size is key, fusion to an additional molecule can be disadvantageous. Albumin-derived affinity proteins (ADAPTs) are a new type of scaffold proteins based on one of the albumin-binding domains of streptococcal protein G, with engineered binding specificities against numerous targets. Here, we engineered this scaffold further and showed that this domain, as small as 6 kDa, can harbor two distinct binding surfaces and utilize them to interact with two targets simultaneously. These novel ADAPTs were developed to possess affinity toward both serum albumin as well as another clinically relevant target, thus circumventing the need for an albumin-binding fusion partner. To accomplish this, we designed a phage display library and used it to successfully select for single-domain bispecific binders toward a panel of targets: TNFα, prostate-specific antigen (PSA), C-reactive protein (CRP), renin, angiogenin, myeloid-derived growth factor (MYDGF), and insulin. Apart from successfully identifying bispecific binders for all targets, we also demonstrated the formation of the ternary complex consisting of the ADAPT together with albumin and each of the five targets, TNFα, PSA, angiogenin, MYDGF, and insulin. This simultaneous binding of albumin and other targets presents an opportunity to combine the advantages of small molecules with those of larger ones allowing for lower cost of goods and noninvasive administration routes while still maintaining a sufficient in vivo half-life.

中文翻译：

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用于同时靶标结合和白蛋白相关半衰期延长的小型双特异性亲和蛋白

白蛋白结合融合伴侣经常被用作体内通常会很快从循环中清除的小治疗分子的半衰期延长。然而，在小尺寸是关键的应用中，融合到额外的分子可能是不利的。白蛋白衍生亲和蛋白 (ADAPT) 是一种新型支架蛋白，基于链球菌蛋白 G 的白蛋白结合结构域之一，具有针对众多靶点的工程结合特异性。在这里，我们进一步设计了这个支架，并表明这个小至 6 kDa 的结构域可以拥有两个不同的结合表面，并利用它们同时与两个目标相互作用。这些新的 ADAPT 被开发为对血清白蛋白以及另一个临床相关靶点具有亲和力，从而避免了对白蛋白结合融合伴侣的需求。要做到这一点，我们设计了一个噬菌体展示文库，并使用它成功地选择了针对一组靶标的单域双特异性结合物：TNFα、前列腺特异性抗原 (PSA)、C 反应蛋白 (CRP)、肾素、血管生成素、髓源性生长因子（MYDGF）和胰岛素。除了成功识别所有靶标的双特异性结合剂外，我们还证明了由 ADAPT 与白蛋白和五个靶标（TNFα、PSA、血管生成素、MYDGF 和胰岛素）中的每一个组成的三元复合物的形成。白蛋白和其他靶点的这种同时结合提供了一个机会，可以将小分子的优点与大分子的优点结合起来，从而降低商品成本和无创给药途径，同时仍然保持足够的体内半衰期。